archives@tulane.edu / Previous work has revealed a presynaptic cytosolic Na+-dependent regulation on vesicular glutamate content and mEPSC amplitude via activating vacuolar Na+/H+ exchangers (NHEs) expressed on the synaptic vesicles, suggesting a presynaptic determinant of quantal size for synaptic strength. However, it remains unknown how spike activities control intracellular Na+ at the axon terminals and how the fluctuation of presynaptic Na+ during activities modulates quantal content and contributes to synaptic strength. I studied these questions using the calyx of Held, a giant glutamatergic synapse. With two-photon Na+ imaging, I found that presynaptic Na+ substantially accumulated during spike firing in a frequency and duration-dependent manner. This spike-induced elevation of presynaptic Na+ gradually increased EPSC amplitude by solely affecting vesicular glutamate filling, which was further confirmed as increased amplitude of asynchronous released vesicles, but without affecting the size of readily releasable pool or neurotransmitter release probability. This Na+-dependent modulation of EPSC amplitude resulted in a change of the reliability of transferring presynaptic spike to postsynaptic firing. Finally, blockade of NHEs reduced both EPSC amplitude and reliability of synaptic signaling, suggesting that NHEs are required for presynaptic Na+ regulation of synaptic transmission. Recent studies demonstrated that a non-inactivation cation channel NALCN (Na+ leak channel, non-selective), characterized as a major Na+ leak channel, is widely expressed in the central nervous system. Immunostaining revealed the expression of NALCN channel at the calyceal terminals. In line with a role of NALCN in controlling the cell excitability, calyces with conditional knockout (cKO) of NALCN exhibited a more hyperpolarized resting membrane potential compared with the wildtype (WT) calyces. Blockade of NALCN with a non-specific blocker gadolinium (Gd3+) induced a reduction of basal Na+ level and mEPSC amplitude in the WT but not in cKO group, suggesting the involvement of presynaptic NALCN channels in regulating the vesicular glutamate content. More importantly, two-photon Ca2+ imaging showed that NALCN channels were permeable to Ca2+, and Gd3+ decreased the basal Ca2+ level in WT but not cKO calyces. The Ca2+ permeability was further confirmed by reduced sensitivity of mEPSC frequency in response to increased extracellular Ca2+ concentration in cKO and reduced initial release probability in response to application of Gd3+ to block NALCN channels in WT group. Finally, Gd3+ induced a stronger reduction of EPSC amplitude in WT group compared to cKO group. Overall, these data indicate that NALCN channels regulate glutamate transmission through modulation of both quantal size and initial release probability. / 1 / Dainan Li

The entorhinal cortex is reciprocally connected with both neocortical sensory areas and the hippocampal formation, and is thought to play a pivotal role in learning and memory. Changes in synaptic strength are thought to provide the major neurophysiological basis for memory formation, but little is known about synaptic plasticity in the entorhinal cortex. The objectives of this research were to provide a basis for the interpretation of evoked potentials recorded from the entorhinal cortex following pyriform (primary olfactory) cortex stimulation 𝘪𝘯 𝘷𝘪𝘷𝘰, and to determine the conditions under which synaptic enhancements in this pathway may occur and contribute to lasting changes in the processing of olfactory information. The synaptic currents which generate field potentials in the entorhinal cortex following pyriform cortex and medial septal stimulation were first localized to the superficial layers of the entorhinal cortex using current source density analysis techniques in the anesthetized rat. This allowed changes in the strength of these synaptic inputs to be monitored in the awake rat by measuring evoked field potential amplitudes at a single cortical depth. Long-term synaptic potentiation (LTP) in this pathway was reliably induced following stimulation of the pyriform cortex with either epileptogenic stimuli, or with prolonged subconvulsive high-frequency trains. Further, stimulation which results in short-term frequency potentiation effects, was found to increase the amount of LTP induced. Concurrent stimulation of the medial septum at a frequency similar to that of the endogenous theta rhythm also resulted in a cooperative enhancement of the LTP produced. Computational modelling techniques were then used to formalize the heterosynaptic contribution of frequency potentiating medial septal inputs to Hebbian synaptic modification in entorhinal cortex. These results indicate that the frequency of rhythmic activity in sensory afferents and the activity of the medial septum may play critical roles in the regulation of synaptic plasticity in the entorhinal cortex. / Thesis / Doctor of Philosophy (PhD)

No / Activity-dependent gene transcription and protein synthesis underlie many forms of learning-related synaptic plasticity. At excitatory glutamatergic synapses, the immediate early gene product Arc/ Arg3.1 couples synaptic activity to postsynaptic endocytosis of AMPA-type glutamate receptors. Although the mechanisms for Arc induction have been described, little is known regarding the molecular machinery that terminates Arc function. Here, we demonstrate that the RING domain ubiquitin ligase Triad3A/RNF216 ubiquitinates Arc, resulting in its rapid proteasomal degradation. Triad3A associates with Arc, localizes to clathrin-coated pits, and is associated with endocytic sites in dendrites and spines. In the absence of Triad3A, Arc accumulates, leading to the loss of surface AMPA receptors. Furthermore, loss of Triad3A mimics and occludes Arc-dependent forms of synaptic plasticity. Thus, degradation of Arc by clathrin-localized Triad3A regulates the availability of synaptic AMPA receptors and temporally tunes Arc-mediated plasticity at glutamatergic synapses. / A final draft copy of this article is not yet available.

Synapses vary widely in the probability of transmitter release. For instance, in response to an action potential the phasic synapses of the crayfish have a 100-1000-fold higher release probability than tonic synapses. The difference in release probability is attributed to differences in the exocytotic machinery such as the degree of “zippering” of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and molecular approaches to determine if the zippered state of SNAREs associated with synaptic vesicles and the interaction between the SNARE complex and Complexin influence the probability of release at the synapse. I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by SNARE interaction (zippering) or open to proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-terminal half of crayfish VAMP’s SNARE motif (Vc peptide) designed to interfere with SNARE complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-terminal end exposed under resting conditions. I used a peptide containing part of the crayfish Complexin central α-helix domain to interfere with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic evoked release and inhibited tonic evoked release under low stimulation but attenuated release at both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual function under low synaptic activity but only promoted release under high synaptic activity. The results showed that the zippered state of the SNARE complex does not determine initial release probability as a similar zippered SNARE complex structure under resting conditions is common to both phasic and tonic synapses. However, Complexin may have a role in influencing the initial release probability of a synapse. Therefore, the interaction between the SNARE complex and Complexin is important for release but other factors contribute more significantly to synaptic strength.

Synapses vary widely in the probability of transmitter release. For instance, in response to an action potential the phasic synapses of the crayfish have a 100-1000-fold higher release probability than tonic synapses. The difference in release probability is attributed to differences in the exocytotic machinery such as the degree of “zippering” of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex. I used physiological and molecular approaches to determine if the zippered state of SNAREs associated with synaptic vesicles and the interaction between the SNARE complex and Complexin influence the probability of release at the synapse. I used three Botulinum neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by SNARE interaction (zippering) or open to proteolytic attack. Under low stimulation conditions, the light-chain fragment of botulinum B (BoNT/B-LC) but not BoNT/D-LC or tetanus neurotoxin (TeNT-LC) cleaved VAMP and inhibited evoked release at both phasic and tonic synapses. In addition, a peptide based on the C-terminal half of crayfish VAMP’s SNARE motif (Vc peptide) designed to interfere with SNARE complex zippering at the C-terminal end inhibited release at both synapses. The susceptibility of VAMP to only BoNT/B-LC and interference by the Vc peptide indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end with the C-terminal end exposed under resting conditions. I used a peptide containing part of the crayfish Complexin central α-helix domain to interfere with the interaction between Complexin and the SNARE complex. The peptide enhanced phasic evoked release and inhibited tonic evoked release under low stimulation but attenuated release at both synapses under intense stimulation. Therefore, Complexin appeared to exhibit a dual function under low synaptic activity but only promoted release under high synaptic activity. The results showed that the zippered state of the SNARE complex does not determine initial release probability as a similar zippered SNARE complex structure under resting conditions is common to both phasic and tonic synapses. However, Complexin may have a role in influencing the initial release probability of a synapse. Therefore, the interaction between the SNARE complex and Complexin is important for release but other factors contribute more significantly to synaptic strength.

No description available.