The first sensory synapse is the site where sensory afferent fibers make synaptic connections with second order neurons. Somatic and craniofacial afferents terminate in spinal cord dorsal horn (SDH) and caudal spinal trigeminal nucleus (CSTN). Neurotransmitter release from first order nerve terminals regulates ascending sensory transmission. Several lines of evidence indicate that plasticity in the spinal cord dorsal horn underlies secondary hyperalgesia. The sensory receptors, Transient Receptor Potential (TRP) channels, are expressed not only at peripheral terminals, but also at the central terminals of sensory neurons. While the role of these channels at the periphery is detecting environmental stimuli, their function at central terminals is not fully understood. Furthermore, TRP channel expression has been shown in CNS nuclei like hippocampus that are not tightly linked to somatosensation. In this study, I first determined the functionality of TRP channels at the first sensory synapse and hippocampus using pharmacological activators. I then determined if putatively endogenous TRP channel activators modulate synaptic transmission at the first sensory synapse. Lastly, I determined if recordings that respond to capsaicin demonstrate synaptic plasticity in either hippocampus or spinal cord, in an attempt to attribute synaptic plasticity mechanisms to TRPV1 activity at glutamatergic terminals. I have used slice patch-clamp technique to record miniature, spontaneous and evoked currents in lamina II neurons of spinal cord dorsal horn, CSTN and hippocampus. In lamina II neurons of SDH and CSTN, capsaicin, a TRPV1 agonist, robustly increased the frequency of mEPSCs and sEPSCs in a dose dependant manner. Although capsaicin increased m/sEPSC frequency, eEPSC amplitude, which reflects synchronous action potential propagation at glutamatergic terminals, was markedly depressed by capsaicin. Our studies indicate capsaicin inhibits action potential dependant transmission at central terminals. Resiniferatoxin (RTX) is a TRPV1 agonist that displays higher potency (>100 fold) compared to capsaicin, and deactivation with this agonist is minimal. RTX also depressed eEPSC amplitude in lamina II neurons of SDH and CSTN; unexpectedly, RTX increased m/sEPSC frequency to lesser extent compared to capsaicin. The TRPA1 agonist, N-methyl maleimide (NMM), increased s/mEPSC frequency in lamina II neurons; however, NMM did not depress eEPSC amplitude like capsaicin and RTX. It is possible that inhibition of nerve terminal firing is a unique property of TRPV1 agonists compared to other noxious chemicals. To justify a physiological relevance for nociceptive TRP channel expression at the first sensory synapse, I studied the effect of endogenous TRP channel agonists on synaptic transmission at the first sensory synapse. Anandamide (AEA) is an agonist of CB1/CB2 and TRPV1 receptors; it is less potent at TRPV1 receptors than capsaicin. AEA increased sEPSC frequency in 70% of neurons, whereas the remainder of neurons showed a decrease in sEPSC frequency. Unlike capsaicin and RTX, anandamide did not dramatically depress eEPSC amplitude. Methyl glyoxal (MG) is a putative TRPA1 agonist produced during conditions of hyperglycemia. MG increased the frequency of sEPSCs in SDH lamina II neurons. I next used high frequency synaptic stimulation (HFS-100 Hz, 1s) to model synaptic activity during pain transmission. HFS induced a modest increase in sEPSC frequency and minimally changed eEPSC amplitude; patches that showed HFS modulation also responded to capsaicin. In studying the role of TRP channels in modulating synaptic transmission at central synapses, I finally performed experiments in hippocampus with 2 objectives; 1) to determine extent of capsaicin responsiveness as an indicator of TRPV1 functionality, and 2) to evaluate synaptic plasticity in response to HFS. Capsaicin effect on sEPSC frequency in CA1 and CA3 neurons was minimal in comparison to its effect in dorsal horn neurons. HFS at schaffer collateral region caused LTP in CA1 neurons that was more pronounced than for spinal cord. In conclusion, TRP channels are expressed at central terminals of nociceptors where they modulate glutamatergic transmission. Studying their role at the first sensory synapse enhances our understanding of nociceptive transmission, and this study suggests this receptor for a target for intervening in pathological pain transmission at the level of spinal cord.

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Molecular and genetic analysis of synaptic signaling in Drosophila has yielded many insights into nervous system development, properties of synaptic transmission, and how long-lasting changes in neurons occur. Synaptic signaling components required for synaptic transmission and pathways leading to nervous system plasticity are typically conserved from insects to humans. The role of proteins and genes in synaptic function in flies can be analyzed from the level of a single synapse to complex behaviors in the whole organism. Because of a fully sequenced genome and the ease of mutagenesis in flies, genetic screens have been useful in identifying novel regulators of synaptic transmission and long-term memory.In flies, conditional mutations affecting synaptic transmission at nerve terminals often lead to temperature sensitive paralysis. In a screen for mutations that interact with Drosophila shibirets mutants, the stoned gene was identified as a regulator of synaptic vesicle cycling. Stoned encodes two neuronally expressed proteins, stonedA and B, which are required for synaptic vesicle recycling and normal synaptic transmission. However, the exact functions of the two stoned proteins are not fully understood. We investigate distinct roles of the stoned proteins here and show that stoned has a novel role in synaptic growth.Memory in flies can be divided into genetically distinct phases based on the requirement for protein synthesis and activation of the transcription factor CREB. Novel regulators of long-term olfactory avoidance memory were isolated in a mutant screen in flies. Mutants in the Drosophila gene lk6, homologous to the translational regulator MNK, have defects in long-term olfactory avoidance memory. We find that lk6 is highly expressed in the fly nervous system, and is activated by and functions downstream of Ras/ERK signaling in fly neurons. Insights provided here from Drosophila add to the evidence that MNK may be the link between ERK signaling and the regulation of translation in long-term plasticity.Ultimately, understanding synaptic function has therapeutic potential to aid in alleviation of nervous system dysfunction. Insight into the molecular pathways underlying plasticity and long-term memory gained from studies in flies, mollusks, and rodents has been pivotal in the development of potential drugs to aid in memory deficits in humans.

The crayfish, Procambarus clarkii, has a multitude of ideal sites in which synaptic transmission may be studied. Its opener muscle, being innervated by a single excitatory neuron is a good model for studying the structure/function of neuromuscular junctions since the preparation is identifiable from animal to animal and the nerve terminals are visible using a vital dye. This allows ease in finding a suitable site to record from in each preparation and offers the ability to relocate it anatomically. Marking a recorded site and rebuilding it through electron microscopy gives good detail of synaptic struture for assesment.In the first of these studies, low output sites known as stems (which lie between varicosities) were used to reduce n (number of release sites) in order to minimize synaptic complexity so individual quantal events could be analyzed by their unique parameters (area, peak, tau, rise time and latency). This was in attempt to uncover specific quantal signatures that could be traced back to the structure of the area recorded. It was found that even at stem regions synaptic structure is still complex having multiple synapses each of which could harbor a number of AZs. This gives insight as to how quantal analysis should be treated. Even low output synapses n must be treated at the AZ level.Synaptic depression was studied at the crayfish extensor muscle. By depressing the phasic neuron and recording from the muscle it appears thatdepression is a presynaptic phenomenon. The use of 5-HT gave insight to vesicular dynamics within the nerve terminal, by delaying depression and increasing maximum EPSP amplitude. TEM of phasic nerve terminals reveals no change in numbers of dock or RRP vesicles. Short term facilitation and vesicular dynamics were studied with the use of 5-HT and a neurotoxin TBOA, which blocks the glutamate transporter. In this study I showed differential mechanisms that control RRP and RP vesicles. By blocking glutamate reuptake, the RRP is depleted as shown by reduced EPSPs, but recovered with 5-HT application. The understanding of vesicle dynamics in any system has relevance for all chemical synapses.

abstract: Angelman syndrome (AS) is a neurodevelopmental disorder characterized by developmental delays, intellectual disabilities, impaired language and speech, and movement defects. Most AS cases are caused by dysfunction of a maternally-expressed E3 ubiquitin ligase (UBE3A, also known as E6 associated protein, E6-AP) in neurons. Currently, the mechanism on how loss-of-function of the enzyme influences the nervous system development remains unknown. We hypothesize that impaired metabolism of proteins, most likely those related to E6-AP substrates, may alter the developmental trajectory of neuronal structures including dendrites, spines and synaptic proteins, which leads to disrupted activity/experience-dependent synaptic plasticity and maturation. To test this hypothesis, we conducted a detailed investigation on neuronal morphology and electrophysiological properties in the prefrontal cortex (PFC) layer 5 (L5) corticostriatal pyramidal neurons (target neurons). We found smaller soma size in the maternal Ube3a deficient mice (m-/p+; 'AS' mice) at postnatal 17-19 (P17-19), P28-35 and older than 70 days (>P70), and decreased basal dendritic processes at P28-35. Surprisingly, both excitatory and inhibitory miniature postsynaptic currents (mEPSCs and mIPSCs) decreased on these neurons. These neurons also exhibited abnormalities in the local neural circuits, short-term synaptic plasticity and AMPA/NMDA ratio: the excitatory inputs from L2/3 and L5A, and inhibitory inputs from L5 significantly reduced in AS mice from P17-19; Both the release probability (Pr) and readily-releasable vesicle (RRV) pool replenishment of presynaptic neurons of the target neurons were disrupted at P17-19 and P28-35, and the change of RRV pool replenishment maintained through adulthood (>P70). The AMPA/NMDA ratio showed abnormality in the L5 corticostriatal neurons of PFC in AS mice older than P28-35, during which it decreased significantly compared to that of age-matched WT littermates. Western Blot analysis revealed that the expression level of a key regulator of the cytoskeleton system, Rho family small GTPase cell division control protein 42 homolog (cdc42), reduced significantly in the PFC of AS mice at P28-35.These impairments of synaptic transmission and short-term synaptic plasticity may account for the impaired neuronal morphology and synaptic deficits observed in the PFC target neurons, and contribute to the phenotypes in AS model mice. The present work reveals for the first time that the E6-AP deficiency influences brain function in both brain region-specific and age-dependent ways, demonstrates the functional impairment at the neural circuit level, and reveals that the presynaptic mechanisms are disrupted in AS model. These novel findings shed light on our understanding of the AS pathogenesis and inform potential novel therapeutic explorations. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2017

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