Despite their acknowledged efficacy, currently available antidepressants still demonstrate undesirable side effects, shortfalls in effectiveness and a delayed onset of action. All these agents act via monoaminergic mechanisms, although recent studies have begun to note the potential role of the cholinergic system as well as the amino acid pathways in affective isorders. It has been suggested that glutamate NMDA receptor activation may be involved in hippocampal degeneration seen in patients with depression, as well as contributing as a molecular target for the antidepressant action of known antidepressant drugs. Glutamate either separately or via the release of nitric oxide, regulates the release of various transmitters in the brain critical for affective state, e.g. monoamines (noradrenaline, dopamine), indoleamines (5HT), y-aminobutyric acid (GABA) and acetylcholine. The aim of this study was to investigate N-methyl-D-aspartate (I\IMDA) and muscarinic M1 receptor characteristics and also GABA and acetylcholine levels in a genetic animal model of depression, the Flinders Sensitive Line (FSL) rat, with respect to its control, viz. Flinders Resistant Line (FRL) rat, thereby establishing a possible role for the amino acid and cholinergic pathways in the hippocampus and frontal cortex, two brain areas implicated in depression. In addition, anxietylike behaviours were assessed using the open field and social interaction tests. A sensitive liquid chromatography tandem mass spectrometer (LC/MS/MS) method was used in the quantification of acetylcholine as well as high performance liquid chromatography with electrochemical detection (HPLG-EGD) for the quantification of GABA in the above-mentioned brain areas of FSL and FRL rats. NMDA and muscarinic M1 receptor characteristics were expressed in terms of receptor denSity (Bmax) and affinity (Kd) values and were performed using [3H]-MK801 (27.5 Gi/mmol) and quinuclidinyl benzilate (52.0 Gilmmol) for NMDA and M1 receptors, respectively. In addition, to provide evidence for face validity, behavioural assessments were routinely performed using the open field test and social interaction test. Significantly elevated levels of acetylcholine were found in the frontal cortex but with significantly reduced levels in the hippocampus of FSL rats. Cortical and hippocampal muscarinic receptor binding characteristics remained unchanged, while no differences with regard to GABA levels and NMDA receptor binding characteristics were noted in these brain areas. In concordance with studies from the literature, aversive and locomotor behaviour as measured in the open field test, provided evidence of anxiogenic behaviour in the FSL rat, evinced by significantly less social interaction than their FRL counterparts. In addition, evidence for a lack in general activity of the FSL rat in the open field was also noted. Our data therefore suggest the presence of a cholinergic dysfunction in both the frontal cortex and hippocampus of the FSL rat, although this is not accompanied by simultaneous changes in muscarinic M1 receptor binding in key limbic brain regions. Although increased cholinergic drive is a recognised characteristic of FSL rats and is representative of the model's' construct validity, we suggest that the depressive phenotype of these animals is not related to altered cholinergic activity in a single brain region, but instead involves various limbic brain regions, possibly being more dependent on opposing cholinergic deficits in the cortex and hippocampus. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2008.

Despite their acknowledged efficacy, currently available antidepressants still demonstrate undesirable side effects, shortfalls in effectiveness and a delayed onset of action. All these agents act via monoaminergic mechanisms, although recent studies have begun to note the potential role of the cholinergic system as well as the amino acid pathways in affective isorders. It has been suggested that glutamate NMDA receptor activation may be involved in hippocampal degeneration seen in patients with depression, as well as contributing as a molecular target for the antidepressant action of known antidepressant drugs. Glutamate either separately or via the release of nitric oxide, regulates the release of various transmitters in the brain critical for affective state, e.g. monoamines (noradrenaline, dopamine), indoleamines (5HT), y-aminobutyric acid (GABA) and acetylcholine. The aim of this study was to investigate N-methyl-D-aspartate (I\IMDA) and muscarinic M1 receptor characteristics and also GABA and acetylcholine levels in a genetic animal model of depression, the Flinders Sensitive Line (FSL) rat, with respect to its control, viz. Flinders Resistant Line (FRL) rat, thereby establishing a possible role for the amino acid and cholinergic pathways in the hippocampus and frontal cortex, two brain areas implicated in depression. In addition, anxietylike behaviours were assessed using the open field and social interaction tests. A sensitive liquid chromatography tandem mass spectrometer (LC/MS/MS) method was used in the quantification of acetylcholine as well as high performance liquid chromatography with electrochemical detection (HPLG-EGD) for the quantification of GABA in the above-mentioned brain areas of FSL and FRL rats. NMDA and muscarinic M1 receptor characteristics were expressed in terms of receptor denSity (Bmax) and affinity (Kd) values and were performed using [3H]-MK801 (27.5 Gi/mmol) and quinuclidinyl benzilate (52.0 Gilmmol) for NMDA and M1 receptors, respectively. In addition, to provide evidence for face validity, behavioural assessments were routinely performed using the open field test and social interaction test. Significantly elevated levels of acetylcholine were found in the frontal cortex but with significantly reduced levels in the hippocampus of FSL rats. Cortical and hippocampal muscarinic receptor binding characteristics remained unchanged, while no differences with regard to GABA levels and NMDA receptor binding characteristics were noted in these brain areas. In concordance with studies from the literature, aversive and locomotor behaviour as measured in the open field test, provided evidence of anxiogenic behaviour in the FSL rat, evinced by significantly less social interaction than their FRL counterparts. In addition, evidence for a lack in general activity of the FSL rat in the open field was also noted. Our data therefore suggest the presence of a cholinergic dysfunction in both the frontal cortex and hippocampus of the FSL rat, although this is not accompanied by simultaneous changes in muscarinic M1 receptor binding in key limbic brain regions. Although increased cholinergic drive is a recognised characteristic of FSL rats and is representative of the model's' construct validity, we suggest that the depressive phenotype of these animals is not related to altered cholinergic activity in a single brain region, but instead involves various limbic brain regions, possibly being more dependent on opposing cholinergic deficits in the cortex and hippocampus. / Thesis (M.Sc. (Pharmacology)--North-West University, Potchefstroom Campus, 2009.

Introdução: A anemia aguda tem sido associada com distúrbios neurofisiológicos e cognitivos em pacientes saudáveis. Evidências experimentais sugerem que a hemodiluição pode aumentar lesões cerebrais, limitando o suprimento de oxigênio ao tecido cerebral. No entanto, o mecanismo exato pelo qual as lesões cerebrais ocorrem em pacientes anêmicos ainda não está claramente definido. O objetivo desse estudo foi avaliar os precursores da apoptose neuronal Bax, Bcl-x no córtex frontal, atividade das caspases 3 e 9 na fração citosólica do hipocampo e na fração mitocondrial do córtex frontal, assim como a fragmentação do DNA na fração nuclear e mitocondrial do córtex frontal, após hemodiluição normovolêmica aguda. Métodos: Vinte e quatro porcos foram anestesiados e randomizados em 4 grupos de 6 animais: Controle, hemodiluição normovolêmica aguda (HNA) com hematócrito alvo de 15% (Ht 15%), HNA com hematócrito alvo de 10% (Ht 10%) e hipóxia-hipóxica (HH). A HNA foi realizada com 1ml de hidroxetil amido (130/0,4) por ml de sangue retirado, até o hematócrito alvo desejado (10 ou 15%). O HH consistiu de ventilação com baixa fração expirada de O2 (FiO2), sendo de 6% por 60 minutos, servindo como grupo controle positivo. Os animais do grupo controle não sofreram nenhuma dessas intervenções. As proteínas pró-apoptótica Bax e anti-apoptótica Bcl-x foram avaliadas por Western blotting nas frações nucleares e mitocondriais do córtex frontal. A atividade das caspases 3 e 9 foi avaliada nas frações mitocondrial e citosólica do hipocampo por espectrofluorometria. A fragmentação do DNA foi avaliada por eletroforese nas frações nuclear e mitocondrial do córtex frontal. Os dados foram comparados por análise de variância (ANOVA) seguida por teste de Tukey (p<0,05). Resultados: Não foi observada diferença significativa entre os grupos controle, Ht 15% e Ht 10% em relação à proteína pró-apoptótica Bax nas frações nuclear e mitocondrial. Entretanto, o grupo HH foi significativamente diferente dos grupos controle e Ht 15% na fração nuclear e de todos os grupos na fração mitocondrial. Não foi encontrada diferença significativa em relação à Bcl-x. A atividade das caspases 3 e 9 nas frações mitocondrial e nuclear foi diferente no grupo HH quando comparado com os demais grupos. Não foi observada diferença significativa em relação à fragmentação do DNA entre os grupos controle, Ht 15% e Ht 10%. Conclusão: A avaliação dos precursores da apoptose demonstrou que a hemodiluição normovolêmica aguda com hematócrito alvo de 15% e 10% não induziu apoptose, sugerindo que a oxigenação cerebral foi preservada / Background: Acute anemia has been associated with neurophysiologic and cognitive dysfunctions in healthy patients. Experimental evidences suggest that hemodilution may increase cerebral lesions, limiting oxygen supply to the brain tissue. Nevertheless, the exact mechanisms through which cerebral lesions occur in anemic patients havent been clearly defined. Therefore, the objective of the present study was to evaluate neuronal apoptosis precursors Bax, Bcl-x in the frontal cortex, caspase 3 and 9 activity in the mitochondrial and cytosolic fractions of the hippocampus, even as DNA fragmentation in the mitochondrial and nuclear fractions of the frontal cortex after acute normovolemic hemodilution. Methods: Twenty four pigs were anesthetized and randomized into 4 groups of 6 animals: sham, acute normovolemic hemodilution (ANH) to reach a hematocrit of 15% (Ht 15%), ANH to reach a hematocrit of 10% (Ht 10%) and hypoxic-hipoxia (HH). ANH was performed with 1ml hydroxyethyl starch 130/0.4 (HES) per ml of blood withdrawn to the desired target hematocrit (10 or 15%). HH consisted of ventilation with low fraction of inspired oxygen (FiO2) of 6% for 60 minutes, serving as a positive control group. Sham animals were not involved in any of these interventions. Pro-apoptotic Bax and anti-apoptotic Bcl-x proteins were evaluated by Western blotting in nuclear and mitochondrial fractions of the frontal cortex and activities of caspases-3 and-9 were evaluated in the mitochondrial and cytosolic fractions of the hippocampus by spectrofluorometry. DNA fragmentation was evaluated by electrophoresis in the mitochondrial and nuclear fraction. Data were compared by analysis of variance (ANOVA) followed by Tukeys test (p<0.05). Results: No statistical significance was found among sham, Ht 15% or Ht 10% groups regarding pro-apoptotic protein Bax, in nuclear or mitochondrial fractions. However, group HH presented significant difference from sham and Ht 15% groups in the nuclear fraction and from all groups in the mitochondrial fraction. No statistical significance was found with Bcl-x. The activities of caspases-3 and-9 in cytosolic and mitochondrial fractions were statisticaly different in group HH when compared with all other groups. No statistical significance was found in relation to DNA fragmentation among sham, Ht 15% or Ht 10%. Conclusion: The evaluation of apoptosis precursors demonstrated that ANH with target hematocrit 15% and 10% did not induce neuronal lesion, suggesting that cerebral oxygenation was preserved

The development of cholinergic neurons in mouse frontal cortex was studied both in vivo and in vitro by immunocytochemistry with an antibody to choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine synthesis. While cortical cholinergic neurons have previously been characterized in rat cortex, up until very recently, intrinsic cortical cholinergic neurons were considered to be absent in mouse, and little is known about their development or phenotypic characteristics. The present study found no ChAT-positive neurons in mouse frontal cortex on postnatal day 0 (P0, the day of birth). On P7 there were few, faintly stained, ChAT-positive neurons. The numerical density of ChAT-positive neurons increased substantially with age, from none on P0, to 9.2 + 1.4 on P7, to 14.8 + 0.9 on P16, and 41.6 + 3.9 in adulthood. Considering that the numerical density of total neurons decreases during this postnatal period, the data represent a marked developmental increase in the percentage of cholinergic neurons. The development of cholinergic neurons showed very similar timelines in rat and mouse frontal cortex. Cultures prepared from mouse frontal cortex on embryonic day 16 were maintained for 25, 76, or 100 days in vitro (div). The percentage of ChAT-positive neurons was considerably higher than in vivo, ranging from a mean 28% to 31% across the three age (div) groups. With increasing age of the cultures, the numerical density of total neurons and ChAT-positive neurons decreased while the percentage of ChAT-positive neurons did not change significantly. These observations suggest some temporal stability in the cultures. Using dual immunofluorescence, ChAT-positive neurons were tested for colocalization with GAD or TH. The majority of ChAT-positive neurons colocalized with GAD, both in vitro and in vivo. However, ChAT did not colocalize with TH, either in vitro or in vivo. Our comparison of intact frontal cortex and cultures suggest that while the percentage of cholinergic neurons was greater in the cultures, the cholinergic neurons developed phenotypic similarities in vitro and in vivo.

MDD is a severe and debilitating disorder that is associated with a growing global economic burden due to reduced workplace productivity along with increased healthcare resource utilization. Furthermore, depression markedly enhances the risk for suicide, mortality that is especially worrisome given that 30% of depressed individuals have an inadequate response to current antidepressants. This inadequacy of antidepressants necessitates the discovery of a better understanding of the pathobiology of MDD. Most current antidepressants work through monoamine neurotransmitters, and their relative efficacy in depression led to the now dated monoamine-deficiency hypothesis. The limited usefulness of antidepressants has led to a reinvigorated search for other pathologies in depression that might yield clues for the development of better drug treatments. In this regard, a strong association has been found between oxidative stress and MDD. Our lab recently found increased DNA oxidation and elevated poly(ADP)ribose polymerase (PARP1) gene expression in the brain from donors that had MDD at the time of death. Besides DNA damage repair, PARP1 mediates several downstream inflammatory effects that may contribute to pathology in MDD. In fact, our lab has demonstrated that PARP-1 inhibition produces antidepressant-like effects in rodents, suggesting that PARP-1 inhibitors hold promise as a novel antidepressant drug. While our lab had previously demonstrated elevated PARP1 gene expression in the frontal cortex in MDD, whether PARP1 protein levels were also increased in depression had not been verified. My thesis research was performed to determine whether PARP1 protein expression was also elevated in the brain in MDD. I studied primarily the hippocampus because it is part of the limbic (mediating emotion) system of the brain and because previous research has shown numerous other pathologies in the hippocampus. My study was carried out simultaneously as others in our lab were measuring PARP1 protein levels in frontal cortex in MDD. This latter work was important since the lab’s previous work had observed elevated PARP1 gene expression in the frontal cortex, rather than in the hippocampus which was not previously studied. Hippocampal and frontal cortical brain sections were cut from frozen blocks of both MDD and psychiatrically normal control brain donors for these studies. PARP1 protein levels were estimated by assisted-imaging software. The findings herein demonstrate that levels of PARP1 immunoreactivity are significantly elevated in the frontal cortex of MDD donors as compared to control donors. However, there was no change in PARP1 immunoreactivity in the hippocampus in MDD.

Schizophrenia postmortem brain is characterized by γ aminobutyric acid downregulation and by decreased dendritic spine density in frontal cortex. Protracted L-methionine treatment exacerbates schizophrenia symptoms, and our earlier work (Tremolizzo et al. and Dong et al.) has shown that L-methionine decreases reelin and GAD67 transcription in mice which is prevented by co-administration of valproate. In this study, we observed a decrease in spine density following L-methionine treatment, which was prevented by co-administration of valproate. Together with our earlier findings conducted under the same experimental conditions, we suggest that downregulation of spine density in L-methionine-treated mice may be because of the decreased expression of reelin and that valproate may prevent spine downregulation by inhibiting the methylation induced decrease in reelin.

Projections from medial agranular cortex to brain stem in rat were determined by use of the anterograde tracers Phaseolus vulgaris leucoagglutinin, or wheat germ agglutinin conjugated horseradish peroxidase. Axonal trajectories were also followed by means of the Wiitanen modification of the Fink-Heimer degeneration technique. AGm was identified on the basis of its cytoarchitectonics. AGm projected to the anterior pretectal nucleus, the rostral interstitial nucleus of the medial longitudinal fasciculus, the medial accessory oculomotor nucleus of Bechterew, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch, the nucleus cuneiformis and subcuneiformis, intermediate and deep superior collicular layers, the paramedian pontine reticular formation (reticularis pontis oralis and caudalis, and reticularis gigantocellularis), and raphe centralis superior. Differences in connections between rostral and caudal injections were observed: pontine and medullary projections were lighter from the rostral portion of AGm than from the more caudal portions of AGm. The heaviest projections to the anterior pretectal nucleus were from the caudal portion of AGm. The subcortical projections were very similar to those described for the frontal eye field in monkeys, and the majority of them targeted areas thought to be involved in coordination of gaze with head and neck movements. Thus AGm in rats may contain the homologue of the primate frontal eye fields.

The lateral prefrontal cortex (LPFC) and the anterior cingulate cortex (ACC) are two key regions of the frontal executive control network. Ascending cholinergic pathways differentially innervate these two functionally distinct cortices to modulate arousal and motivational signaling for higher-order functions. The action of acetylcholine (ACh) in sensory cortices is constrained by layer, anatomical cell type, and subcellular localization of distinct receptors, but little is known about the nature and organization of frontal-cholinergic circuitry in primates. In this dissertation, we characterized the anatomical localization of muscarinic acetylcholine receptors (mAChRs), m1 and m2–the predominant subtypes in the cortex–and their expression profiles on distinct cell types and pathways in ACC and LPFC of the rhesus monkey, using immunohistochemistry, anatomical tract-tracing, whole cell patch-clamp recordings, and single nucleus RNA sequencing. In the first series of studies (Chapter 2), we used immunohistochemistry and high-resolution confocal microscopy to reveal regional differences in m1 and m2 receptor localization on excitatory pyramidal and inhibitory neuron subpopulations and subcellular compartments in ACC (A24) versus LPFC (A46) of adult rhesus monkeys (Macaca mulatta; aged 7-11 yrs; 4 males and 2 females). The ACC exhibited a greater proportion of m2+ inhibitory neurons and a greater density of presynaptic m2+ receptors localized on inhibitory (VGAT+) terminations on pyramidal neurons compared to the LPFC. This result suggests a greater cholinergic suppression of GABAergic neurotransmission in ACC. In a second set of experiments (Chapter 3), we examined the heterogeneity of m1 and m2 laminar expression in functionally distinct ACC areas A24, A25, and A32. These differ in their connections with higher order cortical areas and limbic structures, such as the amygdala (AMY). The density of m1+ and/or m2 expressing (m1+/m2+) pyramidal neurons was significantly greater in A24 compared to A25 and to A32, while A25 exhibited a significantly greater density of m2+VGAT+ terminals. In addition, we examined the substrates for cholinergic modulation of long-range cortico-limbic processing using bidirectional neural tracers to label one specific subtype, the AMY-targeting projection neurons in these ACC areas. Compared to A24 and A32, the limbic ventral A25 had a greater density of m1+/m2+ AMY-targeting pyramidal neurons across upper layers 2-3 and deep layers 5-6, suggesting stronger cholinergic modulation of amygdalar outputs. Lastly (Chapter 4), we assessed the functional effects of cholinergic modulation on excitatory and inhibitory synaptic activity as well as the molecular signatures related to m1 and m2 receptor expression. In experiments using in vitro whole-cell patch-clamp recordings of layer 3 pyramidal neurons in ACC and LPFC, we found that application of the cholinergic agonist carbachol (CCh) significantly decreased the frequency of excitatory postsynaptic currents (EPSCs) to a greater extent in ACC A24 than in LPFC A46. Using single nucleus RNA sequencing, we found that enriched m1 and m2 transcriptional profiles in distinct cell-types and frontal areas (ACC A24 and LPFC A46) had differentially expressed genes associated with down-stream signaling cascades related to synaptic signaling and plasticity. Together, these data reveal the anatomical, functional, and transcriptomic neural substrates of diverse cholinergic modulation of local excitatory and inhibitory circuits and long-range cortico-limbic pathways in functionally-distinct ACC and LPFC frontal areas that are important for cognitive-emotional integration.

O estrógeno é importante para o desenvolvimento de redes neuronais. Assim, investigamos mecanismos celulares relacionados à neuroproteção, através da sinalização rápida mediada pelo GPER-1 em cultura mistas e enriquecida de neurônios submetidas ou não à privação de glicose/oxigênio (PGO). Mostramos que as células corticais em cultura expressam o receptor GPER-1 e esta marcação encontra-se dispersa tanto no citosol como no núcleo. Nossos resultados mostraram que a proteção, via sinalização estrogênica, foi dependente da composição celular. A ausência da sinalização via GPER-1 previamente à PGO aumentou a morte celular induzida pela PGO, sugerindo que o bloqueio desta sinalização via GPER-1 pode estar relacionado ao pior prognóstico de lesões isquêmicas, e a suplementação com G1 no meio de cultura durante a privação e reperfusão atenuaram estes efeitos. Além disso, nossos resultados apontam para a influência das células da glia como mediadores do papel neuroprotetor, via sinalização estrogênica não-nuclear, neste contexto de privação de glicose/oxigênio. / Estrogen is important to the development of neural networks. Thus, we investigated the cellular mechanisms related to neuroprotection through the rapid signaling mediated by GPER-1 in mixed culture and enriched neurons submitted or not to glucose/oxygen deprivation (OGD). We showed that cortical cell cultures express GPER-1 receptor and this are dispersed both in the cytosol and the nucleus. Our results showed that protection via estrogen signaling was dependent on the cellular composition. The lack of a signaling pathway GPER-1 before OGD increased cell death induced by OGD, suggesting that blocking of GPER-1 signaling pathway could be related to poor prognosis of ischemic lesions and G1 supplementation of culture media during deprivation and reperfusion attenuated these effects. In addition, our results point to the influence of glial cells as mediators of the neuroprotective role via non-nuclear estrogen signaling in this context of glucose/oxygen deprivation.