Many recent studies on the segmental motor control system have employed spike-triggered-averaging (STA) and other forms of cross-correlation to either attribute CNS, reflex, or direct motor effects to the impulses of a single (reference) neuronal spike train or to explore conditions under which pairs of neural units show temporal correlations in their discharge. Our experience with these techniques suggested the need for a control procedure that tests for synchrony between the reference and other spike trains such as to: (1) either preclude that the observed effects are due to spike trains other than or in addition to the reference train; or (2) give insight into the conditions leading to correlated discharge between two units. A motor unit synchronization test based on analysis of EMG waveforms has already been described. We have modified this test for the detection of synchrony between either afferent or efferent signals by analysis of averaged muscle nerve signals rather than EMG waveforms. Our procedure involves use of a multi-unit muscle nerve recording that serves as the input to a signal averager triggered by a spike train from either: (1) a motor unit's EMG; (2) a dorsal root filament or ganglion cell; or (3) a ramdom trigger source. With appropriate delay of the muscle nerve signal input, the non-rectified average of the trigger signal's waveform is compared to the rectified average which contains this waveform together with contributions of all other active unitary events. Additionally, the rectified average is compared to a "randomly" triggered average of the same input signal. On the basis of these recordings, it can be determined, within certain boundary conditions, whether or not any other unitary events are in synchrony with the reference event. Such synchronization is expressed quantitatively in the form of a synchronization index (SI). We evaluated the efficacy of the SI by electronic simulation procedures and by comparing its use to that of a cross-correlation procedure that tests for synchrony on the basis of crosscorrelograms computed between two simultaneously recorded spindle afferent spike trains during brief stretch of a passive muscle at progressively increasing amplitudes (5 - 100um). These experiments revealed that the SI is a sensitive test of afferent synchrony in the passive muscle provided the spike trains of interest have a signal-to-noise (S/N) ratio > 0.2 in the muscle nerve recording and that it is recognized that the detectable degree of synchronization of a non-reference event is a function of its S/N ratio. For tests on the active muscle, the force levels must remain low. Otherwise increased neuronal activity in the muscle nerve recording decreases the S/N ratio of individual spike trains. Thus, despite restrictive (but predictable) boundary conditions, the SI test can contribute importantly to select conclusions drawn from cross-correlation studies.

The moth Manduca sexta has been a common model for the study of the insect olfactory systems. The neuronal architecture in the antennal lobes (ALs) of insects and in the olfactory lobes of vertebrates is similar in structure and development. In Manduca, as in other olfactory systems, sensory receptor neurons send axons into the AL where they form synapses with local interneurons (LNs) and projection neurons (PNs) within the structural units of glomeruli. Here, I present the morphological development of one type of interneuron, the uniglomerular projection neuron (uPN), in normal AL development and in AL development in the absence of olfactory receptor neurons (ORNs). Using fluorescent-dye labeling of uPNs and confocal microscopy, my results show that in the absence of ORNs, uPN dendritic arborization is uncharacteristic of that in normally developing ALs, reinforcing the concept that afferent input guides the development of architecture in sensory areas of the brain.

The establishment of neuronal polarity constitutes a central phase in neuronal development and synaptogenesis. In my thesis, I study factors that regulate the development of neuronal polarity and its relationship with neurotransmitter receptor expression. For my experiments, I have investigated the development of sensory neurons from neonatal rat nodose ganglia in culture. Sensory neurons have a pseudo-unipolar morphology, do not extend dendrites, and are devoid of synaptic connections on their somata. However, nodose neurons form synapses de novo in cultures, and I show that the neurons have retained the ability to extend dendrites. Extrinsic factors control dendrite extension by these neurons: the ganglionic satellite cells inhibit the growth of dendrites and induce the neurons to develop a unipolar morphology. In the absence of satellite cells, nodose neurons establish a new multipolar morphology and, in response to nerve growth factor (NGF), extend several dendrites. However, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) do not induce the neurons to extend dendrites, but promote the expression of properties typical of nodose neurons in vivo. / As nodose neurons acquire a new dendrite-axonal polarity in the presence of NGF, they increase the density of functional neuronal nicotinic acetylcholine receptors (nAChRs) on their somato-dendritic domains. To learn more about the relationship between dendrites extension and nAChR gene expression, I have examined the changes in transcript levels of nAChR subunits in neonatal rat sympathetic neurons developing in culture. I show that the developmental pattern of nAChR subunit expression in the cultured neurons follows closely that of sympathetic neurons developing in vivo, with the exception of one specific subunit $ alpha sb7$. I show that the increase in $ alpha sb3$ mRNA levels correlates well with an increase in the density of functional nAChRs on the neurons. In addition, my results suggest that these increases are regulated by mechanisms intrinsic to neonatal sympathetic neurons. On the other hand, the changes in $ alpha sb7$ gene expression, which correlate with changes in $ alpha$-bungarotoxin binding, are activity-dependent and regulated by a calcium/calmodulin-dependent protein kinase pathway. The results presented in this thesis provide insights on how neurons are influenced in their extension of dendrites and how this extension affects neurotransmitter receptor expression.

No description available.

No description available.

Behaviors are a product of underlying neural circuits, yet there is a paucity of mechanistic information about how nervous systems contribute to the repeated evolution of similar behaviors. Theoretical studies have predicted that the same behavioral output can be generated by neural circuits with different properties. Here, we test the theory in biological circuits by comparing the central pattern generator (CPG) circuits underlying swimming behaviors in nudibranchs (Mollusca, Gastropoda, Euthyneura, Nudipleura). In comparative studies of neural circuits, neurotransmitter content can serve as landmarks or molecular markers for neuron types. Here, we created a comprehensive map of GABA-immunoreactive neurons in six Nudipleura species. None of the known swim CPG neurons were GABA-ir, but they were located next to identifiable GABA-ir neurons/clusters. Despite strong conservation of the GABA-ergic system, there were differences, particularly in the buccal ganglia, which may represent adaptive changes. We applied our knowledge of neurotransmitter distribution along with morphological traits to identify the neuron type Si1 in Flabellina, a species that swims via whole body left-right (LR) flexions and in Tritonia, a dorsal-ventral (DV) swimming species. Si1 is a CPG member of the LR species Melibe, whereas its homologue in the LR species Dendronotus is not. In Flabellina, Si1 was part of the LR CPG and despite having similar synaptic connections as Flabellina and Melibe, Si1 in Tritonia was not part of its DV swim CPG. Side by side circuit comparison of Flabellina, Melibe and Dendronotus revealed different combinations of circuit architecture and modulation resulting in different circuit configurations for LR swimming. This includes differences in the role and activity pattern of Si1, sensitivity to curare and the effect of homologues of C2, a DV CPG neuron, on the LR motor pattern. These results collectively reveal three different circuit variations for generating the same behavior. It suggests that the neural substrate from which behaviors arise is phylogenetically constrained. While this neural substrate can be configured in multiple different ways to generate the same outcome, the possibilities are finite and, as seen here, similar structural and functional neural motifs are used in the evolution of these circuits.

The Distal-less homeobox (Dlx) genes encode homeodomain transcription factors found in all animals of the phylum Chordata. These genes are involved in early vertebrate development of limbs, sensory organs, branchial arches and the forebrain (telencephalon and diencephalon). The mouse and human genomes each have six Dlx genes organized into convergently transcribed bigene clusters (Dlx1/2, Dlx3/4 and Dlx5/6). In the forebrain, Dlx1/2 and Dlx5/6 genes play essential roles in GABAergic neuron proliferation, migration and survival. Each bigene cluster includes a short intergenic region (~3.5-16kb) harboring cis-regulatory elements (CREs) that control expression of the Dlx genes. The Dlx1/2 intergenic region harbors the I12b/I12a CREs, while Dlx5/6 includes I56i/I56ii. In determining the regulatory roles of the CREs on Dlx activity and forebrain development, I have characterized the phenotypic changes that occur in mice that have an I56i enhancer deletion. I have also characterized mice with double deletions of I56i and I12b as well as mice that harbored an I12b deletion and have a SNP in the I56i enhancer (vI56i). Mutant mice with a single targeted deletion of I56i are viable, fertile and do not show obvious developmental defects. These mice have significant decreases in Dlx5/6, Gad1/Gad2 and Evf-2 expression in the forebrain and have defects related to GABAergic neuron development. The ΔI56i mutants demonstrate a behavioral phenotype related to anxiety and learning deficits. Mice that lack the I12b enhancer and have the vI56i do not show morphological abnormalities but have severely disrupted Dlx expression. When mice are homozygous for the I56i and I12b enhancer deletion, they do not survive past post natal day 5 and exhibit a dwarfed body size. These mice look weak and seem to have limited motor ability. In characterizing mice with targeted deletions of highly conserved Dlx enhancers, we will have a better understanding of forebrain development.

This abstract sent to supervisor 5/4/12 Tyrosine hydroxylase is the enzyme which controls the ratelimiting step in the synthesis of noradrenaline. In order to discover whether the activity of tyrosine hydroxylase might serve as an indicator of noradrenergic function in the brain, two preparations for the direct measurement of TH activity in rat brain regions by a tritium-release method were employed: synaptosomal suspensions prepared from pinchedoff nerve terminals, and partially solubilised enzyme preparations prepared from frozen homogenates or synaptosomal suspensions and assayed at saturating concentrations of cofactor and tyrosine. There was evidence of an increase in tyrosine hydroxylase activity in hippocampal synaptosomes of rats killed immediately after a mild electrical footshock. Activation of synaptosomal enzyme activity was also found after single doses of clonidine and parachloroamphetamine, and after repeated handling; and single doses of morphine and of yohimbine appeared to lower tyrosine hydroxylase activity. Repeated administration of drugs such as clonidine, desipramine and 2-deoxyglucose, however, did not affect tyrosine hydroxylation rate. A preliminary finding , suggesting differences in synaptosomal tyrosine hydroxylase activity related to experience with different reinforcement schedules (continuous reward vs. partial reward) in a runway experiment, was not substantiated in later experiments; nor was there any difference between the synaptosomal tyrosine hydroxylase activity of naive controls and rats given repeated daily shocks for a week. The saturated TH assay performed on solubilised enzyme was, as predicted, unresponsive to the short term stimulation effects detected with the synaptosomal assay. However, other changes, such as a reduced maximal hydroxylation rate after repeated desipramine administration, and an increased rate several weeks after a course of electrical stimulation of the septal area, were established with the saturated assay. Although the changes in stimulated rats were associated with increased behavioural tolerance to stress, e.g. resistance to extinction of a running response in a runway, other experiments in which the behavioural stress-tolerance was induced by behavioural methods alone showed no accompanying changes in TH activity. Measures of synaptosomal and saturated soluble TH activity appear to constitute independent indicators of noradrenergic function. It seems that synaptosomal tyrosine hydroxylase activity is not, as anticipated, controlled by the firing rate of locus coeruleus neurones; but it may be subject to local regulation in noradrenergic terminals. The results are discussed in the context of theoretical aspects of the regulation of noradrenaline synthesis in the brain, and the mechanisms underlying physiological responses to stress and behavioural tolerance to stress.

The responses and morphology of nine sound-sensitive interneurons are described in the cricket Teleogryllus oceanicus. Each of the neurons receives direction-specific input in the prothoracic ganglion, and each projects at least one interganglionic axon. Five of the neurons respond best to high frequencies ($>$10 kHz); four are most sensitive to low frequencies (3-10 kHz). Responsiveness to model calling songs was examined in addition to testing sensitivity to wind and light. Anatomical observations reveal that seven of the neurons receive auditory input via polysynaptic pathways, and that at least five of the neurons have morphology consistent with them providing input to mesothoracic motor neurons which are involved in behavioral responses to sound. Correlations between structure, topographic organization, and spectral sensitivity were found. The structure of one previously identified, auditory neuron was examined and found to change during late post-embryonic life. This represents a novel developmental pattern.

Dopamine (DA) is a key catecholamine neurotransmitter involved in motor control, cognition, and neuroendocrine regulation. Reduced DA transmission is associated with Parkinson’s disease, depression, and anhedonia. An overexpression of the dopamine transporter in mice (DAT-tg) results in a 40% reduction in extracellular DA, and can be classified as a genetic model of hypodopaminergia. Reserpine treatment depletes extracellular DA, and is a pharmacological model of hypodopaminergia. The aim of this study was to determine morphological and proteomic changes to medium spiny neurons (MSNs), which receive dopaminergic input, as a consequence of reduced DA transmission. To achieve this, MSNs were fluorescently labelled using a diolistics method and immunofluorescence. There were no observable changes to morphology or proteomic profile of MSNs in DAT-tg animals. Reserpine treatment resulted in reduced spine density in MSNs. DAT-tg animals may present a level of DA depletion that is below the threshold to induce morphological changes to MSNs.