Synaptic Transmission: Modulation, Plasticity And Effect

综合神经科学实验室（LIN）突触药理学部分的研究重点是确定神经调节和可塑性的机制，以及酒精和其他滥用药物对这些神经元功能的影响。正在进行的研究正在继续我们对酒精对NMDA和非NMDA谷氨酸受体功能的影响的研究。我们比较了乙醇对突触和非突触NMDA受体的抑制作用，因为这些不同位置的受体在神经可塑性和神经毒性中似乎起着不同的作用。使用小鼠海马微岛/自噬制备观察到的结果表明，突触和非突触NMDA受体对急性酒精抑制同样敏感。我们已经观察到乙醇对该受体的抑制是时间依赖性的，目前的实验旨在确定暴露于乙醇0.5 - 0.2分钟产生的增强抑制的分子机制。我们还将使用自断制备来确定乙醇是否会改变NMDA受体突触池重新填充的速率。这些实验有可能揭示乙醇是否对NMDA受体的细胞内运输有快速影响。最近的研究结果还表明，改变ampa型谷氨酸受体脱敏的突变改变了这些受体的乙醇抑制作用。这些结果的一般模式是，减少脱敏的突变会减少酒精抑制，而增强脱敏的突变会增强酒精抑制。作为与UNTHSC Tina Machu博士实验室正在进行的合作的一部分，我们还在旨在确定5-HT3受体对神经递质5-羟色胺的酒精敏感性的分子基础的研究方面取得了进展。改变5-HT3A受体亚基（通常为亮氨酸）293位的氨基酸特性，可减少或消除乙醇和三氯乙醇对受体的增强作用。这种氨基酸残基位于受体的第二跨膜区域，位于假定的α螺旋的非孔面侧。这个位置类似于作为醇的一部分的氨基酸残基。结合位点?GABAA和甘氨酸受体是5-HT3受体的近亲。293位的大部分氨基酸取代完全破坏了乙醇和三氯乙醇的增强作用。只有异亮氨酸替代保存酒精的效果。因此，没有观察到氨基酸侧链性质与酒精敏感性之间的明确关系。所有的氨基酸取代对通道动力学都有深远的影响，并具有增加开放通道状态稳定性和增加部分激动剂在受体上的功效的一致效果。这些发现表明这种残基在控制通道门控中起关键作用。酒精敏感性的破坏很可能是由于酒精增强作用被阻断的结果，因为酒精增加了开放通道状态的稳定性。根据这些发现，目前还不可能确定L293是否是酒精的一部分？结合位点?在5-HT3受体中

The focus of research in the Laboratory for Integrative Neuroscience (LIN), Section on Synaptic Pharmacology is the determination of mechanisms underlying neuromodulation and plasticity and the effects of alcohol and other drugs of abuse on these neuronal functions. Ongoing studies are continuing our examination of alcohol effects on NMDA and non-NMDA glutamate receptor function. We have compared ethanol inhibition at synaptic and non-synaptic NMDA receptors because receptors at these different locations appear to have differential roles in neuroplasticity and neurotoxicity. Results observed using a microisland/autapse preparation from mouse hippocampus indicate that synaptic and non-synaptic NMDA receptors are equally sensitive to acute alcohol inhibition. We have observed that ethanol inhibition of this receptor is time-dependent, and current experiments are aimed at determining molecular mechanisms involved in enhanced inhibition produced by exposure to ethanol for 0.5 -0 2 min. We will also use the autapse preparation to determine if ethanol alters the rate of re-filling of the synaptic pool of NMDA receptors. These experiments have the potential to reveal if ethanol has rapid effects on the intracellular trafficking of NMDA receptors. Recent findings also indicate that mutations that alter desensitization of AMPA-type glutamate receptors alter ethanol inhibition of these receptors. The general pattern of these results is that mutations that reduce desensitization reduce alcohol inhibition, while inhibition is enhanced by mutations that enhance desensitization. We have also made progress on studies aimed at determining the molecular basis of alcohol sensitivity of the 5-HT3 receptor for the neurotransmitter serotonin as part of an ongoing collaboration with the laboratory of Dr. Tina Machu at UNTHSC. Altering the amino acid identity at position 293 in the 5-HT3A receptor subunit (normally a leucine) reduces or abolishes potentiation of the receptor by ethanol and trichloroethanol. This amino acid residue lies in the 2nd transmembrane domain of the receptor on the non-pore-facing side of the presumed alpha helix. This position is analogous to amino acid residues that have been proposed as part of an alcohol ?binding site? in the GABAA and Glycine receptors, close molecular cousins of the 5-HT3 receptor. The large majority of amino acid substitutions at position 293 completely disrupted potentiation by ethanol and trichloroethanol. Only substitution of isoleucine preserved alcohol effects. Thus, no clear relationship between amino acid sidechain properties and alcohol sensitivity was observed. All of the amino acid substitutions had profound effects on channel kinetics with a consistent effect of increasing the stability of the open channel state, and increasing the efficacy of partial agonists at the receptor. These findings suggest that this residue has a key role in control of channel gating. The disruption of alcohol sensitivity is very likely a consequence of occlusion of alcohol potentiation given that alcohols increase open channel state stability. In light of these findings it is not possible at this time to determine if L293 is part of an alcohol ?binding site? in the 5-HT3 receptor. We have continued studies of synaptic plasticity in dorsal striatum, as well as our investigations of similar plastic changes in hippocampus and amygdala. In past studies our laboratory and others have shown that endocannabinoids, endogenous lipid metabolites that activate cannabinoid receptors, act as retrograde signals that produce short and long-term changes in neurotransmitter release probability. During the past year we have characterized several new forms of cannabinoid-dependent synaptic plasticity. Studies in striatal brain slices have revealed that endocannabinoid-dependent long-term synaptic depression (LTD) can be induced by sustained afferent stimulatiion at a moderate frequency (10 Hz). This form of LTD is unique among endocannabinoid-dependent forms of plasticity in that is does not appear to require retrograde signaling. This finding indicates that different forms of synaptic plasticity can be brought about by different frequencies of synaptic activation within the physiological range of firing rates of striatal neurons and their cortical afferents. We have continued our studies in the hippocampal formation of postnatatal development of endocannabinoid-dependent depolarization-induced suppression of inhibition (DSI), a short-term decrease in neurotransmitter release stimulated by retrograde endocnnabinoid signaling. These studies have led to the realization that DSI requires ?priming? by metabotropic glutamate receptors (mGluRs) early in development (P7-P10), but becomes independent of mGluRs at later developmental stages (P15-P20). Furthermore, we have found that sustained low frequency synaptic activation can prime DSI and induces a long-term depression of inhibitory synaptic transmission. Both of these forms of plasticity are dependent on mGluR activation. We believe that these mGluR- and endocannabinoid-dependent forms of synaptic plasticity ultimately enhance excitatory synaptic transmission by reducing synaptic inhibition. These mechanisms may play roles in spatial learning and memory involving the hippocampus. We have also continued to explore the molecular mechanisms involved in endocannabinoid production leading to DSI and LTD in amygdala neurons using newly-implemented techniques for isolating neurons with attached GABAergic synaptic boutons. Depolarization of the isolated postsynaptic neuron produces presynaptic depression that is dependent on endocannabinoids and CB1 cannabinoid receptors. We have evidence for the existence of two separable components of synaptic depression in this preparation. The first component persists for 10s of seconds and resembles DSI. This early component is dependent on increased postsynaptic calcium for its induction. The second component is independent of postsynaptic calcium increases, but requires activation of mGluR5. This later-developing depression begins ~30 seconds after depolarization and persists for the duration of the recording. The properties of this delayed, mGluR-dependent component of synaptic depression resemble amygdalar LTD. These findings indicate that DSI and LTD require only a postsynaptic neuron and a functional presynaptic terminal, and thus retrograde signaling is a simple and highly localized phenomenon. The use of this preparation should allow us to gain more accurate information about the onset and duration time courses of DSI and LTD. The exquisite pharmacological and physiological control afforded by this preparation will allow us to better characterize the molecular mechanisms involved in DSI and LTD. Endocannabinoid-dependent synaptic plasticity in amygdala has been implicated in extinction of fear conditioning, and thus it is important to understand the mechanisms involved in this plasticity. Biochemical studies aimed at understanding the intracellular signals that link receptor activation to induction of plasticity, and determining the mechanisms involved in long-lasting depression and DSI are continuing. We are currently examining effects of CB1 activation on phosphorylation and function of presynaptic vesicle-associated proteins to begin to understand how this receptor may produce lasting alterations in neurotransmitter secretion. We are also examining ethanol effects on neuronal survival and differentiation. Studies to date indicate that neurotoxic effects of ethanol in cerebellar granule neurons involve inhibition of the intracellular signaling enzymes PI3 kinase, ERK and protein kinase C. These several lines of research should allow to gain a better understanding of synaptic mechanims of learning and addiction.