Nestin is a type VI intermediate filament protein that marks proliferative cells in the central and peripheral nervous system of vertebrates during development and adulthood. Nestin is not only expressed in progenitor cells of neuronal tissues but is also present in muscle, heart, lung, pancreas and skin follicle tissues. The goal of this thesis is to investigate and characterize the nestin protein in goldfish and relate nestin expression to neuroregeneration and brain plasticity events in the adult goldfish forebrain. Currently little is known about nestin function and regulation in vertebrates, especially in fish. In this study we used Rapid amplification of cDNA ends PCR (RACE-PCR) to isolate goldfish nestin mRNA. We uncovered several different mRNA transcripts. PCR analysis and sequencing further identified three different nestin transcripts of 4003, 2446, and 2126 nucleotides with a predicted protein length of 860, 274, and 344 amino acids respectively. We next applied a multiple-antigenic peptide (MAP) strategy to generate a polyclonal goldfish-specific nestin antibody against a 23 amino acid sequence located at the N-terminal end of goldfish nestin. Western blotting revealed the existence of three different nestin protein isoforms (nestin A, B and C); the first report of nestin isoforms in teleost species. Nestin expression and distribution in the goldfish brain is complex and revealed both individual and tissue-dependent variations. The most remarkable finding following principal component analysis of the western blot data was the uniqueness of the pituitary, hypothalamus and telencephalon. These tissues are proliferative in nature containing progenitor and proliferative cellular pools that are involved in important biological axes such as the motor and reproductive axis. Interestingly, all three tissues were able to change their proliferative cellular profile of nestin protein expression to alleviate the detrimental effects of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon administration. The toxin MPTP destroys dopamine neurons in the fish brain leading to motor deficits and reproductive difficulties. The incorporation of 5-bromo-2’-deoxyuriding (BrdU) into newly synthesized DNA revealed an upregulation of BrdU immunolabeling following MPTP administration in the area telencephali pars dorsalis (Vd) and along the ventricular surface area of the telencephalon suggesting the generation of new neurons in the adult central nervous system. This thesis reports novel nestin isoforms and illustrates regenerative events occurring in the goldfish telencephalon following a neurotoxic insult. This work provides a framework for future investigations of the differential roles and regulation of the nestins to better understand seasonal neuronal plasticity, neuronal regeneration and neuronal circuitry in teleost.

The third intracellular loop (IL3) and cytoplasmic tail (CT), which are the most divergent regions between human D1-class dopaminergic receptors (hD1R and hD5R), have been implicated in modulating their subtype-specific functional phenotypes. The importance of the IL3 for Guanine nucleotide-binding protein (G-protein) coupling and specificity has long been acknowledged in the G-protein-coupled receptor (GPCR) field. However, the exact role the central region of the IL3, notably the N- and C-terminal moieties, plays in GPCR receptor functionality remains unclear. Studies in our laboratory indicated that the IL3/N-terminal moiety of hD1-class receptors appears to be critical for facilitating agonist-independent and dependent activation of hD1R and hD5R. Furthermore, the IL3/C-terminal portion of hD1-class receptors constrains the receptor in the inactive state and reduces receptor affinity for agonists and G-protein coupling. I put forward the following hypothesis: 1. The functional properties of hD1-class receptors are regulated via a molecular micro-switch present within the IL3 central region modulating the functional properties of the receptor distinctly, 2. The functional differences between D1R and D5R require structural elements from both N- and C-terminal halves of the IL3 central region, and 3. The molecular interplay between the N- and C-terminal halves of the IL3 central region is dependent on the amino acid chain length and content. Herein, I have employed site-directed mutagenesis, and alanine replacement approaches to analyze comprehensively the structural determinants within the N- and C-terminal moieties of the IL3 central region that regulate ligand binding and G-protein coupling properties of hD1-class receptors. Moreover, my Ph.D. research aimed to pinpoint whether the IL3 length and/or structural motif(s) regulate ligand binding and activation properties of hD1R and hD5R. The results of my study highlight the importance of structural elements from both the proximal and distal segments of the IL3/central region of hD1-class receptors for the ligand binding and receptor activation status. Additionally, my results underline the significance of preserving the length of the IL3 regardless of the amino acid content. This study also shows the pivotal role played by a phenylalanine residue, F2646.27, in the signaling properties of hD1R. Notably, mutating F2646.27 leads to a mutant hD1R with characteristics resembling those of constitutively active mutant GPCRs. Unraveling the amino acid/amino acids constraining the receptor in the inactive state will perhaps provide an attractive target for drug design. Future work aims at developing drugs that particularly bind to the intracellular face of hD1R and improving selectivity towards hD1R may prove useful in limiting the side effects associated with the conventional therapy of brain disorders such as in the case of L-DOPA induced dyskinesia (LID) seen in individuals suffering from Parkinson’s disease.

Cariprazine is a novel dopamine (DA) and serotonin (5-HT) partial agonist with an in vitro receptor affinity profile that endows it with the potential to be used successfully in the treatment of both unipolar and bipolar disorders. The objective of this study was to determine whether in vitro findings with cariprazine lead to functional alterations of monoamine systems in the intact rat brain. In vivo electrophysiological recordings were carried out in male Sprague-Dawley rats under chloral hydrate anesthesia. Dorsal raphé nucleus (DRN), locus coeruleus (LC), and hippocampus cornu ammonis region 3 (CA3) pyramidal neurons were recorded and cariprazine was administered systemically by intravenous injection or locally through iontophoresis. In the DRN, cariprazine induced a complete inhibition of the firing of 5-HT neurons, which was fully reversed by the selective 5-HT1A antagonist WAY100.635. In the LC, the inhibitory effect of the preferential 5-HT2A agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) was reversed by cariprazine with an ED50 value of 67 µg/kg, i.v., and it did not block the inhibitory effect of the α2-adrenergic agonist clonidine. In the hippocampus, when cariprazine was administered by iontophoresis, it inhibited the firing of pyramidal neurons, but it did not dampen the suppressant effect of 5-HT. These results indicate that, in vivo, cariprazine acts as a 5-HT1A agonist in the DRN, as an antagonist on 5-HT2A receptors controlling the firing of NE neurons, and is a full agonist at 5-HT1A receptors located on pyramidal neurons of the hippocampus. The modulatory actions of cariprazine on the 5-HT and NE systems may contribute to its reported effectiveness in depressive episodes.

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Behavioural sensitization models are useful for understanding many disorders, including obsessive-compulsive disorder and drug addiction. Many of these models are produced by sensitization of dopamine neurotransmission, resulting in behaviours which include increased locomotor activity. Alterations to dopamine-mediated locomotor sensitization may be possible via activation of serotonergic neurotransmission, and there is evidence to suggest this may be through repeated activation of serotonin 1A receptors. The current study examines the development of locomotor sensitization in an animal model via repeated exposure of both a dopamine (D2R/D3R) and serotonin 1A (5-HT1A) agonist. To examine this, male Long-Evans rats were exposed to 10 injections of a combination of different doses of quinpirole and 8-OH-DPAT and tested in activity chambers for locomotor stimulation (measured by total distance travelled). Animals were then exposed to challenges of quinpirole, and 8-OH-DPAT and tested again for locomotor activity. Results showed that high doses of quinpirole or 8-OH-DPAT induced locomotor sensitization. However, when the two drugs were co-administered, 8-OH-DPAT displayed some initial disruption of quinpirole-induced sensitization. Animals sensitized to either quinpirole or 8-OH-DPAT did show higher locomotion when challenged with the drug to which they were sensitized. However, simultaneous quinpirole and 8-OH-DPAT sensitization seemed to prevent maximal responding when challenged with quinpirole. In all, our data suggests that sensitization to quinpirole and 8-OH-DPAT is occurring via separate neural mechanisms, with 5-HT1A agonism interfering with development of dopaminergic (D2R) sensitization. / Thesis / Master of Science (MSc)

Nicotine (NIC) abuse involves activation of midbrain dopamine (DA) neurons and NIC addiction involves neuroadaptive changes in the mesolimbic DA reward system. GABA neurons in the midbrain ventral tegmental area (VTA) express α4β2-containing nicotinic acetylcholine receptors (nAChRs), whose activation increases GABAergic input to DA neurons. However, this initial effect is decreased after chronic NIC treatment (as in the case of smokers) by inducing nAChR desensitization. Thus, GABA neuron inhibition results in increased DA release in limbic structures such as the nucleus accumbens. To support this hypothesis, we evaluated the effects of acute and chronic NIC on GAD-67 positive neurons in the VTA of GAD GFP mice using in vivo and in vitro electrophysiological methods. In in vivo studies in naïve mice, stimulation of the peduncopontine tegmental nucleus (PPT) activated VTA GABA neurons orthodromically and antidromically. Orthodromic activation of VTA GABA neuron spikes by PPT stimulation was blocked by the nAChR mecamylamine (1 mg/kg). Acute systemic NIC (0.15-0.5 mg/kg IV) had mixed overall effects on VTA GABA neuron firing rate, but in situ microelectrophoretic application of NIC produced a brisk and consistent enhancement (200-500 %) of VTA GABA neuron firing rate that showed no acute tolerance or sensitization with repeated, periodic current application. Local NIC activation was blocked by systemic administration of mecamylamine. Compared to 12 day chronic saline injections, chronic NIC injections (2 mg/kg IP/day) significantly increased VTA GABA neuron firing rate. In in vitro studies, compared to 12 day chronic saline injections, chronic NIC injections decreased DA neuron firing rate. In addition, chronic NIC increased DA neuron, but decreased GABA neuron GABA-mediated sIPSCs. These findings demonstrate that there is reciprocal innervation between the PPT and VTA and that cholinergic input from the PPT is excitatory on VTA GABA neurons. Moreover, VTA GABA neurons are excited by acute NIC and sensitize to chronic NIC, suggesting that α4β2 nAChR subunits on these neurons may play an important role in the adaptations to chronic NIC. Thus, quantitative molecular studies are ongoing to determine specific alterations in nAChRs on VTA GABA neurons to chronic NIC.

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