universidade de faculdade ciências tamento de biologia a...
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BEHAVFOOD A
VIOURAL E
AND WAT
Joã
Me
U
Depar
EFFECTS
TER REGA
NA
ão Pedro
strado e
Univers
Faculda
rtament
OF CHRO
ARDING A
AÏVE C57
o da Co
em Biol
idade d
ade de C
to de Bi
ONIC ADM
ANXIETY A
7BL/6N
sta Alva
ogia Hu
2011
e Lisboa
Ciências
ologia A
MINISTRA
AND DEPR
MALE MI
ares Vie
umana e
a
s
Animal
ATION OF
RESSION
ICE
egas Nu
e Ambie
IMIPRAM
PARADIG
nes
ente
MINE IN
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BEHAV
AND
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D WATER R
Joã
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Depa
EFFECTS OF
REGARDIN
ão Pedro
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Univers
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F CHRONIC
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C57BL
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sidade de
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sta Alva
r. Tatyana
rno: Dr. Jo
logia Hu
2011
e Lisboa
Ciências
ologia An
STRATION
PRESSION
LE MICE
ares Vie
a Strekalo
odi Pawlus
mana e A
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N OF IMIPR
N PARADIG
egas Nu
ova, MD, P
ski, Ph.D
Ambient
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a
“No one is poor except he who lacks knowledge”
Rabi Hyman Krustofski, The Simpsons, 1991
b
Acknowledgements
To my Family, who has supported and given me good advice through the whole process of my
Masters (especially while abroad) and throughout my life, to whom I owe being able to proceed
with my studies and do what I like.
To Prof. Dr. Orlando Luís, Prof. Dr. Luís Vicente and Prof. Dr. José Carlos Dionísio for
awakening my interest for animal physiology, behaviour, neurobiology and neurochemistry, which
were the driving force to start researching in this field.
To Dr. Tatyana Strekalova, for accepting me as her master student, for enduring in thorough
reviews of my work over and over (and over) again even on tight schedule, for all the constructive
critics, for her trust and involvement in so many projects, for all I have learnt from her, for support
in Maastricht and all opportunities she has organized for my training in the Netherlands and Lisbon
and for making me grow both as an individual and as a scientist.
To Dr. Jodi Pawluski for accepting co-orientation of the thesis, for always being available to help,
for all the observations, remarks and suggestions that improved this work and for all I have learned
from her.
To Prof. Dr. Deodália Dias and Prof. Dr. Ana M Crespo, for all the support, availability and for
allowing me to have part of my thesis done abroad.
To Professor Dr. Harry WM Steinbusch, for welcoming me to Maastricht University, for all the
support, the activities and advice given.
To Luís Marques, for his technical support and for sharing working space at Faculty of Sciences of
the University of Lisbon and to Marisela Martínez Claros, for all the guidance, experience,
patience and advice given me during my stay in Maastricht University.
To Prof. Dr. Ana Isabel Moura Santos, for all the support, guidance, help and thanks to whom the
execution of other projects during this year at Faculdade de Ciências Médicas da Universidade
Nova de Lisboa was possible.
c
To Dr. René F. Jansen, for introducing me to the forced swim test analysis with an on-
development phase software, for sharing ideas and experience.
To Internationale Stichting Alzheimer Onderzoek (ISAO)(the Netherlands) grant N 09501 to Dr
Tatyana Strekalova, for travelling grants and for supporting my stay in Maastricht; to Technosmart
(Roma, Italy) and Dr. Giacomo dell’Omo, for the travelling grants to Amsterdam and equipment.
To Dr. Álvaro Tavares and Dr. Cláudia Florindo for their most valuable advices on how to work
in and how to manage a scientific laboratory, on helping me to become self-sufficient and
perfectionist in everything I do.
To Margarida Correia and Andreia Valença, for being great friends, for their infinite patience
and all their help (this was also their work, they agreed on having me writing it) and for making life
in Maastricht so much easier.
To Julie Vignisse for her great friendship, sense of humour, for all her help and good moments both
in Lisbon and Maastricht. To Rodrigo C. Marques, a great friend, “muito doido”, who made
working and living far from home much lighter, easier and funnier.
To all my friends in Maastricht who made me feel like home and have fun too. Zé, Sarina, Nikki,
Berta, Carmen, Fabien, Henry, Miguel, Samsiya, Alejandro, Pablo and Leonidas, dank u!!!
To Francisco JM Teodósio, for all the “broments”, brainstormings, and good advice, for making
me stay focused on what mattered most and helping me not to forget to relax some other time. To
João FS Josué and Carlos TA Lopes for supporting and encouraging me all the way, for making
up time when I needed their help, for the good time in Maastricht and for being great friends.
A very special thanks to Margarida for all the love, help, support, patience, advice, criticism, sense
of humour and encouragement.
I also thank to all other friends and professors who contributed to this long journey for
knowledge and for being present in my life.
d
Abstract
Depression is a heterogeneous disorder with a lifetime prevalence of 15% that encompasses
high disability, social and financial burden. More studies are needed for better treatment of this
disorder, therefore pre-clinical models are important for the development of new drug candidates
and the improvement of our knowledge on available therapies. Here, the effects of the
administration via food or drinking water of a low dose of imipramine, a tricyclic antidepressant,
which is used as a reference compound in animal models of depression were tested. The objectives
were to assess whether 1) these two methods of drug delivery are effective in the induction of
behavioural effects in mouse models of depression and anxiety and 2) a selected dose of imipramine
elicits, at the same time, general effects on locomotion, water intake and body weight, which were
previously described for a higher drug dose in the same study design and considered as signs of
toxicity. To address the first question, naive C57BL6 mice were treated with imipramine (7
mg/kg/day) for 4 weeks either with drinking water and food and tested in the sucrose preference,
forced swim and tail suspension tests. Body weight, open field locomotion and novel cage activity
were evaluated in order to address the second question. First, the results of this work suggest
positive answer to both questions. Second, more pronounced effects of the antidepressant on both
types of behavioural read-outs were found in mice treated via drinking water. The present work
shows that both ways of imipramine dosing are effective in mice and that the employed dose elicits
anti-depressant and anti-anxiety effects. However, as the applied treatment results in changes in
animals’ weight, locomotion and drinking patterns that may compromise behavioural measurements
during pre-clinical studies which use it, the usefulness of such treatment in antidepressant drug
development is questioned.
Key words: Depression, imipramine, food, water, pre-clinical studies.
e
Resumo
As doenças de foro psiquiátrico são entidades clínicas complexas e heterogéneas com um
forte pendor hereditário, como é o caso da depressão e das manifestações patológicas de ansiedade.
Estas apresentam uma incidência de 20.8% e 28.8% respectivamente e uma prevalência ao longo da
vida de 15%, o que corresponde a mais de 20 milhões de pessoas afectadas por ano, acarretando
uma grande morbilidade e fardo social que corresponde a encargos financeiros superiores a 80
biliões de dólares anuais, considerando apenas os Estados Unidos. Estudos estatísticos da
Organização Mundial de Saúde prevêem que a depressão seja a segunda maior causa de
desabilitação e morbilidade em 2020, unicamente suplantada pela condição de isquémia cardíaca.
Apesar de se tratar de entidades clínicas distintas há uma elevada taxa de co-ocorrência entre
as diversas manifestações de ansiedade patológica e depressão. De acordo com um estudo
americano conduzido por Kessler e colaboradores (1994, 2004, 2005a, 2005b, 2006), 58% dos
indivíduos que experienciam depressão co-manifestam ansiedade patológica e 67% dos sujeitos
ansiosos compartilham episódios depressivos. A debilidade causada por estes estados propicia ou
está associada também à co-incidência com patologias cardiovasculares (15-25%), asma (43%),
síndromes gastrointestinais (10-25%), dor crónica (11-27%), artrite reumatóide (25-35%),
fibromialgia (60%), obesidade (37%), cancro (10-30%) e suicídio (10-15%), este último
correspondendo a cerca de um terço do total de ocorrências nos Estados Unidos.
A exposição a stresse e situações traumáticas é considerada como o principal factor de
predisposição para a ocorrência de ansiedade e depressão, razão pela qual muitos dos modelos
animais desenvolvidos compreendem uma avaliação da resposta etológica face a paradigmas como
sejam os de desespero/inescapabilidade, exposição imprevisível, stresse crónico moderado,
preferência/recompensa ou de conflito aproximação/evicção.
O tratamento destas condições patológicas no ser humano envolve farmacoterapia
suplementada por psicoterapia e medidas auxiliares como exercício físico, embora nos modelos
animais mais utilizados, dos quais 95% são roedores, apenas a primeira abordagem é sustentável
sendo a única mensurável e possível de ser modelada. O emprego de tratamento pré-clínico em
modelos animais possibilita avaliar o efeito de novos medicamentos ou expandir o conhecimento
sobre a acção de drogas actuais.
A abordagem farmacológica da depressão reside no uso de antidepressivos. Os mais comuns
pertencem a duas classes: tricíclicos e inibidores selectivos da re-internalização de serotonina. Dos
primeiros destaca-se a imipramina, descoberta no fim da década de 1950 por Ronald Kuhn, que
actua sobre a serotonina, norepinefrina, epinefrina e dopamina, mas que comporta efeitos
secundários sérios maioritariamente pela sua acção anticolinérgica. Dos segundos, introduzidos no
f
final da década de 1980, realçam-se o citalopram e a fluoxetina, especializados no bloqueio da re-
internalização da serotonina, apresentando um melhor perfil de tolerância e menos efeitos
secundários. Foram efectuados estudos em que se verificou que os antidepressivos também
manifestam, em muitos casos, acção ansiolítica.
A farmacoterapia para as diversas manifestações de ansiedade patológica recorre ao uso de
ansiolíticos como as benzodiazepinas e azapironas, as quais substituíram os barbitúricos, utilizados
essencialmente pelos seus efeitos sedativos, mas que recorrentemente comportavam efeitos
secundários graves e apresentavam uma elevada taxa de mortalidade associada.
A selecção de uma adequada via de administração e dose são essenciais pois podem influir
directamente no desempenho dos animais em situações de teste. Administrações por injecção, seja
intraperitoneal ou endovenosa permitem um efeito mais rápido do agente farmacológico, mas
requerem manipulação acrescida dos animais, causa desconforto, picos de concentração e comporta
o risco de infecção, enquanto outras abordagens mais naturais como através da comida ou bebida,
sustentam a auto-administração e um consumo mais ou menos regular, com a desvantagem de uma
mais lenta cinética de absorção e um menor controlo sobre a dose real administrada.
Neste trabalho foram testados em murganhos da estirpe C57BL/6 os efeitos da administração
através de comida ou de água de uma dose baixa de imipramina, um antidepressivo tricíclico
actualmente utilizado como tratamento de referência em modelos animais para o estudo da
depressão.
Os objectivos do presente estudo foram avaliar se 1) ambas as vias de administração descritas
são eficazes na indução de efeitos comportamentais em modelos de ansiedade e depressão em
murganhos; 2) uma dose determinada de imipramina evidencia, em simultâneo, efeitos sobre a
locomoção geral, consumo de soluções e alterações de peso nos animais, à semelhança do que foi
previamente descrito aquando do uso de uma dose mais elevada do mesmo composto num estudo
com delineamento semelhante, os quais foram interpretados como indicadores de toxicidade.
Após uma semana de ambientação ao laboratório e de uma avaliação etológica e fisiológica
inicial, 23 murganhos da estirpe C57BL/6 foram dispostos em três grupos experimentais, um sem
tratamento, outro em que uma dose de imipramina é distribuída através da homogeneização com
comida e um terceiro em que a mesma dose é dissolvida em água durante um período de quatro
semanas nas quais os animais foram sujeitos a uma bateria de testes. Findo o período de tratamento
seguiu-se uma avaliação fisiológica e etológica final, seguida da qual os animais foram sacrificados
de acordo com as normas europeias, legislação portuguesa e boas práticas estabelecidas pela
Sociedade Portuguesa de Ciência em Animais de Laboratório.
Para responder à primeira questão, os animais foram submetidos aos testes de consumo e
preferência de sacarose, de natação forçada de Porsolt, de suspensão pela cauda (depressão), da
g
arena circular elevada e da caixa preta-e-branca (ansiedade). A avaliação da variação do peso,
locomoção em arena aberta e da nova residência foram indicadores utilizados para responder à
segunda questão.
Os resultados obtidos sugerem uma resposta positiva a ambas as questões. Primeiro, o
tratamento com imipramina resultou na redução da duração dos períodos de flutuação e
imobilidade, dois parâmetros característicos de comportamentos de desespero e do tipo depressivo,
em conjunto com o incremento no tempo que os animais despenderam nas áreas expostas da arena
circular elevada e da caixa branca-e-preta, denotando também um efeito ansiolítico. Segundo, os
dados comportamentais obtidos demonstram um efeito mais pronunciado deste antidepressivo
quando administrado através da água.
Sumariamente, os dados obtidos sugerem que o tratamento com uma baixa dose de
imipramina quer através da comida, quer da água seja eficaz como método de administração
farmacológico e como tratamento antidepressivo/ansiolítico. Contudo, mostram também uma
alteração em parâmetros fisiológicos e comportamentais como o peso, locomoção geral e consumo
de líquidos que podem influir negativamente e ser responsáveis por artefactos em avaliações
etológicas realizadas em estudos pré-clínicos que utilizem imipramina como antidepressivo-padrão.
A conveniência e validade do recurso ao tratamento crónico com imipramina como antidepressivo-
padrão em estudos pré-clínicos de depressão, ainda que em doses reduzidas são então questionadas.
Trabalhos futuros podem expandir estes resultados a uma análise bioquímica e morfológica
sobre acção nos tecidos (actualmente em curso) e ter como objectivos descobrir qual a razão para
uma maior eficácia do tratamento quando administrado através da água ou se o efeito antidepressivo
que se verifica no teste de natação forçada se deve à uma inibição de memória como sugerido por
alguns autores.
Palavras-chave: Depressão, imipramina, comida, água, estudos pré-clínicos.
h
Table of contents
Acknowledgements .............................................................................................................................. b
Abstract ................................................................................................................................................ d
Resumo ................................................................................................................................................ e
Table of contents .................................................................................................................................. h
List of abbreviations............................................................................................................................. k
Figure index ........................................................................................................................................ m
Table index ........................................................................................................................................... o
Chapter I – Introduction ..................................................................................................................... 15
1.1. Objective of the study.......................................................................................................... 15
1.2. Modelling anxiety and depression in rodents ...................................................................... 15
1.3. Effects of antidepressants and anxiolytics in rodent models ............................................... 16
1.4. Methods of drug delivery in animal models ........................................................................ 17
1.4.1. Invasive methods of systemic delivery ........................................................................ 17
1.4.2. Non-invasive methods of systemic delivery ................................................................ 18
1.5. Imipramine, its biochemistry and metabolism .................................................................... 19
1.5.1. Action of imipramine on brain function ....................................................................... 19
1.5.2. Toxicity and side-effect profile .................................................................................... 19
1.5.3. The use of imipramine in animal models of anxiety and depression ........................... 20
1.6. Delivery of imipramine in animal models of depression .................................................... 20
1.7. Tests of evaluation of antidepressant- and anxiolytic-like effects of antidepressants in
rodent models ................................................................................................................................. 21
1.7.1. Methods of assessment of hedonic status in animal models ........................................ 21
1.7.2. Novel cage test ............................................................................................................. 21
1.7.3. Open field test .............................................................................................................. 22
1.7.4. Dark-light box .............................................................................................................. 22
1.7.5. Elevated plus maze ....................................................................................................... 23
i
1.7.6. Elevated / O-maze test ................................................................................................. 24
1.7.7. Forced swim test / Porsolt test ..................................................................................... 24
1.7.8. Tail suspension test ...................................................................................................... 25
Chapter II – Material and Methods .................................................................................................... 26
2.1 General conditions of the experiment ................................................................................. 26
2.2 Experiment schedule ........................................................................................................... 26
2.3 Treatment with imipramine via food and water .................................................................. 27
2.4 Behavioural study ................................................................................................................ 27
2.4.1 Body weight .................................................................................................................. 27
2.4.2 Assessment of sucrose consumption and preference in the two-bottle test .................. 28
2.4.3 Assessment of exploratory and vertical behaviour in the novel cage test ..................... 28
2.4.4 Assessment of anxiety-like behaviour in the elevated O-maze test .............................. 28
2.4.5 Assessment of anxiety-like behaviour in the dark-light box test .................................. 29
2.4.6 Assessment of anxiety-like behaviour in the open-field test......................................... 29
2.4.7 Assessment of depressive-like behaviour in the forced-swim test ................................ 29
2.4.8 Assessment of depressive-like behaviour in the tail suspension test ............................ 29
2.5 Humane endpoints and ethical justification of the study .................................................... 30
2.6 Statistical analysis ............................................................................................................... 30
Chapter III – Results and discussion .................................................................................................. 31
3.1. Results ................................................................................................................................. 31
3.1.1. Effects of treatment with imipramine on body weight ................................................. 31
3.1.2. Effects of treatment with imipramine on Sucrose test parameters ............................... 32
3.1.3. Effects of treatment with imipramine on Novel cage activity ..................................... 36
3.1.4. Effects of treatment with imipramine on parameters of the Dark-light box test .......... 37
3.1.5. Effects of treatment with imipramine on behaviour in the Elevated O-maze test ....... 41
3.1.6. Effects of treatment with imipramine on the Open-field behaviour ............................ 45
j
3.1.7. Effects of treatment with imipramine in the Forced swim test .................................... 57
3.1.8. Effects of treatment with imipramine in the tail suspension test ................................. 58
3.2. Discussion ........................................................................................................................... 59
3.2.1. Effects of treatment with imipramine on body weight ................................................. 59
3.2.2. Effects of treatment with imipramine on Sucrose test parameters ............................... 61
3.2.3. Effects of treatment with imipramine on Novel cage activity ..................................... 62
3.2.4. Effects of treatment with imipramine on parameters of the Dark-light box test .......... 63
3.2.5. Effects of treatment with imipramine on behaviour in the Elevated O-maze test ....... 64
3.2.6. Effects of treatment with imipramine on the Open-field behaviour ............................ 65
3.2.7. Effects of treatment with imipramine in the Forced swim test .................................... 66
3.2.8. Effects of treatment with imipramine in the tail suspension test ................................. 67
Chapter IV – Conclusions, relevance of the data for the field and outlook ....................................... 68
4.1. What else could have been done or be improved? .............................................................. 68
Chapter V – Literature cited............................................................................................................... 70
Tables ................................................................................................................................................. 84
k
List of abbreviations
Ø Diameter
5-HT Serotonin (5-hydroxytryptamine)
5-HT1A Serotonin (5-hydroxytryptamine) receptor 1A
APA American Psychiatric Association
ACTH Adrenocorticotropic hormone
BDNF Brain-derived neurotrophic factor
C / CTR Control group
Ca2+ Calcium
B Baseline (see Results)
CI Confidence interval
CRH Corticotropin-releasing hormone
D1, D2 Day 1, Day 2 (see Results)
DES Desipramine
dH2O Distilled water
DLB Dark-light box test
DNA Deoxyribonucleic acid
EPM Elevated plus maze
E End-treatment (see Results)
EU European Union
EZM Elevated zero maze
FDA Food and Drugs Administration
FST Forced swim test or Porsolt’s test
GluR1 Glutamate receptor 1
Fig. Figure
HPA Hypothalamus-pituitary-adrenals
i.p. Intraperitoneal (injection)
i.v. Intravenous (injection)
IMI Imipramine
IMI-food, IF Group treated with imipramine in food
IMI-drink, ID Group treated with imipramine in water
K+ Potassium
MAOI Monoamine oxidase inhibitor
Na+ Sodium
l
NE Norepinephrine
OFT Open field test
p.o. Per os / gavage
RH Relative humidity
SEM Standard error of the measurement
SNRI Selective serotonin and noradrenaline reuptake inhibitor
SSRI Selective serotonin reuptake inhibitor
TCA Tricyclic antidepressants
TST Tail suspension test
w/v Weight per volume
W2-W5 Weeks 2-5.
m
Figure index
Figure 1: Chemical structure of imipramine........................................................................... 19
Figure 2: Timeline of the experiment...................................................................................... 27
Figure 3: Effects of imipramine treatment on body weight.................................................... 31
Figure 4: Effects of imipramine treatment on average water intake....................................... 33
Figure 5: Effects of imipramine treatment on the average intake of sucrose solution............ 34
Figure 6: Effects of imipramine treatment on average preference sustained per group relatively
to sucrose intake, in percentage (%)........................................................................ 35
Figure 7: Effects of imipramine treatment on average number of rearings in the novel cage test
................................................................................................................................. 37
Figure 8: Effects of imipramine treatment on dark-light box test average latency to exit to the
lit area...................................................................................................................... 38
Figure 9: Effects of imipramine treatment on dark-light box test average number of exits... 39
Figure 10: Effects of imipramine treatment on dark-light box test average total time spent in the
lit area...................................................................................................................... 40
Figure 11: Effects of imipramine treatment on O-maze test average latency to exit to the open
arms.......................................................................................................................... 41
Figure 12: Effects of imipramine treatment on O-maze test average number of exits............. 43
Figure 13: Effects of imipramine treatment on O-maze test average total time spent in open
arms ......................................................................................................................... 44
Figure 14: Effects of imipramine treatment on average distance travelled in the open field test
................................................................................................................................. 45
Figure 15: Effects of imipramine treatment on average instant speed in the open field test.... 47
Figure 16: Effects of imipramine treatment on average number of line crossings in the open
field test................................................................................................................... 48
n
Figure 17: Effects of imipramine treatment on the average number of entries to the peripheral
zone of the open field............................................................................................... 49
Figure 18: Effects of imipramine treatment on the average number of entries to the intermediate
zone of the open field............................................................................................... 50
Figure 19: Effects of imipramine treatment on the average number of entries in the central zone
of the open field....................................................................................................... 51
Figure 20: Effects of imipramine treatment on the mean number of entries per zone, per group,
per week.................................................................................................................. 52
Figure 21: Distribution of mean time spent in peripheral zone................................................ 53
Figure 22: Distribution of mean time spent in the intermediate zone....................................... 54
Figure 23: Distribution of mean time spent in the central zone................................................ 55
Figure 24: Distribution of the mean number of entries per zone, per group, per week............ 56
Figure 25: Forced swim test..................................................................................................... 57
Figure 26: Tail suspension test, day 1...................................................................................... 58
Figure 27: Tail suspension test, day 2....................................................................................... 59
o
Table index
Table 1: Parallels of human symptoms of depression in rodents............................................... 84
Table 2: Behavioural tests used for measurement of animal parallels of depression- and anxiety-
like states .............................................................................................................. 85
Table 3: Effects of imipramine in animal models....................................................................... 88
Table 4: Routes of drug administration in experimental animals............................................... 92
Table 5: Side-effects of imipramine in patients.......................................................................... 93
Table 6: Effect of imipramine on the average number of entries in the open field test............. 94
Table 7: Effect of imipramine on the average time spent out in the open field test................... 95
15
Chapter I – Introduction
1.1. Objective of the study
Pre-clinical studies which use depression and anxiety paradigms often require the modelling
of prolonged administration of drugs which are used in clinic, in experimental animals. In this work,
the effects of a low dose (7 mg/kg/day) of the tricyclic antidepressant imipramine delivered for 4
weeks via dosing with food and drinking water to three-month old naïve C57BL/6N male mice
were assessed in order to estimate the efficacy of drug administration.
Some antidepressant drugs which are used in animal models of depression and anxiety,
including tricyclic antidepressants (TCA), have toxic-like effects on mice during chronic
administration. This includes imipramine, delivered in C57BL6 at the dose 15 mg/kg/day for 4
weeks. In the present study, the question whether a low dose of imipramine (7 mg/kg/day)
administrated either with food and drinking water induces such effects was also addressed by
measuring several basal physiological variables and behaviours.
1.2. Modelling anxiety and depression in rodents
Depression and anxiety are complex, multifactorial disorders with many emotional and
psychological features that render them difficult to be studied as a whole (Cryan et al., 2002). The
commonly adopted strategy is to model single endophenotypic differences, i.e., to decompose the
disorder to specific, clear-cut behavioural outputs relevant to the disease state as opposed to a whole
syndrome or a holistic approach (Geyer and Markou, 1995; Cryan et al., 2002).
Mice and rats are broadly used in animal models for pre-clinical studies of anxiety and
depression. Rats emerged as a tool partly because they are relatively cheap and easy to breed,
perform well in many of the cognitive and operant tasks of modern behavioural pharmacology and
the application of invasive techniques is easier, as is the screening for toxicity testing new
compounds (Cryan and Holmes, 2005). The ascent of the use of mice in pre-clinical studies started
with the progress in genetic engineering, which rendered high variability and availability of
genotypes, also comprising the advantage of even cheaper breeding, housing and dosing of these
animals (Holmes et al., 2004; Tecott, 2003; Cryan and Holmes, 2005).
Not all symptoms of depression and anxiety can be adequately modelled in small rodents
(Table 1). Cryan, Holmes (2005) and others (Trull and Rich, 1999; Geyer and Markou, 1995) have
proposed that the only acceptable and sufficient criteria for an animal model to be considered as
valid are those which ensure strong predictive validity and a reliability of the behavioural readout
that are stable across different laboratories. Other criteria, such as construct and discrimination
validity, defended by McKinney and Bunney (1969), might have heuristic and desirable value, but
16
Wistar rat (left) and C57BL/6 mice (right) are two of the most broadly used laboratory strains.
are not mandatory (Cryan et al., 2002).Tests for anxiety-like and depression-like behaviours come
with idiosyncrasies and limitations, hence none of them provides the ideal model for the disorder of
interest. A battery of tests comprising various tasks and addressing different animal parallels of a
depressive-like state provides both stronger support for a reliable anxiety-related and depression-
related phenotypes and hints on the neurobiology which underlies specific drug action related to
these pathological states in mammals (Cryan et al., 2002; Table 2).
1.3. Effects of antidepressants and anxiolytics in rodent models
The main approaches for the treatment of anxiety and mood disorders in humans comprise
pharmacotherapy, psychotherapy and supportive measures, such as deep brain and vagus
stimulation among other methods. With this regard, the antidepressants and anxiolytics have been
studied most extensively (Cryan et al., 2002; Bschor and Adli, 2008). Many paradigms are available
for the purposes of mimicking these mood disorders in pre-clinical studies.
Antidepressant medications have been used for the treatment of depression from the time
when imipramine, a tricyclic antidepressant (TCA) with properties of a serotonin and
norepinephrine re-uptake inhibitor was proposed to be used in depressed patients in late 1950s by
Kuhn (Kuhn, 1958). Since then, selective serotonin and/or noradrenaline reuptake inhibitors
(SSRI/SNRI), monoamine oxidase inhibitors (MAOI) and pre-synaptic autoceptors (Bschor and
Adli, 2008) were developed as new classes of pharmacotherapy for depression (Cryan et al., 2002).
The SSRI are usually the first line antidepressants for the pharmacological treatment of
depression (93% over 164 million prescriptions in 2008) because their efficacy is close to that of
TCA but with lesser side effects and a broader therapeutic range (Petersen et al., 2002; Bschor and
Adli, 2008). Hence, a list of the American Food and Drugs Administration (FDA) for approved
pharmacotherapy of depression contains mostly SSRIs (Diefenbach and Goethe, 2006). Some
antidepressants can also be used for the treatment of anxiety disorders.
In pre-clinical models of anxiety and depression, both SSRI and TCA are used in the
validation of animal parallels of depressive disorder for their face validity and pharmacological
sensitivity
imipramine
delivery m
paradigms
1.4. Me
Vario
models; ea
Invasive m
although th
can be up t
accuracy o
an animal.
1.4.1. InvInvas
os (Lipinsk
2004), osm
1975; Glot
1982; Pott
2008) and
2000). Cu
Invasintravemetho
(reviewed
e in model
methods and
(Table 4).
ethods of d
ous invasiv
ach of them
methods are
hey cause a
to 6 weeks o
of the dosing
vasive met
sive system
ki et al., 20
motic minipu
tzbach and
tenger et al.
intramuscu
urrently, mo
ive methoenous injec
ods of drug d
in Cryan
s of anxiety
doses in se
drug delive
ve or non-in
m has advan
e used to a
a discomfort
or even long
g and provi
thods of sy
mic delivery
008), nasal a
umps (Lipin
Preskorn, 1
., 2000; Ga
ular injection
ouse model
ods of systctions (centrdelivery in a
et al., 2005
y and depr
veral strain
ery in anim
nvasive me
ntages and d
achieve mo
t to an anim
ger. Non-in
ide slower o
ystemic de
of antidepr
administrati
nski et al., 2
1982), subcu
ambarana et
ns (Nagy an
ls of depre
temic drugre) and impanimal mod
5; Murphy,
ression, diff
ns of mice, b
mal model
ethods of d
drawbacks
ore precise
mal, especia
nvasive meth
onset of the
elivery
ressant med
ion (Berlin
2008), as w
utaneous, in
t al., 2001;
nd Johansse
ssion mostl
g delivery plantation odels.
, 2008; Ta
ferent resea
but also in r
s
drug admini
as it is sum
dosing an
ally with ch
hods are con
erapeutic eff
dications can
et al., 1996
well as intrav
ntraperitone
Dhingra an
en, 1977; Su
ly employ
to animalof osmotic
able 2). Wi
arch groups
ats, mainly
istration are
mmarized in
d faster ef
hronic applic
nsidered to
fect, but cau
n be achiev
6; Illum, 20
venous (i.v.
eal (i.p.; Gl
nd Sharma,
utfin et al.,
i.p. injecti
l models. Iminipumps
ith regards
assessed a
in behaviou
e employed
n the Table
ffect of the
cation, whi
have limita
uses less dis
ved through
03; Graff a
; Nagy and
lotzbach and
2005; Lipi
1984; Potte
ons, accord
Intraperitons (right) are
17
the use of
a variety of
ural despair
d in animal
es 3 and 4.
e treatment,
ch duration
ations in the
scomfort to
gavage/per
and Pollack,
Johansson,
d Preskorn,
inski et al.,
enger et al.,
ding to the
neal (left), e invasive
7
f
f
r
l
.
,
n
e
o
r
,
,
,
,
,
e
18
Administration via food (left) and via water (right).
literature available. Recently, osmotic mini pumps became more broadly used. Aside from the risk
of surgical complications and high costs, they are believed to provide controlled and constant
release of the drug with high accuracy of dosing and minimize daily handling of animals which can
interfere with an experiment (Alves, 2009).
1.4.2. Noninvasive methods of systemic delivery Non-invasive delivery of antidepressant medications comprises of the administration of the
drug with food or drinking solutions. For these purposes drugs are usually diluted in drinking
solutions as water or milk (David et al., 2007; Becker et al., 2010) mixed with palatable solutions,
usually sweetened or fatty (Levine et al., 2003) or dipped directly in the back of an animal’s tongue
(Nagy and Johansson, 1975; Sutfin et al., 1984; Pottenger et al., 2000). Some authors consider the
latter method as invasive due to the aggressive handling and forced feeding required in the
execution of this technique.
A compound can also be mixed with powdered food. This approach is mainly employed for
drugs which have poor solubility and in situations where the drugs have to be delivered for a very
long period. Nowadays there are some commercially available food pellets already supplemented
with drugs. These non-invasive methods are recommended in chronic drug treatment as animals can
feed naturally and “self-administer” drugs which do not have an aversive taste, smell or texture.
Chronic treatment with such compounds may lead to a decrease in food and liquid intake and
weight loss and insufficiently homogenized drugs with the food/drink might result in overdose.
19
Figure 1: Chemical structure of imipramine.
1.5. Imipramine, its biochemistry and metabolism
Imipramine (IMI; (C19H24N2)) is a tertiary amine
absorbed by the intestinal lumen. Readily n-deaminated, has its
rings hydroxylated in the liver via cytochrome P450 into a
secondary amine, desipramine (DES), which is 10x up to 50x
pharmacologically more active (Richelson, 2001; Villela et al.,
2002; Madrígal-Bujaidar et al., 2008). Due to its liver
metabolism, actual bioavailability for IMI corresponds to only
50-60% of the initially delivered dose. Imipramine reaches
maximum concentration in plasma within one to three hours,
while desipramine takes between four to eight hours to reach its
peak values, therefore being less toxic and better tolerated
(Villela et al., 2002). Excretion of imipramine/desipramine
involves glucoronation dependant on UDP-glucoronyl-
transferase and deamination to inactivated monoamines by
isoenzyme P450/2D6, which are then excreted by the urinary
tract (Villela et al., 2002). Imipramine half-life is 29 hours and
desipramine is 21-23h (Richelson, 2001).
1.5.1. Action of imipramine on brain function Imipramine blocks a reuptake of serotonin (5-HT) and norepinephrine (NE) into pre-synaptic
neurons. In the brain regions as the locus ceruleus, chronic but not acute administration leads to
accumulation of NE at the synapse and decreases firing of noradrenergic neurons via activation of
the inhibitory α2-adrenergic receptors, thus resulting in reduced secretion of corticotropin-releasing
factor (CRH). This reduction has an antidepressant effect by decreasing HPA axis hyperactivity
(Boyle et al., 2005) and lowering circulating levels of cortisol in humans and corticosterone in
rodents (Michelson et al., 1997). Imipramine blocks central and peripheral acetylcholine muscarinic
receptors, acting as anticholinergic substrate (Woolf et al., 2007), inhibiting serotonin 5-HT1A at the
hypothalamus (Boyle et al., 2005), but also histamine, α1/α2-adrenergic and β-adrenergic receptors
as well as K+/Na+ adenosintriphosphatase (Villela et al., 2002; Bschor and Adli, 2008).
1.5.2. Toxicity and sideeffect profile As a TCA, imipramine has a narrow therapeutic dosage interval. In humans, the minimum
toxicity-associated dose was of 100 mg/day and minimum lethal dose observed was of 200 mg/day
whereas minimum desipramine toxicity was at 1000-1200 mg/day (Villela et al., 2002). As for
20
rodents, lethal dosage is of 38 mg/kg i.v. for mice, 25 mg/kg in rats and 15 mg/kg in rabbits
(Azima, 1959). There is a regular incidence of side effects in patients treated with imipramine, up to
70% (Azima, 1959; Table 5). The toxicity and side-effect profile are difficult to assess
behaviourally without physiological monitoring, reason by which this issue will not be addressed in
this work.
1.5.3. The use of imipramine in animal models of anxiety and depression Since its discovery in the late 1950s by Kuhn, imipramine has been used in animal
experimentation mainly due to its antidepressant-like effect. In rodent models, IMI is able to revert
chronic helpless behaviour in the forced swim test (FST) and tail suspension test (TST) as well as
improve scores in other behavioural tests for anxiety and depression, reduces depression-induced
sleep architecture modifications either at acute or chronic administrations (Gervasoni et al., 2002),
produces acute analgesic effect (Yanagida et al., 2008; Table 2, Table 3). At a molecular level,
imipramine upregulates glutamate receptor 1 (GluR1)(Du et al., 2004), increases BDNF
concentration in the hippocampus (Tsankova et al., 2006) and has muscle-relaxing effect by
inhibiting voltage-sensitive Ca2+ channels, reducing Ca2+ uptake and availability, diminishing
muscle contractility and tonus (Becker et al., 2004). Although the use of a low dose (7 mg/kg) has
been reported in the literature (Cryan et al., 2005), results are inconsistent and do not reflect a
chronic treatment situation. The effects of this particular dose administrated to small rodents via
food and water was absent from the literature consulted for the present work, which was aimed to
address these questions.
1.6. Delivery of imipramine in animal models of depression
The most common method for the delivery of imipramine to animal models is intra-peritoneal
injection (Table 3). Different groups have shown an antidepressant-like action of imipramine (7.5-
64 mg/kg) on both rats and mice of different strains in behavioural despair paradigms, such as the
forced swim test (Porsolt et al., 1977, 1978; Bai et al., 2001; David et al., 2001, 2003; Bourin et al.,
2003; Castagné et al., 2006, 2009; Assis et al., 2009; Bessa et al., 2009; Gutierrez-García and
Contreras, 2009) and tail suspension test (Steru et al., 1985, 1987; Teste et al., 1993; O’Neill et al.,
1996; Liu and Gershenfeld, 2001; Winterhoff et al., 2003; Ripoll et al., 2003).
While a bolus of intra-peritoneal administration is well known to evoke above mentioned
antidepressant-like effects, the action of chronic treatment with imipramine with food and water on
the FST and TST is less known. Different groups have shown an antidepressant-like action of
imipramine delivered with water on hedonic deficit, another parameter of depressive-like behaviour
in rodents, by restoring to normal the intake of palatable solutions, which is decreased during
21
depressive-like states (Papp et al., 2000; Becker et al., 2010). Besides, imipramine, administrated
per os, was found to evoke an anxiolytic-like effect in the novelty suppressed feeding test (David et
al., 2007). These effects are paralleled by an increase of the neurogenesis in imipramine-treated
groups, as shown by an increased number of BrdU-tagged cells in the subgranular zone (David et
al., 2007) and dentate gyrus (An et al., 2008) of the hippocampus in animals treated with this drug
when compared to depressed controls..
1.7. Tests of evaluation of antidepressant and anxiolyticlike effects of
antidepressants in rodent models
Many animal models used for testing mice for anxiety and depression-like behaviour
precondition animals by exposing them to stress (Cryan and Holmes, 2005). Among these tests are
paradigms for locomotion and exploratory behaviours such as the open field test (OFT) and the
novel cage test (NCT), anxiety paradigms as the dark-light box test (DLB), elevated plus maze test
(EPM) and elevated O-maze test, as well as behavioural despair paradigms, such as the forced swim
test (FST) or Porsolt test and the tail suspension test (TST).
1.7.1. Methods of assessment of hedonic status in animal models Anhedonia, defined as loss of interest to pleasure (Hamilton, 1966, Klein, 1974), is one of the
core symptoms of depression that can be mimicked in animals (Wise, 2002; Kornetsky, 2004;
Nestler, 2005). Consumption and/or preference for palatable food or solutions and intracranial self-
stimulation (ICSS) can be used as a measure of sensitivity to a reward stimulus (Zacharko et al.,
1984; Strekalova et al., 2004; Nestler, 2005).
1.7.2. Novel cage test The novel cage test, developed by Strekalova (2001), presents a testing environment with a
low level of aversion since the mice are introduced to an arena which essentially has the same
features as the home cage. In this paradigm, an animal is placed in the centre of the novel cage for 5
min. Vertical locomotion, rearing activity (standing on posterior limbs) scores denote a normal,
healthy behaviour, opposed to hypo- or hyperactivity in this test which often correlate with
abnormal state of different kinds (Prut and Belzung, 2003; Marques et al., 2008). Excessive
locomotion in this test was shown to parallel anxiety like state and toxic effects of drugs, whereas
reduced activity was found to correlate with a depressive like state, usually induced by stress
(Strekalova et al., 2004; Marques et al., 2008).
22
1.7.3. Open field test The open field test is a modification of Hall’s original apparatus described in 1934, and
usually consists of a white, square arena with tall walls and lit from above (Hall, 1934). In this test,
locomotion (distance travelled, average instant speed, number of line crossings), time spent in the
central, intermediate or peripheral zones of the apparatus are scored (Hall, 1934; Prut and Belzung,
2003; Kalueff et al., 2007). If an apparatus is divided into four quadrants, each crossing or rearing
comprises one unit of exploratory activity (Crawley, 1985; Prut and Belzung, 2003). Anxiolytics,
and antidepressants with anxiolytic-like effects, increase the number of transitions and time spent in
more exposed areas of the test, without increasing the general locomotion (speed, distance) of the
animal (Lister, 1990; Deussing, 2006). Although its sensitivity to anxiolytic drugs is essential, many
authors regard it as inconsistent (Deussing, 2006).
1.7.4. Darklight box The dark-light box test, conceived by Crawley and Goodwyn in the 1980s is based on the
inner conflict between innate aversion of rodents to brightly illuminated areas and the spontaneous
exploratory behaviour in response to mild stressors as are novel environment and light (Crawley
and Goodwyn, 1980; Crawley, 1985; Shimada et al., 1995; Bourin and Hascoët, 2003).
A natural conflict situation occurs when an animal is exposed to an unfamiliar environment or
novel object. The conflict is between the tendency to explore and the initial tendency to avoid the
unfamiliar (neophobia). The exploratory activity, thus, is determined by these two opposite
motivations (Bourin and Hascoët, 2003). The display comprises two sections: one white, lit,
corresponding to 2/3 of the total area and a smaller (1/3), black area covered by a lid, where the
animals are placed in the beginning of the test. These two sections are connected by an opening of
variable dimensions, usually wider rather than taller (Bourin and Hascoët, 2003). In this test,
Sucrose test display (left) and a mouse being tested in the novel cage test (right).
23
increased time and number of exploratory behaviours in the exposed, lit part, increased number of
transitions and a smaller latency for the first dark to white transition without an increase in
spontaneous locomotion is considered to reflect anxiolytic activity (Shimada et al., 1995; Bourin
and Hascoët, 2003). Some antidepressants have anxiolytic-like properties thus showing similar
effects to those described above.
Apparatuses used for testing anxietylike behaviour in small rodents: The open field
arena (a), elevated plus-maze (b), elevated O-maze (c) and light/dark box (d) apparatuses are
used for testing anxiety-like behaviour in small rodents. Image source: Cryan and Holmes,
2005.
1.7.5. Elevated plus maze The elevated plus maze derives from a work of Montgomery (1955), validated as an animal
model of anti-anxiety in rats by Pellow and co-workers (1985) and in mice by Lister (1987). The
apparatus, placed at a determined height over the floor surface, consists of a plus-shaped maze
withstanding two open arms and two enclosed arms connected by a central square platform. The
open space and elevation of the maze produce the anxiogenic stimulus which is targeted by
anxiolytics drugs and antidepressant drugs with anxiolytic profiles (Lister, 1990). Less anxious
animals spend more time in open arms, register an increased number of exits to open arms, head-dip
24
and stretch-out more times, having also a smaller initial latency to exit secluded arm (Carola et al.,
2002). The EPM is sensitive to anxiogenic and anxiolytic drugs, is easy to perform and score but is
difficult to discriminate between decreased anxiety-related avoidance from increased novelty-
seeking behaviour (Holmes, 2001; Deussing, 2006) or interpret the behaviour on the central square
platform (Shepherd et al., 1994).
1.7.6. Elevated / Omaze test The O-maze was projected by Shepherd in 1992 as a modification of the EPM. This design
comprises an annular black platform with two opposite closed quadrants and two open quadrants
that precludes the ambiguity of scoring of time spent by an animal in a central square (Shepherd,
1992; Shepherd et al., 1993; Shepherd et al., 1994; Kulkarni et al., 2007). The increased number of
exits to open arms, of stretch-outs and head-dipping behaviours, the lower latency to exit from
closed arm and increased time spent in open arms are considered as signs of decreased anxiety
(Shepherd et al., 1994; Kulkarni et al., 2007). Compounds with an anxiolytic-like profile enhance
these behaviours whilst anxiogenic drugs have the opposite effect.
1.7.7. Forced swim test / Porsolt test The forced swim test, developed by Porsolt in 1977 in rats, relies on the premise that an
animal when forced to swim in a situation from which there is no possible escape, after a period of
vigorous activity will eventually cease to move altogether, making only the movements necessary to
keep its head above water level (Porsolt, 1977; Porsolt et al., 1978). The modified forced swim test
uses a larger tank with enough depth so as an animal cannot reach the bottom of the apparatus with
its tail (Cryan et al., 2002; Petit-Demouliere et al., 2005). These modifications allow to measure
horizontal movement (swimming), vertical movement (climbing or thrashing), latency of
immobility and time immobile (Cryan et al., 2002).
Antidepressants lead to a longer latency to immobility, to a reduction of total time spent
immobile and to increased movement in treated animals. This test is sensitive to antidepressant
treatment, but not to anxiolytic drugs and perhaps is the most broadly used paradigm for screening
new drug candidates for antidepressant-like activity (Deussing, 2001). Hypothermia is a risk
associated with this test when water temperature is not well controlled. Another confound which
can occur with the testing of TCA in this model regards its interaction with the acquisition of
contextual memory, which is impaired after a single administration of imipramine and other drugs
of this class. As in the original Porsolt test, scoring of floating behaviour in the FST is carried out
24 h after the first swimming session.
Appara
test (lef
Holmes
1.7.8. TaiIn th
suspended
attempts to
lower laten
automated
computer s
Ripoll et a
Antid
2001; Ripo
and predic
between st
et al., 200
several adv
with conte
uncommon
not suitabl
atuses for
ft) and forc
s, 2005.
il suspens
he tail susp
by its tail o
o escape an
ncy to imm
version of
software sco
l., 2003).
depressants
oll et al., 20
ctive to anti
trains used
05). In com
vantages: th
extual mem
n for geneti
e for the tes
evaluation
ced swim t
ion test
pension tes
over a hang
nd despair-
mobility are
f the TST u
ores the dur
s reduce im
003; Cryan
idepressant
and betwee
mparison to
he TST mod
mory and ca
ically modif
sting in the
n of behav
test or Por
st, develop
ging surface
-associated
considered
ses a strain
ration and i
mmobility in
et al., 2005
action and
en laborator
the FST, i
del does no
n be used
fied animal
TST as anim
vioural des
rsolt test (r
ed by Ster
e, and the a
immobility
to reflect a
n gauge atta
intensity of
n both FST
5). The TST
insensitive
ries (Deussin
t is less re
ot cause hyp
in animals
s (Liu and
mals climb
spair in sm
right). Imag
ru and col
animal alter
y states. Lo
a greater de
ached to the
f the behavi
and TST p
T is regarde
e to anxioly
ng, 2001; L
producible,
pothermia u
with impai
Gershenfeld
on their tail
mall roden
ges obtaine
laborators
rnates betwe
onger perio
egree of beh
e tip of the
ours (Liu a
paradigms
ed to be gen
ytics, to hav
Liu and Ger
, cannot be
unlike the F
ired motor
d, 2001). So
ls (Ripoll et
nts: Tail sus
ed from Cr
in 1985, a
een engagin
ods of imm
havioural d
tail and tog
and Gershen
(Liu and G
nerally high
ve high repr
rshenfeld, 2
e used in ra
ST, does in
function w
ome mouse
t al., 2003).
25
spension
ryan and
a mouse is
ng in active
mobility and
despair. The
gether with
nfeld, 2001;
Gershenfeld,
ly sensitive
roducibility
001; Cryan
ats, but has
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which is not
e strains are
5
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26
Chapter II – Material and Methods
2.1 General conditions of the experiment
All protocols were in accordance to the European Union’s Directive 86/609/EEC and Council
Directive 93/119/EC, Portuguese law Law-Decrees DL129/92 (July 6th), DL197/96 (October 16th)
and Ordinance Port.131/97 (November 7th). This project was approved by the Ethical Committee of
the Faculdade de Ciências da Universidade de Lisboa.
a) Animals
For this project, twenty-three (23) C57BL/6N male mice three months old were supplied by
Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 2780-156 Oeiras, Portugal.
Mice were housed individually in 24x14x13cm, solid floor, wire grid, polycarbonate cages
under a reverse 12:12h hour light:dark cycle (lights on 21:00), at 21±2°C, 55±5% RH, food and
water ad libitum, otherwise noted. Cages were cleaned and animals were weighed once per week,
including wood scrapes for bedding, hand tissue as enrichment and refill of water and commercial
chow supplies. The animals were single-housed since preference tests have to be performed
individually for accurate acquaintance of consumption levels but also due to the fact that the used
strain is characterized by aggressive behaviour between males when housed in groups, thus largely
increasing behavioural variability in the experiment.
b) Reagents
Imipramine Hydrochloride (Sigma-Aldrich I7379-5G) was used to prepare food pellets and
water solution with imipramine. Regular commercial sugar was used for preference tests.
2.2 Experiment schedule
This experimental work was performed over the course of nine weeks, divided into four
periods (Fig. 2). During week 1, animals were allowed to adapt to individual housing and new
facilities. During weeks 2 and 3 basal water and food consumption were measured and mice were
tested in open field, dark-light box and sucrose preference for establishment of baseline behaviour.
During weeks 4 to 7, mice were treated with imipramine and weekly tested in the novel cage, open
field and for sucrose preference. On the last two weeks (8, 9), post-treatment behavioural
assessments in the forced swim test, tail suspension test, O-maze, dark-light box and open field
were made and animals were sacrificed.
After the adaptation period, the animals were assigned into three experimental groups: control
(distilled water and normal chow, n=7), “IMI-food” (distilled water and imipramine-prepared food,
We
n=8), and “
balanced u
order to m
tests took p
Figure
2.3 Tre
For t
to powder
hand, simi
was weigh
3 days in
weighing t
adjusted to
estimated
mg/kd/day
due to pho
days.
2.4 Beh
2.4.1 BoBody
administra
could pote
with a 0.1g
eek 1
•Adaptation period
“IMI-drink”
upon initial
minimize art
place from t
2: The time
eatment w
treatment w
by a comm
lar in size a
hed and then
a raw, dai
the bottles (
o a dose 7 m
during 3 da
y. Bottles w
otosensitivit
havioural
dy weight
y weight is
tion of ant
entially affe
g resolution
We
•••••
” (imiprami
parameters
tefacts in b
the onset of
eline of the
with imipra
with food, im
mercial blen
and shape to
n dissolved
ily consump
(week 2); th
mg/kg/day.
ays by wei
with imipram
ty, the food
study
s a useful i
tidepressant
ect this para
n scale when
eeks 2,3
• EZM• DLB• H2O intake•Food intake• Sucrose 1%
ine in distill
s of sucros
behavioural
f the dark ph
experiment
amine via
mipramine w
nder). Disti
o commerc
in tap wate
ption of wa
he mean of
Similarly, f
ghing consu
mine were c
pellets and
indicator an
ts, behaviou
ameter in th
n no behavio
We
•
led water an
se intake an
testing wit
hase of the l
t.
food and w
was weighed
illed water
ial pellets.
er. Drug con
ater was m
these value
for drug adm
umed food
covered wit
d water with
nd therefore
ural testing
his experim
oural tests w
eek 4
•Treatment
nd normal c
nd water co
th imiprami
light cycle (
water
d and mixed
was added
Regarding t
ncentration
measured in
es was take
ministration
(week 2) a
th tinfoil as
h treatment
e was week
g, food rest
ment. Anima
were perform
We
•••
•
chow, n=8)
onsumption
ine-treated
(9:00h).
d with comm
d and food
treatment w
was calcula
experimen
n and amou
n with food,
and then ad
s the drug i
were prepa
kly evaluat
triction, cha
als were we
med.
eeks 5‐7
•Treatment •Weekly OFT• Weekly Novecage• Weekly Sucrose 1%
. Groups of
, and body
animals. B
mercial cho
pellets wer
with water, i
ated as follo
ntal group o
unt of imipr
, daily food
djusted to a
is photosens
ared fresh e
ted in this
anges in liq
eighed onc
el
W
27
f mice were
weight, in
Behavioural
ow (reduced
re made by
imipramine
ows: during
of mice by
ramine was
intake was
a dose of 7
sitive. Also
every 3 to 5
study. The
quid intake
e per week
Weeks 8,9
•FST (2 days) x2•TST (2 days) x2•OFT x2•DLB•EZM
•Euthanas
7
e
n
l
d
y
e
g
y
s
s
7
o
5
e
e
k
ia
28
2.4.2 Assessment of sucrose consumption and preference in the twobottle test During this 10h test mice were given a free choice between two bottles, one with 1% w/v
sucrose solution and another with distilled water, starting with the onset of the dark (active) phase
of the animals’ cycle (9h00). To prevent possible effects of side-preference in drinking behaviour,
the position of the bottles in the cage was switched in the middle of the test (at t=5h, or 14h). For
the same reason, the position of the bottles was also randomized between cages. Bottles must be
placed gently in the mouse cage, in a position close to horizontal to keep the animals undisturbed
and to avoid liquid spillage.
No previous food or water deprivation was applied before the test. In order to balance the air
temperature between the room and the drinking bottles, these were filled and weighed in advance
during preceding evening and kept in the same room where the test took place. This measure
prevents the physical effect of liquid leakage resulting from growing air temperature and pressure
inside the bottles when filled with liquids, which are cooler than the room air. With this method, the
error of measurement of liquid intake does not exceed 0,1ml (Strekalova, 2008). Falcon tubes
(50ml) were used as bottles. Total intake of fluids, water and sucrose solution consumption were
simultaneously estimated in all groups by weighting the bottles with 0.1g resolution scales. The
preference for sucrose was calculated as a percentage of the consumed sucrose solution (VS) over
the total intake of liquids (Total (T) = VWater + VS), using the formula:
Sucrose Preference (%) = x 100
2.4.3 Assessment of exploratory and vertical behaviour in the novel cage test In the novel cage test mice were introduced into a standard plastic cage with the floor surface
covered with fresh sawdust. The number of exploratory rearings (animal standing on hindlimbs)
was assessed under red light (25 lux) during a 5 min period by visual observation. The light source
is placed at about 30 cm from the setup in a lateral position due to intense heat generation and
characteristics of the lamp. Video recording was done on the side of the cage.
2.4.4 Assessment of anxietylike behaviour in the elevated Omaze test This apparatus consists of a black circular path (runway width 5.5cm, Ø = 46cm) with two
opposing compartments protected by walls made of polyvinyl-chloride (height = 10cm, width =
3mm) and two open sectors of equal size. The maze is elevated 20cm above the ground and
illuminated from above with determined light intensity (25 lux). Animals were placed inside one of
the two closed quadrants for a total time of 5 minutes and the latency to the first exit to an open
29
arm, total number of exits to the open arms and total duration of time spent in open arms were
registered. The test was recorded with a web camera.
2.4.5 Assessment of anxietylike behaviour in the darklight box test The dark/light box is consists of two plexiglas compartments, one black/dark (15x20x25cm)
covered by a black plexiglas lid (15x20cm) and one white (30x20x25cm), lit from above (25 lux),
connected by an opening (7x5cm). Mice were placed into the dark compartment, from where they
could visit the lit box, illuminated by determined white light. Behaviours were recorded by a
recording digital camera on top of apparatus, and also by hand. Total time spent in the lit area,
number of visits to this anxiety-related compartment and latency to exit from the unlit compartment
was scored over a total test time of 5 min.
2.4.6 Assessment of anxietylike behaviour in the openfield test Two open field arenas (50x50x40cm) were made of wood covered by white plastic. Boxes
were evenly illuminated with white light (25 lux). Animals were placed in the centre of the arena.
Behaviours are recorded for 10 minutes with a video camera and subsequently analyzed with
AnyMaze v.4.70 (Stoelting Co., Wood Dale, IL 60191, USA). Tracked behaviours include total
time spent in centre, mid and peripheral arena, number of line crossings, average speed and total
locomotion.
2.4.7 Assessment of depressivelike behaviour in the forcedswim test Mice were placed in a transparent pool (20x15x30cm) filled with warmed tap water (25°C,
height 18 cm) and lit by white light (25 lux) for 6 min. The test was recorded sideways and
behavioural scoring was done manually. Latency to begin floating, defined as the time between
introduction of a mouse into the pool and the very first moment of complete absence of directed
movements of animals’ head and body and total time spent immobile were scored.
2.4.8 Assessment of depressivelike behaviour in the tail suspension test In the tail suspension test, animals were hung by the last 1-2 cm of their tails. For this
procedure, duct-tape, a clip and a suspended projected iron bar were used, 20cm above a surface.
Animals were tested for 6 minutes. White light was placed above the apparatus (25 lux), and video
recording from the side. Latency to immobilization and total immobilization time were measured.
The used strain has the particularity of spontaneously climbing by their tails when facing this
paradigm. Whenever that was the case animals had to be withdrawn from the test as recommended
in the literature (Mayorga and Lucki, 2001).
30
2.5 Humane endpoints and ethical justification of the study
At the end of experimental protocol animals were sacrificed as required by the EU directive
86/609/EEC. The animal sacrifice was performed by an accredited and trained person, one animal at
a time in a separate room with visual, olfactory and sound isolation from where the remaining
animals were housed. Mice were injected intraperitoneally with an overdose of sodium pentothal (a
barbiturate, commercial name Eutasil®). When loss of consciousness was verified, death was
confirmed by cervical dislocation. Animal bodies were processed for incineration.
This work helps to define better conditions for drug research and development which are
important for human health and well being, as well as to reduce the number of animals in potential
studies by increasing the accuracy of the experiments. All efforts were undertaken to prevent
unnecessary suffering of mice.
2.6 Statistical analysis
A Shapiro-Wilk normality test with 95% confidence interval (CI) was performed to check if
the data followed a Gaussian distribution. To compare data from all groups, whenever normality
was verified, two-tailed unpaired t-test with 95% CI was used. When normality was not verified, a
Mann-Whitney test was performed, also two-tailed with 95% CI (GraphPad Prism version 5.01 for
Windows, GraphPad Software, San Diego, California, USA, www.Figurepad.com).
31
Chapter III – Results and discussion
3.1. Results
3.1.1. Effects of treatment with imipramine on body weight Before treatment, all groups shown similar weight averages (Control(C)=26.39g; IMI-
food(IF)=26.67g and IMI-drink (ID)=27.36g; Fig. 3A). Baseline weight differences between groups
are not statistically significant difference (CxIF: p=0.8717 F=0.359; CxID: p=0.6093, F=1.854;
IFxID: p=0.7311, F=1.364). After treatment, while control mice barely changed average weight (C:
27.23g), both treatment groups sustained weight loss (IF: 24.99g; ID: 25.01g; Fig. 3A). Post-
treatment weight differences between groups are also not statistically significant (CxIF: p=0.0747,
F=1.562; CxID: p=0.0856, F=1.135; IFxID: p=0.9806, F=1.388). Although the absolute change in
bodyweight between initial and final measurements were not significant (Fig. 3B), relative changes
show otherwise (Fig. 3C, 3D).
Body Weight
0
10
20
30
40
Baselin
eEnd
A
Wei
ght (
g)
Body Weight
0
10
20
30
40 B
Control
IMI-fo
od
IMI-d
rink
Wei
ght (
g)
B E B E B E
Weight difference (g)
Control
IMI-fo
od
IMI-d
rink
-20
-10
0
10
20
*
*
C
Diff
eren
ce (g
)
Weight difference (%)
Control
IMI-fo
od
IMI-d
rink
-40
-20
0
20
40 *
*
D
Diff
eren
ce (%
)
Control Imi-food Imi-drink
32
Figure 3: Effects of imipramine treatment on body weight. A – Baseline and end-treatment
average weight comparison between groups; B – Baseline and end-treatment average weight
comparison within groups (left: baseline, right: end-treatment); C – Within group average
weight difference, in gram; D – within group average weight difference between groups, in
percentage. Dotted lines correspond to a variation of 0±10% bodyweight, IMI-food – group
treated with imipramine in food, IMI-drink – group treated with imipramine in water, *p<0.05, B=baseline, E=end treatment, bars = SEM.
If these changes are expressed in gram, control mice gained an average +0.8429g, whereas
groups treated with imipramine in food and in water lost -1.686g and -2.350g in average,
respectively (Fig. 3C). These changes are not statistically significant if the two treatment groups are
compared (p=0.6097, F=2.438) but are so when matched to control mice (CxIF: p=0.0252,
F=1.110; CxID: p=0.0249, F=2.196). On other hand, if changes are expressed in percentage (Fig.
3D), control group weight gain corresponds to an average +3.116%, whereas groups treated with
imipramine in food and water show significant average reductions of -6.424% and -9.239%,
respectively. Although the relative weight differences between the two imipramine-treated groups
are not significant, there are statistically significant difference between these groups and control
animals (CxIF: p=0.0230, F=1.100; CxID: p=0.0254, F=2.859; IFxID: p=0.5795, F=2.598).
Thus, both methods of treatment with imipramine reduced body weight with a more
pronounced effect in mice treated with drinking water.
3.1.2. Effects of treatment with imipramine on Sucrose test parameters A comparison of water consumption during baseline and after-treatment conditions showed
that control and imipramine-treated mice have no differences in this parameter (C: p=0.3581,
F=2.338; IF: p=0.9541; F=1.409; Fig. 4A). Repeated measurement of imipramine-treated groups
revealed significant increase of water intake in a course of treatment (ID: p=0.0482, F=1.689).
Weekly average water intake comparison between groups shows no statistically significant
differences with the exception of the two imipramine-treated groups during the third week of
treatment (p=0.0151, F=2.219; Fig. 4B). It also shows a trend for the group treated with imipramine
in water to have the highest water intake and the group treated with imipramine in food to have the
lowest water consumption among experimental groups.
33
0
2
4
6
8
10
*
Control
IMI-f
ood
IMI-d
rink
A
Wat
er in
take
(mL)
B B BE E E
Baseli
ne
Week 2
Week 3
Week 4
0
2
4
6
8
10
*
B
Wat
er c
onsu
mpt
ion
(mL)
Control
IMI-fo
od
IMI-d
rink
0
2
4
6
8
10
*
*
CW
ater
con
sum
ptio
n (m
L)
B B BW2 W2W2 W3 W3W3 W4W4W4
Figure 4: Effects of imipramine treatment on average water intake. Sucrose intake (Fig. 5)
and preference sustained per group relatively to sucrose intake, in percentage (%) is also
presented (Fig. 6). A – Average baseline (left bar) and end-treatment (right bar) water
consumption; B – Average water consumption per week between groups; C – Average water
consumption between weeks for each group. IMI-food – group treated with imipramine in
food, IMI-drink – group treated with imipramine in water, *p<0.05, B=baseline, E=end
treatment, W2-4=weeks 2 to 4, bars = SEM.
Group comparison with water consumption during treatment reveals no significant differences
between baseline and after-treatment measurements in control mice (p>0.05; Fig. 4C). These
differences were observed between the second and third weeks of treatment in the group treated
with imipramine in food (p=0.0337, F=2.528) and between the first and final observations
(p=0.0482, F=1.689) in the group treated with imipramine in water.
Control Imi-food Imi-drink
34
A comparison of groups during baseline and end-treatment observations in sucrose
consumption showed that all groups increase of solution intake (Fig. AA). A difference is not
significant in both control mice and the group treated with imipramine via food (C: p=0.5010,
F=6.145; IF: p=0.2223; F=1.432), and were significant in the group treated with imipramine in
water (ID: p=0.0008, F=1.993). Also, significant differences were found in mean sucrose intake
values between imipramine-treated groups in the final measurement (p=0.0345, F=1.268).
0
2
4
6
8
10
***#
Control
IMI-f
ood
IMI-d
rink
A
Sucr
ose
solu
tion
inta
ke (m
L)
B B BE E E
Baseli
ne
Week 2
Week 3
Week 4
0
2
4
6
8
10
******
B
Sucr
ose
cons
umpt
ion
(mL)
Control
IMI-fo
od
IMI-d
rink
0
2
4
6
8
10
§$&
C
Sucr
ose
cons
umpt
ion
(mL)
B BB W2 W2 W2W3 W3W3 W4 W4W4
Figure 5: Effects of imipramine treatment on the average intake of sucrose solution. A –
Average baseline (left bar) and end-treatment (right bar) sucrose consumption; B – Average
sucrose consumption per week between groups; C – Average sucrose consumption between
weeks for each group. IMI-food – group treated with imipramine in food, IMI-drink – group
treated with imipramine in water, ***p<0.001, *p<0.05, #p<0.05 vs. IMI-drink, §p<0.001 vs.
week 2, $p<0.01 vs. week 3, &p<0.001 vs. week 4, B=baseline, E=end-treatment, W2-
4=weeks 2-4, bars = SEM.
Control Imi-food Imi-drink
35
Weekly measurement of average sucrose intake shows a lack of statistically significant
differences between the groups, with an exception of the two imipramine-treated groups at the
second (p=0.0002, F=1.361) and fourth (p=0.0345, F=1.268) weeks of treatment (Fig. 5B). It also
shows a trend for the group treated with imipramine via water to display the highest sucrose intake
among the groups. A within group comparison of sucrose consumption dynamics between weeks
shows no significant differences in control group or in mice treated with imipramine with food
(p>0.05); these were observed between the first and all other weeks in the group treated with
imipramine in water (IDBaselinexIDW2: p=0.0001, F=1.455; IDBaselinexIDW3: p=0.0054, F=1.173;
IDBaselinexIDW4: p=0.0008, F=1.993; Fig. 5C).
0
20
40
60
80
100
Control
IMI-f
ood
IMI-d
rink
A
Pref
eren
ce (%
)
BBB E E E
Baseli
ne
Week 2
Week 3
Week 4
0
20
40
60
80
100 B
Pref
eren
ce (%
)
Control
IMI-fo
od
IMI-d
rink
0
20
40
60
80
100 C
#$
Pref
eren
ce (%
)
B BB W2 W2W2 W3 W3W3 W4 W4 W4
Figure 6: Effects of imipramine treatment on average preference sustained per group relatively to sucrose intake, in percentage (%). A – Average baseline (left bar) and end-treatment (right bar) sucrose preference; B – Average sucrose preference per week between groups; C – Average sucrose preference between weeks for each group. IMI-food – group treated with imipramine in food, IMI-drink – group treated with imipramine in water, #p<0.01 vs. week 3, $p<0.05 vs. week 4, B= baseline; E=end treatment, W2-W4=week 2-4, bars = SEM.
Control Imi-food Imi-drink
36
A comparison of the baseline and after-treatment measurements of sucrose preference
consumption did not reveal any differences between the groups (C: p=0.5712, F=1.646; ID:
p=0.5660; F=1.208; Fig. 6A). Non-significant increase was found in the group treated with
imipramine in water (IF: p=0.4528, F=4.672).Weekly average sucrose preference comparison
between groups shows no statistically significant differences either. A trend for elevated sucrose
preference was found in the group treated with imipramine with food (Fig. 6B).
A within group comparison of sucrose preference dynamics between weeks did not identify
significant differences in control mice and in the group treated with imipramine in water (p>0.05;
Fig. 6C). Such differences were observed between the second and last weeks of treatment in the
group treated with imipramine in food, which had higher values of this parameter at these time
points (IFW2xIFW3: p=0.0016, F=1.089; IFW2xIDW4: p=0.0182, F=1.096).
3.1.3. Effects of treatment with imipramine on Novel cage activity The effects of imipramine on exploratory rearings in the novel cage test are presented on the
Figure 7. A comparison of baseline and after-treatment scores of rearings in the novel cage test
revealed a lack of difference between the measurements (Fig. 7A). Control mice and the group
treated with imipramine in water show non-significant decrease in number of exploratory rearings
(C: p=0.1030, F=1.528; ID: p=0.0744; F=1.341). The group treated with imipramine via water did
not show such trend (IF: p=0.9496, F=2.960).
Control
IMI-fo
od
IMI-d
rink
0
20
40
60 A
W2# of
rea
rings
W4 W2 W4 W2 W4
Control Imi-food Imi-drink
37
Week 2
Week 3
Week 4
0
20
40
60
* ** ** *
#
B#
of r
earin
gs
Control
IMI-fo
od
IMI-d
rink
0
20
40
60 C§
# of
rea
rings
W2 W2W2 W3 W3W4W3 W4 W4
Figure 7: Effects of imipramine treatment on average number of rearings in the novel cage
test. A – Average baseline (left bar) and end-treatment (right bar) number of rearings; B –
Average number of rearings per week between groups; C – Average sucrose preference
between weeks for each group. IMI-food – group treated with imipramine in food, IMI-drink
– group treated with imipramine in water, **p<0.01 vs. IMI-drink, *p<0.05 vs. IMI-drink,
#p<0.05 vs. control, §p<0.05 vs. weeks 2 and week 4, W2-4=Weeks 2-4, bars = SEM.
Weekly scoring of number of rearings in the groups shows statistically significant differences
between control mice and the group treated with imipramine in water during all time points
(CTRW2xIDW2: p=0.0122, F=1.418; CTRW3xIDW3: p=0.0022, F=4.289; CTRW4xIDW4: p=0.0102,
F=1.616; Fig. 7B). This was the case during the first week of treatment for both imipramine-treated
groups (IFW2xIDW2: p=0.0012, F=2.727) and control mice and the group treated with imipramine in
the third week of treatment (CTRW3xIFW3: p=0.0465, F=1.196). A within group comparison of
average number of rearings dynamics during the treatment period did not identify significant
differences in control mice and in the group treated with imipramine in water (p>0.05; Fig. 7C).
Such differences were observed in the group treated with imipramine via food between at the
second and third week of dosing (IFW2xIFW3: p=0.0016, F=1.089) and third and fourth weeks
(IFW3xIFW4: p=0.0182, F=1.096).
3.1.4. Effects of treatment with imipramine on parameters of the Darklight box test In this test, the latency to exit, number of transitions and total time spent out in the lit are
assessed in the dark-light box apparatus (Fig. 8-10). Results compare baseline and post-treatment
measurements of these behaviours in each of the three experimental groups.
38
Baseli
ne
Post-tes
t0
50
100
150
200A
Late
ncy
(s)
0
50
100
150
200
*
Contro
l
IMI-fo
od
IMI-d
rink
B
Late
ncy
(s)
E EEB
BB
Control
IMI-fo
od
IMI-d
rink
-200
-100
0
100
200*
*
C
Late
ncy
diffe
renc
e (s
)
Figure 8: Effects of imipramine on treatment dark-light box test average latency to exit to the
lit area. A – Baseline and post-treatment average latency comparison between groups; B –
Baseline (left) and post-treatment (right) average latency for each group. C – Within group
average latency difference, in seconds, comparing baseline and post-treatment scores, IMI-
food – group treated with imipramine in food, IMI-drink – group treated with imipramine in
water, *p<0.05, B=baseline, E=end-treatment, bars = SEM.
A comparison of baseline and post-treatment latencies of the first exits between groups did
not result to any statistically significant differences found (CTRBaselinexIFBaseline: p=0.3967, F=2.847;
CTRBaselinexIDBaseline: p=0.0601, F=17.89; IFBaselinexIDBaseline: p=0.1187, F=1.455; CTREndxIFEnd:
p=0.8048, U=22; CTREndxIDEnd: p=0.3357; U=19; IFEndxIDEnd: p=0.8665, U=26; Fig. 8A). A
comparison of repeated measurements of this parameter within groups shows a non-significant
increase in both control mice and in the group treated with imipramine via food (C: p=0.2038,
Control Imi-food Imi-drink
39
F=26.11; IF: p=0.2265, F=8.421; Fig. 8B). A statistically significant increase in this parameter was
observed in mice treated with imipramine in water (p=0.0493, F=16.26).
The relative average increase or reduction in latency time that was observed in each of three
groups is presented on the Figure 8C. The group treated with imipramine in water is significantly
different from the other two groups in this behavioural measure (CTRxID: p=0.0297, F=1.200;
IFxID: p=0.0217, F=1.632).
Baslin
e
Post-tes
t0
5
10
15
20A
# of
exi
ts
0
5
10
15
20
*
Control
IMI-fo
od
IMI-d
rink
B
# of
exi
ts
B BB E EE
Control
IMI-fo
od
IMI-d
rink
-20
-10
0
10
20 * C
Diff
eren
ce (#
)
Figure 9: Effects of imipramine treatment on dark-light box test average number of exits. A –
Baseline and post-treatment average number of exits comparison between groups; B –
Baseline (left) and post-treatment (right) average number of exits for each group; C – Within
group average number of exits difference, in seconds, comparing baseline and post-treatment
scores. IMI-food – group treated with imipramine in food, IMI-drink – group treated with
imipramine in water, *p<0.05, B=baseline, E=end-treatment, bars = SEM.
A comparison of baseline and post-treatment values of the number of exits between groups
did not revealed any statistically significant differences (CTRBaselinexIFBaseline: p=0.4888, F=4.338;
CTRBaselinexIDBaseline: p=0.3332, F=3.556; IFBaselinexIDBaseline: p=0.8646, F=1.220; CTREndxIFEnd:
p=0.5107, F=1.401; CTREndxIDEnd: p=0.0521; F=1.414; IFEndxIDEnd: p=0.0519, F=1.980; Fig. 9A).
Control Imi-food Imi-drink
40
On other hand, a comparison between average number of exits within groups shows a significant
reduction in control mice (p=0.0285, F=1.745), a non-significant reduction in the group treated with
imipramine via food (p=0.3583, F=1.625) and a non-significant increase in the group treated with
imipramine via water (p=0.4624, F=2.880; Fig. 9B).
The relative average increase or reduction in the number of exits of the dark-light box
observed in each of three groups is presented on the Figure 9C. The only statistically significant
difference was found between control mice and the group treated with imipramine via water
(p=0.0389, F=4.047).
A comparison of the baseline and post-treatment average time spent in the lit compartment of
the dark-light box did not reveal statistically significant differences between the groups
(CTRBaselinexIFBaseline: p=0.6864, F=2.027; CTRBaselinexIDBaseline: p=0.3166, F=4.120;
IFBaselinexIDBaseline: p=0.5182, F=2.032; CTREndxIFEnd: p=0.7973, F=1.312; CTREndxIDEnd:
p=0.1147; F=1.499; IFEndxIDEnd: p=0.1538, F=1.143; Fig. 10A).
Baseli
ne
Post-tre
atmen
t0
50
100
150
200A
Tim
e sp
ent i
n lit
are
a (s
)
0
50
100
150
200
*
Contro
l
IMI-fo
od
IMI-d
rink
B
Tim
e sp
ent
in li
t are
a (s
)
B B BE EE
Control
IMI-fo
od
IMI-d
rink
-200
-100
0
100
200 C
Diff
eren
ce ti
me
spen
tin
lit a
rea(
s)
Figure 10: Effects of imipramine treatment on dark-light box test average total time spent in
the lit area. A – Baseline and post-treatment average total time spent in the lit area comparison
between groups; B – Baseline (left) and post-treatment (right) average total time spent in the
Control Imi-food Imi-drink
41
lit area for each group; C – Within group total time spent in the lit area difference, in seconds,
comparing baseline and post-treatment scores. IMI-food – group treated with imipramine in
food, IMI-drink – group treated with imipramine in water, *p<0.05, B=baseline, E=end-
treatment, bars = SEM
A comparison between average time spent in the lit compartment of the dark-light box within
groups shows a significant reduction of this behaviour in control mice (p=0.0373, F=4.221), a non-
significant reduction in the group treated with imipramine via food (p=0.1464, F=1.578) and a non-
significant increase in the group treated with imipramine via water (p=0.6073, F=1.463; Figure.
10B). The relative average increase or reduction in the time spent in the lit area of the dark-light
box observed in each of three groups is shown on the Figure 10C, where no statistically significant
differences are observed.
3.1.5. Effects of treatment with imipramine on behaviour in the Elevated Omaze test In this test, the latency to exit, number of transitions and total time spent in the open
quadrants were evaluated (Fig. 11-13). Baseline and post-treatment assessments for each of the
three experimental groups were also compared.
Baseli
ne
Post-tre
atmen
t0
50
100
150
200 A
Late
ncy
(s)
Control
IMI-fo
od
IMI-d
rink
0
50
100
150
200 B
Late
ncy
B BB E EE
Control
IMI-fo
od
IMI-d
rink
-200
-100
0
100
200C
Late
ncy
diffe
renc
e (s
)
Control Imi-food Imi-drink
42
Figure 11: Effects of imipramine treatment on O-maze test average latency to exit to the
open arms. A – Baseline and post-treatment average latency comparison between groups; B –
Baseline (left) and post-treatment (right) average latency for each group; C – Within group
average latency difference, in seconds, comparing baseline and post-treatment scores. IMI-
food – group treated with imipramine in food, IMI-drink – group treated with imipramine in
water, B=baseline, E=end-treatment, bars = SEM.
A comparison of the baseline and post-treatment latency, revealed no statistically significant
differences between groups (CTRBaselinexIFBaseline: p=0.9621, F=1.082; CTRBaselinexIDBaseline:
p=0.8859, F=1.105; IFBaselinexIDBaseline: p=0.9217, F=1.196; CTREndxIFEnd: p=0.3046, U=16;
CTREndxIDEnd: p=0.0931; U=13; IFEndxIDEnd: p=1.000, U=28; Fig. 11A). A comparison between
average latency within groups shows a non-significant increase in both control mice (p=0.4913,
F=1.454) and in the group treated with imipramine in food (p=0.8817, F=2.258) as well as a non-
significant reduction in the group treated with imipramine in water (p=0.2578, F=2.488; Fig. 11B).
The relative average increase or reduction in latency time observed in each of three groups is
presented on the Figure 11C; no significant differences are observed (CTRxIF: p=0.5405, F=1.214;
CTRxID: p=0.0890, F=1.982; IFxID: p=0.3474, F=2.407).
43
Baseli
ne
Post-tre
atmen
t0
5
10
15
20 A
# of
exi
ts
Control
IMI-fo
od
IMI-d
rink
0
5
10
15
20 B
B BB E E E
# of
exi
ts
Control
IMI-fo
od
IMI-d
rink
-20
-10
0
10
20 C
Diff
eren
ce (#
)
Figure 12: Effects of imipramine treatment on O-maze test average number of exits. A –
Baseline and post-treatment average number of exits comparison between groups; B –
Baseline (left) and post-treatment (right) average number of exits for each group; C – Within
group average number of exits difference, in seconds, comparing baseline and post-treatment
scores. IMI-food – group treated with imipramine in food, IMI-drink – group treated with
imipramine in water, B=baseline, E=end-treatment, bars = SEM.
A comparison of the baseline and post-treatment scores of exits in the O-maze between
groups revealed lack of such differences (CTRBaselinexIFBaseline: p=0.8444, U=22.50;
CTRBaselinexIDBaseline: p=0.7249, U=24.50; IFBaselinexIDBaseline: p=0.7653, U=25; CTREndxIFEnd:
p=0.0959, F=6.668; CTREndxIDEnd: p=0.0947; F=8.072; IFEndxIDEnd: p=0.9073, F=1.211; Fig.
12A). A comparison of average number of exits within groups shows no changes in control mice
either (p=0.9031, F=1.553), and a not-significant increase in both imipramine-treated groups (IF:
p=0.1421, F=15.41; ID: p=0.2654, F=2.602; Fig. 12B).
Control Imi-food Imi-drink
44
The relative average increase or reduction in latency time observed in each of three groups is
compared in Figure 12C, where no significant differences are observed (CTRxIF: p=0.0915,
F=5.921; CTRxID: p=0.0968, F=4.559; IFxID: p=0.8180, F=1.299).
Baseli
ne
Post-tre
atmen
t0
50
100
150
200
*
A
Tim
e in
ope
n ar
ms
(s)
Control
IMI-fo
od
IMI-d
rink
0
50
100
150
200 B
Tim
e in
ope
n ar
ms
(s)
BB
BEE E
Control
IMI-fo
od
IMI-d
rink
-200
-100
0
100
200 C
Diff
eren
ce o
f tim
e in
open
arm
s (s
)
Figure 13: Effects of imipramine treatment on O-maze test average total time spent in open
arms. A – Baseline and post-treatment average total time spent out comparison between
groups; B – Baseline (left) and post-treatment (right) average total time spent in open arms for
each group; C – Within group total time spent in open arms difference, in seconds, comparing
baseline and post-treatment scores. IMI-food – group treated with imipramine in food, IMI-
drink – group treated with imipramine in water, *p<0.05, B=baseline, E=end-treatment, bars =
SEM
A comparison of the baseline values of time spent in open arms in the O-maze between
groups revealed no differences between the measurements (CTRBaselinexIFBaseline: p=0.8048, U=22;
CTRBaselinexIDBaseline: p=0.6126, U=23; IFBaselinexIDBaseline: p=0.1893, U=16; Fig. 13A). However,
significant differences are observed between control mice and the group treated with imipramine in
water (CTREndxIFEnd: p=0.2769, F=1.856; CTREndxIDEnd: p=0.0410; F=2.590; IFEndxIDEnd:
Control Imi-food Imi-drink
45
p=0.2820, F=1.396). A comparison between average time spent in open arms in the O-maze
revealed no change in the group treated with imipramine in food (p=0.7484, F=1.031) and a non-
significant decrease in both control mice and in the group treated with imipramine in water (C:
p=0.2261, F=9.153; ID: p=0.6331, F=3.676; Fig. 13B). The relative average increase or reduction
in time spent on open arms is observed in each of three groups is presented on Figure 13C. No
significant differences are observed (CTRxIF: p=0.0961, F=1.856; CTRxID: p=0.5639, F=1.120;
IFxID: p=0.2863, F=10.47).
3.1.6. Effects of treatment with imipramine on the Openfield behaviour In this paradigm, the average distance travelled, instant speed, number of line crossings,
number of entries in the exterior, intermediate and central zones of the open field apparatus were
measured, as well as total time spent in each of these three zones (Fig. 14-19, 21-23).
When comparing the baseline and end-treatment observations for the average distance in the
open field test, all groups show a non-significant decrease (C: p=0.6013, F=2.638; IF: p=0.3039;
F=1.206; ID: p=0.5462, F=1.020; Fig. 14A).
Control
IMI-fo
od
IMI-d
rink
0
10
20
30
40
50 A
Dis
tanc
e (m
)
W2 W2W2W2W2 W2
Week 2
Week 3
Week 4
Week 5
0
10
20
30
40
50 B
Dis
tanc
e (m
)
Control
IMI-fo
od
IMI-d
rink
0
10
20
30
40
50 C
******
Dis
tanc
e (m
)
W2 W3 W4 W5 W2 W3 W4 W5 W2 W3 W4 W5
Control Imi-food Imi-drink
46
Figure 14: Effects of imipramine treatment on average distance travelled in the open field
test. A – Average distance covered during first (left bar) and last (right bar) assessments; B –
Average distance covered per group, per week; C – Average distance covered comparison
between weeks, per group. IMI-food – group treated with imipramine in food, IMI-drink –
group treated with imipramine in water, *p<0.05 vs. IFW3, **p<0.01 vs. IFW3, ***p<0.001 vs.
IFW3, W2-W5=Weeks 2-5, bars correspond to SEM.
Weekly average distance comparison between groups shows no statistically significant
differences, although the difference between the two treatment groups during the third week of
treatment can be considered significant, depending on the numerical readout (p=0.0512, F=5.047;
Fig. 14B). It was also noted a trend for the group treated with imipramine in water to have the
highest distance covered. A within group comparison of average distance covered dynamics
between weeks shows no significant differences in control mice and in the group treated with
imipramine in water (p>0.05; Fig. 14C). At the same time, these were observed between the third
and remaining weeks of treatment in the group treated with imipramine in food (IFW2xIFW3:
p=0.0010, F=5.091; IFW3xIFW4: p=0.0004, F=1.039; IFW3xIFW5: p=0.0225, F=6.142).
A comparison of the baseline and after-treatment measurements of the average instant speed
in the distance in the open field test revealed a non-significant decrease of this parameter in all
groups (C: p=0.3888, F=4.940; IF: p=0.2898; F=1.160; ID: p=0.5319, F=1.007; Fig. 15A).
Control
IMI-fo
od
IMI-d
rink
0.00
0.02
0.04
0.06
0.08 A
Spee
d (m
/s)
W5 W2 W5W2 W5W2
Control Imi-food Imi-drink
47
Week 2
Week 3
Week 4
Week 5
0.00
0.02
0.04
0.06
0.08 BSp
eed
(m/s
)
Control
IMI-fo
od
IMI-d
rink
0.00
0.02
0.04
0.06
0.08 C
**
****
Spee
d (m
/s)
W2 W2W4W2 W3W3 W3W4W5 W5 W4 W4
Figure 15: Effects of imipramine treatment on average instant speed in the open field test. A
– Average instant speed during first (left bar) and last (right bar) assessments; B – Average
instant speed per group, per week; C – Average instant speed comparison between weeks, per
group. IMI-food – group treated with imipramine in food, IMI-drink – group treated with
imipramine in water, *p<0.05 vs. IFW3, **p<0.01 vs. IFW3, ***p<0.001 vs. IFW3, W2-
W5=Weeks 2-5, bars correspond to SEM.
Weekly average instant speed comparison shows a lack of statistically significant differences
between groups, although the difference between the two treatment groups during the third week of
treatment is close significant (p=0.0508, F=4.992; Fig. 15B). A within group comparison of
average instant speed dynamics between weeks shows that there were no significant differences in
control mice and in the group treated with imipramine in water (p>0.05; Fig. 15C). Such
differences were observed between the third and remaining weeks of treatment in the group treated
with imipramine in food (IFW2xIFW3: p=0.0011, F=5.086; IFW3xIFW4: p=0.0004, F=1.095;
IFW3xIFW5: p=0.0232, F=5.898).
Control
IMI-fo
od
IMI-d
rink
0
200
400
600 A
# of
cro
ssin
gs
W5 W2 W5 W2 W5W2
Control Imi-food Imi-drink
48
Week 2
Week 3
Week 4
Week 5
0
100
200
300
400
500 B#
of c
ross
ings
Control
IMI-fo
od
IMI-d
rink
0
100
200
300
400
500 C
*
**
# of
cro
ssin
gs
W4 W2W2W2 W5W3W4 W4W3 W3W5 W5
Figure 16: Effects of imipramine treatment on average number of line crossings in the open
field test. A – Average number of line crossings during first (left bar) and last (right bar)
assessments; B – Average number of line crossings per group, per week; C – Average number
of line crossings comparison between weeks, per group. IMI-food – group treated with
imipramine in food, IMI-drink – group treated with imipramine in water, *p<0.05 vs. IFW3, **p<0.01 vs. IFW3, W2-W5=Weeks 2-5, bars correspond to SEM.
A comparison of the baseline and after-treatment observations for the average number of line
crossings in the open field test revealed lack of changes (p=0.9665, F=2.730), whereas the group
treated with imipramine in food demonstrated a non-significant decrease of this parameter
(p=0.6348; F=2.068) and the group treated with imipramine in water reveals a non-significant
increase of this behaviour (p=0.4769, F=2.993; Fig. 16A). Weekly average number of line
crossings comparison was not statistically different between groups during the study (Fig. 16B). At
all time points, group treated with imipramine with water had the highest average number of line
crossings. Within group comparison of average distance covered dynamics between weeks showed
lack of significant differences in control mice and in the group treated with imipramine via water
(p>0.05; Fig. 16C). Such differences were observed between both second and third weeks
(IFW2xIFW3: p=0.0087, F=1.353) and between third and fourth (IFW3xIFW4: p=0.0220, F=1.207)
weeks of treatment in the group treated with imipramine via food.
A comparison of the baseline and after-treatment measurements of the average number of
entries in the peripheral zone of the open field test did not reveal any changes in control mice
(p=0.8966, F=2.425), however, the group treated with imipramine via food demonstrated a non-
significant decrease (p=0.4726; F=1.280) and the group treated with imipramine in water reveals a
non-significant increase (p=0.2164, F=1.227) of this parameter (Fig. 17A).
49
Control
IMI-fo
od
IMI-d
rink
0
10
20
30
40
50 A
W2 W5 W2 W5 W2 W5
# of
ent
ries
perip
hery
Week 2
Week 3
Week 4
Week 5
0
10
20
30
40
50
*
B
# of
ent
ries
perip
hery
Control
IMI-fo
od
IMI-d
rink
0
10
20
30
40
50
*
C
W3 W4W2 W5 W2 W3 W4 W5 W2 W3 W4 W5
# of
ent
ries
perip
hery
Figure 17: Effects of imipramine treatment on the average number of entries to the
peripheral zone of the open field. A – Average number of entries in the peripheral zone during
first (left bar) and last (right bar) assessments; B – Average number of entries in the
peripheral zone per group, per week; C – Average number of entries comparison between
weeks, per group. IMI-food – group treated with imipramine in food, IMI-drink – group
treated with imipramine in water, *p<0.05, W2-W5=Weels 2-5, bars correspond to SEM.
Weekly evaluated average number of entries in the peripheral zone comparison showed that
there were no statistically significant differences between groups, except between the two
imipramine-treated groups in the first assessment, corresponding to the second week of treatment
(p=0.0847, F=1.690; Fig. 17B). A within group comparison of average number of entries in the
peripheral zone of the open field between weeks shows the absence of significant differences in
control mice and in the group treated with imipramine in water (p>0.05); these differences were
observed between the second and third weeks of treatment in the group treated with imipramine in
food (IFW2xIFW3: p=0.0087, F=1.353; Fig. 17C).
Control Imi-food Imi-drink
50
The analysis of the baseline and after-treatment measurements of the average number of
entries in the intermediate zone of the open field test revealed lack of changes in control mice
(p=0.8572, F=2.628), whereas the group treated with imipramine in food displayed a non-
significant decrease of this behaviour (p=0.1505; F=2.598), and the group treated with imipramine
in water had a non-significant increase of entries (p=0.1953, F=9.606; Fig. 18A).
Weekly average number of entries in the intermediate zone comparison shows no statistically
significant differences, except between the two imipramine-treated groups in the first assessment,
corresponding to the second week of treatment (p=0.0144, F=1.918; Fig. 18B).
Control
IMI-fo
od
IMI-d
rink
0
10
20
30
40
50 A
W5 W2 W5 W2W2 W5
# of
ent
ries
inte
rmed
iate
Control
IMI-fo
od
IMI-d
rink
0
10
20
30
40
50 C
W2 W3 W4 W5 W3W2 W4 W5 W3 W4 W5W2
# of
ent
ries
inte
rmed
iate
Week 2
Week 3
Week 4
Week 5
0
10
20
30
40
50
*B
# of
ent
ries
inte
rmed
iate
Figure 18: Effects of imipramine treatment on the average number of entries to the
intermediate zone of the open field. A – Average number of entries in the intermediate zone
during first (left bar) and last (right bar) assessments; B – Average number of entries in the
intermediate zone per group, per week; C – Average number of entries comparison between
weeks, per group. IMI-food – group treated with imipramine in food, IMI-drink – group
treated with imipramine in water, *p<0.05, bars correspond to SEM.
Control Imi-food Imi-drink
51
A within group comparison of average number of entries in the intermediate zone of the open
field between weeks shows a lack of significant differences for each of the three experimental
groups (Fig. 18C). A comparison of the baseline and end-treatment observations for the average
number of entries in the central zone of the open field test revealed a non-significant decrease of
this behaviour in control mice and the group treated with imipramine via food show (C: p=0.7367;
F=3.221; IF: p=0.0720; F=1.070; Fig. 19A). In contrast, the group treated with imipramine in water
reveals a non-significant increase of this variable (p=0.2483, F=2.116).
Control
IMI-fo
od
IMI-d
rink
0
5
10
15 A
W5W2 W2 W2W5 W5
# of
ent
ries
in c
entr
e
Week 2
Week 3
Week 4
Week 5
0
10
20
30
40
50
*
B
# of
ent
ries
in c
entr
e
Control
IMI-fo
od
IMI-d
rink
0
10
20
30
40
50
*
C
W2 W3 W4 W5 W2 W3 W4 W5 W2 W4 W5W3
# of
ent
ries
in c
entr
e
Figure 19: Effects of imipramine treatment on the average number of entries in the central
zone of the open field. A – Average number of entries in the central zone during first (left bar)
and last (right bar) assessments; B – Average number of entries in the central zone per group,
per week; C – Average number of entries comparison between weeks, per group. IMI-food –
group treated with imipramine in food, IMI-drink – group treated with imipramine in water, *p<0.05, W2-W5=Weeks 2-5, bars correspond to SEM.
Control Imi-food Imi-drink
52
Weekly measured number of entries in the central zone comparison shows absence of
statistically significant differences between groups, except between the two imipramine-treated
groups in the first assessment, corresponding to the second week of treatment (p=0.0233, F=8.694;
Fig. 19B). A within group comparison of average number of entries in the central zone of the open
field between weeks shows no significant differences in control mice and in the group treated with
imipramine in water (p>0.05; Fig. 19C). These changes were observed between the second and
third weeks of treatment in the group treated with imipramine in food (IFW2xIFW3: p=0.0224,
F=1.636).
Week 2
Week 3
Week 4
Week 5
0
10
20
30
40
50
* ***
&A
# of
ent
ries
Week 2
Week 3
Week 4
Week 5
0
10
20
30
40
50
***
** ***
***
B
# of
ent
ries
Week 2
Week 3
Week 4
Week 5
0
10
20
30
40
50
*** ** *** **
C
# of
ent
ries
Figure 20: Effects of imipramine treatment on the mean number of entries per zone, per
group, per week. A – Control mice, B – IMI-food, C – IMI-drink. Light bars: peripheral zone,
intermediate bars: intermediate zone, dark bars: central zone, IMI-food – group treated with
imipramine in food, IMI-drink – group treated with imipramine in water, *p<0.05 vs. other
zones, **p<0.01 vs. other zones, ***p<0.001 vs. other zones, &p<0.01 vs. peripheral zone, bars
= SEM.
Control Imi-food Imi-drink
53
The significant preference for the more peripheral areas of the open field when compared to
the central, exposed square is seen in each of the three experimental groups, with exception of
control mice on the second and fourth assessments (Fig. 20A). This figure also shows a difference
between the central zone and both intermediate and peripheral zones. Additional statistical
information with p and F values is shown in Table 6.
A comparison of baseline and after-treatment observations for the average time spent in the
peripheral zone of the open field test, revealed a non-significant increase of this measure in control
mice and the group treated with imipramine with food (C: p=0.1747, F=41.45; IF: p=0.1124,
F=2.428), while the group treated with imipramine via water reveals a non-significant decrease
(p=0.3781, F=1.828; Fig. 21A).
Control
IMI-fo
od
IMI-d
rink
0
150
300
450
600
750 A
W5 W2W2 W5 W2 W5
Tim
e in
per
iphe
ry (s
)
Week 2
Week 3
Week 4
Week 5
0
150
300
450
600
750
*
B
Tim
e in
per
iphe
ry (s
)
Control
IMI-fo
od
IMI-d
rink
0
150
300
450
600
750 C
Tim
e in
per
iphe
ry (s
)
W2 W3 W4 W5 W2 W3 W4 W5 W2 W3 W4 W5
Figure 21: Distribution of mean time spent in peripheral zone. A – Average number of time
spent in the peripheral zone during first (left bar) and last (right bar) assessments; B –
Average number of time spent in the peripheral zone per group, per week; C – Average time
spent out comparison between weeks, per group. IMI-food – group treated with imipramine
Control Imi-food Imi-drink
54
in food, IMI-drink – group treated with imipramine in water, *p<0.05, bars correspond to
SEM.
The analysis of weekly measured average time spent out in the peripheral zone comparison
shows a lack statistically significant differences, except between the two imipramine-treated groups
in the first assessment during the second week of treatment (p=0.0224, F=1.636; Fig. 21B). A
within group comparison of total time spent out in the peripheral zone of the open field between
weeks shows a lack of significant differences between weeks for each of the three experimental
groups(Fig. 21C).
Control
IMI-fo
od
IMI-d
rink
0
50
100
150
200
250 A
Tim
e in
inte
rmed
iate
zon
e (s
)
W2 W2W5 W5 W2 W5
Week 2
Week 3
Week 4
Week 5
0
50
100
150
200
250
*
B
Tim
e in
inte
rmed
iate
zon
e (s
)
Control
IMI-fo
od
IMI-d
rink
0
50
100
150
200
250 C
Tim
e in
inte
rmed
iate
zon
e (s
)
W2 W3 W4 W5 W2 W3 W4 W5 W2 W3 W4 W5
Figure 22: Distribution of mean time spent in the intermediate zone. A – Average number
of time spent in the intermediate zone during first (left bar) and last (right bar) assessments;
B – Average time spent out in the intermediate zone per group, per week; C – Average time
spent out comparison between weeks, per group. IMI-food – group treated with imipramine
in food, IMI-drink – group treated with imipramine in water,*p<0.05, W2-W5=Weels 2-5,
bars correspond to SEM.
Control Imi-food Imi-drink
55
The analysis of the baseline and after-treatment measurements of the average time spent in the
intermediate zone of the open field test revealed a non-significant increase of this behaviour both in
control mice (p=0.5131, F=1.335; Fig. 22A) and in the group treated with imipramine in water
(p=0.1505, F=2.598), whereas the group treated with imipramine in food displayed a non-
significant increase in time spent (p=0.3172, F=1.824). A comparison of weekly evaluated average
time spent out in the intermediate zone found no significant differences between groups, except
between the two imipramine-treated groups in the first assessment, during the second week of
treatment (p=0.0210, F=1.609; Fig. 22B). A within group comparison of total time spent out in the
intermediate zone of the open field between weeks shows an absence of significant differences (Fig.
22C).
Control
IMI-fo
od
IMI-d
rink
0
50
100
150
200
250 A
Tim
e in
cen
tre
(s)
W5 W2W2 W5 W2 W5
Week 2
Week 3
Week 4
Week 5
0
50
100
150
200
250 B
Tim
e in
cen
tre
(s)
Control
IMI-fo
od
IMI-d
rink
0
50
100
150
200
250 C
Tim
e in
cen
tre
(s)
W2
W3
W4 W5 W2 W3 W4 W5 W2 W3 W4 W5
Figure 23: Distribution of mean time spent in the central zone. A – Average number of time
spent in the central zone during first (left bar) and last (right bar) assessments; B – Average
time spent out in the central zone per group, per week; C – Average time spent out
comparison between weeks, per group. IMI-food – group treated with imipramine in food,
Control Imi-food Imi-drink
56
IMI-drink – group treated with imipramine in water, *p<0.05, W2-W5=Weeks 2-5, bars
correspond to SEM
A comparison of baseline and after-treatment observations for the average time spent in the
central zone of the open field test revealed a non-significant decrease of this behaviour in all groups
(C: p=0.7882, F=3.341; IF: p=0.0798, F=3.422; ID: p=0.8351, F=1.616; Fig. 23A). Weekly
measured average time spent out in the central zone comparison shows a lack of statistically
significant differences between groups (Fig. 23B). A within group comparison of average time
spent out in the central zone of the open field between weeks shows a lack of significant differences
for each of the three experimental groups (Fig. 23C).
Week 2
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Figure 24: Distribution of the mean number of entries per zone, per group, per week. A –
Control mice, B – IMI-food, C – IMI-drink. Light bars: peripheral zone, intermediate bars:
intermediate zone, dark bars: central zone, IMI-food – group treated with imipramine in food,
IMI-drink – group treated with imipramine in water,*p<0.05, **p<0.01, ***p<0.001 between all
zones, &p<0.01 vs. other zones, bars = SEM.
Control Imi-food Imi-drink
57
The significant preference for peripheral areas of the open field versus central area was found
in all the three experimental groups, for statistical information see Table 7 (Fig. 24).
3.1.7. Effects of treatment with imipramine in the Forced swim test In this test, the latency to float and total time spent floating were evaluated in the forced
swim test. Measurements of both days of testing were compared for each of the three experimental
groups (Fig. 25).
Day 1
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Figure 25: Forced swim test. A – Comparison of average latency to float between all groups
in the two days of the forced swim test; B – Within group comparison of average latency to
float between day 1 (left bar) and day 2 (right bar) of the forced swim test; C – Comparison of
average total time spent floating between all groups in the two days of the forced swim test; D
– Within group comparison of average total time spent floating between day 1 (left bar) and
day 2 (right bar) of the forced swim test. IMI-food – group treated with imipramine in food,
IMI-drink – group treated with imipramine in water, **p<0.01, ***p<0.001, %p<0.01 vs.
control, #p<0.001 vs. control, &p<0.01 vs. IMI-drink, %p<0.01 vs. control, D1=Day 1,
D2=Day 2, bars = SEM.
Control Imi-food Imi-drink
58
On the first day of the forced swim test, the group treated with imipramine in food had the
highest latency to float while the control group had the lowest one (C=79.3s; ID= (259.5s and
108.8s, respectively; Fig. 25A). In this test, significant differences between the group treated with
imipramine in food and the other two experimental groups were found (CxIF: p=0.0002, F=17.46;
CxID: p=0.0815, F=2.842; IFxID: p=0.0007, F=6.144). The group treated with imipramine via
food showed the lowest total time spent floating in comparison to the other two experimental
groups (C=197.1; IF=38.32; ID=231.9; Fig. 25C). Significant differences are only found for the
group treated with imipramine via food as compared to control mice (p=0.0008, F=4.569) and the
group treated with imipramine in water (p<0.0001, F=1.368).
On the second day of forced swim test, all experimental groups show a reduced latency of
floating in comparison to the first assessment (Fig. 25B). The group treated with imipramine via
food displayed significantly higher latency of floating versus other experimental groups (CxIF:
p<0.0001, F=27.30; CxID: p=0.1573, F=36.85; IFxID: p=0.0015, ID=1.350). Control mice have
shown an increase in total time spent floating on the second day of the forced swim test, in
comparison to Day 1 results (Fig. 25C-D).In contrast to that, such changes were not obvious for
both groups treated with imipramine. The total amount of time spent floating by the group treated
with imipramine via food is significantly lower than values measured in other experimental groups
(CxIF: p<0.0001, F=5.219; CxID: p=0.1258, F=1.056; IFxID: p<0.0001, F=4.944).
3.1.8. Effects of treatment with imipramine in the tail suspension test In this test, the latency to immobilization and total time spent immotile in the forced swim
test were assessed in both days 1 and 2 (Fig. 26-27).
Figure 26: Tail suspension test, day 1. A – latency to immobilization, in seconds; B –
average total time spent immotile, in seconds. IMI-food – group treated with imipramine in
food, IMI-drink – group treated with imipramine in water, Bars = SEM.
Control Imi-food Imi-drink
59
On the first day of the tail suspension test, the group treated with imipramine via food
demonstrated the highest latency of the first episode of immobilization while the other two
experimental groups had similar scores between themselves; no significant differences were found
(Fig. 26). Control mice spent the least time immobile when compared to imipramine-treated that
had similar performance (Fig. 26B). Significant differences were absent between the three
experimental groups.
Control Imi-food Imi-drink0
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Figure 27: Tail suspension test, day 2. A – Latency to immobilization, in seconds; B –
average total time spent immotile, in seconds. *p<0.05, IMI-food – group treated with
imipramine in food, IMI-drink – group treated with imipramine in water, bars = SEM.
On the second day of the tail suspension test, the control group had the highest latency in
comparison to both imipramine-treated groups (Fig. 27); the latter ones non-significantly reduced
this behaviour relatively to the first measurement. The three groups have spent approximately the
same time immobile as in the first day (Fig. 27B). However, a significant increase of the duration of
immobilization was found between control mice and the group treated with imipramine via food
(CxIF: p=0.0395, F=10.95; CxID: p=0.2381, F=2.488; IFxID: p=0.8902, F=27.23).
3.2. Discussion
3.2.1. Effects of treatment with imipramine on body weight Previous studies (Strekalova and Gorenkova, in preparation) have shown that a 4-week
administration of imipramine with food or water at the dose of 15 mg/kg/day resulted in a reduction
of body weight in naïve C57BL/6N mice. In this work, I tested whether a treatment with a lower
dose of this drug (7 mg/kg/day) would exclude such effect of imipramine which can confound other
behavioural testing in pre-clinical models of depression in rodents.
In the beginning of the study all animals had similar body weight (Fig. 3A). After four weeks
of treatment, groups treated with imipramine had a non-significant reduction of body weight in
Control Imi-food Imi-drink
60
comparison to the control group (Fig. 3B). The control group gained body weight during the 4-
week experiment, this difference was significant in comparison to a baseline measurement
(+0.84g/animal, +3.11%). Both imipramine-treated groups, in contrast, lost weight. In comparison
to the baseline values, mice treated with imipramine via food had a mean reduction of -
1.67g/animal, -6.42%, mice treated with imipramine via water had a mean reduction of -
2.35g/animal, -9.24% (Fig. 3C-D).
These data suggest that a low dose of imipramine still affects body weight of naive mice like
the higher dose of this drug and also argue for the validity of selected methods of drug
administration via food and water. A decrease of body weight can confound behavioural testing for
pre-clinical models of depression in rodents since its occurrence in naïve animals can mask a full
antidepressant effect with false positive results and therefore, the application of imipramine even in
low dose as in this work seems to be not convenient for such type of studies.
The drop of body weight in mice treated with imipramine via food observed in this study
could potentially be an effect of the feeding procedure: while control had both food and water ad
libitum, the first group had a limited number of pellets. If this would be the situation, these animals
would have lost weight even more than animals which were dosed via water. As it was not the
case, this explanation is unlikely. Besides, there was sufficient food available for each mouse from
this group of animals throughout the experiment.
The weight reduction as an effect of treatment with imipramine found in this work is in line
with abovementioned results of Strekalova and Gorenkova (in preparation) obtained in C57BL/6N
naïve male mice. Similarly to these data, the administration of imipramine with drinking water
during 3-5 weeks significantly decreased body weight of naïve rats (Von Frijtag et al., 2002). In
rats, 15 daily injections of imipramine (10 or 20 mg/kg) decreased body weight, higher dose also
decreased body weight (Mogensen et al., 1994). In this study, imipramine-induced reduction of
body weight was observed at the lower dose of treatment that might be explained by species-
specific differences and duration of the drug delivery.
While in unstressed rodents treatment with imipramine was reported to result in the weight
reduction, in stressed animals the treatment with this tricyclic antidepressant was shown to result in
the opposite effect, leading to a restoration of body weight which was lost due to stress, as
described by Plaznik and collaborators (1993). In the latter study, imipramine was administrated per
os to rats subjected to 1h -restraint stress or by Kumar and Singh (2007) in a sleep deprivation
procedure in mice. In humans, chronic treatment with imipramine and other tricyclics resulted on an
increase of body weight (Berken et al., 1984; Fernstrom et al., 1986; Shioiri et al., 2003).
A possible explanation of distinct effects of imipramine in naïve and stressed rodents might
be related to its effects on the sympathetic nervous system. It is proposed that in naïve animals
61
imipramine activates sympathetic tone, the activity of which is altered during stress. Besides, the
clearance of imipramine in stressed animals is higher as the metabolic rate is enhanced by stress-
induced sympathetic activation.
Altogether, the present work’s data show that body weight is affected by chronic treatment
with employed imipramine treatment, thus suggesting that the applied treatment delivered either
with water or food was effective and can be employed for drug delivery. On other hand, the
undesired effects of imipramine on body weight rise concerns regarding the use of this drug in pre-
clinical models of depression because of possible false positive results in these studies.
3.2.2. Effects of treatment with imipramine on Sucrose test parameters Anhedonia, a decreased ability to experience pleasure, is depicted as one of the core
symptoms of depression (APA, 2000) which can be measured in rodents by the decrease of intake
and preference of palatable solutions (Willner, 1997; Strekalova et al., 2004; Grippo et al., 2006).
Antidepressants of various classes were reported to normalize the consumption and preference for
those solutions over water in the sucrose test,which is decreased in experimental animals during
depressive-like sates (Muscat et al., 1992; D’Aquila et al., 1997; Konkle et al., 2003; Bekris et al.,
2005; Rygula et al., 2006).
One of the few studies which used naïve animals is the work of Strekalova and Gorenkova (in
preparation) in which a 4-week administration of imipramine with drinking water at the dose of
15 mg/kg/day was found to increase sucrose intake and preference, two commonly used measures
of hedonic status, in C57BL/6N mice. In the current work, the hypothesis whether a treatment with
a lower dose of this drug would evoke any effects on behavioural parameters reflective for a
depressive-like state in naïve mice was tested. This study meant to define conditions of the
treatment with a standard antidepressant which can make possible to avoid confounds in studies
with pre-clinical models of depression.
In the beginning of the experiment, all groups had similar intake of water, sucrose and
preference (Fig. 4-6). After a four-week treatment, the group treated with imipramine via drinking
water had significantly increased water and sucrose intake and not significantly decreased sucrose
preference. A pairwise comparison reveals that the only statistically significant difference found
regards post-treatment sucrose intake between groups treated with imipramine. This difference can
be explained by a more efficient drug delivery with water than the one with food.
In all time points of the study, all groups consumed more sucrose solution than water, which
is consistent with intact rodents do show a preference for palatable solutions (Cunningham et al.,
2003; Tordoff and Bachmanoff, 2003; Strekalova et al., 2004; Bessa et al., 2009; Karatayev et al.,
2009; Fig. 6). These results are supported by a study by Strekalova and Gorenkova (in preparation)
62
where imipramine delivered via drinking water (15 mg/kg/day) over four weeks significantly
increases both water and sucrose intake, revealing that even in low dose, chronic administration of
imipramine increased sucrose intake in naive animals.
Imipramine was demonstrated to restore sucrose preference in anhedonic rodents subjected to
stressful procedures (Von Frijtag et al., 2002; Bekris et al., 2005). Also in accordance to those of
Strekalova and Gorenkova (in preparation) the present results show that a low dose of imipramine
delivered with drinking water but not with food significantly increases water and sucrose intake in
naïve mice without changing preference. Altogether it seems that the use of a stressor is needed is
needed to change preference in a way that can be restored by antidepressants
The increased intake effect elicited by the administration of a low dose of imipramine to naïve
mice is undesirable as it may lead to an inaccurate quantification of the delivered drug, suggesting
that this antidepressant has to be used with caution in mouse models of depression due to possible
confounding effects of what is either a normal behaviour or a result of antidepressant action.
Perhaps, even lower doses have to be considered for such type of experiments.
3.2.3. Effects of treatment with imipramine on Novel cage activity In the novel cage test (NCT), used to assess general activity as well as exploration of a new
environment, mice are placed in a new, identical-to-home cage with small amount of bedding. Mice
with depressive-like syndrome display a reduced number of rearings in the novel cage test
(Strekalova et al., 2004). On other hand, scores of vertical activity are increased in animals with
general hyper-locomotion (Boeck et al., 2010; Zhu et al., 2010). Chronic administration of
imipramine at a dose of 15 mg/kg/day with drinking water significantly affected basal physiological
behaviours including increased vertical exploration in the novel cage test (Strekalova and
Gorenkova, in preparation) that was found in similar studies and interpreted as signs of toxic
effects of applied treatment. In this work, the hypothesis whether a low dose of imipramine
delivered per os would have an effect on the number of rearings in comparison to control group was
tested.
The exploration in the NCT was assessed weekly from the second week of treatment (Fig. 7).
At all time points of the study, the number of rearings observed in mice treated with imipramine via
drinking water is significantly higher than in control group. On the third week, mice treated with
imipramine via food also shown a non-significant increase of the novel cage activity suggesting that
drug administration with water is more efficient.
Other studies resulted in similar outcomes, as for instance, Gao and Cutler (1994) shown that
chronic administration of imipramine delivered with intraperitoneal injections and with drinking
63
water for 12-16 days, in doses 15.8 and 63.2 µmol/kg in mice caused an enhancement of their
activity in novel environment.
The obtained results show that even a low dose of imipramine administrated chronically
delivered either with water and food affects mouse activity by increasing the number of rearings.
Such treatment elicits hyper-arousal behaviour, which is considered to be a sign of toxic effects on
the animals and is not convenient for any applications in pre-clinical studies.
3.2.4. Effects of treatment with imipramine on parameters of the Darklight box test This test is used to assess potential anxiogenic and anxiolytic treatment (Crawley, 1985;
Costall et al., 1988; Kilfoil et al., 1989; Young and Johnson, 1991). The chronic administration of
imipramine at the dose of 15 mg/kg/day delivered with drinking water decreased latency to first
exit, increase the number of transitions between lit and dark areas and of total time spent in the
white compartment (Strekalova and Gorenkova, in preparation), which are suggested indices of
anxiety state (Crawley, 1981; Kilfoil et al., 1989; Onaivi and Martin, 1989; Gorzó et al., 1998;
Bourin and Hascoët, 2003; Strekalova et al., 2004). In this work, the hypothesis whether a low dose
of imipramine delivered orally either by food or with drinking water would evoke such effects was
tested.
These results show that a four-week treatment with imipramine delivered with water
significantly reduces the latency to exit of the dark compartment (Fig. 8). Moreover, a statistically
significant difference is found between mice treated with imipramine in water and the other two
groups, which suggests that unlike the delivery of imipramine with food, the supplementation of
drinking water with imipramine exerts an anxiolytic-like effect (Fig. 8A).
The number of exits observed after the four-week treatment was significantly reduced in
control mice, showing increased anxiety (Fig. 9B). On other hand, the reduced number of exits
observed in the group treated with imipramine in food reveals an interaction between anxiety and a
counter-balancing action of treatment (Fig. 9B). The group treated with imipramine in water was
the only one to increase the number of exits due to an anxiolytic-like effect of treatment (Fig. 9B).
Furthermore, the number of exits is only statistically different between the group treated with
imipramine in water and control mice (Fig. 9A). These data indicate a trend for imipramine to have
a more eminent anxiolytic-like effect at a low dose when delivered with water.
By the end of a four-week treatment, both control mice and the group treated with imipramine
in food have shown a reduction in total time spent in the lit compartment, whereas in mice treated
with imipramine delivered with water this parameter was increased (Fig. 10B). No statistically
significant differences between groups were found (Fig. 10A). These results also suggest a trend for
64
an anxiolytic-like effect of imipramine delivered with water, in agreement with De Angelis (1996)
and Krass and others (2008).
The above results are in line with those of Strekalova and Gorenkova (in preparation) which
shown that the administration of imipramine at a dose of 15 mg/kg/day over 4 weeks results in a
reduction in latency and increases in number of transitions and total time spent in the lit
compartment, suggesting an anxiolytic-like effect. A study by De Angelis (1996) in naïve CD1
female mice also shows a trend for imipramine to reduce anxiety-like behaviours in the dark-light
box at doses of 10-20 mg/kg, while another work by Krass and collaborators (2008) shows an
increase in time spent in the white compartment when imipramine (15 mg/kg) is administrated.
Some investigators, however, report lack of effects of imipramine in the dark-light test (Onaivi and
Martin, 1989) at different doses, both in rats as Pich and Samanin (5-20 mg/kg/day), Gorzó and
colleagues (30mg/kg) and in mice as Shimada and others (5-10 mg/kg). A discrepancy in the
described effects of imipramine on anxiety-related behaviours could be due to fluctuations in the
sensitivity of tested populations of animals to treatment, as suggested by studies in mice with
different individual characteristics of anxiety and emotionality (Freund et al., 1979; Devi and
Reddy, 2005).
The above results suggest that a four-week chronic administration of imipramine at a low
dose to naïve mice can evoke an anxiolytic-like effect in the dark-light box apparatus only if
delivered with water.
3.2.5. Effects of treatment with imipramine on behaviour in the Elevated Omaze test The elevated O-maze appeared as a modification of the elevated plus maze (EPM) as a test
used to quantify anxiety levels in rodents with the main advantage of being circular in shape and
circumventing the behavioural ambiguity created by the central platform in the latter (Shepherd et
al., 1994). The percent time spent in exposed quadrants (Montgomery, 1955; Lister, 1987; Shepherd
et al., 1994; Rodgers et al., 1997), latency to the first transition between quadrants (Rodgers et al.,
1997) and number of transitions (Pellow et al., 1985) are accepted scoring variables to reflect
anxiety in this model. The present work addresses if a 4-week chronic treatment with a low dose of
imipramine delivered to naïve mice with water or food elicits an anxiolytic-like effect.
After a four-week experimental period both the latency to exit to open quadrants and the
number of exits show no statistically significant differences between groups (Fig. 11A, 12A). As
for total time spent in the exposed areas of the O-maze only the group treated with imipramine in
water was significantly different from control mice (Fig. 13A).
These data are in line with those of Mirza and colleagues (2007) who shown that an
administration of 15 mg/kg/day of fluoxetine and 20 mg/kg/day of paroxetine in mice effectively
65
increases time spent in the open quadrants of the zero maze as well as the number of transitions
between quadrants. Another work by Pahkla and collaborators (2000) also shows that in adult rats
the administration of desipramine (10-20 mg/kg) and fluoxetine (10 mg/kg) increase the number of
transition in this test, however failing to show an anxiolytic-like effect in the latency to first exit and
total time spent out.
Altogether these data suggest that the delivery of imipramine at a low dose has anxiolytic-like
behavioural effects in naïve male mice when delivered with water. Nonetheless, the need to achieve
a strong statistical validity requires further experimentation either to confirm the results obtained or
to rule out the validity of the proposed hypothesis.
3.2.6. Effects of treatment with imipramine on the Openfield behaviour The open field test is commonly used to evaluate locomotor responses to drugs as well as
behavioural responses to novelty. Locomotion can be assessed by scoring average speed, number of
line crossings or total distance whereas the number of entries and time spent in each of three zones
(central, intermediate and peripheral) can be used in addition to the classical test as indicators of
anxiety (Prut and Belzung, 2003; Deussing, 2006; Kalueff et al., 2007). In this work, the hypothesis
whether a 4-week treatment with a low dose (7 mg/kg/day) of imipramine delivered with food or
water to naïve mice is able to show anxiolytic-like effects without influencing locomotion was
tested.
These results have shown that a four-week treatment with imipramine evokes no significant
effects on parameters of locomotor behaviour: distance covered, number of line crossings and
average speed in the open field (Fig. 14-16). However, both number of entries and time spent out
parameters show a very significant distinction between the different zones of the apparatus with all
animals choosing to spend more time by the walls than in the intermediate zone, being the central
square the least preferred (Fig. 17-24).
The above data are in line with the literature where the chronic administration of imipramine
up to 20 mg/kg in mice (De Angelis, 1996; Pogorelov et al., 2007) and 30 mg/kg in rats (Réus et
al., 2009; Fortunato et al., 2010) does not elicit behavioural changes in locomotor patterns
(Kulkarni and Dandiya, 1973; Ergun et al., 2008; Chaviaras et al., 2010), number of line crossings
(Gutiérrez-García et al., 2009; Réus et al., 2009; Fortunato et al., 2010) or time spent in central area
(Pogorelov et al., 2007).
These results suggest that animals display the known rodent thigmotaxis where animals prefer
to spend more time by the walls and avoid exposed areas and also indicate that the chronic
administration of a low dose (7 mg/kg/day) of imipramine with food or water to naïve male mice
66
has no significant effect in locomotor behaviour or as an anxiolytic in the open field test, making it
suitable to use in pre-clinical models of depression in rodents.
3.2.7. Effects of treatment with imipramine in the Forced swim test The forced swim test is based upon the observation that rodents eventually develop
immobility when placed in a cylinder of water after they stop active escape-oriented behaviours,
such as climbing or swimming (Cryan et al., 2005). Antidepressant treatments reduce the amount of
immobility (Borsini and Meli, 1988; Rogóz et al., 2005), delay its onset (Castagné et al., 2009), and
increase or prolong active escape behaviours displayed during the FST (Frankowska et al., 2007;
Hayashi et al., 2011). In this work, the hypothesis whether the effects of a low dose of imipramine
on latency to immobility and total time spent immobile was tested.
Data from both days of testing show a statistically significant increased in latency to
immobility and reduction in total time spent floating in the group treated with imipramine in food
when compared to both control mice and the group receiving treatment in water (Fig. 25).
Comparing the two days of testing, while on one hand control mice and the group treated with
imipramine in water show a significant reduction in the latency to first immobilization, on other
hand only control shown a trend to increase the total time spent immobile between consecutive days
of testing (Fig. 25). These results verify those of Porsolt and others (1977) where on the second day
of testing, animals show a significant decrease in latency to immobilization, interpreted as a signal
of helplessness.
The findings in this work are in line with the literature where the administration of doses as
low as 5 mg/kg (Bai et al., 2001) up to 60 mg/kg significantly increase the total swimming duration,
both in mice (Poleszak et al., 2005; Kulkarni and Dhir, 2007; Shimamura et al., 2007) and rats
(Porsolt et al., 1978; Rybicka and Plaznik, 1998; Kitamura et al., 2002; Rogóz et al., 2005;
Kusmider et al., 2007; Bessa et al., 2009; Gutierrez-García and Contreras, 2009; Réus et al., 2010).
Another study, by Castagné and colleagues (2006), demonstrates that a chronic administration of
imipramine (16-64 mg/kg) to naïve mice significantly increases the latency to immobility in a dose-
dependent manner. Also, several authors (Frankowska et al., 2007; Hayashi et al., 2011) observed
an increase in escape-oriented behaviours but only when a high dose of imipramine was
administrated (30-60 mg/kg).
The reduction of total time spent immobile and the increase in latency to immobilization on
both testing days in the group treated with imipramine in food seen in this work suggests an
effective antidepressant action, not present when treatment is delivered with water.
67
3.2.8. Effects of treatment with imipramine in the tail suspension test The test is based on the fact that animals subjected to the short-term stress of being suspended
by their tail in a restricted space from which there is no possibility of an escape, eventually cease to
struggle, surrendering themselves to the experimental conditions, developing an immobile posture
regarded as despair or helplessness (Kulkarni and Dhir, 2007; Cunha et al., 2008), which is known
to be significantly reduced by antidepressants (David et al., 2001; Ripoll et al., 2003). In this work
the hypothesis whether a low dose of imipramine delivered either with food or water is able to
increase the latency time to immobility and reduce the total time animals spent immobile was
tested.
Concerning the latency to immobilization parameter, we found no differences between groups
(Fig. 26A, 27A). Between two consecutive testing days, there was a significant reduction of this
parameter in the group treated with imipramine in food. The total immobility time was increased in
the imipramine-treated groups when compared to control mice, but only on second day the group
treated with imipramine in food was significantly different from control (Fig. 26B, 27A). Both a
reduction in latency and an increase in total immobility time were unexpected in imipramine-treated
groups. This can either be due to an intrinsic effect of treatment where the administration of a low
dose of imipramine has a depressant-like effect or to human error in the execution of the protocol.
Since the invention of the tail suspension test in 1985 by Stéru and colleagues, several authors
have reported an antidepressant-like action of imipramine, which significantly reduces total
immobility time in different strains of mice (Klodzinska et al., 1999; Bai et al., 2001; Liu and
Gershenfeld, 2003; Ripoll et al., 2003; Mason et al., 2009) without affecting locomotor activity
(Popik et al, 2003; Belozertseva et al, 2007). The effective dose range is controversial with some
groups which obtained the results at low doses, such as 1 mg/kg (Cunha et al., 2008), 2 mg/kg
(Steru et al., 1985) or 5 mg/kg (Kulkarni and Dhir, 2007). Majority of reports demonstrated effects
when doses 15-60 mg/kg were used. (Bai et al., 2001; David et al., 2001; Popik et al., 2003; Liu et
al., 2003; Ripoll et al., 2003; Crawley et al., 2005; Belozertseva et al., 2007; Mason et al., 2009),
with the highest ones (>30 mg/kg) inducing a significant hyperthermia (David et al., 2001; Liu et
al., 2003).
Analysing both obtained data and the literature, the unexpected results regarding an increase
of total immobility time in the groups treated with imipramine are most likely due to human errors
in the execution of the protocol and no effect can be attributed to treatment.
68
Chapter IV – Conclusions, relevance of the data for the field and outlook
In this work, a low dose of imipramine was chronically delivered to naïve mice either via food
or drinking water in a study with a typical design for pre-clinical evaluation of behavioural effects
of a pharmacological drug with an antidepressant or anxiolytic profile.
The main goals were to assess whether 1) these two methods of drug delivery are effective in
the induction of behavioural effects in mouse models of depression and anxiety and 2) a selected
dose of imipramine elicits, at the same time, general effects on locomotion, water intake and body
weight, which were previously shown for the higher drug dose in the same study design and
considered as signs of toxicity. The sucrose preference, forced swim and tail suspension tests were
employed to address the first question whereas the measurement of body weight, open field
locomotion and novel cage activity were evaluated in order to address the second question.
First, the results of this work suggest positive answer to both questions. Obtained data
evidence the induction of behavioural effects in mouse models of depression and anxiety and that a
chronic treatment with a low dose of imipramine delivered with food and water. Also, this treatment
in naïve male C57BL/6 mice elicited general effects on locomotion, water intake and body weight.
Second, more pronounced effects of imipramine on both types of behavioural read-outs were
found in mice treated via drinking water on the majority of measured variables. The delivery of
imipramine with food affected basal physiological variables in a similar way to the administration
of the drug with water did. It reduced body weight, but did not elevate the liquid intake, locomotion
in the open field and novel cage vertical activity, a sign of arousal.
Thus, on one hand, our results show that both ways of imipramine dosing are effective in
mice, and that the employed dose induces the anti-depressant and anti-anxiety effects. However,
applied treatment results in changes in animals’ weight, locomotion and drinking that may
compromise behavioural measurements during pre-clinical studies which use it. This suggests that
the application of imipramine in pre-clinical models of depression, where long dosing is employed
is not convenient as may result to artefacts in behavioural tests.
4.1. What else could have been done or be improved?
The present work shows that the delivery of a chronic treatment with imipramine with food
and with water is possible, although different behavioural effects are associated with the way of
administration. On other hand, a low dose of this drug was effective in mice and evokes the
antidepressant and anxiolytic effects. However, applied treatment also results in increased
immobilization behaviour and this effect needs an explanation.
69
Complementary biochemical and neuroanatomical measurements are planned to be done in
order to obtain better understanding of the behavioural effects of the chronic administration of low
doses of imipramine in naïve C57BL/6N male mice. Blood sampling at scheduled time points and
analysis of circulating levels of imipramine in the blood, stress hormones as ACTH, CRH or
cortisol is advised. Post-mortem brain extraction and evaluation of imipramine concentrations
would be of high relevance as well, such studies are under way. This protocol could also be tested
on other classes of TCA. The number of animals used in subsequent experiments should be
increased and testing other strains of mice would be of high interest as well.
70
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Tables
Table 1: Parallels of human symptoms of depression in rodents* Human symptoms Simulation in animals
Markedly diminished interest to pleasures in
daily activities (anhedonia)
Intracranial self stimulation, progressive ratio
responding for positive reward, consumption of
palatable solutions and food, social withdrawal
test.
Large changes in appetite or body weight Difficulty in mimicking in animals.
Insomnia or excessive sleeping EEG analyses of sleep
Psychomotor agitation or lowness of movement Difficulty in handling, presence of observable
stereotypies, abnormal locomotion scores (OFT)
Fatigue or loss of energy Reduced activity in the home cage
Low ability to concentrate Impaired working and spatial memory
Difficulty in performing simple tasks leading to
poor personal hygiene Poor coat condition, impaired nest building
Feelings of worthlessness and guilt Cannot be modelled
Recurrent thoughts of suicide Cannot be modelled *Adapted from Cryan and Holmes, 2005
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Table 2: Behavioural tests used for measurement of animal parallels of depression- and anxiety-like states 1. Depression Test Measures Characteristics Effect of antidepressants Observations References Forced swim test (FST) I, H Depressed animals
spend more time immobile, floating
IMI 10 mg/kg (not 5 mg/kg) reduces immobility time and increase latency time in dose-dependent way
α2-adrenergic receptors knock-out render C57BL/6 mice insensitive to chronic IMI administration. Chronic administration of antidepressants can cause hypothermia
David et al., 2001; Schram et al., 2001; Rogóz et al., 2004; Kitamura et al., 2008; Castagné et al., 2009.
Tail suspension test (TST) I, H Depressed animals spend more time immobile
IMI 16 mg/kg reduces immobility time, engagement in escape pursuit and increase latency to immobility
Degree of response to CIT administration depends on mouse strain. Not suitable for use in rats.
Cryan & Holmes, 2005; Crawley et al., 2005
Resident-intruder model Sd Depressed animals become subservient, passive, resilient, poor grooming and fur state
IMI engages fighting behaviour and increased aggressivity, normal grooming
Histone acetylation in the hippocampus seems to be crucial for chronic IMI treatment to work
Rilke et al., 2001; Strekalova et al., 2004; Krishnan & Nestler, 2008
Sucrose consumption test (SCT) A Depressed animals do not prefer palatable solutions over water
IMI (10 mg/kg) reverts preference of palatable solutions over water
Decrease sucrose consumption ≤65% is a criteria for anhedonia
Strekalova et al., 2004; Bessa et al., 2009
Open space swim test (OSST) I, H Same as forced swim test but with repeated measures
Same as forced swim test. IMI 10 mg/kg i.p. is able to revert depression-like phenotype.
Females are more prone to show depression-like behaviours, as in humans. Allows rats to test various escape approaches
Sun & Alkon, 2006
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Table 2 (cont.): Behavioural tests used for measurement of animal parallels of depression- and anxiety-like states 2. Anxiety Test Measures Characteristics Effect of antidepressants Observations References Elevated plus maze (EPM) Aoe Anxious animals
restrict exploration in open arms
Anxiolytic and IMI (20 mg/kg) action increases number and time of exploration of open arms
Pollier, 2000; Cole & Rodgers, 1995
Open-field test (OFT) Aoe Anxious animals walk close to the walls of the box
Anxiolytic action increases amount of time and crossing central area. In rats neither 5 nor 10 mg/kg prove effective
Rogóz et al., 2004
Dark-light paradigm Aoe Anxious animals have aversion for lit spaces
SSRI promote increased exploration of lit areas and transition scores between areas
Fish et al., 2005; Crawley & Goodwyn, 1980
Burying behaviour test (BBT) Ano Anxious animals tend to bury potentially harmful or strange objects
Anxiolytic action reduces burying behaviour and increases time spent around object
Suitable for both rats and mice
Fish et al., 2005
Ultrasound vocalization test (USV) Sa Pups separated from dams perform ultrasound vocalizations and reduce rolling behaviour
Chronic treatment with SSRI reduces USV and increases rolling behaviour
Cryan & Holmes, 2005; Fish et al., 2005
Elevated zero maze (EZM) Aoe Anxious animals have aversion for lit, open spaces
Increases the number of exits and time spent in lit areas
Strekalova et al., 2004
87
Table 2 (cont.): Behavioural tests used for measurement of animal parallels of depression- and anxiety-like states 2. Anxiety Test Measures Characteristics Effect of antidepressants Observations References Elevated open platform Aoe Anxious animals
freeze IMI (0.1-10 mg/kg) has no effect
Myata et al., 2007
Object exploration test Ano Anxious animals do little exploration of novel objects in cage
IMI (8 mg/kg) reduces latency to object exploration and increases time around object
Van Miegem et al., 2009
Punished rewards test Pa Suppression of drinking
Anxiolytics reduce suppression of drinking
Suitable only for rats Cryan & Holmes, 2005
I = inescapability; H = helplessness; A = anhedonia; Sd = social defeat; Aoe = aversion of open spaces; Sa = separation anxiety; Ano = aversion to new objects; Pa = pain anxiety; i.p. = intraperitonial; IMI = imipramine
88
Table 3: Effects of imipramine in animal models
Reference Via Dose Model Effect
Assis et al, 2009 i.p. 60’ 10 mg/kg
Rat No effect in the FST
20 mg/kg Reduces immobility in the FST
30 mg/kg
Azima, 1959 i.v. 38 mg/kg Mouse
Lethal dose 25 mg/kg Rat 15 mg/kg Rabbit
Bai et al., 2001 i.p. 15’
2.5 mg/kg
Mouse
No significant effect on immobility in the FST or TST 5 mg/kg Reduces immobility in the FST but not in the TST
10 mg/kg Reduces immobility in the FST and TST
15 mg/kg 30 mg/kg
Reduces immobility in the TST but not in the FST 45 mg/kg
Bekris et al., 2005 i.p. 30’ 10 mg/kg Rat Weight loss, increased sucrose consumption
Bourin et al., 1996 i.p. 30’ 4 mg/kg
Mouse Increases time spent in white compartment in the dark-light box test.
32 mg/kg Decreases immobility time in the FST
Bessa et al., 2009 i.p. 30’ 10 mg/kg Rat Increased sucrose consumption, stressed with imipramine have similar immobility time to unstressed rats in FST and TST
Castagné et al., 2006 i.p. 24h, 4h, 30’ 8 mg/kg
Rat, mouse Reduces immobility in FST and TST, but more in rat 16 mg/kg 32 mg/kg
89
Table 3 (cont.): Effects of imipramine in animal models
Castagné et al., 2009 i.p. 30’
8 mg/kg
Mouse
No significant change in latency and immobility/floating behaviour (FST, TST)
16 mg/kg Increases latency to immobility and floating (FST, TST) 32 mg/kg Significantly decreases immobility/floating and increases latency to
immobility/floating (FST, TST). 64 mg/kg
David et al., 2001 i.p. 30’
4 mg/kg
Mouse No significant differences
8 mg/kg 16 mg/kg
Significantly reduces immobility in FST and TST 32 mg/kg
David et al., 2003 i.p. 30’
1 mg/kg
Mouse No significant effect in reducing immobility in the FST
2 mg/kg 4 mg/kg 8 mg/kg
16 mg/kg Significantly reduces immobility in the FST in NMRI mouse strain
David et al., 2007 In water 20 mg/kg Mouse Reduces anxiety and increases BrdU-marked cells in subgranular zone
Grecksch et al., 1997
i.p. 60’ 20 mg/kg Rat Restores normal learning performance after Bulbectomy Slow release
polymers 3.75 mg/kg
Gutierrez-Garcia and Contreras, 2009 i.p. 60’ daily
2.5 mg/kg
Mouse
No significant effect found in OFT or FST
5 mg/kg Reduces the number of line crossings in the OFT for the second and third week of testing and reduces total immobility score in the FST
Kulkarni and Dhir, 2007 i.p. 30’
2 mg/kg
Mouse
No significant effects in the FST or TST 5 mg/kg
Significantly reduces total immobility score in the FST and TST 10 mg/kg 20 mg/kg
90
Table 3 (cont.): Effects of imipramine in animal models
Liu, Gershenfeld, 2001 i.p. 30’ 30 mg/kg Mouse Significant reduction in immobility in the TST
Naudon et al., 2007 i.p. 30’ 10 mg/kg Rat Impairs spatial memory
O’Neill et al., 1996 s.c. 30’
3 mg/kg
Mouse No significant effect in the TST
10 mg/kg Reduces immobility in the TST 30 mg/kg
Papp et al., 2000 In water 10 mg/kg Rat Restores preference for sucrose after chronic mild stress
Porsolt et al., 1978 i.p. 30’
7.5 mg/kg
Rat Reduces rat immobility in the FST
15 mg/kg Reduces immobility in FST and decreases locomotion in OFT 30 mg/kg
Ripoll et al., 2003 i.p. 30’
1 mg/kg
Mouse
No effect over immobility in the TST 2 mg/kg 4 mg/kg Reduces immobility for NMRI strain in the TST 8 mg/kg Reduces immobility for Swiss, NMRI, DBA/2 and C57BL/6 strains
in the TST 16 mg/kg
Song et al., 2006 i.p.30’
5 mg/kg
Mouse No significant effect on memory, food intake, weight loss
10 mg/kg Significant reduction of food consumption and body weight 20 mg/kg
Steru et al., 1985; Steru et al., 1987 i.p. 30’
0.125 mg/kg
Mouse
No significant effect found on immobility in the TST 0.25 mg/kg 0.5 mg/kg 1 mg/kg 2 mg/kg
Reduces immobility in the TST 4 mg/kg 8 mg/kg
16 mg/kg
91
Table 3 (cont.): Effects of imipramine in animal models
Teste et al., 1993 i.p. 60’
2 mg/kg
Mouse
No significant effect in the TST 4 mg/kg 8 mg/kg
16 mg/kg Reduces immobility in the TST 32 mg/kg
64 mg/kg No significant effect in the TST
Winterhoff et al., 2003 p.o. 60’
1 mg/kg
Mouse
No effect found in the TST 3 mg/kg
Reduces immobility in the TST 10 mg/kg 30 mg/kg
92
Table 4: Routes of drug administration in experimental animals Route of administration Advantages Disadvantages Literature
Drink Natural approach, does not require trained personnel to administer treatment, easy to deliver
Some drugs are not soluble in fluids, may change taste or odour, perceived by animals, lack of precision on drug intake
David et al., 2007; Becker et al., 2010
Food / Oral Natural approach, does not require trained personnel to administer treatment, easy to deliver
Some drugs modify the food taste or smell or are aggressive to the gastrointestinal tract. Slow absorption. Lack of precision on drug intake
Nagy and Johansson, 1977; Meisch, 2001; Pottenger, 2000; Sutfin et al., 1984; Nagy and Johansson, 1975; Moeller and Jorgenstern, 2008; Lipinski et al., 2008
Forced feeding / gavage Food directly delivered to stomach Requires trained personnel, risk of damaging tissues with the cannulae Lipinski et al., 2008
Intramuscular Faster onset of therapeutic effect, possibility of smaller doses, easy to deliver
Requires trained personnel, not indicated for antidepressives as can cause tissue damage
Sutfin et al., 1984; Nagy and Johansson 1975, 1977.
Intraperitoneal Easy to deliver, fast onset of therapeutic effect, bigger amounts of drug
In fat animals absorption is very slow, drug concentration peaks
Pottenger et al., 2000; Glotzbach and Preskorn, 1982, Dhingra and Sharma, Gambarana et al., 2001;
Intravenous Fastest onset of therapeutic effect, small doses. Bypasses 90% hepatic metabolism
Risk of embolism, infection, necrosis, overdose, drug concentration peaks
Nagy and Johansson, 1975; Glotzbach and Preskorn, 1982
Nasal / inhalation Bypass of the blood-brain barrier Not used for antidepressants, requires anaesthetized animal
Berlin et al., 1996, Graff and Pollack, 2004; Illum, 2003, Moeller and Jorgenstern, 2008.
Osmotic pumps Constant, determined delivery rate, good for chronic treatments Requires surgery
Lipinski et al., 2008
Subcutaneous Extended release profile, good for less soluble drugs, slow, constant release of drug
Requires precise execution, limited volume, can damage tissues, depends on biochemical properties
Kadir, 1992; McLennan et al., 2005
93
Table 5: Side-effects of imipramine in patients*
A. Mild side effects
1) Anticholinergic symptoms: tachycardia, mydriasis, dry mouth, warm dry skin, delayed
gastric emptying, slowed intestinal peristalsis, urinary retention, confusion and agitation.
2) Increased variation of appetite and weight
3) Increased fatigue, sedation, weakness, restlessness, dizziness, drowsiness
4) Constipation, nausea, epigastric distress
5) Increased intraocular pressure, blurred vision
B. Severe poisoning
1) Cardiac complications (arrhythmias and conduction disturbances, abrupt changes in
consciousness, convulsions, hypotension)
2) Pulmonary distress (respiratory depression, sudden apnoea, aspiration pneumonia,
oedema)
3) DNA damage (excess of micronuclei, increased formation of ROS)
4) Seizures
ROS – Reactive oxidative stress. *Adapted from; Anderson et al., 1998; Heard et al., 2001; Richelson, 2001; Pascher and Kecskmeti, 2004; Woolf et
al., 2007; Bschor and Adli, 2008; Flanagan, 2008, Verdu et al., 2008.
94
Out – Peripheral zone of the open field apparatus; In – Intermediate zone of the open field apparatus; Centre – Central zone of the open field apparatus.
Table 6: Effect of imipramine on the average number of entries in the open field test
Week 2 Week 3 Week 4 Week 5 p F p F p F p F
Control Out x In 0.5642 1.875 0.6313 1.389 0.3622 1.897 0.4296 1.730
Out x Centre 0.0650 6.241 0.1627 14.48 <0.001 6.440 1.000 1.000 In x Centre 0.0449 11,70 0.0813 20.12 0.0002 12.60 0.0044 14.34
IMI-food Out x In 0.0706 1.409 0.4378 1,716 0.2897 2.115 0.3780 2.087
Out x Centre 0.0001 4.403 0.0034 7.510 0.0008 3.845 0.0002 5.266 In x Centre <0.0001 6.204 0.0024 12,89 0.0007 8.133 0.0004 10.99
IMI-food Out x In 0.1255 1.241 0.5504 2.005 0.4303 1.382 0.3825 1.869
Out x Centre <0.0001 22.66 0.0039 6.492 0.0002 22.62 0.0056 6.834 In x Centre <0.0001 28.13 0.0059 13.02 <0.0001 31.26 0.0032 12.77
95
Out – Peripheral zone of the open field apparatus; In – Intermediate zone of the open field apparatus; Centre – Central zone of the open field apparatus.
Table 7: Effects of imipramine on the average time spent out in the open field test
Week 2 Week 3 Week 4 Week 5 p F p F p F p F
Control Out x In 0.0004 36.96 0.0747 1.557 <0.0001 1.508 <0.0001 1.190
Out x Centre 0.0002 872.2 0.0468 1.337 <0.0001 21.98 <0.0001 70.31 In x Centre 0.0123 23.60 0.7228 1.165 <0.0001 14.60 0.0007 59.07
IMI-food Out x In <0.0001 1.251 <0.0001 1.068 <0.0001 1.464 <0.0001 1.338
Out x Centre <0.0001 26.72 <0.0001 176.9 <0.0001 14.72 <0.0001 37.65 In x Centre 0.0001 21.36 0.0125 165.7 <0.0001 10.06 <0.0001 28.13
IMI-food Out x In <0.0001 1.230 <0.0001 1.187 <0.0001 1.011 <0.0001 1.233
Out x Centre <0.0001 66.16 <0.0001 70.43 <0.0001 90.74 <0.0001 74.84 In x Centre 0.0017 53.78 0.0011 59.36 <0.0001 91.78 0.0009 60.72
96
“To be conscious of our ignorance is a great step to knowledge”
Benjamin Disraeli (1804-1881), Sybil, 1845