Robert A. Nichols

Gabapentin inhibits presynaptic Ca 2+ influx and synaptic transmission in rat hippocampus and neocortex

Gabapentin is a widely used drug with anticonvulsant, antinociceptive and anxiolytic properties. Although it has been previously shown that Gabapentin binds with high affinity to the alpha(2)delta subunit of voltage-operated Ca(2+) channels (VOCC), little is known about the functional consequences of this interaction. Here, we investigated the effect of Gabapentin on VOCCs and synaptic transmission in rat hippocampus and neocortex using whole-cell patch clamp and confocal imaging techniques. Gabapentin (100-300 microM) did not affect the peak amplitude or voltage-dependency of VOCC currents recorded from either dissociated or in situ neocortical and hippocampal pyramidal cells. In contrast, Gabapentin inhibited K(+)-evoked increases in [Ca(2+)] in a subset of synaptosomes isolated from rat hippocampus and neocortex in a dose-dependent manner, with an apparent half-maximal inhibitory effect at approximately 100 nM. In hippocampal slices, Gabapentin (300 microM) inhibited the amplitude of evoked excitatory- and inhibitory postsynaptic currents recorded from CA1 pyramidal cells by 30-40%. Taken together, the results suggest that Gabapentin selectively inhibits Ca(2+) influx by inhibiting VOCCs in a subset of excitatory and inhibitory presynaptic terminals, thereby attenuating synaptic transmission.

Functional Nicotinic Acetylcholine Receptors Containing α6 Subunits Are on GABAergic Neuronal Boutons Adherent to Ventral Tegmental Area Dopamine Neurons

Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR α6 subunit message is abundant in the VTA, but α6*–nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well understood. Here, we report evidence for α6β2*–nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. α-Conotoxin (α-Ctx) MII labeling coupled with immunocytochemical staining localizes putative α6*–nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature IPSCs (mIPSCs) mediated by GABAA receptors. These increases are abolished by α6*–nAChR-selective α-Ctx MII or α-Ctx PIA (1 nm) but not by α7 (10 nm methyllycaconitine) or α4* (1 μm dihydro-β-erythroidine)–nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR β2 knock-out mice. Moreover, ACh induces an α-Ctx PIA-sensitive elevation in intraterminal Ca2+ in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nm nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 d of systemic nicotine exposure. Collectively, these results indicate that α6β2*–nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic α6β2*–nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.

Direct Observation of Serotonin 5‐HT3 Receptor‐Induced Increases in Calcium Levels in Individual Brain Nerve Terminals

Abstract: Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5‐HT3 receptor, a ligand‐gated ion channel. The 5‐HT3 receptor agonist‐induced calcium level changes in individual synaptosomes were compared with responses evoked by K+ depolarization. Using the fluorescent dye fluo‐3 to measure relative changes in internal free Ca2+ concentration ([Ca2+]i), K+‐induced depolarization resulted in variable but rapid increases in apparent [Ca2+]i among the individual terminals, with some synaptosomes displaying large transient [Ca2+]i peaks of varying size (two‐ to 12‐fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca2+]i (two‐ to 2.5‐fold over basal levels). Agonist activation of 5‐HT3 receptors induced slow increases in [Ca2+]i (rise time, 15–20 s) in a subset (∼5%) of corpus striatal synaptosomes, with the increases (averaging 2.2‐fold over basal) being dependent on Ca2+ entry and inhibited by millimolar external Mg2+. We conclude that significant increases in brain nerve terminal Ca2+, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand‐gated ion channels.

Mechanisms in the regulation of neurotransmitter release from brain nerve terminals : current hypotheses

Depolarization of the nerve terminal leads to the influx of calcium through voltage-sensitive CaZ+-chan nels (VSCC), which, in turn, triggers the release of neurotransmitter by exocytosis of intraterminal vesicles in which the neurotransmitter is stored (1,2). In recent years, there has been a substantial increase in our understanding of the ionic events involved during depolarization of brain nerve terminals, especially the resultant activation of Ca z+channels (3-14). In addition, it appears that Ca z+ entry during sustained nerve terminal stimulation can activate processes, secondary to exocytosis, which alter on-going neurotransmitter release. However, the molecular mechanisms by which the increased cytosolic Ca z+ causes exocytosis and the subsequent modulation of on-going neurotransmitter release are not as well understOod. On nerve-terminal stimulation, a number of proteins undergo marked changes in their state of phosphorylation (15,16,17) and the actin cytoskeleton undergoes changes in polymerization state (18,19). It has been proposed that these two stimulation-induced changes are secondary processes involved in neurotransmitter release modulation. It is the purpose of this review to examine the

Antibodies against the extracellular domain of the 5-HT3 receptor label both native and recombinant receptors.

We have developed polyclonal antibodies (pAb120) against a peptide corresponding to a region within the extracellular domain of the 5-hydroxytryptamine3 (5-HT3) receptor subunit, thus permitting, for the first time, localization of 5-HT3 receptors at the cell surface in intact (non-permeabilized) systems. The antibodies are both specific and sensitive: pAb120 recognized as little as 63 ng of protein from HEK293 cells expressing recombinant 5-HT3 receptors, whilst Western blots of recombinant 5-HT3 receptors purified from Sf9 cells revealed two bands at 48 and 54 kDa, and native 5-HT3 receptors from N1E-115 cell membranes produced a broad band at 50-54 kDa with a smaller band at 35 kDa. These bands were also labelled by antibodies against the intracellular loop of the 5-HT3 receptor. Immunofluorescent labelling revealed a ring of intense fluorescence in the plasma membrane of non-permeabilized HEK293 cells expressing recombinant 5-HT3 receptors. Studies on native 5-HT3 receptors revealed that pAb120 could recognize 5-HT3 receptors on presynaptic terminals isolated from rat striatum, and immunohistochemical studies in rat brain sections revealed labelling of cell bodies, dendrites and varicose axons in hippocampus, cortex and lateral hypothalamus; all of these areas have been reported to express 5-HT3 receptors. We conclude that pAb120 is a highly specific and sensitive antiserum that will assist in clarifying fundamental questions about 5-HT3 receptor neurobiology.

Nicotinic receptors co-localize with 5-HT3 serotonin receptors on striatal nerve terminals

Nicotinic acetylcholine receptors and 5-HT(3) serotonin receptors are present on presynaptic nerve terminals in the striatum, where they have been shown to be involved in the regulation of dopamine release. Here, we explored the possibility that both receptor systems function on the same individual nerve terminals in the striatum, as assessed by confocal imaging of synaptosomes. On performing sequential stimulation, nicotine (500 nM) induced changes in [Ca(2+)](i) in most of the synaptosomes ( approximately 80%) that had previously responded to stimulation with the 5-HT(3) receptor agonist m-chlorophenylbiguanide (mCPBG; 100 nM), whereas mCPBG induced [Ca(2+)](i) responses in approximately half of the synaptosomes that showed responses on nicotinic stimulation. The 5-HT(3) receptor-specific antagonist tropisetron blocked only the mCPBG-induced responses, but not the nicotinic responses on the same synaptosomes. Immunocytochemical staining revealed extensive co-localization of the 5-HT(3) receptor with the alpha4 nicotinic receptor subunit on the same synaptosomes, but not with the alpha3 and/or alpha5 subunits. Immunoprecipitation studies indicate that the 5-HT(3) receptor and the alpha4 nicotinic receptor subunit do not interact on the nerve terminals. The presence of nicotinic and 5-HT(3) receptors on the same presynaptic striatal nerve terminal indicates a convergence of cholinergic and serotonergic systems in the striatum.

β-Amyloid activates presynaptic α7 nicotinic acetylcholine receptors reconstituted into a model nerve cell system: involvement of lipid rafts

Beta amyloid (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease. Aβ is the major constituent of senile plaques, but there is a significant presence of Aβ in the brain in soluble forms. The results of functional studies indicate that soluble Aβ interacts with the α7 nicotinic acetylcholine receptor (nAChR) complex with apparent high affinity. However, conflicting data exist as to the nature of the Aβ–α7 nAChR interaction, and whether it is the result of specific binding. Moreover, both agonist‐like and antagonist‐like effects have been reported. In particular, agonist‐like effects have been observed for presynaptic nAChRs. Here, we demonstrate Aβ1‐42‐evoked stimulatory changes in presynaptic Ca2+ level via exogenous α7 nAChRs expressed in the axonal varicosities of differentiated hybrid neuroblastoma NG108‐15 cells as a model, presynaptic system. The Aβ1‐42‐evoked responses were concentration‐dependent and were sensitive to the highly selective α7 nAChR antagonist α‐bungarotoxin. Voltage‐gated Ca2+ channels and internal Ca2+ stores were both involved in Aβ1‐42‐evoked increases in presynaptic Ca2+ following activation of α7 nAChRs. In addition, disruption of lipid rafts by cholesterol depletion led to substantially attenuated responses to Aβ1‐42, whereas responses to nicotine were largely intact. These results directly implicate the nicotinic receptor complex as a target for the agonist‐like action of pico‐ to nanomolar concentrations of soluble Aβ1‐42 on the presynaptic nerve terminal, including the possible involvement of receptor‐associated lipid rafts. This interaction probably plays an important neuromodulatory role in synaptic dynamics.

Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K+ ‐induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 mM Ba2+ could substitute for 1 mM Ca2+ in evoking the release of endogenous glutamate. In addition, Ba2+ was found to evoke glutamate release in the absence of K+‐induced depolarization. Ba2+ (1–10 mM) depolarized synaptosomes, as measured by voltage‐sensitive dye fluorescence and [3H]‐tetraphenylphosphonium cation distribution. Ba2+ partially inhibited the increase in synaptosomal K+ efflux produced by depolarization, as reflected by the redistribution of radiolabeled 86Rb+. The release evoked by Ba2+ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura‐2, cytosolic [Ca2+] increased during stimulation by approximately 200 nM, but cytosolic [Ba2+] increased by more than 1 μM. Taken together, our results indicate that Ba2+ initially depolarizes synaptosomes most likely by blocking a K+ channel, which then activates TTX‐sensitive Na+ channels, causing further depolarization, and finally enters synaptosomes through voltage‐sensitive Ca2+channels to evoke neurotransmitter release directly. Though Ba2+‐evoked glutamate release was comparable in level to that obtained with K+‐induced depolarization in the presence of Ca2+, the apparent intrasynaptosomal level of Ba2+ required for a given amount of glutamate release was found to be several‐fold higher than that required of Ca2+.

Role of Key Aromatic Residues in the Ligand-binding Domain of α7 Nicotinic Receptors in the Agonist Action of β-Amyloid

Background: β-Amyloid activates presynaptic nicotinic receptors, regulating nerve terminal Ca2+. Results: Mutating tyrosine 188 but not tyrosine 195 in the ligand-binding domain of α7-nicotinic receptors eliminates activation by β-amyloid. Conclusion: Tyrosine 188 in the ligand-binding domain of α7-nicotinic receptors plays a key role in β-amyloid-induced activation of the receptors. Significance: Direct activation of α7-nicotinic receptors may represent a neuromodulatory function of β-amyloid. Soluble β-amyloid (Aβ) resides in certain regions of the brain at or near picomolar concentration, rising in level during the prodromic stage of Alzheimer disease. Recently, we identified the homomeric α7 nicotinic acetylcholine receptor (α7-nAChR) as one possible functional target for picomolar Aβ. This study was aimed at addressing which residues in α7-nAChRs potentially interact with Aβ to regulate the presynaptic function of this receptor. Site-directed mutagenesis was carried out to study the key aromatic residues in the mouse α7-nAChR agonist-binding pocket. Mutations of tyrosine188 resulted in a decrease in activation of presynaptic α7-nAChRs by ACh and Aβ but with no change in response to nicotine, indicating the critical role of Tyr-188 in presynaptic regulation by Aβ. Coimmunoprecipitation additionally revealed direct binding of Aβ to α7-nAChRs and to the Tyr-188 mutant receptor. In contrast, mutations of Tyr-195 in α7-nAChR led to decreased activation by nicotine without apparent effects on ACh- or Aβ-induced responses. Agonist-induced responses of Tyr-93 mutant α7-nAChRs indicated possible interactions of nicotine and Aβ with its hydroxyl group, but there was no change in presynaptic responses after mutation of Trp-149. All of the mutants were shown to be expressed on the plasma membrane using cell surface labeling. Together, these results directly demonstrate an essential role for the aromatic residue Tyr-188 as a key component in the agonist binding domain for the activation of α7-nAChRs by Aβ.

Dopamine receptor regulation of Ca2+ levels in individual isolated nerve terminals from rat striatum: comparison of presynaptic D1‐like and D2‐like receptors

We have directly observed the effects of activating presynaptic D1‐like and D2‐like dopamine receptors on Ca2+ levels in isolated nerve terminals (synaptosomes) from rat striatum. R‐(+)‐SKF81297, a selective D1‐like receptor agonist, and (–)‐quinpirole, a selective D2‐like receptor agonist, induced increases in Ca2+ levels in different subsets of individual striatal synaptosomes. The SKF81297‐ and quinpirole‐induced effects were blocked by R‐(+)‐SCH23390, a D1‐like receptor antagonist, and (–)‐sulpiride, a D2‐like receptor antagonist, respectively. SKF81297‐ or quinpirole‐induced Ca2+ increases were inhibited following blockade of voltage‐gated calcium channels or sodium channels. In a larger subset of synaptosomes, quinpirole decreased baseline Ca2+. Quinpirole also inhibited veratridine‐induced increases in intrasynaptosomal Ca2+ level. Immunostaining confirmed the presynaptic expression of D1, D5, D2 and D3 receptors, but not D4 receptors. The array of neurotransmitter phenotypes of the striatal nerve endings expressing D1, D5, D2 or D3 varied for each receptor subtype. These results suggest that presynaptic D1‐like and D2‐like receptors induce increases in Ca2+ levels in different subsets of nerve terminals via Na+ channel‐mediated membrane depolarization, which, in turn, induces the opening of voltage‐gated calcium channels. D2‐like receptors also reduce nerve terminal Ca2+ in a different but larger subset of synaptosomes, consistent with the predominant presynaptic action of dopamine in the striatum being inhibitory.