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.

No description available.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "December 2002." Typescript. Vita. Includes bibliographical references (leaves 121-148). Also issued on the Internet.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 100-123).

Transient receptor potential (TRP) channels play an important role in sensory and nonsensory functions. TRPVanilloid 1 and TRPVanilloid 4 are proposed to be involved in inflammation-induced pain. TRPV1 is extensively studied and it is specifically involved in inflammatory thermal hypersensitivity. Mechanical hypersensitivity is one of the significant components of nociception. Several receptors have been proposed to underlie mechanosensation. The molecular entities responsible for mechanosensation are not fully understood. In this study, I have characterized the properties of TRPV4, a putative mechanosensitive ion channel expressed in dorsal root ganglion (DRG) neurons and nonsensory tissues. First, I have investigated the expression and function of TRPV4 and TRPV1 in the DRG neuronal cell bodies as well as their central terminals and determined the modulation by protein kinase C (PKC). Both TRPV4 and TRPV1 are expressed in DRG and laminae I and II of the spinal dorsal horn (DH). Ca2+ fluorescence imaging and whole-cell patch-clamp experiments showed that both capsaicin-induced TRPV1 response and 4alpha-phorbol 12, 13-didecanoate (4alpha-PDD)-induced TRPV4 response were observed in a proportion of the same DRG neurons, suggesting their co-expression. Incubation of DRG neurons with phorbol 12, 13-dibutyrate (PDBu), a PKC activator, resulted in a significantly greater potentiation of TRPV4 currents than TRPV1 currents. In HEK cells heterologously expressing TRPV4, PDBu potentiated TRPV4-mediated single-channel current activity. In patch-clamped DH neurons, the application of 4alpha-PDD at the first sensory synapse increased the frequency but not the amplitude of the miniature excitatory postsynaptic currents (mEPSCs), suggesting a presynaptic locus of action. 4alpha-PDD-induced increase in the frequency of mEPSC was further facilitated by PDBu. These results suggest that TRPV4 in the central terminals modulates synaptic transmission and is regulated by PKC. Second, I have studied the mechanosensitivity of TRPV4 in cell-attached patches by applying direct mechanical force via the patch pipette. In TRPV4 expressing HEK cells, the application of negative pressure evoked single-channel current activity in a reversible manner and the channel activity was enhanced after incubation with PDBu. TRPV4 has been shown to be activated by hypotonicity. Here I show that negative pressure exaggerated hypotonicity-induced single-channel current activity. However, in similar experimental conditions, cells expressing TRPV1 did not respond to mechanical force. TRP channels are also expressed in non-sensory regions and the role of these channels is not fully understood. Both TRPV4 and TRPV1 are expressed in the hippocampus. Using whole-cell patch-clamp techniques, I have found that 4alpha-PDD increased the frequency, but not the amplitude of mEPSCs in cultured hippocampal neurons, suggesting a presynaptic site of action. Interestingly, the application of capsaicin had no effect on synaptic transmission in hippocampal neuronal cultures. Finally, I have investigated the expression and function of TRP channels in diabetes because TRP channels have been shown to be involved in peripheral neuropathy as well as vascular complications in diabetes. ROS production plays a critical role in the progress of diabetes. I propose that lower levels of ROS up-regulate the expression TRP channels in the early stages of diabetes, leading to hyperalgesia, and higher levels of ROS or chronic exposure to ROS down-regulate TRP channels in the late stages of diabetes, resulting in hypoalgesia. I have found that the expression of TRPV1 and phospho p38 (p-p38) MAPK was increased in DRG of streptozotocin (STZ)-injected diabetic and non-diabetic hyperalgesic mice. An increase in TRPV1 and p-p38 MAPK levels was induced by STZ or H2O2 treatment in stably TRPV1 expressing HEK cells, suggesting the involvement of STZ-ROS-p38MAPK pathway. TRPV4 has been reported to be involved in vasodilatation by shear stress in blood vessels. Here, I have demonstrated that TRPV4 is expressed in lymphatic endothelial cells (LECs). Treatment with low concentration of H2O2 enhanced the expression of TRPV4 at mRNA and protein levels in LECs, suggesting that mild levels of ROS up-regulate TRPV4 expression. In diabetes, beta cell dysfunction is responsible for decreased insulin release. TRPV4 is expressed in RINm5F (beta cell line), islets and pancreas. It has been shown that hypotonicity induced insulin release in beta cell lines, which was mediated by activation of stretch-activated channels, raising the possibility of the involvement of TRPV4, a mechanosensitive channel. Therefore, I have studied the functional role of TRPV4 in beta cells. Incubation with 4alpha-PDD enhanced insulin release in RINm5F cells, suggesting TRPV4 regulates insulin secretion from pancreatic beta cells. Since TRPV4 expression levels are decreased in diabetes, insulin secretion from beta cells may be impaired. In summary, TRPV1, a thermosensitive channel, and TRPV4, a mechanosensitive channel, contribute to thermal and mechanical hyperalgesia, respectively in the early stage of DPN through their up-regulation by ROS-p38 MAPK and insulin/IGF-1 pathways. Due to the mechanical sensitivity of TRPV4 channel, the up-regulation in the early stage and down-regulation in the late stage may be involved in the development of vascular complications and regulation of insulin release in diabetes.

No description available.

Die Funktion unseres Zentralnervensystems beruht auf der zeitlich präzisen Übertragung elektrischer Signale zwischen Neuronen. Diese synaptische Übertragung findet in weniger als einer tausendstel Sekunde statt. Eine schnelle und hochfrequente Signalübertragung erweitert die Kodierungskapazität und beschleunigt die Verarbeitung von Informationen. Obwohl viele der an synaptischer Übertragung beteiligten Prozesse und Proteine bekannt sind, ist das Verständnis der Mechanismen, die für eine schnelle und hochfrequente Signalübertragung verantwortlich sind, bisher unvollständig. Um die Mechanismen hochfrequenter synaptischer Übertragung zu untersuchen, wurden in dieser Arbeit prä- und postsynaptische Patch-Clamp Ableitungen an der zerebellären Moosfaser-Körnerzell-Synapse in akuten Hirnschnitten der Maus eingesetzt. Es zeigte sich, dass diese Synapse präsynaptische Aktionspotenziale mit einer Frequenz über einem Kilohertz feuern kann und dass Informationen in diesem Frequenzbereich an die postsynaptische Zelle übertragen werden können. Hierbei vermitteln besonders schnelle Natrium- und Kalium-Kanäle eine extrem kurze Dauer der Aktionspotenziale, die dennoch metabolisch relativ effizient sind. Schnelle Kalzium-Kanäle und eine schwache präsynaptische Kalzium-Pufferung ermöglichen eine synchrone Vesikelfreisetzung mit hohen Frequenzen. Zusätzlich greift die Präsynapse auf einen großen Vorrat an freisetzbaren Vesikeln zurück, dessen Auffüllung besonders schnell stattfindet. Aufgrund der hochfrequenten synaptischen Übertragung ist die Moosfaser- Körnerzell-Synapse ideal, um zu untersuchen, wie schnell die auf eine Vesikelfreisetzung folgende Endozytose vonstatten geht. Mit optimierten, hochauflösenden Kapazitätsmessungen konnte an der Moosfaser-Körnerzell- Synapse eine sehr schnelle Endozytose nach einzelnen Aktionspotenzialen gezeigt werden. Die hohe Geschwindigkeit der Endozytose unterstützt somit eine hochfrequente synaptische Übertragung. Diese schnelle Endozytose wird durch die Moleküle Dynamin und Actin vermittelt und ist unabhängig von einer Wirkung von Clathrin. Stärkere Stimuli wie längere Depolarisationen evozieren eine langsamere Form der Endozytose, die zusätzlich Clathrin-abhängig ist. Durch die mechanistische Beschreibung hochfrequenter Signalübertragung an einer zentralen Synapse erweitern die Ergebnisse der vorliegenden Arbeit unser Verständnis von synaptischer Übertragung und Informationsverarbeitung im Zentralnervensystem.

by Hu Xiao Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 103-111). / Abstracts in English and Chinese. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Basic Physiology of the Nervous System --- p.1 / Chapter 1.1.1 --- Membrane Potential and Its Propagation --- p.2 / Chapter 1.1.2 --- Cellular Communication --- p.3 / Chapter 1.2 --- Background of Neural Modeling --- p.5 / Chapter 1.2.1 --- Models for Membrane --- p.5 / Chapter 1.2.2 --- The Models for Gap Junctions --- p.9 / Chapter 1.2.3 --- A Study on the Pulse Train --- p.11 / Chapter 1.3 --- Main Purposes of the Study --- p.14 / Chapter 1.4 --- Organization of Thesis --- p.15 / Chapter 2 --- Electrical Synaptic Model --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Model Description --- p.19 / Chapter 2.2.1 --- An Introduction of the Active Membrane Model --- p.19 / Chapter 2.2.2 --- The Electrical Synaptic Model --- p.25 / Chapter 2.3 --- Numerical Calculation --- p.32 / Chapter 2.4 --- Simulation Results --- p.37 / Chapter 2.5 --- Discussion --- p.44 / Chapter 3 --- Analysis of the Synaptic Model --- p.46 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Time Constant Analysis --- p.48 / Chapter 3.2.1 --- Junctional Time Constant in Bennette's Model --- p.48 / Chapter 3.2.2 --- The Junctional Time Constant in Our Model --- p.52 / Chapter 3.3 --- Model Reconstruction --- p.57 / Chapter 3.4 --- Discussion --- p.62 / Chapter 4 --- Action Potential Train Transmission Analysis --- p.69 / Chapter 4.1 --- Theoretical Analysis on the Refractory Period at the Post-membrane --- p.70 / Chapter 4.1.1 --- Introduction of Membrane Threshold and Refractory Period --- p.71 / Chapter 4.1.2 --- Stochastic Models of Neuron Firing --- p.73 / Chapter 4.1.3 --- Effect of Refractory Period on the p.d.f. of Poisson Process --- p.78 / Chapter 4.2 --- Simulation of the Action Potential Train Transmission --- p.85 / Chapter 4.2.1 --- Effects of the Model Parameter on the Action Potential Train Transmission --- p.90 / Chapter 4.2.2 --- Effects of the Refractory Period of the Post-membrane on the Action Potential Train Transmission --- p.94 / Chapter 4.3 --- Results --- p.96 / Chapter 4.3.1 --- Section Summary --- p.98 / Chapter 5 --- Conclusions and Future Studies --- p.99 / Chapter 5.1 --- Conclusions of Major Contributions --- p.99 / Chapter 5.2 --- Topics for Future Studies --- p.101

Frequenin (Frq) and its mammalian homologue, Neuronal Calcium Sensor 1 (NCS-1), are calcium-binding proteins, which regulate neurotransmitter release. However, reports are contradictory, and little is known about Frq's cellular mechanisms. The Drosophila nervous system can be used to gain a better understanding of the function of Frq. There are two Frq-encoding genes in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Loss-of-function phenotypes were studied using three different procedures: creating a deletion designed to remove the entire frq1 gene and part of the frq2 gene; using an interfering C-terminal peptide to prevent Frq binding to its intracellular targets; and using RNAi to reduce frq1 and frq2 transcript levels. Deletion of the entire frq1 gene and part of the frq2 gene resulted in impaired neurotransmitter release and enhanced nerve terminal growth. To discriminate chronic from acute loss-of-function effects, the effects of transgenic expression and forward-filling an interfering C-terminal peptide into presynaptic terminals were compared. In both cases, a reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations during development. The chronic treatment also enhanced nerve terminal growth. Conversely, gain-of-function conditions yielded an increase in quantal content and a reduction in nerve terminal growth. Frqs' effects on transmitter output were not due to changes in the number of active zones, nor were they due to changes in the size of the readily releasable pool of vesicles. Oregon Green 488 BAPTA-1 conjugated to 10 kDa Dextran was forward-filled into presynaptic boutons to detect changes in presynaptic Ca2+ signals. Ca2+ responses to presynaptic nerve impulses demonstrated that Frq modulates neurotransmitter release by regulating Ca2+ entry. Gain-of-function phenotypes remained present in a PI4KB null background, demonstrating that Frq's effects were not due to an interaction with PI4KB. All effects seen for all studies were identical for both Frqs, indicating that the two Frq proteins are likely functionally redundant. Overall, Frqs have two distinct functions: one on neurotransmission, primarily by regulating Ca2+ entry, and another on axonal growth and synaptic bouton formation.

Frequenin (Frq) and its mammalian homologue, Neuronal Calcium Sensor 1 (NCS-1), are calcium-binding proteins, which regulate neurotransmitter release. However, reports are contradictory, and little is known about Frq's cellular mechanisms. The Drosophila nervous system can be used to gain a better understanding of the function of Frq. There are two Frq-encoding genes in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Loss-of-function phenotypes were studied using three different procedures: creating a deletion designed to remove the entire frq1 gene and part of the frq2 gene; using an interfering C-terminal peptide to prevent Frq binding to its intracellular targets; and using RNAi to reduce frq1 and frq2 transcript levels. Deletion of the entire frq1 gene and part of the frq2 gene resulted in impaired neurotransmitter release and enhanced nerve terminal growth. To discriminate chronic from acute loss-of-function effects, the effects of transgenic expression and forward-filling an interfering C-terminal peptide into presynaptic terminals were compared. In both cases, a reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations during development. The chronic treatment also enhanced nerve terminal growth. Conversely, gain-of-function conditions yielded an increase in quantal content and a reduction in nerve terminal growth. Frqs' effects on transmitter output were not due to changes in the number of active zones, nor were they due to changes in the size of the readily releasable pool of vesicles. Oregon Green 488 BAPTA-1 conjugated to 10 kDa Dextran was forward-filled into presynaptic boutons to detect changes in presynaptic Ca2+ signals. Ca2+ responses to presynaptic nerve impulses demonstrated that Frq modulates neurotransmitter release by regulating Ca2+ entry. Gain-of-function phenotypes remained present in a PI4KB null background, demonstrating that Frq's effects were not due to an interaction with PI4KB. All effects seen for all studies were identical for both Frqs, indicating that the two Frq proteins are likely functionally redundant. Overall, Frqs have two distinct functions: one on neurotransmission, primarily by regulating Ca2+ entry, and another on axonal growth and synaptic bouton formation.