Thesis advisor: Michael A. McDannald / The ability to appropriately respond to threats is critical for survival. Disruptions in the neural circuits underlying threat responding are studied in animal models and have clinical implications for anxiety disorders in humans. Pavlovian fear conditioning has been extensively used to study the behavioral and neural basis of defensive systems for threat in animals. In a typical procedure, a cue is paired with foot shock, and subsequent cue presentation elicits freezing, a behavior linked to predator detection. Studies have since shown a fear conditioned cue can elicit locomotion, a behavior that - in addition to jumping, and rearing - is linked to imminent or occurring predation. Yet, the full neural circuit for conditioned, activity-promoting behaviors (e.g. locomotion, jumping, and rearing) remains unclear. The overarching goal of this dissertation is to demonstrate that a fear conditioned cue elicits a variety of defensive behaviors and to probe the neural circuit responsible for the expression of such activity-promoting defensive behaviors. To address the lack of research on activity-promoting defensive behaviors, I conducted experiments to observe multiple behaviors during fear discrimination over a baseline of reward seeking and constructed temporal ethograms of behavior. To improve efficiency in behavior scoring for future projects, I devised and trained a machine learning pipeline using convolutional neural networks. To aid in the understanding of the full neural circuit for activity-promoting defensive behaviors, I investigated the role of dopaminergic neurons of the ventral tegmental area in the expression of the defensive behaviors we observed during fear discrimination. Ultimately, the findings in this dissertation contribute to our general understanding of fear behavior in animals and may inform therapeutic strategies for anxiety disorders. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Psychology and Neuroscience.

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Nocturnal rodents were censused every two weeks from January 1975 until September 1976 on two 1.69 ha (4.13 ac) live-trap grids. Grids were located about 48 kilometers (30 miles) south of Tucson AZ in a cresosote bush-cactus-mesquite-grassland ecotone. All nocturnal rodents, except Dipodomys merriami, were removed from one of the grids beginning in May 1975 and continuing biweekly until September 1976. Effects on the population biology of D. merriami were subsequently analyzed. Density, home range, weight changes, production of young, sex ratio and minimum residence (time between first and last capture) of D. merriami were analyzed on each grid. Effects of removal on D. merriami were minimal. Density on the removal grid increased immediately after removal began; however, this effect decreased with time, as numbers of d. merriami decreased on both grids. Total heteromyid density on the control grid also decreased during the experiment. Removal caused no significant effect on home range. A similar, consistently inverse relationship between home range and density occurred on both grids. Mean weight for both reproductively active and inactive males and females was not significantly different following removal. The number of juveniles increased slightly after removal began, but production of young on both grids was similar, and low. The two populations exhibited different sex ratios for four months after removal began, with males being caught more frequently on the removal grid. Minimum residence times were similar on both grids. It appears that the relatively increased availability of reources had only a temporary effect on D. merriami. In the longer perspective, D. merriami seems to have exhibited what is probably an evolved response to increased resources; i.e., long-term changes in population parameters occur slowly, and only when improved conditions persist for relatively long periods of time.

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Grâce à un modèle expérimental de la trypanosomose humaine Africaine (THA ou maladie du sommeil), le rat infecté par Trypanosoma brucei brucei, nous avons examiné l’implication du monoxyde d’azote (NO) dans le développement de cette pathologie. Des variations opposées de la concentration de ce composé ont été observées chez les animaux infectés, au niveau des compartiments périphérique et central : le NO diminue au niveau du sang et augmente au niveau cérébral. Ces changements sont dépendants de la NO-synthase inductible (iNOS). Au niveau périphérique, la diminution du NO qui survient favorise l’installation du parasite car la pression trypanocide de ce composé est diminuée. Dans cette situation, la L-arginine, le substrat à la base de la synthèse du NO, est utilisée pour la synthèse de polyamines, des composés nécessaires à la croissance du parasite. Ces mécanismes sont très probablement déclenchés par le trypanosome via ses facteurs solubles. Au niveau cérébral, la synthèse du NO est aussi soumise à des régulations qui impliquent l’arginase et la NG, NG-diméthylarginine diméthylaminohydrolase (DDAH). Tandis que l’activité de l’arginase demeure constante, celle de la DDAH augmente au cours de l’infection en accord avec les données des western-blot et des amino-acides. Cette augmentation, qui dépend essentiellement de l’isoforme DDAH-2, conduit à une augmentation du NO cérébral dont les propriétés sont trypanocides. Ces changements, contraires à ceux observés en périphérie, sont défavorables à la survie du trypanosome au niveau du cerveau. Ils pourraient constituer une protection supplémentaire contre l’entrée des trypanosomes dans cet organe / By way of an experimental model of human African trypanosomiasis (HAT or sleeping sickness), the rat infected by Trypanosoma brucei brucei, we examined the involvement of nitric oxide (NO) in the development of this pathology. In the infected animals, opposite variations in NO concentration were observedeither at peripheral or brain compartments: NO decreases in blood but increases in brain. These changes are dependent on the activity of the inducible NO-synthase (iNOS). In periphery, the decrease observed in NO concentration favors the parasite entrance because the trypanocidal pressure exerted by NO is decreased. In such a situation, L-arginine, the substrate conducing to the synthesis of NO, is employed for the synthesis of polyamines, a category of compounds necessary for the parasite growth. It is likely that above mechanisms might be triggered by parasites. In brain, NO synthesis is submitted to additive complex regulatory processes implying arginase and NG, NG-dimethylarginine dimethylaminohydrolase (DDAH). While the arginase activity remains constant, that of DDAH increases throughout the infection process in keeping with western-blot and amino acids data. This increase, depending mainly on DDAH-2 isoform, lasts in a brain NO increase which enhance the trypanocidal pressure. Above changes, opposite to those observed in periphery, are not favorable to the parasite survival in brain. They might constitute an additive protection against the parasite entry in this organ

L’objectif de cette thèse est de développer des méthodes IRM permettant d’étudier l’impact d’une ischémie focale transitoire sur le cerveau de rat nouveau-né. Les techniques utilisées sont l’imagerie à contraste renforcé par le manganèse (MEMRI), l’imagerie du tenseur de diffusion (DTI) ainsi que de façon préliminaire l’imagerie Q-ball (QBI). Le MEMRI après injection intra cérébrale a été utilisé afin d’étudier de manière dynamique le tractus cortico-thalamique, en parallèle le DTI a servi de marqueur de la structuration cérébrale. Les résultats ont montré une atteinte du tractus cortico-thalamique ipsi-latéral, sept et quatorze jours après ischémie. De manière générale le DTI a montré une structuration ralentie à la suite de l’ischémie. A partir de ces résultats la faisabilité d’une méthode d’acquisition rapide et de traitement de données Q-ball a été établie puis testée sur un animal immature. Les méthodes mises en place se sont révélées efficaces dans le suivi de la maturation cérébrale dans des conditions normales ainsi que pathologiques, ouvrant des perspectives d’études liées au développement cérébral. / The thesis aim is to develop MRI methods to study the impact of focal transient ischemia in neonatal rat brain. The principal techniques used are MEMRI (Manganese Enhanced MRI), DTI (Diffusion Tensor Imaging) and QBI (Q-Ball Imaging). MEMRI was used to observe in a dynamic way the cortico-thalamic manganese transport combined with the structural informations extracted from the DTI experiments. Results have shown a cortico-thalamic pathway disturbance, at seven and fourteen days after ischemia. Globally DTI results have shown a slowed brain structuration. From these results, the feasibility of a fast acquisition method and the post processing steps of Q-ball protocol was established and applied in an immature rat. The different MRI protocols developed during this thesis have shown good efficiency to follow the rat brain maturation, in healthy and pathological conditions, thus opening new perspectives for brain development studies.

A imobilização produz efeito deletério sobre a musculatura estriada. A avaliação das propriedades mecânicas do músculo esquelético é um parâmetro de mensuração para elucidar se o exercício físico pode constituir importante forma de prevenção ou reversão dos défices decorrentes da imobilização. Seis grupos (GC1, GI, GC2, GIL, GIE, GE), com dez animais cada, foram utilizados com o objetivo de avaliar a interferência do exercício físico por esteira em algumas propriedades mecânicas de modelos animais sujeitos previamente à imobilização do membro posterior. Os animais dos grupos GI, GIL e GIE foram submetidos a vinte e um dias de imobilização gessada do membro posterior direito. Os animais dos grupos GIE e GE foram submetidos a protocolo de treino em esteira por vinte e um dias. Os grupos GC1 e GC2 serviram como controle. Após alcançarem à idade ideal, os animais sofreram eutanásia e os gastrocnêmios das patas direitas foram submetidos a ensaios de tração para análise da carga máxima suportada, deformação na carga máxima e tensão. Os resultados obtidos mostraram que a imobilização exerce papel deletério sobre as propriedades mecânicas do músculo esquelético, a tensão não apresenta necessariamente correlação com a carga máxima e que o protocolo de exercícios adotado neste estudo não foi capaz de restaurar todas as propriedades mecânicas, principalmente a carga máxima. / Immobilization has a deleterious effect on skeletal muscles. The evaluation of the mechanical properties of the skeletal muscle is a parameter used to elucidate if physical activity may prevent or revert the deficits caused by immobilization. Six groups (GC1, GI, GC2, GIL, GIE, GE), with ten animals each, were used to assess the interference of treadmill running on mechanical properties of animal models submitted to plaster cast immobilization of the right hind limb. The animals in groups GI, GIL and GE were submitted to twenty-one days of right hind limb immobilization. Animals in groups GIE and GE were submitted to a treadmill running protocol for twenty-one days. The groups GC1 and GC2 served as controls. The animals underwent euthanasia after reaching the age limit and the gastrocnemius of the right hind limb was submitted to a traction test to analyze the ultimate load, ultimate load deformation and tension. The results showed that immobilization caused a deleterious effect on the muscle\'s mechanical properties and the tension is not necessarily correlated with maximum load. Furthermore the running protocol as used in this study was not able to restore all the normal muscle mechanical properties, mainly the maximum load.

La recherche dans le domaine de la prise en charge de la douleur, notamment chronique, a un besoind’innovation car les traitements disponibles à l’heure actuelle sont pour la plupart anciens et souventliés à des effets indésirables. Il est maintenant admis que les études précliniques de la douleur ont denombreuses limites : pertinence des modèles, utilisation d’une stimulation douloureuse surajoutée,détermination d’un simple seuil ou délai, prise en compte de la seule composante sensoridiscriminative…De ce fait, certaines molécules efficaces chez l’animal et donc prometteuses, n’ontpas eu les effets escomptés chez l’homme. La base de notre travail de recherche s’attache donc àproposer de nouvelles méthodes d’appréciation de la douleur chronique chez l’animal en prenant encompte ses aspects multidimensionnels. De nombreuses études ont mis en évidence une altération dela qualité de vie chez des patients atteints de douleur chronique. Cette altération se caractérisenotamment par des perturbations émotionnelles et cognitives. Ces paramètres ne sont pas toujours prisen compte chez l’animal dans l’évaluation de traitements antalgiques mais pourrdouleurnt amener denouvelles possibilités et perspectives précliniques. Notre travail a consisté à étudier l’impact de ladouleur sur les comportements spontanés (automatisation du test au formol), la composanteémotionnelle et les capacités cognitives chez le rongeur. Il a été complété par l’exploration du rôle del’amygdale dans les mécanismes impliqués dans ces modifications comportementales.L’amélioration du test au formol a été réalisée dans le but de visualiser au mieux les comportementsspécifiques observés lors d’une douleur aiguë de type inflammatoire. Notre adaptation a permis, chezles mêmes animaux, de pouvoir dissocier l’effet antalgique et l’effet sédatif d’une molécule à l’aided’une méthode automatisée plus rapide et moins subjective.Parallèlement, nous avons apprécié l’impact de la douleur chronique sur la composante émotionnelleet les performances cognitives dans deux modèles de douleur chronique (inflammatoire etneuropathique). Les animaux souffrant de douleur chronique inflammatoire présentent desperturbations plus importantes que les animaux neuropathiques, perturbations pouvant être amélioréespar un traitement pharmacologique. Des études mécanistiques utilisant des micro-injections demorphine au niveau de l’amygdale ont souligné une implication importante du complexe basolatéraldans ces composantes émotionnelles et cognitives de la douleur.Ces nouvelles approches comportementales pourrdouleurnt permettre de mieux caractériser l’impact globalde la douleur chronique chez l’animal et de compléter la batterie de tests couramment utilisés enpréclinique. Ceci pourrait déboucher sur une transposition plus réaliste des résultats obtenus chezl’animal à l’homme, et donc conduire à une meilleure prédictibilité clinique de l’efficacité destraitements. Enfin, la mise en évidence de nouvelles cibles thérapeutiques innovantes implique l’étudedes mécanismes responsables de ces altérations comportementales. / Research in the field of pain management, including chronic pain management, needs innovationbecause available treatments are mostly old and often associated with many side effects. It is now wellrecognized that preclinical studies on pain have many limitations: the relevance of the models, the useof imposed painful stimulations, determination of simple thresholds or delays, taking into account thesensory-discriminative component of pain alone… Indeed, some molecules that are efficient inanimals and that are considered as promising, didn’t have the desired effect in humans. Therefore, thebasis of our research aims to propose new methods to assess chronic pain in animals taking intoaccount its multidimensional aspects. Many studies have shown impaired quality of life in patientssuffering from chronic pain. This alteration is characterized by emotional and cognitive disturbances.These components of pain are not always taken into account in animal when studying analgesictreatments, but could bring new preclinical possibilities and perspectives. Our work consisted instudying the impact of pain on spontaneous behaviours (automated formalin test), emotionalcomponent and cognitive capacities in rodents. This work has been completed by the exploration ofthe role of the amygdala in the mechanisms underlying those behavioural modifications.Improvement of the formalin test was conducted in order to better visualize the specific behaviorsobserved during an acute inflammatory pain. Our adaptation has allowed dissociating the analgesicand sedative effect of a molecule in a same animal, using an automated method which is faster and lesssubjective than the manual method.In the meantime, we assessed the impact of chronic pain on the emotional and cognitive performancesin two models of chronic pain (inflammatory and neuropathic). Animals suffering from chronicinflammatory pain have more important impairments than animal suffering from neuropathic pain,impairments that can be improved with a pharmacological treatment. Mechanistic studies using microinjectionsof morphine in the amygdala have emphasized an important involvement of the basolateralcomplex in these emotional and cognitive components of pain.These new behavioural approaches may help better characterize the overall impact of chronic pain inanimals and complete the battery of tests commonly used in preclinical studies. This could lead to amore realistic transposition of the results obtained from animals to humans, and thus lead to betterpredictability for the clinical efficacy of treatments. Finally, the identification of new targets forinnovative therapies involves the study of mechanisms responsible for these behavioral impairments.

A linhagem Wistar Audiogenic Rat (WAR), um modelo genético de epilepsia, apresenta diversas comorbidades. Dados prévios demonstraram níveis cerebrais elevados de proteína tau fosforilada (pTau), uma das principais características anatomopatológicas da doença de Alzheimer (DA). Estudos recentes sugerem que a DA seja um terceiro tipo de diabetes. O objetivo deste estudo foi validar a linhagem WAR como um possível modelo para o estudo da DA relacionada com alterações na regulação da via de sinalização da insulina. Para isso, foram selecionados 63 animais machos adultos e envelhecidos (n=28 Wistars, 14 Wistars Hannover e 21 WARs). Os animais foram submetidos ao labirinto aquático de Morris (LAM). Após o LAM, foram eutanasiados e tiveram seus hipocampos dissecados. Avaliou-se a expressão de betaamilóide (A?), tau, pTau (S396), receptor de insulina (IR), receptor de insulina -1 fosforilado em serina 312 (pIRS-1 S312) e glicogênio sintase quinase 3 alfa e beta (pGSK-3?/? S21/9) fosforiladas pelo método de Western blotting nos hipocampos ventral (HV) e dorsal (HD) e/ou confirmados por imunoistoquímica. Os animais da linhagem WAR adultos e envelhecidos apresentaram maior latência para encontrar a plataforma de fuga durante o período de treinamento e maioria dos retestes no LAM. Os animais WAR adultos apresentaram diminuição da expressão de A? e IR no HV, aumento da expressão de pGSK-3?/? (S21/9) no HD e aumento do número de células positivas para pIRS-1 (S312) no hipocampo. Os animais envelhecidos apresentaram aumento da expressão de pTau (S396) nos HV e HD, diminuição de A? e IR no HV e aumento da expressão de pGSK-3?/? (S21/9) nos HV e HD. Apesar de os animais WAR terem sido selecionados geneticamente para estudar epilepsia, nossos achados sugerem a coexistência de alterações comportamentais e moleculares características de DA e resistência central à insulina, podendo se somar ao fenótipo inicialmente selecionado. Portanto, outros estudos moleculares e histológicos são necessários para melhor compreensão dos dados encontrados e para possível validação da linhagem WAR como um modelo para estudar a relação entre DA e resistência central à insulina. / The Wistar Audiogenic Rat (WAR), a genetic model of epilepsy, presents many comorbidities. Previous data suggested elevated brain levels of phosphorylated tau (pTau), one of the the main hallmarks of Alzheimer\'s Disease (AD). Recent studies have hypothesized AD as the third type of diabetes. Therefore, the aim of this study was to validate the WAR strain as a model to study AD and correlate with alterations in the insulin signaling pathway regulation. To this end, we selected 63 male adult and aged animals (n=28 Wistars, 14 Wistars Hannover and 21 WARs). The animals were subjected to the Morris Water Maze (MWM) test. After the MWM, the rats were euthanized and their ventral (VH) and dorsal (DH) hippocampi were dissected for Western blot analysis in order to measure the expression levels of A?, tau, pTau (S396), insulin receptor (IR), alpha and beta phosphorylated glycogen synthase kinase 3 (pGSK-3?/? Ser21/9), and phospho-insulin receptor substrate-1 (pIRS-1 S312) in the ventral (VH) and dorsal hippocampus (DH) by means of the Western blotting method. The results were confirmed by immunohistochemical analysis. Both adult and aged WARs showed longer latency to find the escape platform during the learning period and most retests of the MWM. The adult WARs presented lower A? and IR expression in the VH, higher pGSK-3?/? (S21/9) expression in the DH, and higher levels of positive cells for pIRS-1 (S312) in the hippocampus. The aged WARs presented higher pTau (S396) expression in both VH and DH, lower A? and IR expression in the VH, higher pGSK-3?/? (S21/9) expression in both VH and DH. Although the WAR animals were primarily selected to study epilepsy, our findings in this study suggest the coexistence of an AD-like phenotype and central insulin resistance in addition to the initially selected epilepsy phenotype. Therefore, other molecular and histological studies are needed to better understand our data and to validate the WAR strain as a model to study the relationship between AD and central insulin resistance.

My PhD research studies, described in this thesis, were designed to document the temporal development of mechanical allodynia, a hallmark symptom of painful diabetic neuropathy (PDN), as well as opioid hyposensitivity using two different rat models of diabetes mellitus (DM). Specifically, the studies were conducted using the streptozotocin (STZ)-diabetic rat model of chemically-induced Type 1 diabetes in two different rat strains, as well as the Zucker Diabetic Fatty (ZDF) rat genetic model of Type 2 diabetes. Additionally, a longitudinal investigation of the effect of basal insulin replacement therapy to restore euglycaemia from 7-days post-STZ administration, on the development of mechanical allodynia in the hindpaws of the STZ-diabetic Wistar rat model of PDN, was conducted. The studies herein also included a longitudinal study to document the temporal development of mechanical allodynia and opioid hyposensitivity in the ZDF rat, which also examined the influence of dietary composition on the time course for the development of mechanical allodynia in the hindpaws, together with opioid hyposensitivity in these animals. In the final section of this thesis, the experiments were designed to examine possible mechanisms that may contribute to the development of opioid hyposensitivity in ZDF diabetic rats. These experiments involved the quantification of opioid receptor messenger ribonucleic acid (mRNA) gene expression as well as μ-opioid receptor (MOP-r) functional responses in tissues collected from 29-wk old diabetic ZDF rats relative to 7-wk old pre-diabetic control ZDF animals. In Chapter One, diabetes mellitus and more specifically its longterm complication, PDN, the focus of this doctoral research program, has been reviewed. Specifically, possible pathogenic mechanisms underlying mechanical allodynia, the relevant diabetic rodent models of PDN, use of insulin replacement therapy in diabetic rodents and its impact on hallmark symptoms of PDN, role of opioid pharmacology, the comparative efficacy of opioids in the treatment of PDN, and possible mechanisms that may underpin the development of opioid hyposensitivity in PDN, including the impact of altered excitatory neurotransmitters, have been reviewed. In Chapter Two, a preliminary study was conducted to investigate the efficacy of 4-wks treatment with Linplants (subcutaneous (s.c.) sustained-release bovine insulin implants) alone and in combination with ActRapid® (s.c. human insulin; 0.05 U to 3.5 U/100 g/day) with respect to glycaemic control in STZ-diabetic Wistar rats, and on acute diabetes characteristics for a 5-wk post-STZ administration period. Briefly, STZ-diabetic rats were divided into three groups: (1) rats which received no insulin treatment, (2) rats which were implanted with one s.c. Linplant at Day 7 post-STZ administration, and (3) rats which received one s.c. Linplant plus a once-daily injection of ActRapid® once diabetes was confirmed at 7-days post-STZ administration. The findings were that following implantation of a single Linplant at Day 7 post-STZ administration, euglycaemia was achieved in 50% of STZ-diabetic rats, with glycaemic control maintained for up to 4-wks post-implantation. Furthermore, once-daily injection of ActRapid™ to animals whose blood glucose levels (BGLs) were not well-controlled through use of Linplants alone, failed to achieve euglycaemia. It is possible that the ActRapid™ doses administered were not sufficient to achieve euglycaemia, and that increasing the doses may provide more effective glycaemic control. However, doubling the mean ActRapid™ dose from 1.63 (+ 0.3) U administered at Day 28 to 2.56 (+ 0.6) U administered at Day 34 post-STZ administration effectively only reduced BGLs by 1.3 mM to 11.6 + 1.6 mM. This suggests that although administering additional large doses of ActRapid™ to STZ-diabetic rats may eventually achieve euglycaemia, this method would presumably not be a more efficient method in achieving euglycaemia compared with the use of dosage-adjustable s.c. Linplants. Group (1) STZ-diabetic rats which were not treated with insulin developed diabetic signs including polydipsia, hyperphagia, decreased rate of body weight gain, and mechanical allodynia. Group (2) rats in which insulin treatment from 7-days post-STZ administration restored euglycaemia and reversed polydipsia and hyperphagia, were protected against the development of mechanical allodynia and reduced weight gain for the 5-wk study duration, while rats from Group (3) with incomplete glycaemic control developed levels of polydipsia, hyperphagia, reduced weight gain and mechanical allodynia intermediate between rats in Groups (1) and (2). These findings collectively suggest a direct correlation between the level of glycaemic control and the extent to which mechanical allodynia, a defining symptom of PDN, develops. In Chapter Three, the findings from the preliminary 5-wk study in Chapter Two were used to design a 24-wk longitudinal study of the temporal development of mechanical allodynia and opioid hyposensitivity in STZ-diabetic Wistar rats for comparison with the findings of a similar study previously undertaken by our laboratory using STZ-diabetic Dark Agouti rats (Nielsen et al, 2007). Additionally, this study examined the effects of tight glycaemic control achieved through the use of insulin implants as a means of potentially preventing the development of mechanical allodynia and opioid hyposensitivity for up to 24 weeks in STZ-diabetic Wistar rats. Briefly, STZ-diabetic rats were divided into 3 groups: (1) non-insulin treated STZ-diabetic Dark Agouti rats to provide comparison data with our laboratory’s previously published data in this rat strain (Nielsen et al, 2007), (2) non-insulin treated STZ-diabetic Wistar rats to examine possible between-species differences, and (3) STZ-diabetic Wistar rats which were treated with adjustable-dose s.c. Linplants from Day 7 post-STZ administration to maintain euglycaemia for the remainder of the 24-wk study period. In this 24-wk longitudinal study in STZ-diabetic rats, body weight, 24-hr water intake, paw withdrawal thresholds (PWTs) and BGLs were monitored at fortnightly intervals in all animals in order to document possible temporal changes in the development of diabetic signs and mechanical allodynia in the hindpaws respectively. STZ-diabetic rats underwent 6-wkly opioid antinociceptive testing, using single bolus doses of each of morphine and oxycodone with a 2-3 day washout period between individual opioids in order to assess the potential influence of both diabetes and glycaemic control on opioid potency in these animals. The findings demonstrate that non-insulin treated STZ-diabetic rats of both strains exhibited a decreased rate of body weight gain and polydipsia, as well as progressive development of mechanical allodynia in the hindpaws and loss of morphine potency. Importantly, STZ-diabetic Wistar rats which were treated with insulin to maintain euglycaemia from Day 7 post-STZ administration failed to develop these diabetic symptoms for the duration of the 24-wk study period, highlighting the importance of chronic hyperglycaemia in the development of mechanical allodynia and morphine hyposensitivity in the STZ-diabetic rodent model of PDN. The research described in Chapter Four involved a 22-wk longitudinal study of the development of diabetes and its longterm sensory nerve complications, viz mechanical allodynia and opioid hyposensitivity, in the ZDF rodent model of Type 2 diabetes commencing at 7-wks of age. This study also examined the influence of four different diets fed to separate groups of ZDF rats from 7-wks age, on the time course for the development of diabetes, mechanical allodynia in the hindpaws and opioid hyposensitivity in these animals. Briefly, ZDF rats were sub-divided into four dietary groups, each of which was fed one of the four following diets for 22-wks commencing at 7-wks of age, viz: (a) Purina 5008™, (b) a domestically-produced rat chow of similar composition to Purina 5008 (termed Purina Composition diet), (c) a Diabetogenic diet, or (d) Standard Rat Chow. All rats underwent once-fortnightly measurement of BGLs, body weight, 24-hr water intake, and measurement of PWTs in the hindpaws. Additionally, ZDF rats underwent opioid antinociceptive testing, similar to that previously described for STZ-diabetic rats (Chapter Three), to investigate the influence of diabetes and dietary composition on the antinociceptive potency of single bolus doses of morphine and oxycodone administered at 6-weekly intervals over a 22-wk study period. The afore-mentioned data were compared with the respective data obtained from the pre-diabetic control group of ZDF rats that were euthanised at 7-wks of age prior to the development of hyperglycaemia. The results demonstrate that the ZDF rat develops mechanical allodynia in the hindpaws and opioid hyposensitivity in a temporal fashion, in a manner similar to that previously documented for the STZ-diabetic Wistar rat model of Type 1 diabetes (Chapter Three). For the four diets assessed, there did not appear to be significant differences between dietary groups with respect to the time course and extent of development of hyperglycaemia, mechanical allodynia or opioid hyposensitivity in the ZDF rat model of PDN. The study described in Chapter Five investigated the effect of both diabetes and dietary composition on opioid receptor mRNA expression in tissue samples collected from the five groups of ZDF rats used in the behavioural studies described in Chapter Four and outlined above. Briefly, mRNA expression for each of the - (MOP), - (DOP), and - (KOP) receptors were quantified in mid-brain and spinal cord tissues prepared from 29-wk old diabetic ZDF rats maintained on one of four diets from 7-wks age, and compared with the respective expression levels in samples prepared from pre-diabetic ZDF rats euthanised at 7-wks of age. Overall, the findings suggest that diabetes does not alter opioid receptor mRNA expression in the mid-brain or spinal cord of diabetic ZDF rats at 29-wks of age relative to the corresponding levels of mRNA expression in the mid-brain and spinal cord of pre-diabetic ZDF rats at 7-wks of age. Hence, the marked reduction in the anti-allodynic potency of morphine and oxycodone observed in diabetic ZDF rats at 29-wks of age relative to that observed in pre-diabetic ZDF rats at 7-wks of age (Chapter Four) does not appear to be associated with a decrease in opioid receptor mRNA expression. In Chapter Six, the effect of both advanced diabetes and dietary composition on opioid-agonist stimulated [35S]GTPγS binding was examined in spinal cord tissue membranes from the ZDF rat. Specifically, [35S]GTPγS binding assays were used to assess the ability of a -opioid ligand (DAMGO) to stimulate -opioid receptor coupling to inhibitory G proteins in homogenates prepared from spinal cord samples of 29-wk old ZDF rats maintained on one of four different diets from 7-wks age (Chapter Four), relative to [35S]GTPγS binding in homogenates prepared from spinal cord samples of pre-diabetic 7-wk old ZDF rats. As specific MOP agonist-stimulated [35S]GTPγS binding was significantly decreased in spinal cord homogenates from diabetic ZDF rats at 29-wks of age relative to that for pre-diabetic ZDF rats (7-wks), this may contribute, at least in part, to the morphine hyposensitivity observed in diabetic ZDF rats at 29-wks of age relative to the pre-diabetic ZDF group. However, closer examination of these data revealed that specific MOP agonist-stimulated [35S]GTPγS binding above basal did not differ significantly between the pre-diabetic group and the longterm diabetic group of ZDF rats. Instead, there was significantly lower basal [35S]GTPγS binding in the spinal cord of ZDF rats at 29-wks c.f. 7-wks of age. Together, the findings suggest that impaired basal G-protein function rather than impaired coupling of MOP-r to its inhibitory G-protein may, at least in part, underpin -opioid agonist hyposensitivity in 29-wk ZDF rats. Finally, Chapter 7 contains a brief description of the main conclusions and discussion of the relevance of this doctoral research project, including potential future research directions.