É conhecido que o sistema colinérgico, via receptor nicotínico a7, atua como atenuador do processo inflamatório. O lítio é utilizado no tratamento do transtorno afetivo bipolar e, em microdoses, foi benéfico para o tratamento da doença de Alzheimer. O objetivo desse estudo foi avaliar os efeitos do tratamento crônico com microdoses de lítio para a formação de mediadores inflamatórios em cérebros de camundongos jovens, após injeção intraperitoneal de LPS, e se os receptores a7 estavam envolvidos nesse processo. Após sete meses de tratamento, foi observado que o grupo lítio-LPS apresentou formação da memória espacial, diferentemente do grupo água-LPS. Oito dias após a injeção de LPS, os níveis de IL-6, TNF-a, IL-10 e pGSK-3b/GSK-3b estavam iguais em todos os grupos, evidenciando que o processo inflamatório já estava terminado. Além disso, não houve diferença com relação à densidade de CAT e a7. Não foi possível comprovar ou excluir o efeito anti-inflamatório das microdoses de lítio. Portanto, o desenho experimental deverá ser ajustado. / It is known that the cholinergic system, via a7 nicotinic receptors, acts as attenuator of the inflammatory process. Lithium is used in the treatment of bipolar disorder and microdosing was beneficial for the treatment of Alzheimer\'s disease. The aim of this study was to evaluate the effects of chronic treatment with lithium microdoses for the formation of inflammatory mediators in the brain of young mice after intraperitoneal injection of LPS, and if a7 receptors were involved in this process. After seven months of treatment, animals treated with lithium-LPS presented spatial memory, while water-LPS animals did not. Eight days after the induction of acute inflammation levels of IL-6, TNF-a, IL-10 and pGSK-3b/GSK-3b were the same in all groups, indicating that the inflammatory process had been completed. Still, there was no difference with respect to the density of CAT and a7. It was not possible to confirm or exclude the anti-inflammatory effect of microdoses of lithium. So, the experimental design will be adjusted.

A common organizational feature of any nervous system is the existence of groups of neurons that share a set of common traits but that can be further divided into individual neuron types and subtypes. Understanding the mechanistic basis of neuron type and subtype diversification processes will constitute a major step toward understanding brain development and evolution. In this dissertation, I have explored the mechanistic basis for the specification of motor neuron classes in the nematode C. elegans which serves as a paradigm for neuron diversification processes. Cholinergic motor neurons in the C. elegans ventral nerve cord share common traits, but are also comprised of many distinct classes, each characterized by unique patterns of effector gene expression (e.g. motor neuron class-specific ion channels, signaling molecules, and neurotransmitter receptors). Both the common as well as class-specific traits are directly activated by the terminal selector of cholinergic motor neuron identity, the EBF/COE-like transcription factor UNC-3. Via forward genetic screens to identify mutants that are defective in class specification, I have discovered that the diversification of UNC-3/EBF-dependent cholinergic motor neurons is controlled by distinct sets of phylogenetically conserved, motor neuron class-specific transcriptional repressors. One such repressor is in fact a novel gene previously uncharacterized in C. elegans or any nervous systems and is now named bnc-1. By molecularly dissecting the cis-regulatory region of effector genes, I found that the repressor proteins prevent UNC-3/EBF from activating class-specific effector genes in specific motor neuron subsets via discrete binding sites that are adjacent to those of UNC-3/EBF. And by using CRISPR/Cas9-mediated genome engineering to tag repressor proteins with inducible degrons, I demonstrate that these repressors share the important feature of being continuously required throughout the life of the animal to counteract, in a class-specific manner, the function of the UNC-3/EBF terminal selector that is active in all motor neuron classes. I propose that the strategy of antagonizing the activity of broadly acting terminal selectors of neuron identity in a neuron subtype-specific manner may constitute a general principle of neuron subtype diversification.

All complex organisms possess a nervous system which they use to monitor environmental and internal stimuli. In higher vertebrates, the nervous system is comprised of billions of cells which form highly plastic neural networks from their synapses. These large neural circuits modulate complex behaviors. The nematode roundworm Caenorhabditis elegans uses a small but highly-interconnected nervous system to carry out complex behaviors. The nervous system of C. elegans is a tractable model to determine the effects of changes on a nervous system at the systemic, cellular, genetic, and molecular levels. The C. elegans male’s nervous system detects environmental conditions, mating cues, attractants, repellents, and the location and composition of possible food sources and integrates these inputs to compute the decision of whether or not to mate. Mating behavior in the C. elegans male is regulated at a number of steps by cholinergic signaling from various sensory and sensory-motor neurons, but a comprehensive model of how cholinergic signaling controls this circuit has not yet been elucidated. Previous studies have thoroughly dissected the cellular structure, neural connectivity, and signaling pathways of the male’s peripheral circuits located in the genital regions of the animal’s tail. However, no studies have been conducted to determine what role the cephalic cholinergic neurons have in regulating mating behavior. I hypothesized that cephalic cholinergic neurons exert regulatory control over the male-specific mating circuit. I inserted the transmembrane light-activated ion pore Channelrhodopsin-2 fused to YFP and expressed from the Punc-17small promoter into these neurons and selectively stimulated them using high-intensity blue light. Stimulation induced mating-like behaviors in the male tail consistent with behaviors seen during copulation with a hermaphrodite. Using behavioral assays, I demonstrated that these behaviors were male-specific and only occurred after direct stimulation in the absence of a hermaphrodite. Incidence of mating-like behaviors increased significantly as the worm aged, and the mating circuit retained a memory of the stimulus, indicated by the latency between stimulation and onset of mating-like behaviors. Brief food deprivation, which normally downregulates excitability of the mating circuit via UNC-103 ERG-like K+ channels, caused an unexpected increase in the number of blue light-stimulated behaviors displayed. Pharmacological assays using acetylcholine (ACh) agonists showed that stimulation of the cephalic cholinergic neurons increased propensity for spicule protraction in the presence of an ACh agonist, and partially restored the decline in spicule protraction associated with temporary food deprivation. I sought to identify the cephalic cholinergic neuron or neurons responsible for regulating mating-like behavior in the tail circuits. I looked for a reduction in mating-like behaviors after stimulation after removal of a cephalic cholinergic neuron pair via laser micro-ablation. Two cholinergic and chemosensory neuron pairs in the inner labial sensilla (IL2L/R and IL2VL/R) appear to generate and/or relay the signal that induces mating-like behaviors in the tail. I hypothesize that these neurons sense environmental cues before the male contacts a mate, and modulate lasting motivational changes within the male mating circuit.

The nervous system coordinates a sequence of muscle movements to give rise to animal behaviors. In complex invertebrates or lab-studied vertebrates, due to the large number of cells in their nervous systems and the complexities of their behaviors, it is difficult to address how circuits process information to direct each motor output of the behavior. In this dissertation, I used the Caenorhabditis elegans male copulation behavior as a model to address how a compact circuit coordinates different behavioral programs. Insertion of a male copulatory organ into a suitable mate is a conserved and necessary behavioral step for most terrestrial mating. However, the detailed molecular and cellular mechanisms for this distinct social interaction have not been elucidated in any animal. During mating, the C. elegans male cloaca is positioned over the hermaphrodite’s vulva as he attempts to insert his copulatory spicules repetitively. Rhythmic spicule thrusts cease when insertion is sensed. Circuit components consisting of sensory/motor neurons and sex muscles for these steps have been previously identified, but it was unclear how their outputs are integrated to generate a coordinated behavior pattern. Here, I show that contraction of the male oblique muscles is required to sustain genital contact between the sexes. These muscles are innervated by the postcloacal sensilla (p.c.s.) sensory/motor neurons, which secret ACh to activate the levamisole-sensitive AChR and the ACR-16-containing ionotropic AChR on the oblique muscles. For spicules to rhythmically thrust during genital contact, activity of the oblique muscles and the gubernacular muscles is transmitted to the spicule protractor muscles instantaneously via gap junctions between these muscles and causes shallow protractor contractions. The rhythmic protractor contractions eventually switch to sustained contraction, as the SPC sensory-motor neurons integrate information of spicule position at the vulva with inputs from the hook and cloacal sensilla. The ERG-like K+ channel, UNC-103, which decreases the spicule circuit excitability, is likely to set a threshold requirement for integration of these inputs, so that sustained spicule muscle contraction is not stimulated by fewer inputs. In addition, I demonstrate that a cholinergic signaling pathway mediated by a muscarinic acetylcholine receptor, GAR-3, is used to enhance the ionotropic AChRs-mediated fast synaptic transmission in the copulation circuit. GAR-3 is expressed in multiple cells of the copulation circuit, but mainly in the cholinergic p.c.s. neurons and SPC neurons. Activation of GAR-3 is coupled to Gαq to trigger downstream signal transduction events that modulate neurotransmitter release from these neurons. Males with a loss-of-function allele of the gar-3 gene are defective in inserting their spicules into the hermaphrodite’s vulva efficiently. Since the p.c.s. neurons regulate the male’s contact with the hermaphrodite’s vulva, and the SPC neurons are required for spicule insertion during mating, GAR-3 probably facilitates male mating behavior via enhancing synaptic transmission from these neurons to their postsynaptic partners.

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 153-165).

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that are recognized as global environmental contaminants and a potential health risk. They have been shown to elicit neurodevelopmental toxicity through disruption of the cholinergic neurotransmitter system in rodent models, but the effects of environmentally relevant exposures in wildlife species are unknown. The objective of this study was to assess the effects of the commercial pentabrominated diphenyl ether mixture DE-71 on cholinergic parameters in captive mink (Mustela vison) following dietary exposure of adult females and in utero, lactational and dietary exposure of their offspring. Adult females were fed diets containing 0, 0.1, 0.5 or 2.5 μg/g DE-71 from four weeks prior to breeding through weaning of their kits at six weeks of age. A portion of the weaned kits were maintained on their respective diets through 27 weeks of age. Cholinergic neurochemical biomarkers, including muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (mAChR) binding, cholinesterase (ChE) activity and acetylcholine (ACh) concentration, were assayed in the cerebral cortex, and ChE activity measured in the plasma. Results indicated no significant effects of DE-71 on cholinergic parameters in the cerebral cortex, but a 3-fold increase in ChE activity in the plasma of adult females in the 2.5 μg/g DE-71 group. There were also no direct effects of DE-71 on mAChR or mAChR binding or ChE activity in the enzyme and receptor fractions from the whole brain of untreated mink following in vitro exposure to 0-23.6 nM DE-71. This study demonstrated that environmentally relevant exposures to DE-71 did not affect key parameters of the cholinergic neurotransmitter system in the brain of captive mink.

The pedunculopontine tegmental nucleus (PPTg) has been proposed to be critical in drug and food reward. It is a major source of cholinergic inputs to the substantia nigra and ventral tegmental area, areas important in reward, and is believed to modulate activity of dopamine neurons through a cholinergic mechanism. The firing rate of cholinergic PPTg cells is regulated by muscarinic autoreceptors that can be blocked to increase cell firing. If the PPTg modulates dopamine neurons involved in reward, then a muscarinic antagonist microinjected into the PPTg should be rewarding. To test this hypothesis, bilateral microinjections of scopolamine methyl bromide (5 $ mu$g or 20 $ mu$g) or 0.9% saline were used as reward treatments in the conditioned place preference test. On the test day, rats from both doses of drug avoided the drug-paired chamber, which suggests that cholinergic PPTg cells are not involved in reward.

Activation of muscarinic acetylcholine receptors (mAChRs) has pronounced effects on GABAergic interneurons, including depolarization of their resting membrane potential, and increasing their action potential and vesicular release frequency. Moreover, postsynaptic mAChR activation in hippocampal pyramidal neurons reduces the expression of the K+-Cl- cotransporter (KCC2). However, whether mAChR activation modulates the expression of disinhibition-mediated synaptic plasticity has not been examined. I induced inhibitory long-term potentiation (LTP) by applying coincident pre/postsynaptic stimulation in the hippocampus. This plasticity was characterized by an increase in the postsynaptic potential (PSP) amplitude and a depolarization in the inhibitory postsynaptic potential (IPSP) reversal potential; characteristics of disinhibition-mediated LTP (dmLTP). Activation of mAChRs during this plasticity induction protocol prevented the expression of dmLTP via a presynaptic downregulation of transmitter release. This was concluded from evidence that the PSP amplitude and IPSP reversal potential were unaltered, and paired-pulse depression occurred following plasticity induction in the presence of mAChR activation.

In order to assess cognitive function in multiple sclerosis (MS) and the effect of cholinergic modulation, experiments were conducted using functional magnetic resonance imaging (fMRI) to assess the brain activation during cognitive tasks. A study comparing the processing of verbal working memory with an N-back task found that patients showed smaller increase in activation than healthy controls with greater task difficulty, suggesting a reduced functional reserve. Controls and patients showed differences of correlations between brain regions activated. Interactions between prefrontal regions may provide an adaptive mechanism that could limit clinical expression of the disease distinct from recruitment of novel processing regions. The effect of Rivastigmine on the cognitive processing in MS patients was tested in a longitudinal study, involving serial fMRI scans. Changes in the brain activation patterns were demonstrated with drug administration, without any changes in behavioural measures. Rivastigmine may act to increase the functioning of the normal neural network reducing the need for previously recruited compensatory mechanisms in MS patients. A study on healthy subjects examined the effect of cholinergic inhibition on cognitive processing and brain activation. Changes in functional activation due to Hyoscine during verbal working memory were found analogous to that in MS patients without any changes in behavioural measures. Processes that potentially impair brain cognitive function may recruit similar compensatory functional adaptive mechanisms. Studies on rats and MS patients explored the effect of Rivastigmine on the relationship of the BOLD fMRI signal with the underlying neural activity. Rivastigmine may be influencing the cortical excitability after direct cortical stimulation but showed only a small effect on the BOLD signal under more physiological neural activity. The neural activity in response to visual stimulation is slightly increased with Rivastigmine in MS patients, a change not detected with functional imaging. These studies suggest that changes in BOLD signal do represent sufficiently large changes of underlying neural activity in the presence of Rivastigmine. The relationship of damage in MS to measures of connectivity was studied using diffusion tensor imaging (DTI). Correlation was found between measures of connectivity and callosal size, a measure of fibre loss. The distribution of lesions was spatially correlated with changes in connectivity due to MS. Thus DTI could be utilized to explore the connectivity changes associated with MS, and the relationship with changes in functional activation.

Activation of muscarinic acetylcholine receptors (mAChRs) has pronounced effects on GABAergic interneurons, including depolarization of their resting membrane potential, and increasing their action potential and vesicular release frequency. Moreover, postsynaptic mAChR activation in hippocampal pyramidal neurons reduces the expression of the K+-Cl- cotransporter (KCC2). However, whether mAChR activation modulates the expression of disinhibition-mediated synaptic plasticity has not been examined. I induced inhibitory long-term potentiation (LTP) by applying coincident pre/postsynaptic stimulation in the hippocampus. This plasticity was characterized by an increase in the postsynaptic potential (PSP) amplitude and a depolarization in the inhibitory postsynaptic potential (IPSP) reversal potential; characteristics of disinhibition-mediated LTP (dmLTP). Activation of mAChRs during this plasticity induction protocol prevented the expression of dmLTP via a presynaptic downregulation of transmitter release. This was concluded from evidence that the PSP amplitude and IPSP reversal potential were unaltered, and paired-pulse depression occurred following plasticity induction in the presence of mAChR activation.