The mechanisms underlying the generation of rapid eye movement (REM) sleep are poorly understood. Despite a lack of direct support, neurons maximally active during REM sleep (REM sleep-active) located in the pedunculopontine tegmental nucleus (PPTn) are hypothesized to generate this state and its component phenomenology. This hypothesis has never been directly tested, since the results of selectively inhibiting this cell-group have never been determined. Using microdialysis, electrophysiology, histochemical and pharmacological methods in freely-behaving rats (n=22) instrumented for sleep-wake state and respiratory muscle recordings, I selectively inhibited REM sleep-active PPTn neurons. Contrary to the prevailing hypothesis, I showed that REM sleep-active PPTn neurons suppress REM sleep by limiting the frequency of its onset. These neurons also shape the impact of REM sleep on breathing. REM sleep-active PPTn neurons restrain behavioural activation of upper-airway musculature during REM sleep, while depressing breathing rate and respiratory activation of the upper-airway musculature across sleep-wake-states.

The mechanisms underlying the generation of rapid eye movement (REM) sleep are poorly understood. Despite a lack of direct support, neurons maximally active during REM sleep (REM sleep-active) located in the pedunculopontine tegmental nucleus (PPTn) are hypothesized to generate this state and its component phenomenology. This hypothesis has never been directly tested, since the results of selectively inhibiting this cell-group have never been determined. Using microdialysis, electrophysiology, histochemical and pharmacological methods in freely-behaving rats (n=22) instrumented for sleep-wake state and respiratory muscle recordings, I selectively inhibited REM sleep-active PPTn neurons. Contrary to the prevailing hypothesis, I showed that REM sleep-active PPTn neurons suppress REM sleep by limiting the frequency of its onset. These neurons also shape the impact of REM sleep on breathing. REM sleep-active PPTn neurons restrain behavioural activation of upper-airway musculature during REM sleep, while depressing breathing rate and respiratory activation of the upper-airway musculature across sleep-wake-states.

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The reinforcing properties of nicotine depend partly on cholinergic projections from the pedunculopontine tegmental (PPTg) and laterodorsal tegmental (LDTg) nuclei to midbrain dopamine neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Neuronal activation was investigated using Fos expression in these areas following acute (0, 0.1, 0.4, 0.8mg/kg) or chronic systemic nicotine (0, 0.1, 0.4, 0.8, 1.0mg/kg given once per day for 5 days). We also examined co-localization of Fos expression in bNOS and TH positive neurons to determine what populations of neurons were activated by nicotine. Acute nicotine resulted in dose related Fos expression, with the biggest increase seen after 0.4mg/kg nicotine, but no co-localization occurred with bNOS in the PPTg/LDTg. Surprisingly, nicotine also failed to activate midbrain dopamine neurons. After animals were sensitized to nicotine there was a similar dose response curve in Fos expression, but the biggest increase was seen after 0.8mg/kg nicotine. Chronic nicotine, like acute, also preferentially activated non-cholinergic neurons in the LDTg and PPTg and non-dopamine neurons in the SNc and VTA. Further experiments looked at the mechanisms of Fos expression after nicotine administration. Fos expression in the LDTg/PPTg and SNc/VTA was suppressed after d-amphetamine, despite an increase in locomotor activity, suggesting that the increased Fos expression after chronic nicotine was not simply due to the locomotor activating effects of sensitized nicotine. Blocking autoreceptors in the dopaminergic midbrain by haloperidol pre-treatment did not increase Fos expression in dopamine neurons indicating that the inhibitory mechanism was not dependent on local autoreceptors. Novel methods of visualising and lesioning GABA neurons in the mesopontine tegmentum and midbrain were also examined. The data suggest that the mechanisms by which dopamine is involved in the pharmacological actions of passively administered nicotine are more complex than was first thought and that the role of non-dopamine neurons in the VTA (possibly GABA or glutamate containing) are also important.

Orientadora: Profa. Dra. Marcela Bermudez Echeverry / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Neurociência e Cognição, 2016. / A Doença de Parkinson (DP) é caracterizada por uma degeneração progressiva dos neurônios dopaminérgicos na substância negra e pela presença de 4 sinais cardinais, bradicinesia, rigidez muscular, tremor em repouso e instabilidade postural. Sua etiologia ainda é desconhecida, no entanto, o aumento do estresse oxidativo é uma das hipóteses a ser analisada. Existe um interesse cada vez maior em compreender o papel funcional e comportamental do estriado dorsolateral (CPU-DL), principalmente sobre o controle do equilíbrio postural. Dessa forma, o presente projeto se propôs a avaliar o efeito de diferentes toxinas, 6-hidroxidopamina (6-OHDA), L-butionina-sulfoximina (BSO) e a rotenona, sobre o CPU-DL e a resposta compensatória do sistema nitrérgico de forma tempo dependente. Foram utilizados ratos Wistar adultos, todos os procedimentos foram aprovados pelo CEUA-UFABC (Protocolo 035/2014). O Estudo 1 foi separado em 2 fases, no experimento 1.1 os animais foram divididos em 3 grupos, os quais receberam microinjeções no CPU-DL direito de 6- OHDA, BSO ou salina, sendo avaliados comportamentalmente 5 dias após a cirurgia e sacrificados no 6º dia. No experimento 1.2 os animais foram divididos em 3 grupos, os quais foram submetidos a uma dupla lesão, recebendo 6-OHDA no CPUDL + salina no PPTg, 6- OHDA no CPU-DL + BSO no PPTg ou salina no CPU-DL + salina no PPTg. Os animais do experimento 1.2 foram avaliados entre os 15º e 44º dias pós cirúrgico, sendo eutanasiados no 45º. No segundo estudo desenvolvido, os animais foram divididos em 5 grupos, os quais receberam 2 microinjeções no CPU-DL direito de rotenona (3mM, 5mM ou 9mM), DMSO (25% - veículo) ou salina, sendo avaliados entre os dias 15º e 44º pós cirúrgicos e sacrificados no 45º dia. Foram utilizados na avaliação comportamental os testes: comportamento rotatório induzido por apomorfina e anfetamina, teste de suspensão na grade, teste da cauda suspensa, teste do cilindro, equilíbrio vertical e labirinto em cruz elevado. Vinte e quatro horas após as análises comportamentais os animais foram sacrificados e procedimentos para analise imunohistoquímica foram realizados. No experimento 1.1 não se detectou alteração comportamental significativa, entretanto, no experimento 1.2 observou-se uma redução na performance dos grupos tratados em relação ao controle salina. Nossos resultados sugerem que tanto a 6-OHDA como o BSO, quando aplicados no CPU-DL, diminuem a expressão de TH+ na região CPU-DL e CPU-dorsomedial (CPU-DM), com aumento da expressão para a proteína nNOS no CPU-DL, sendo que o grupo 6-OHDA também mostrou aumento no CPUDM e núcleo accumbens. Por outro lado, um aumento escasso na expressão da proteína nitrotirosina foi observado no grupo BSO, no CPU-DM e CPU-ventrolateral (CPU-VL). A avaliação com maior intervalo de tempo realizada com as mesmas toxinas, indicou um possível papel neuroprotetor da injeção de BSO no PPTg contra o efeito neurotóxico da 17 injeção de 6- OHDA na CPU-DL. O segundo estudo desenvolvido corrobora uma potencial qualidade preditiva da microinjeção estriatal de rotenona como modelo de hemiparkinsonismo, pois foi capaz de mimetizar sintomas motores relacionados ao equilíbrio postural (rotarod, grade e equilíbrio vertical) e imunohistoquímicos, com aumento da expressão de nNOS e diminuição da expressão da TH+ de forma dose dependente no CPU e na substância negra. Nossos resultados sugerem que determinadas neurotoxinas, embora pouco exploradas, quando administradas via intra-estriatal, revelaram-se potencialmente efetivas para a indução de alterações no equilíbrio postural em modelos experimentais; corroborando portanto, uma possível interação do sistema dopaminérgico nigro-estriatal atrelado a um desbalanço do sistema nitrérgico, responsável pelo estresse oxidativo/nitrosativo relacionado aos processos neurodegenerativos. Neste sentido novos estudos devem ser conduzidos para uma melhor compreensão e elucidação da etiopatogenia da DP, além de novas perspectivas para futuras estratégias terapêuticas. / Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra and the presence of four cardinal signs, bradykinesia, muscular rigidity, resting tremor, and postural instability. Its etiology is still unknown, however, increased oxidative stress is one of the hypotheses to be analyzed. There is a growing interest in understanding the functional and behavioral role of the dorsolateral striatum (CPU-DL), mainly on the control of postural balance. Thus, this project aimed to evaluate the effect of different toxins, 6-hydroxydopamine (6-OHDA), L-buthionine sulfoximine (BSO) and rotenone on the CPU-DL and the compensatory response nitrergic system time dependent manner. Adult Wistar rats were used, all procedures were approved by CEUA-UFABC (Protocol 035/2014). The first study was separated into two phases in the experiment 1.1 the animals were divided into 3 groups, which were microinjected on the CPU-DL right of 6- OHDA, BSO or saline, being evaluated behaviorally 5 days after surgery and sacrificed on the 6th day. In the 1.2 experiment the animals were divided into 3 groups, which were subjected to a double lesion, receiving 6-OHDA in CPUDL + saline in PPTg, 6-OHDA-CPU-DL + BSO - PPTg or saline + saline. The animals of the experiment 1.2 were evaluated between 15 and 44 days after surgery, being euthanized after 45 days. In the second study developed the animals were divided into 5 groups, which were microinjected the CPU-DL right to rotenone (3 mM, 5 mM or 9mm), DMSO (25% - vehicle) or saline, were evaluated between days 15 and 44 post surgical and sacrificed on the 45th day. Were used for behavioral evaluation tests: rotational behavior induced by apomorphine and amphetamine, the grid test, elevated body swing test, drum test, vertical maze and balance in high cross. Twenty-four hours after the behavioral tests the animals were sacrificed and immunohistochemical analysis procedures were performed. For first study, was not detected behavioral change in the 1.1 experiment, however in the 1.2 experiment, there was a reduced performance of the treated groups compared to saline control on the rotarod test. Nevertheless, our results suggest that both 6- OHDA as BSO, when applied to the CPU-DL decrease TH + expression on the CPU-DL and CPU-DM area with increased expression of nNOS protein in the CPU-DL , and the 6-OHDA group also showed an increase in CPU-DM and nucleus accumbens. Still, a scarce increase in the expression of nitrotyrosine protein was observed in the CPU DM and VL on BSO group. The results of 1.2 suggests a possible neuroprotective role of BSO injection in PPTg after neurotoxic effect of 6-OHDA injection the CPU-DL. To the second study, our findings suggest a potential predictive quality of rotenone striatal microinjection as hemiparkinsonismo model because it was able to mimic motor symptoms related to postural balance (rotarod, grid and vertical balance) and immunohistochemical, an increase of nNOS expression and decreased expression of TH+ dose dependent manner in the CPU and the substantia nigra. Our results suggest that neurotoxins, though little exploited when administered intra-striatal pathway, have proved to be potentially effective for inducing changes in the postural balance in experimental models; corroborating therefore a possible interaction of the nigrostriatal dopaminergic system linked to an imbalance nitrergic system, responsible for the oxidative stress/nitrosative related to neurodegeneration. In this sense new studies should be conducted to better understand and elucidate the pathogenesis of PD, as well as new perspectives for future therapeutic strategies.

Cholinergic inputs from mesopontine tegmentum activate midbrain dopamine (DA) neurons via M5 muscarinic receptors. The M5 receptor is important for mesopontine stimulation-induced accumbal or striatal DA efflux, brain stimulation reward or morphine-induced conditioned place preference (CPP). M5 receptor knockout (KO) mice show 40-50% less morphine-induced locomotion. Pedunculopontine tegmental nucleus (PPT) lesions in rodents block morphine CPP, but are ineffective after 18 hours food deprivation, opiate dependence, or intra-VTA BDNF. Based on these findings, we investigated whether acute food deprivation or intra-VTA BDNF alters morphine-induced locomotion (3 and 10 mg/kg, i.p.) in C57BL/6 M5 KO mice. Non-deprived M5 KOs showed reduced morphine-induced locomotion, suggesting M5 receptors partly mediate morphine-induced locomotion. Morphine-induced locomotion was reversed in food-deprived mice, suggesting the stimulant effects of morphine were altered to bypass the PPT. Unexpectedly, intra-VTA BDNF infusions were ineffective in altering morphine-induced locomotion. Additionally, M5 KOs receiving intra-VTA saline showed no deficits in morphine-induced locomotion.

Cholinergic inputs from mesopontine tegmentum activate midbrain dopamine (DA) neurons via M5 muscarinic receptors. The M5 receptor is important for mesopontine stimulation-induced accumbal or striatal DA efflux, brain stimulation reward or morphine-induced conditioned place preference (CPP). M5 receptor knockout (KO) mice show 40-50% less morphine-induced locomotion. Pedunculopontine tegmental nucleus (PPT) lesions in rodents block morphine CPP, but are ineffective after 18 hours food deprivation, opiate dependence, or intra-VTA BDNF. Based on these findings, we investigated whether acute food deprivation or intra-VTA BDNF alters morphine-induced locomotion (3 and 10 mg/kg, i.p.) in C57BL/6 M5 KO mice. Non-deprived M5 KOs showed reduced morphine-induced locomotion, suggesting M5 receptors partly mediate morphine-induced locomotion. Morphine-induced locomotion was reversed in food-deprived mice, suggesting the stimulant effects of morphine were altered to bypass the PPT. Unexpectedly, intra-VTA BDNF infusions were ineffective in altering morphine-induced locomotion. Additionally, M5 KOs receiving intra-VTA saline showed no deficits in morphine-induced locomotion.

Based on the belief that it is a locomotor control structure, the pedunculopontine tegmental nucleus (PPTg) has been considered a potential target for deep brain stimulation (DBS) for Parkinson's disease (PD) patients with symptoms refractory to medication and/or stimulation of established target sites. To date, a number of patients have been implanted with PPTg electrodes with mostly disappointing results. Exact target site in PPTg, possible mechanisms of PPTg-DBS and likely potential benefits need to be systematically explored before consideration of further clinical application. The research described here approaches these questions by (i) investigating the role of the PPTg in gait per se; (ii) developing a refined model of PD that mimics the underlying pathophysiology by including partial loss of the PPTg itself; (iii) adapting a wireless device to let rats move freely while receiving DBS; and (iv) investigating the effect of DBS at different sites in the PPTg on gait and posture in the traditional and refined model of PD. Underlining the concern that understanding the PPTg as a locomotor control structure is inadequate, the experiments showed that neither partial nor complete lesions of PPTg caused gait deficits. The refined model showed hardly any differences compared to the standard one, but the effect of DBS in each was very different, highlighting the need to take degeneration in the PPTg into consideration when investigating it as a DBS target. The differential results of anterior and posterior PPTg-DBS show the critical importance of intra-PPTg DBS location: Anterior PPTg electrodes caused severe freezing and worsened gait while some gait parameters improved with stimulation of posterior PPTg. The results suggest mechanisms of PPTg-DBS beyond the proposed activation of over-inhibited PPTg neurons, including aggravation of already dysfunctional inhibitory input by anterior PPTg-DBS and activation of ascending projections from posterior PPTg to the forebrain.