Postnatal upregulation of alpha4 and alpha3 nicotinic receptor subunits in the brain of alpha7 nicotinic receptor-deficient mice

The nicotinic receptor subtypes are important for several physiological functions in brain and may therefore play a critical role in brain development. The alpha7 nicotinic receptors which have high Ca2+ permeability are important for cognitive, neuroprotective and trophic functions. In this study, the brain development and the expression of alpha4, alpha3, alpha7, alpha5 and beta2 nicotinic receptors were investigated in the brains of alpha7 deficient (alpha7 -/-), alpha7 heterozygous null (alpha7 +/-) and alpha7 wild-type (alpha7 +/+) mice from postnatal days (P) 7-84. The specific binding of [3H] cytisine and [3H] epibatidine, as well as the expressions of alpha4 and alpha3 nicotinic receptor subunits at mRNA and protein levels, were significantly increased in the cortex and hippocampus of alpha7 -/- and alpha7 +/- mice compared with alpha7 +/+ mice. Furthermore, the alpha4 and alpha3 nicotinic acetylcholine receptor (nAChR) subunits appeared to co-assemble with the alpha5 nAChR subunit in these above brain regions of these mice. No significant change in synaptophysin level was observed. These data suggest that increased levels of alpha4, alpha3-containing nAChRs, co-assembled with the alpha5 nAChR subunit, may contribute to the normal brain development of alpha7 -/- and alpha7 +/- mice.     

Inactivation of alpha7 ACh receptors and activation of non-alpha7 ACh receptors both contribute to long term potentiation induction in the hippocampal CA1 region

Acute and chronic nicotine exposure differentially facilitate the induction of long-term potentiation (LTP), a synaptic model of learning and memory, in the hippocampal CA1 region. The mechanisms underlying these effects of nicotine, however, are unknown. In the present study, both nicotinic acetylcholine receptor (nAChR) agonists and an alpha7 nAChR antagonist facilitated the induction LTP in the hippocampal CA1 region of naive rat. Furthermore, chronic nicotine treatment lowered the threshold for induction of LTP, and acute application of nicotinic agonists, but not an alpha7 antagonist, further facilitated LTP induction in the chronic-nicotine-treated hippocampus. These results suggest not only that both activation of non-alpha7 nAChRs and inactivation of alpha7 nAChRs contribute to LTP induction, but also that chronic-nicotine-mediated facilitation of LTP induction is due to chronic-nicotine-induced desensitization of alpha7 nAChRs.     

Antisense knockdown of the rat alpha7 nicotinic acetylcholine receptor produces spatial memory impairment

Selective and brain penetrating pharmacological antagonists for use in clarifying a role of alpha7 nicotinic acetylcholine receptors (nAChR) in behavioral paradigms are presently unavailable. Studies in alpha7 knock-out mice (KO) have not revealed convincing changes in behavioral phenotype, in particular measures of cognition that include contextual fear conditioning and spatial memory, which may be due to compensatory developmental changes. Therefore, an antisense oligonucleotide (aON) targeted toward the 3'- and 5'-UTR coding regions of the rat alpha7 nicotinic acetylcholine receptor was used. Following central injection of aON into the lateral ventricle of Long Evans rats for 6 days, treated rats exhibited a significant 42% and 25% decrease in alpha7 nAChR densities in hippocampus and cortex, respectively, as measured by [(3)H]-methyllycaconitine (MLA) binding. There was no change in alpha4beta2 densities measured by [(3)H]-cytisine binding. Acquisition of Morris Water Maze (MWM) performance, a measure of spatial memory, was impaired in aON-treated rats. In addition, a reduction in target platform crossings during a subsequent probe-trial was observed. These data demonstrate the ability of this aON to reduce hippocampal and cortical alpha7 nicotinic receptor densities associated with impaired MWM performance and support the specific involvement of the alpha7 nAChR in spatial learning and memory, a phenotype not affected in alpha7 KO mice.     

Activation of nicotinic acetylcholine receptors increases the frequency of spontaneous GABAergic IPSCs in rat basolateral amygdala neurons

The basolateral amygdala (BLA) is a critical component of the amygdaloid circuit, which is thought to be involved in fear conditioned responses. Using whole cell patch-clamp recording, we found that activation of nicotinic acetylcholine receptors (nAChRs) leads to an action potential-dependent increase in the frequency of spontaneous GABAergic currents in principal neurons in the BLA. These spontaneous GABAergic currents were abolished by a low-Ca2+/high-Mg2+ bathing solution, suggesting that they are spontaneous inhibitory postsynaptic currents (sIPSCs). Blockade of ionotropic glutamate receptors did not prevent this increased frequency of sIPSCs nor did blockade of alpha7 nAChRs. Among the nAChR agonists tested, cystisine was more effective at increasing the frequency of the sIPSCs than nicotine or 1,1-dimethyl-4-phenyl piperazinium iodide, consistent with a major contribution of beta4 nAChR subunits. The nicotinic antagonist, dihydro-beta-erythroidine, was less effective than d-tubocurarine in blocking the increased sIPSC frequency induced by ACh, suggesting that alpha4-containing nAChR subunits do not play a major role in the ACh-induced increased sIPSC frequency. Although alpha2/3/4/7 and beta2/4 nAChR subunits were found in the BLA by RT-PCR, the agonist and antagonist profiles suggest that the ACh-induced increase in sIPSC frequency involves predominantly alpha3beta4-containing nAChR subunits. Consistent with this, alpha-conotoxin-AuIB, a nAChR antagonist selective for the alpha3beta4 subunit combination, inhibited the ACh-induced increase in the frequency of sIPSCs. The observations suggest that nicotinic activation increases the frequency of sIPSCs in the BLA by acting mainly on alpha3beta4-containing nicotinic receptors on GABAergic neurons and may play an important role in the modulation of synaptic transmission in the amygdala.     

The role of neuronal nicotinic acetylcholine receptor subunits in autonomic ganglia: lessons from knockout mice

Neuronal nicotinic acetylcholine receptors (nAChR), composed of 12 subunits (alpha2-alpha10, beta2-beta4), are expressed in autonomic ganglia, playing a central role in autonomic transmission. The repertoire of nicotinic subunits in autonomic ganglia includes alpha3, alpha5, alpha7, beta2 and beta4 subunits. In the last 10 years, heterologous expression studies have revealed much about the nature of neuronal nAChRs. However, there is only limited understanding of subunit actions in autonomic system. Functional deletions of subunit by gene knockout in animals could overcome these limitations. We review recent studies on nAChRs on autonomic ganglia for physiological and pharmacological properties and potential locations of the subunits.     

Differential nicotinic acetylcholine receptor subunit expression in the human hippocampus

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of alpha and beta subunits with specific structural, functional and pharmacological properties. In this study the distribution of alpha3, alpha4, alpha7, beta2 and beta4 nAChR subunits in the human hippocampus was investigated using immunohistochemistry. Most pyramidal neurons, pre-alpha cells of the entorhinal cortex and dentate granule cells were immunoreactive for all subunits. Small islands of alpha7 immunoreactive cells were present in the outer presubiculum. alpha4 and beta2, and alpha3, alpha4 and beta2 immunoreactive fibre tracts were present in the stratum radiatum and subiculum, respectively, suggesting nAChRs may play a role in modulating inputs to the hippocampus via Schaffer collaterals and along the perforant pathway. Some astrocytes were immunoreactive for alpha3, alpha7 and beta4 subunits. Immunoreactivity to all subunits was noted in association with blood vessels. These results indicate the involvement of multiple nAChR subtypes in the modulation of both neuronal and non-neuronal functions in the human hippocampus.     

alpha7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not beta-amyloid-induced neurotoxicity

The alpha7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, beta-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from alpha7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, "binge" ethanol and beta-amyloid. Knockout of the alpha7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to beta-amyloid induced toxicity, however, was unaffected by alpha7 nAChR gene null mutation. Further, beta-amyloid did not inhibit the binding of the highly alpha7-selective radioligand, [(125)I]alpha-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited alpha7 nAChR function. These data suggest that alpha7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by beta-amyloid. It is hypothesized that inhibition of alpha7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.     

Acetylcholine causes an increase of intracellular calcium in human sperm

Sperm nicotinic acetylcholine receptors (nAChRs) can influence motility and the initiation of acrosome reaction (AR). We report that AR initiation by acetylcholine (ACh) in capacitated human sperm requires both Na+ and Ca2+ in the external medium. Pre-incubation with 50 microM 3-quinuclidinyl benzilate (QNB) or 50 nM strychnine failed to inhibit the ACh-initiated AR, demonstrating that muscarinic AChRs and nAChRs containing alpha9 subunits do not mediate this event. Choline (2.5, 5 and 10 mM), a highly specific but low potency agonist of the alpha7 nAChR initiated AR, with its effect blocked by the nAChR antagonist methyllycaconitine (MLA). ACh (50-400 microM) stimulated a small transient rise in the intracellular Ca2+ in sperm populations loaded with FURA-2, with 200 microM ACh being maximal (146 nM +/- 23 SEM). The nAChR antagonists, alpha-bungarotoxin (alpha-BTX) and MLA, reduced the ACh-initiated Ca2+ rise by 75 and 78%, respectively, demonstrating the majority of the rise is mediated through nAChRs containing alpha7 or alpha9 subunits. Single cell imaging studies using FLUO-3 resolved two patterns of ACh-stimulated Ca2+ increase in the sperm head: 94% of responding sperm displayed a rise (59.6% +/- 5.7 SEM increase from resting fluorescence intensity), returning to resting levels over a period of 2-3 min. The remaining sperm (6%) displayed a sharp spike of Ca2+ ( approximately 1 min; 86% +/- 4.3 SEM change in fluorescence intensity), followed by abrupt loss of fluorescence, a pattern suggestive of AR. A Ca2+ influx in the sperm midpiece appeared to accompany the Ca2+ influx seen in the head. These observations confirm an ionotropic role for nAChRs in sperm function.     

Rat nicotinic acetylcholine receptor alpha2beta2 channels: comparison of functional properties with alpha4beta2 channels in Xenopus oocytes

Rat hippocampal interneurons express diverse subtypes of nicotinic acetylcholine receptors (nAChRs), including alpha7- and non-alpha7-containing receptors. Although the major subtype of non-alpha7 nAChRs in the hippocampus is thought to be composed of alpha4beta2 subunits, the molecular makeup of these non-alpha7 receptors is likely to be more complicated. Previously, we reported a high level of expression of the alpha2 nAChR subunit in individual rat hippocampal CA1 stratum oriens interneurons. In addition, the non-alpha7 nAChRs from these neurons are less sensitive to block by dihydro-beta-erythroidine (DHbetaE; the broad spectrum non-alpha7 nAChR antagonist) than that expected for alpha4beta2 receptors. We studied the functional properties of rat alpha2beta2 channels expressed in Xenopus oocytes using two-electrode voltage-clamp, and compared these to those properties of the more widely expressed and studied alpha4beta2 channels. Dose-response curves for both receptor subtypes indicated that there are at least two different affinity sites for ACh, the fractional contribution of which depended on the ratio of injected RNA. DHbetaE blocked both receptor subtypes, although the sensitivity to block of alpha4beta2 channels was significantly higher than that for alpha2beta2. Finally, the current-voltage (I-V) relationship for the alpha2beta2 channels more strongly rectified than for the alpha4beta2 channels. These data suggest that functional properties, in particular the sensitivity to block by DHbetaE, might be useful indicators to differentiate between native alpha4beta2 and alpha2beta2 channels. In addition, these data suggest that the relative balance between the high- and low-affinity components being determined by the relative levels of the alpha and beta subunits might be a general property of the heteromeric non-alpha7 nAChRs. By comparing the properties of expressed nAChRs with those of the native channels, we might be able to learn what role alpha2-containing nAChRs may be playing in forming functional channels in the hippocampus.     

Nicotinic acetylcholine receptors of adrenal chromaffin cells

In the adrenal medulla, acetylcholine released by the sympathetic splanchnic nerves activates neuronal-type nicotinic acetylcholine receptors (nAChRs) on the membrane of chromaffin cells which liberate catecholamines into the bloodstream in preparation for the fight and flight reactions. On adrenal chromaffin cells the main class of nAChRs is a pentameric assembly of alpha3 and beta4 subunits that forms ion channels which produce membrane depolarization by increasing Na+, K+ and Ca2+ permeability. Homomeric alpha7 nicotinic receptors are expressed in a species-dependent manner and do not contribute to catecholamine secretion. Chromaffin cell nAChRs rapidly activate and desensitize with full recovery on washout. nAChR activity is subjected to various types of dynamic regulation. It is allosterically modulated by the endogenous neuropeptide substance P that stabilizes receptors in their desensitized state, thus depressing their responsiveness. The full-length peptide CGRP acts as a negative allosteric modulator by inhibiting responses without changing desensitization, whereas its N-terminal fragments act as positive allosteric modulators to transiently enhance nAChR function. nAChR expression increases when cells are chronically exposed to either selective antagonists or agonists such as nicotine, a protocol mimicking the condition of chronic heavy smokers. In this case, large upregulation of nAChRs occurs even though most of the extra nAChRs remain inside the cells, creating a mismatch between the increase in total nAChRs and increase in functional nAChRs on the cell surface. These findings highlight the plastic properties of cholinergic neurotransmission in the adrenal medulla to provide robust mechanisms for adapting catecholamine release to acute and chronic changes in sympathetic activity.     

Young and older good learners have higher levels of brain nicotinic receptor binding

Neuronal alphabeta heteromeric and alpha7 homomeric nicotinic acetylcholine receptors (nAChRs) were compared in 4- and 27-month rabbits selected for learning proficiency. Sixty 4- and 60 27-month rabbits received the alpha7 nAChR agonist (MEM-3389), galantamine, or vehicle during training in trace eyeblink classical conditioning. Brain tissue from the best and worst young and older learners was analyzed with radioligand binding. Vehicle-treated 4- and 27-month good learners had higher alphabeta heteromeric nAChR binding in hippocampus and temporal-parietal cortex than poor learners, and this result was replicated in both age groups of rabbits treated with galantamine. Results indicate that anatomically more numerous nAChRs or functional activation of a greater number of nAChRs may characterize animals demonstrating optimal learning. During normal aging the expression of high-affinity binding sites declines. Age-related changes in the expression of hippocampal alphabeta heteromeric nAChRs may account for some of the documented age-related impairment in learning. However, individual differences in alphabeta heteromeric nAChRs also exist early in life, as better learning in 4-month rabbits was associated with significantly higher binding.     

Central role of fibroblast alpha3 nicotinic acetylcholine receptor in mediating cutaneous effects of nicotine

Smoking is associated with aberrant cutaneous tissue remodeling, such as precocious skin aging and impaired wound healing. The mechanism is not fully understood. Dermal fibroblasts (DF) are the primary cellular component of the dermis and may provide a target for pathobiologic effects of tobacco products. The purpose of this study was to characterize a mechanism of nicotine (Nic) effects on the growth and tissue remodeling function of DF. We hypothesized that the effects of Nic on DF result from its binding to specific nicotinic acetylcholine receptors (nAChRs) expressed by these cells and that downstream signaling from the receptors alters normal cell functioning, leading to changes in skin homeostasis. Using RT-PCR and Western blotting, we found that a 24-hour exposure of human DF to 10 micro M Nic causes a 1.9- to 28-fold increase of the mRNA and protein levels of the cell cycle regulators p21, cyclin D1, Ki-67, and PCNA and a 1.7- to 2-fold increase of the apoptosis regulators Bcl-2 and caspase 3. Nic exposure also up-regulated expression of the dermal matrix proteins collagen type Ialpha1 and elastin as well as matrix metalloproteinase-1. Mecamylamine (Mec), the specific antagonist of nAChRs, abolished Nic-induced alterations, indicating that they resulted from a pharmacologic stimulation of nAChRs expressed by DF. To establish the relevance of these findings to a specific nicotinergic pathway, we studied human DF transfected with anti-alpha3 antisense oligonucleotides and murine DF from alpha3 nAChR knockout mice. In both cases, lack of alpha3 was associated with alterations in fibroblast growth and function that were opposite to those observed in DF treated with Nic, suggesting that the nicotinic effects on DF were mostly mediated by alpha3 nAChR. In addition to alpha3, the nAChR subunits detected in human DF were alpha5, alpha7, beta2, and beta4. The exposure of DF to Nic altered the relative amounts of each of these subunits, leading to reciprocal changes in [(3)H]epibatidine-binding kinetics. Thus, some of the pathobiologic effects of tobacco products on extracellular matrix turnover in the skin may stem from Nic-induced alterations in the physiologic control of the unfolding of the genetically determined program of growth and the tissue remodeling function of DF as well as alterations in the structure and function of fibroblast nAChRs.     

Differential inhibition of rat alpha3* and alpha7 nicotinic acetylcholine receptors by tetrandrine and closely related bis-benzylisoquinoline derivatives

The patch-clamp technique was used to investigate the effects of bis-benzylisoquinoline alkaloids on two of the major neuronal nicotinic acetylcholine receptors (nAChRs), the alpha3-containing nAChR (alpha3*nAChR) endogenously expressed in PC12 cells and the rat alpha7-nAChR heterologously expressed in GH4C1 cells. Tetrandrine and hernandezine reversibly inhibited both receptors displaying half-maximal inhibitory concentrations (IC50) of 8.1 microM and 5.8 microM for alpha3*nAChR and 407.4 nM and 372.2 nM, respectively, for alpha7-nAChR. E6-berbamine completely inhibited the alpha3*nAChR with an IC50 of 5.1 microM, but only partially inhibited the alpha7-nAChR at concentrations up to 30 microM. Tetrandrine inhibition of alpha3*nAChR was functionally non-competitive. All three compounds displaced radiolabelled methyllycaconitine ([3H]-MLA) binding to alpha7-nAChR providing some evidence of competitive antagonism. The results demonstrate that these alkaloids are nAChRs antagonists, with tetrandrine and hernandezine displaying selectivity for one of the major neuronal subtype, the alpha7 nAChR. The different potencies and multiple modes of action on nAChRs may help to better understand the pharmacology of these receptors and to aid in novel drug design.     

NMDA and AMPA receptors contribute to the nicotinic cholinergic excitation of CA1 interneurons in the rat hippocampus

In the hippocampus, glutamatergic inputs to pyramidal neurons and interneurons are modulated by alpha7* and alpha3beta4* nicotinic acetylcholine receptors (nAChRs), respectively, present in glutamatergic neurons. This study examines how nicotinic AMPA, and NMDA receptor nAChR activities are integrated to regulate the excitability of CA1 stratum radiatum (SR) interneurons in rat hippocampal slices. At resting membrane potentials and in the presence of extracellular Mg2+ (1 mM), nicotinic agonists triggered in SR interneurons excitatory postsynaptic currents (EPSCs) that had two components: one mediated by AMPA receptors, and the other by NMDA receptors. As previously shown, nicotinic agonist-triggered EPSCs resulted from glutamate released by activation of alpha3beta4* nAChRs in glutamatergic neurons/fibers synapsing directly onto the neurons under study. The finding that CNQX caused more inhibition of nicotinic agonist-triggered EPSCs than expected from the blockade of postsynaptic AMPA receptors indicated that this nicotinic response also depended on the AMPA receptor activity in the glutamatergic neurons synapsing onto the interneuron under study. Nicotinic agonists always triggered action potentials in CA1 SR interneurons. In most interneurons, these action potentials resulted from activation of somatodendritic AMPA receptors and alpha7* nAChRs. In interneurons expressing somatodendritic alpha4beta2* nAChRs, activation of these receptors caused sufficient membrane depolarization to remove the Mg2+-induced block of somatodendritic NMDA receptors; in these neurons, nicotinic agonist-triggered action potentials were partially dependent on NMDA receptor activation. Removing extracellular Mg2+ or clamping the neuron at positive membrane potentials revealed the existence of a tonic NMDA current in SR interneurons that was unaffected by nAChR activation or inhibition. Thus integration of the activities of nAChRs, NMDA, and AMPA receptors in different compartments of CA1 neurons contributes to the excitability of CA1 SR interneurons.     

Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus

The superficial superior colliculus (sSC) is a key station in the sensory processing related to visual salience. The sSC receives cholinergic projections from the parabigeminal nucleus, and previous studies have revealed the presence of several different nicotinic acetylcholine receptor (nAChR) subunits in the sSC. In this study, to clarify the role of the cholinergic inputs to the sSC, we examined current responses induced by ACh in GABAergic and non-GABAergic sSC neurons using in vitro slice preparations obtained from glutamate decarboxylase 67-green fluorescent protein (GFP) knock-in mice in which GFP is specifically expressed in GABAergic neurons. Brief air pressure application of acetylcholine (ACh) elicited nicotinic inward current responses in both GABAergic and non-GABAergic neurons. The inward current responses in the GABAergic neurons were highly sensitive to a selective antagonist for alpha3beta2- and alpha6beta2-containing receptors, alpha-conotoxin MII (alphaCtxMII). A subset of these neurons exhibited a faster alpha-bungarotoxin-sensitive inward current component, indicating the expression of alpha7-containing nAChRs. We also found that the activation of presynaptic nAChRs induced release of GABA, which elicited a burst of miniature inhibitory postsynaptic currents mediated by GABA(A) receptors in non-GABAergic neurons. This ACh-induced GABA release was mediated mainly by alphaCtxMII-sensitive nAChRs and resulted from the activation of voltage-dependent calcium channels. Morphological analysis revealed that recorded GFP-positive neurons are interneurons and GFP-negative neurons include projection neurons. These findings suggest that nAChRs are involved in the regulation of GABAergic inhibition and modulate visual processing in the sSC.     

β-淀粉样蛋白和胆固醇对大鼠大脑类似阿尔茨海默病病理学进程和尼古丁受体的影响

目的探讨β-淀粉样蛋白（Ag）和胆固醇与阿尔茨海默病（AD）病理学进程和神经型尼古丁受体的关系。方法Wistar大鼠经侧脑室注射Aβ1-42（1mg／ml,5μl／只）来建立类似AD动物模型,并饲以5％胆固醇饮食。采用银染、免疫组织化学、水迷宫、Western blot和逆转录聚合酶链反应（RT-PCR）等方法检测实验动物脑组织形态学、学习记忆能力、神经型尼古丁受体亚单位表达水平等方面的改变。结果Aβ侧脑室注射引起大鼠脑组织中AB沉积;高胆固醇饮食造成大鼠明显高胆固醇血症（血胆固醇浓度较对照组增高21％～24％）;Aβ侧脑室注射引起大鼠学习记忆力降低,导致尼古丁受体α3、α4和α7亚单位蛋白水平降低（较对照组分别降低46％、39％及51％）,并引起酊亚单位mRNA表达水平升高41％-73％;高胆固醇饮食能够明显增强Aβ引起的脑组织中Aβ沉积和星形胶质细胞增多、学习记忆能力降低及尼古丁受体表达改变。结论Aβ能明显引起大鼠大脑病理学改变,降低大鼠学习记忆能力和影响神经型尼古丁受体的表达;高胆固醇饮食可增强Aβ的神经毒性作用及对尼古丁受体的影响。     

Dorsal root ganglion neurons express multiple nicotinic acetylcholine receptor subtypes

Although nicotinic agonists can modulate sensory transmission, particularly nociceptive signaling, remarkably little is known about the functional expression of nicotinic acetylcholine receptors (nAChRs) on primary sensory neurons. We have utilized molecular and electrophysiological techniques to characterize the functional diversity of nAChR expression on mammalian dorsal root ganglion (DRG) neurons. RT-PCR analysis of subunit mRNA in DRG tissue revealed the presence of nAChR subunits alpha2-7 and beta2-beta4. Using whole cell patch-clamp recording and rapid application of nicotinic agonists, four pharmacologically distinct categories of nicotinic responses were identified in cultured DRG neurons. Capacitance measurements were used to divide neurons into populations of large and small cells, and the prevalence of nicotinic responses was compared between groups. Category I (alpha7-like) responses were seen in 77% of large neurons and 32% of small neurons and were antagonized by 10 nM methyllycaconitine citrate (MLA) or or 50 nM alpha-bungarotoxin (alpha-BTX). Category II (alpha3beta4-like) responses were seen in 16% of large neurons and 9% of small neurons and were antagonized by 20 microM mecamylamine but not 10 nM MLA or 1 microM DHbetaE. Category II responses had a higher sensitivity to cytisine than nicotine. Two other types of responses were identified in a much smaller percentage of neurons and were classified as either category III (alpha4beta2-like) or category IV (subtype unknown) responses. Both the alpha7-like and alpha3beta4-like responses could be desensitized by prolonged applications of the analgesic epibatidine.