Stroke is the third leading cause of death and the primary cause of adult long-term disability in Canada. Despite advances in rehabilitation research, stroke survivors experience an unusually high incidence of depressive symptoms which undermine recovery outcomes by reducing patient motivation levels. Human and animal studies have linked the incidence of post-stroke depression and the extent of prefrontal cortex (PFC) damage. The PFC and the lateral habenula (LHb) are limbic structures that are strongly connected to the serotonergic dorsal raphe nucleus (DRN), a key neuronal hub for mood regulation. We hypothesized that PFC stroke produces a depressive phenotype by triggering maladaptive reorganization in mood-related networks. We used viral and optogenetic strategies to functionally characterize PFC and LHb projections to DRN. Moreover, we found that PFC stroke causes a time-dependent remodeling of LHb inputs to DRN 5-HT neurons which results in altered postsynaptic glutamate receptor number and subunit composition. This remodeling likely reflects a homeostatic upregulation of LHb-DRN synapses in response to stroke-induced challenge to network activity. Since these synapses encode stress and aversion, potentiation of this pathway could contribute to depressive symptoms following stroke. However, more work will be needed to identify the behavioral and network-level consequences of altered LHb-DRN dynamics. Thus, a deeper understanding of circuit mechanisms implicated in post-stroke depression will provide insights into this disease and open new treatment avenues to improve recovery.

Indiana University-Purdue University Indianapolis (IUPUI) / Serotonin (5-HT) is heavily implicated in severe anxiety and trauma-related disor-ders, such as panic and post-traumatic stress disorders. Overall, site-specific pharmacolog-ical manipulations show that while 5-HT enhances anxiety-associated/avoidance behaviors in the amygdala, 5-HT inhibits panic-associated escape behaviors in the perifornical hypo-thalamus region (PeFR). Yet, our understanding of how specific serotonergic networks and co-transmitters regulate these conditions, but also other aspects of innate panic (e.g., car-dioexcitation or thermal response that occur during a flight or escape response) or condi-tioned fear behaviors is still elusive. Therefore, utilizing circuit-based gain- and loss-of-function approaches to selectively manipulate amygdala- and PeFR-projecting sero-tonergic systems, we hypothesize that specific serotonergic networks projecting to the amygdala and PeFR respectively enhance conditioned fear responses and attenuate innate panic-associated behaviors and physiological responses. There are two main chapters in this dissertation. In Chapter III, retrograde tracing revealed that the amygdala-projecting neurons from dorsal Raphe (DR) were almost exclusively serotonergic (92-95%) concen-trated in the dorsal/ventral (DRD/DRV) DR, with few non-serotonergic neurons. While selective lesioning of this network with saporin toxin (SAP) facilitated the extinction of conditioned fear behavior, selective optogenetic activation of amygdala-projecting DRD/DRV cell bodies using intersectional genetics reduced extinction of conditioned fear behavior and enhanced anxiety avoidance. In Chapter IV, retrograde tracing showed that the PeFR was innervated by equally selective serotonergic networks concentrated in the lateral wings DR (lwDR) and median Raphe (MR). Contrasting with the results from the amygdala-innervating 5-HT system, lesioning the PeFR-projecting serotonergic network from lwDR/MR was accompanied by reduced extinction of conditioned fear behavior, in-creased anxiety avoidance, and increased CO2-induced panic (elevated escape responses and enhanced cardioexcitation). Conversely, selective activation of lwDR/MR serotonergic terminals in the PeFR decreased anxiety-associated behaviors; inhibited CO2-induced panic, and induced unconditioned and conditioned place preferences. The circuit-based ap-proach data presented here show that amygdala- and PeFR-projecting 5-HT neurons com-prise distinct circuits underlying opposite roles enhancing anxiety/fear responses in the amygdala and dampening fear/panic responses in the PeFR. The identification of distinct circuits controlling anxiety, fear, and panic responses is a fundamental step towards the development of more effective therapies for psychiatric conditions such as anxiety and trauma-related disorders. / 2021-11-04

The Dorsal Raphe Nucleus (DRN) is a complex brain region that has been implicated in disorders such as anxiety and depression. The DRN is divided into subregions through its rostrocaudal and mediolateral axis. It has been reported that after a single restraint session there is differential spatial activation of nitric oxide synthase (NOS) across the DRN. The temporal profile of NOS activity during acute stress is not known but it is important because duration of acute stress is associated with different general responses. In this report rats were restrained for 1, 3, or 6 hours and nicotinamide adenine phosphate diaphorase (NADPH-d) was stained as an index to NOS activity to determine the spatio-temporal profile of NOS throughout a 6 hour restraint. Astrocyte reactivity was also measured to determine whether NOS activation correlated with GFAP expression since astrocytes react to neural activity and store and release l-arginine, the precursor for nitric oxide production. The results showed that the DRN had a dynamic response to acute restraint stress, most notably in the caudal lateral wings where activation increased after 3 hours of restraint (p = > 0.001) but neuron count decreased after 6 hours (p = 0.040). Astrocytes did not correlate with NOS activation but they showed spatio-temporal differences as well whereas they were more active in the rostral half of the DRN. In conclusion, the present study suggests that NOS produced in the DRN may have a role in prolonged exposure to acute stress and that subregions show differential NOS activation.

Diversas evidências indicam que o núcleo dorsal da rafe (NDR), principal fonte de inervação serotonérgica para estruturas límbicas, é uma estrutura heterogênea composta por populações de neurônios serotonérgicos anatômica e funcionalmente distintas, as quais têm sido diferentemente implicadas na fisiopatologia dos transtornos de ansiedade, como o transtorno do pânico. Fármacos antidepressivos são a primeira escolha para o tratamento de tais transtornos e o atraso para a manifestação dos efeitos terapêuticos desses fármacos tem sido, consistentemente, associado a alterações na neurotransmissão serotonérgica. Entretanto, ainda é desconhecido se estas alterações podem ser heterogêneas entre as diferentes sub-regiões do NDR e é este o foco de investigação do presente estudo. Inicialmente, foi investigado o perfil de ativação neuronal das diferentes sub-regiões do NDR, através da avaliação da expressão de proteína Fos, em ratos submetidos à tarefa de fuga no modelo do labirinto em T elevado, após administração aguda ou crônica com a fluoxetina ou a imipramina. Nestes mesmos animais, foram também avaliado o número de células triptofano hidroxilase positivas. Para fins comparativos, o efeito do tratamento com a imipramina em animais submetidos à esquiva inibitória também foi avaliado. A análise imunoistoquímica indicou que a tarefa de fuga promoveu um aumento na expressão da proteína Fos em neurônios nãoserotonérgicos localizados na sub-região das asas laterais do NDR. Efeito este que não foi observado em animais tratados cronicamente com a fluoxetina e a imipramina. Além disso, o tratamento prolongado com a fluoxetina, mas não com a imipramina, foi capaz de aumentar a ativação de neurônios serotonérgicos nesta mesma sub-região. Diferente do que foi observado na tarefa de fuga, a tarefa de esquiva inibitória promoveu um aumento na ativação de neurônios serotonérgicos nas sub-regiões DRC, DRD e DRV. Perfil que não foi observado nos grupos aguda e cronicamente tratados com imipramina. Além disso, somente o tratamento prolongado com a imipramina promoveu uma diminuição no recrutamento de neurônios não-serotonérgicos em diversas sub-regiões do NDR. Diante do resultado imunoistoquímico observado no experimento com a fluoxetina, avaliamos a participação dos autorreceptores 5-HT1A das asas laterais no efeito observado. Através da técnica whole-cell patch clamp, em uma linhagem de camundongos transgênicos que apresenta neurônios serotonérgicos fluorescentes, foi avaliada a responsividade destes receptores após o tratamento com a fluoxetina. Os resultados indicam que os animais tratados cronicamente com fluoxetina apresentam aumento na excitabilidade basal com diminuída sensibilidade ao agonista de receptores 5-HT1A, 8-OHDPAT. Assim, é possível sugerir que o efeito antipânico da fluoxetina administrada cronicamente parece estar relacionado com uma redução na ativação de neurônios não-serotonérgicos e um aumento no recrutamento de neurônios serotonérgicos localizados nas asas laterais, sendo que esta última pode ser explicada pela dessensibilização dos autorreceptores 5-HT1A. Com relação à imipramina, é possível sugerir que o efeito panicolítico promovido pelo tratamento prolongado pode ser devido à diminuição no recrutamento de neurônios não-serotonérgicos das asas laterais. Já para o efeito ansiolítico, tanto a diminuição no recrutamento de neurônios não-serotonérgicos em diferentes sub-regiões do NDR, quanto a diminuição na ativação de neurônios serotonérgicos no DRC, DRD e DRV parecem estar envolvidas. / A wealth of evidence indicates that the dorsal raphe nucleus (DR) is a heterogeneous structure, composed of anatomically and functionally distinct populations of serotonergic neurons, which have been differently implicated in the pathophysiology of anxiety, such as panic disorder. Antidepressant drugs are the first choice in treatment of anxiety disorders, and the delay for the therapeutic effect have consistently been associated with changes in serotonergic neurotransmission within the DR. However, it is unknown whether these alterations can be heterogeneous among the different subregions of the DR, and this is the focus of investigation of the present study. First, it was investigated the profile of neuronal activation of different subregions of the DR - by using the evaluation of Fos protein expression of rats exposed to the escape task in the elevated T-maze test, after acute or chronic administration of fluoxetine or imipramine. In the same animals, it was also investigated the number of positive triptophan hidroxylasis cells. For comparative reasons, it was evaluated the behavioral and immunohistochemical effects of imipramine treatment on inhibitory avoidance acquisition in the elevated T-maze, a response associated with anxiety. The results of the immunohistochemical analysis indicated that animals exposed to escape behavior exhibited higher expression of Fos protein in non-serotonergic neurons in the DR lateral wings. This effect was not observed in fluoxetine or imipramine chronically treated animals. Moreover, chronic treatment with fluoxetine, but not imipramine, was able to increase the activation of serotonergic neurons on this subregion. On the other hand, the inhibitory avoidance task promoted an increase in the activation of serotonergic neurons in the sub-regions DRC, DRD, and DRV. This profile was not observed after acute or chronic treatment with imipramine. Additionally, only the long-term treatment with imipramine showed a decrease in the activation of non-serotonergic neurons in different subregions of the DR. Based on the results obtained with fluoxetine experiment, we evaluated the role of the 5-HT1A autoreceptors located in the lateral wings. For this, we used the whole-cell patch clamp technique in a transgenic mouse line, which exhibit fluorescence in serotonergic neurons. The results indicate that the animals treated chronically with fluoxetine presented an increase in the basal excitability, with lower responsivity to the 5-HT1A agonist - 8-OH-DPAT. Altogether, the results suggest that the anti-panic effect caused by chronic fluoxetine treatment is associated with a reduction in the activation of non-serotonergic neurons, and an increase in the recruitment of non-serotonergic neurons in lateral wings. This last observation seems to be related to a 5-HT1A autoreceptor desensitization in the lateral wings. Regarding imipramine, this panicolytic effect caused by chronic administration of this drug seems to be related to a decreased activation of the non-serotonergic neurons in the lateral wings. Finally, the anxiolytic effect, of imipramine may be associated to a decrease in the recruitment of non-serotonergic neurons in different DR subregions, allied with a decreased activation of serotonergic neurons in the DRC, DRD and DRV.

Diversas evidências indicam que o núcleo dorsal da rafe (NDR), principal fonte de inervação serotonérgica para estruturas límbicas, é uma estrutura heterogênea composta por populações de neurônios serotonérgicos anatômica e funcionalmente distintas, as quais têm sido diferentemente implicadas na fisiopatologia dos transtornos de ansiedade, como o transtorno do pânico. Fármacos antidepressivos são a primeira escolha para o tratamento de tais transtornos e o atraso para a manifestação dos efeitos terapêuticos desses fármacos tem sido, consistentemente, associado a alterações na neurotransmissão serotonérgica. Entretanto, ainda é desconhecido se estas alterações podem ser heterogêneas entre as diferentes sub-regiões do NDR e é este o foco de investigação do presente estudo. Inicialmente, foi investigado o perfil de ativação neuronal das diferentes sub-regiões do NDR, através da avaliação da expressão de proteína Fos, em ratos submetidos à tarefa de fuga no modelo do labirinto em T elevado, após administração aguda ou crônica com a fluoxetina ou a imipramina. Nestes mesmos animais, foram também avaliado o número de células triptofano hidroxilase positivas. Para fins comparativos, o efeito do tratamento com a imipramina em animais submetidos à esquiva inibitória também foi avaliado. A análise imunoistoquímica indicou que a tarefa de fuga promoveu um aumento na expressão da proteína Fos em neurônios nãoserotonérgicos localizados na sub-região das asas laterais do NDR. Efeito este que não foi observado em animais tratados cronicamente com a fluoxetina e a imipramina. Além disso, o tratamento prolongado com a fluoxetina, mas não com a imipramina, foi capaz de aumentar a ativação de neurônios serotonérgicos nesta mesma sub-região. Diferente do que foi observado na tarefa de fuga, a tarefa de esquiva inibitória promoveu um aumento na ativação de neurônios serotonérgicos nas sub-regiões DRC, DRD e DRV. Perfil que não foi observado nos grupos aguda e cronicamente tratados com imipramina. Além disso, somente o tratamento prolongado com a imipramina promoveu uma diminuição no recrutamento de neurônios não-serotonérgicos em diversas sub-regiões do NDR. Diante do resultado imunoistoquímico observado no experimento com a fluoxetina, avaliamos a participação dos autorreceptores 5-HT1A das asas laterais no efeito observado. Através da técnica whole-cell patch clamp, em uma linhagem de camundongos transgênicos que apresenta neurônios serotonérgicos fluorescentes, foi avaliada a responsividade destes receptores após o tratamento com a fluoxetina. Os resultados indicam que os animais tratados cronicamente com fluoxetina apresentam aumento na excitabilidade basal com diminuída sensibilidade ao agonista de receptores 5-HT1A, 8-OHDPAT. Assim, é possível sugerir que o efeito antipânico da fluoxetina administrada cronicamente parece estar relacionado com uma redução na ativação de neurônios não-serotonérgicos e um aumento no recrutamento de neurônios serotonérgicos localizados nas asas laterais, sendo que esta última pode ser explicada pela dessensibilização dos autorreceptores 5-HT1A. Com relação à imipramina, é possível sugerir que o efeito panicolítico promovido pelo tratamento prolongado pode ser devido à diminuição no recrutamento de neurônios não-serotonérgicos das asas laterais. Já para o efeito ansiolítico, tanto a diminuição no recrutamento de neurônios não-serotonérgicos em diferentes sub-regiões do NDR, quanto a diminuição na ativação de neurônios serotonérgicos no DRC, DRD e DRV parecem estar envolvidas. / A wealth of evidence indicates that the dorsal raphe nucleus (DR) is a heterogeneous structure, composed of anatomically and functionally distinct populations of serotonergic neurons, which have been differently implicated in the pathophysiology of anxiety, such as panic disorder. Antidepressant drugs are the first choice in treatment of anxiety disorders, and the delay for the therapeutic effect have consistently been associated with changes in serotonergic neurotransmission within the DR. However, it is unknown whether these alterations can be heterogeneous among the different subregions of the DR, and this is the focus of investigation of the present study. First, it was investigated the profile of neuronal activation of different subregions of the DR - by using the evaluation of Fos protein expression of rats exposed to the escape task in the elevated T-maze test, after acute or chronic administration of fluoxetine or imipramine. In the same animals, it was also investigated the number of positive triptophan hidroxylasis cells. For comparative reasons, it was evaluated the behavioral and immunohistochemical effects of imipramine treatment on inhibitory avoidance acquisition in the elevated T-maze, a response associated with anxiety. The results of the immunohistochemical analysis indicated that animals exposed to escape behavior exhibited higher expression of Fos protein in non-serotonergic neurons in the DR lateral wings. This effect was not observed in fluoxetine or imipramine chronically treated animals. Moreover, chronic treatment with fluoxetine, but not imipramine, was able to increase the activation of serotonergic neurons on this subregion. On the other hand, the inhibitory avoidance task promoted an increase in the activation of serotonergic neurons in the sub-regions DRC, DRD, and DRV. This profile was not observed after acute or chronic treatment with imipramine. Additionally, only the long-term treatment with imipramine showed a decrease in the activation of non-serotonergic neurons in different subregions of the DR. Based on the results obtained with fluoxetine experiment, we evaluated the role of the 5-HT1A autoreceptors located in the lateral wings. For this, we used the whole-cell patch clamp technique in a transgenic mouse line, which exhibit fluorescence in serotonergic neurons. The results indicate that the animals treated chronically with fluoxetine presented an increase in the basal excitability, with lower responsivity to the 5-HT1A agonist - 8-OH-DPAT. Altogether, the results suggest that the anti-panic effect caused by chronic fluoxetine treatment is associated with a reduction in the activation of non-serotonergic neurons, and an increase in the recruitment of non-serotonergic neurons in lateral wings. This last observation seems to be related to a 5-HT1A autoreceptor desensitization in the lateral wings. Regarding imipramine, this panicolytic effect caused by chronic administration of this drug seems to be related to a decreased activation of the non-serotonergic neurons in the lateral wings. Finally, the anxiolytic effect, of imipramine may be associated to a decrease in the recruitment of non-serotonergic neurons in different DR subregions, allied with a decreased activation of serotonergic neurons in the DRC, DRD and DRV.

Synapses are a fundamental unit of computation in the brain. Far from being passive connections between spiking neurons, synapses display striking short-term dynamics, undergo long-term changes in strength, and sculpt network-level processes in a complex manner. These synaptic dynamics, both in time and across space, may be a fundamental determinant of population-level computations and behavioral output of the brain, yet their role in neuromodulatory circuits is relatively under-explored. First, I developed and validated a set of likelihood-based inference tools to quantify the dynamics of synaptic ensemble composition throughout development. Second, I examined network computations in the serotonergic dorsal raphe nucleus through a dynamical lens, exploring the role of short-term synaptic dynamics at sparse recurrent connections, and of distinct long-range synaptic inputs, in shaping the output of spiking populations. 1. Simulation-based inference of synaptic ensembles. Functional features of synapses are typically inferred by sampling small ensembles of synapses, yet it is unclear if such subsamples exhibit biases. I developed a statistical framework to address this question, using it to demonstrate that common bulk electrical stimulation methods for characterizing the fraction of silent synapses exhibit high bias and variance, and using typical sample sizes, possess insufficient statistical power for accurate inference. I developed and validated a novel synthetic likelihood-based inference approach based on a simulator of the underlying experimental methodology. This new estimator, made available in an object-oriented Python toolbox, reduces bias and variance compared to previously reported methods, and provides a scalable method for examining synaptic dynamics throughout development. These tools were validated by targeted recording from hippocampal CA1 neurons in juvenile mice, where they reveal fundamental tradeoffs between release probability, number of synapses sampled, and statistical power. 2. Synaptic dynamics and population computations in the serotonin system. This part is comprised of two manuscripts. First, in the dorsal raphe nucleus, I uncovered slow, inhibitory recurrent interactions between serotonin neurons that are generated by local serotonin release. These connections were probabilistic, displayed striking short-term facilitation, gated the spiking output of serotonin neurons, and could be activated by long-range excitatory input from lateral habenula, representing threat signals. Targeted physiology and modeling revealed that these recurrent short-term facilitation features generated paradoxical excitation-driven inhibition in response to high-frequency habenula input. These facilitation rules additionally supported winner-take-all dynamics at the population level, providing a contrastive operation between functionally distinct serotonergic ensembles. Behaviorally, activating long-range lateral habenula input to dorsal raphe nucleus generated a transient, frequency-dependent suppression of reward anticipation consistent with these recurrent dynamics, without modulating the underlying reward association itself. These dynamics, we suggest, support sharp behavioral state transitions in changing environments. In a second manuscript, I explored the multiplexing of distinct long-range inputs in serotonergic circuits through spike synchrony. I demonstrated that a population of serotonergic neurons receives input from both lateral habenula and prefrontal cortex. These inputs produced similar subthreshold events, but prefrontal cortex triggered spikes with much higher latencies, supporting a population synchrony code for input identity. These input-specific spike timing patterns could be read out by simple linear decoders with high accuracy, suggesting they could be demultiplexed by downstream circuits receiving sparse innervation by serotonergic axons. We uncovered a novel intracellular calcium conductance in serotonergic neurons that altered the spectral characteristics of membrane voltage in a manner sufficient to generate long-latency, power law-distributed spike times, suggesting a simple dynamical origin for the production of synchronous or asynchronous spiking. This work indicates that serotonergic circuits can multiplex distinct informational streams through population spike synchrony mechanisms. Together, these investigations reveal that the dynamics of short-term facilitation and synaptic ensemble composition can act as the fundamental substrate for flexible computation by spiking networks across the brain.

The relatively few serotonin (5-HT) neurons located in the Dorsal Raphe Nucleus (DRN) give rise to an extensive axonal network modulating a wide-range of brain functions and behaviors. In turn, the DRN receives inputs from several brain regions and therefore exhibits the characteristics of a hub network. While recent technological advancements have provided an unprecedented look at the neurobiology of the DRN, important knowledge gaps remain in understanding how the constellation of synaptic inputs to this region confers 5-HT neurons their unique coding features. As a first step towards characterizing the DRN's input processing strategy, we set out to explore the landscape of dendritic operation operating in DRN 5-HT neurons. Using multi-photon microscopy and in vitro electrophysical recordings, we conducted a morphological and electrophysiological survey of 5-HT neurons where we identified two structurally and morphologically distinct types of glutamatergic synapses both expressing small NMDAR-mediated conductance. Our initial findings provide valuable insights on local rules that govern how synaptic inputs to the DRN are being processed to ultimately confer 5-HT neurons their unique coding features.

A abstinência ao etanol está associada ao transtorno de ansiedade. Várias estruturas e sistemas de neurotransmissores estão envolvidos na mediação deste efeito, entretanto, tais substratos ainda não foram completamente elucidados. Em modelos animais, estudos mostram aumento significativo da neurotransmissão nitrérgica em resposta à abstinência do consumo crônico de etanol. O Núcleo Dorsal da Rafe (NDR) é uma importante região envolvida na gênese da ansiedade e possui grande quantidade da isoforma neuronial da enzima Sintase do Óxido Nítrico (nNOS). Assim, o objetivo deste estudo foi verificar se a inibição da transmissão nitrérgica no NDR atenua os efeitos ansiogênicos induzidos pela abstinência do etanol. Com essa finalidade, ratos Wistar machos foram submetidos a tratamento crônico (21 dias) ou agudo (1 dia) com etanol 6%(v/v) seguido da suspensão abrupta do tratamento por 48hs. Após as 48hrs os animais receberam uma injeção intra-NDR de L-NAME (100nmoles/0,2µL; inibidor não seletivo da NOS) ou 7-NI (7-Nitro-Indazol; 5 nmoles/0,2µL; inibidor seletivo para nNOS) e 5 min. depois foram testados no Labirinto em Cruz Elevado (LCE). Em resumo, os dados mostraram que a abstinência de 48hs. do tratamento crônico com etanol promoveu efeitos ansiogênicos significativos, evidenciados pela redução do número de entradas e tempo gasto nos braços abertos do LCE, o qual foi atenuado pelo tratamento intra-NDR com L-NAME mas não com 7-NI, sugerindo o envolvimento do NO no NDR na ansiedade induzida pela abstinência do etanol / The ethanol withdrawal is linked to the disorder of anxiety. Several structures and neurotransmitter systems are involved in mediating this effect, however, such substrates have not yet been elucidated. In animal models, studies show significant increase in neurotransmission nitrérgica in response to abstinence from chronic consumption of ethanol. The Dorsal Raphe Nucleus (DNR) is an important region involved in the genesis of anxiety and has large amounts of the enzyme Sintase neuronial isoform of nitric oxide (nNOS). The aim of this study was to verify that the inhibition of transmission in the DNR nitrérgica attenuates the anxiogenic effects induced by ethanol withdrawal. For this purpose, male Wistar rats were subjected to chronic treatment (21 days) or acute (1 day) with 6% ethanol (v / v) followed by the abrupt suspension of treatment for 48 hours. 48hrs after the animals received an intra-DNR L-NAME (100nmoles / 0.2 L, non-selective inhibitor of NOS) or 7-NI (7-Nitro-indazol, 5 nmol / 0,2 L; selective inhibitor for nNOS ) and 5 min. then were tested in the elevated plus-maze (EPM). Data obtained showed that ethanol withdrawal elicited significant \"anxiety-like\" behaviors, as revealed by the decrease in the number of entries into and time spent onto the open arms of the EPM., which was attenuated by treatment with intra-DNR L-NAME but not with 7-NI. These findings are indicative of the involvement of the DNR \"anxiety-like\" behaviors induced by withdrawal of ethanol.

O núcleo dorsal da rafe (NDR) é a principal fonte de projeções serotonérgicas que inervam o sistema límbico. Estudos mostram que o NDR é uma estrutura complexa, formada por distintas sub-regiões topograficamente organizadas e que apresentam diferentes propriedades neuroquímicas e funcionais. Tem sido proposto que duas vias serotonérgicas oriundas do NDR, o trato prosencefálico e o trato periventricular, modulam diferentemente a expressão de comportamentos defensivos associados aos transtornos de ansiedade generalizada e de pânico. O presente trabalho investigou se as respostas defensivas de esquiva e de fuga evocadas no modelo do labirinto em T elevado (LTE), relacionadas à ansiedade generalizada e ao pânico, respectivamente, recrutam diferentes sub-regiões do NDR. Na primeira etapa do trabalho, usando a técnica de imunoistoquímica para detecção da proteína Fos e da enzima triptofano hidroxilase, avaliamos a expressão da proteína Fos por neurônios serotonérgicos e não-serotonérgicos em diferentes sub-regiões do NDR, bem como em estruturas mesencefálicas vizinhas ao NDR, ou seja, o núcleo mediano da rafe (NMR) e substância cinzenta periaquedutal (SCP) de ratos em decorrência da expressão dos comportamento de esquiva inibitória ou fuga no LTE. Nossos resultados mostraram que os comportamentos de fuga e de esquiva evocados no LTE recrutam distintas populações neuronais do NDR, do NMR, bem como da SCP. Enquanto neurônios serotonérgicos localizados no nível médio e caudal do NDR, mais precisamente, nas sub-regiões dorsal (DRD), caudal (DRC) e interfascicular (DRI), bem como do NMR, estão envolvidos com a aquisição do comportamento de esquiva inibitória, neurônios não serotonérgicos das asas laterais do NDR/SCP ventrolateral, bem como das colunas, dorsomedial e dorsolateral da SCP participam da expressão da fuga. Na segunda parte deste trabalho, avaliamos os efeitos da administração do agonista de receptores AMPA/cainato, ácido caínico, no DRD, DRC e asas laterais do NDR sobre os comportamentos defensivos avaliados no LTE. Os resultados mostraram que a injeção de ácido caínico, tanto no DRD, quanto no DRC, facilita a aquisição da esquiva inibitória e também prejudica a expressão do comportamento de fuga. Já, a estimulação das asas laterais do NDR com ácido caínico induz a expressão do comportamento de fuga, sem alterar o comportamento de esquiva. Efeito oposto sobre o comportamento de fuga foi observado com a administração nesta região de cloreto de cobalto, um inibidor da transmissão sináptica. Nossos resultados mostraram ainda que a administração de ácido caínico nas asas laterais aumenta a distância percorrida pelos animais em uma arena circular, resultado indicativo da evocação da resposta de fuga. Por fim, avaliamos a o envolvimento da neurotransmissão mediada por receptores GABAA e por receptores CRF1 nas asas laterais do NDR, na expressão do comportamento de fuga expressa no LTE. Os resultados mostram que o bloqueio dos receptores GABAA nas asas laterais facilitou a expressão da fuga. Por outro lado a administração de antalarmina, antagonista de receptores CRF1 não alterou a expressão da resposta de fuga, porém prejudicou a aquisição da esquiva inibitória. Em conjunto, os resultados da primeira etapa indicam o envolvimento de diferentes sub-populações neuronais do NDR na expressão dos comportamentos de esquiva e fuga avaliados no LTE. Nossos resultados também apontam o envolvimento de neurônios serotonérgicos do NMR na expressão da esquiva inibitória, bem como a participação da SCP na resposta de fuga. Os resultados da segunda etapa indicam que neurônios serotonérgicos localizados no DRD e DRC possam dar origem aos tratos prosencefálico e periventricular abordados pela teoria de Deakin & Graeff (1991). Embora as asas laterais do NDR estejam marcantemente envolvidas na expressão/regulação da resposta de fuga, populações neuronais específicas dessa região também estão envolvidas na modulação do comportamento de esquiva inibitória. / The dorsal raphe nucleus (DRN) is the main source of serotonergic projections that innervate the limbic system. A wealth of evidence indicates that the DRN is a complex structure composed by topographically organized sub-regions with distinct functional and neurochemical properties. It have been proposed that two serotonergic pathways originating in the DRN, the forebrain and periventricular tracts, distinctly modulate defensive behaviors associated with generalized anxiety disorder and panic disorder. The present study addressed the hypothesis that the two defensive responses evoked by the elevated T maze (ETM), i.e. escape and inhibitory avoidance which have been related to generalized anxiety and panic disorders, respectively, would recruit different subregions of the DRN. In the first part of this work, the number of doubly-immunostained cells for Fos protein and tryptophan hydroxylase, a marker of serotonergic neurons, was assessed within the rat DRN, median raphe nucleus (MRN) and PAG following inhibitory avoidance and escape performance in the ETM. Our results showed that these two defensive responses recruited distinct neuronal populations within the DRN, MRN and PAG. While serotonergic neurons located at the middle and caudal level of the DRN, specifically within the sub-regions dorsal (DRD), caudal (DRC) and interfascicular (DRI), and the MRN are implicated in the acquistion of inhibitory avoidance, nonserotonergic neurons in lateral wings (lwDR) of the DRN /ventrolateralPAG and the dorsal columns of PAG are implicated in the escape expression. In the second part of this study, we evaluated the effects caused by the administration of AMPA/kainate receptor agonist, kainic acid, into the DRD, DRC and lwDR of rats tested in the ETM. The results showed that injection of kainic acid into DRD and DRC facilitated inhibitory avoidance acquisition and impaired escape expression. On the other hand, stimulation of the lwDR by kainic acid facilitated escape expression, without interfering with inhibitory avoidance acquisition. Opposite effect on escape behavior was observed in this region followed injection of cobalt chloride, a synaptic transmission inhibitor. Our results also showed that administration of higher doses of kainic acid into the lwDR promptly evoked a vigorous escape reaction in animals tested in a circular arena. Finally, we evaluated the involvement of GABAA and CRF1 receptor-mediated neurotransmission in the lwDR in the regulation of the escape behavior measured by the ETM. Our results showed that the GABAA receptor antagonist bicuculine injected into the lwDR favored escape expression, without affecting inhibitory avoidance acquisition. On the other hand, local microinjection of the CRF1 receptor antagonist antalarmin impaired the acquisition of inhibitory avoidance, without changing escape expression. Together, our immunohistochemical results indicate the involvement of distinct DRN neuronal subpopulations in the regulation of escape and inhibitory avoidance responses. Our results also suggested that, while serotonergic neurons within the DRD, DRC, DRI and MRN are involved in inhibitory avoidance acquisition, non-serotonergic neurons of the vlPAG, dlPAG and dmPAG are involved in escape expression. The behavioral results with kainic acid indicated that the DRC and DRD may be the origin of the forebrain and periventricular tracts addressed by Deakin & Graeffs theory (1991). Although the lwDR are markedly implicated in escape regulation, specific neuronal populations within this region may also modulate inhibitory avoidance behavior.

A abstinência ao etanol está associada ao transtorno de ansiedade. Várias estruturas e sistemas de neurotransmissores estão envolvidos na mediação deste efeito, entretanto, tais substratos ainda não foram completamente elucidados. Em modelos animais, estudos mostram aumento significativo da neurotransmissão nitrérgica em resposta à abstinência do consumo crônico de etanol. O Núcleo Dorsal da Rafe (NDR) é uma importante região envolvida na gênese da ansiedade e possui grande quantidade da isoforma neuronial da enzima Sintase do Óxido Nítrico (nNOS). Assim, o objetivo deste estudo foi verificar se a inibição da transmissão nitrérgica no NDR atenua os efeitos ansiogênicos induzidos pela abstinência do etanol. Com essa finalidade, ratos Wistar machos foram submetidos a tratamento crônico (21 dias) ou agudo (1 dia) com etanol 6%(v/v) seguido da suspensão abrupta do tratamento por 48hs. Após as 48hrs os animais receberam uma injeção intra-NDR de L-NAME (100nmoles/0,2µL; inibidor não seletivo da NOS) ou 7-NI (7-Nitro-Indazol; 5 nmoles/0,2µL; inibidor seletivo para nNOS) e 5 min. depois foram testados no Labirinto em Cruz Elevado (LCE). Em resumo, os dados mostraram que a abstinência de 48hs. do tratamento crônico com etanol promoveu efeitos ansiogênicos significativos, evidenciados pela redução do número de entradas e tempo gasto nos braços abertos do LCE, o qual foi atenuado pelo tratamento intra-NDR com L-NAME mas não com 7-NI, sugerindo o envolvimento do NO no NDR na ansiedade induzida pela abstinência do etanol / The ethanol withdrawal is linked to the disorder of anxiety. Several structures and neurotransmitter systems are involved in mediating this effect, however, such substrates have not yet been elucidated. In animal models, studies show significant increase in neurotransmission nitrérgica in response to abstinence from chronic consumption of ethanol. The Dorsal Raphe Nucleus (DNR) is an important region involved in the genesis of anxiety and has large amounts of the enzyme Sintase neuronial isoform of nitric oxide (nNOS). The aim of this study was to verify that the inhibition of transmission in the DNR nitrérgica attenuates the anxiogenic effects induced by ethanol withdrawal. For this purpose, male Wistar rats were subjected to chronic treatment (21 days) or acute (1 day) with 6% ethanol (v / v) followed by the abrupt suspension of treatment for 48 hours. 48hrs after the animals received an intra-DNR L-NAME (100nmoles / 0.2 L, non-selective inhibitor of NOS) or 7-NI (7-Nitro-indazol, 5 nmol / 0,2 L; selective inhibitor for nNOS ) and 5 min. then were tested in the elevated plus-maze (EPM). Data obtained showed that ethanol withdrawal elicited significant \"anxiety-like\" behaviors, as revealed by the decrease in the number of entries into and time spent onto the open arms of the EPM., which was attenuated by treatment with intra-DNR L-NAME but not with 7-NI. These findings are indicative of the involvement of the DNR \"anxiety-like\" behaviors induced by withdrawal of ethanol.