Populations Of Nicotinic Receptors Research Articles

Nicotinic Acetylcholine Receptors Expressed by Striatal Interneurons Inhibit Striatal Activity and Control Striatal-Dependent Behaviors.

Acetylcholine is an important modulator of striatal activity, and it is vital to controlling striatal-dependent behaviors, including motor and cognitive functions. Despite this significance, the mechanisms determining how acetylcholine impacts striatal signaling are still not fully understood. In particular, little is known about the role of nAChRs expressed by striatal interneurons. In the present study, we used FISH to determine which neuronal types express the most prevalent beta2 nicotinic subunit in the mouse striatum. Our data support a common view that nAChR expression is mostly restricted to striatal interneurons. Surprisingly though, cholinergic interneurons were identified as a population with the highest expression of beta2 nicotinic subunit. To investigate the functional significance of beta2-containing nAChRs in striatal interneurons, we deleted them by injecting the AAV-Cre vector into the striatum of beta2-flox/flox male mice. The deletion led to alterations in several behavioral domains, namely, to an increased anxiety-like behavior, decrease in sociability ratio, deficit in discrimination learning, and increased amphetamine-induced hyperlocomotion and c-Fos expression in mice with beta2 deletion. Further colocalization analysis showed that the increased c-Fos expression was present in both medium spiny neurons and presumed striatal interneurons. The present study concludes that, despite being relatively rare, beta2-containing nAChRs are primarily expressed in striatal neurons by cholinergic interneurons and play a significant role in behavior.SIGNIFICANCE STATEMENT A large variety of nAChRs are expressed in the striatum, a brain region that is crucial in the control of behavior. The complexity of receptors with different functions is hindering our understanding of mechanisms through which striatal acetylcholine modulates behavior. We focused on the role of a small population of beta2-containing nAChRs. We identified neuronal types expressing these receptors and determined their impact in the control of explorative behavior, anxiety-like behavior, learning, and sensitivity to stimulants. Additional experiments showed that these alterations were associated with an overall increased activity of striatal neurons. Thus, the small population of nicotinic receptors represents an interesting target for a modulation of response to stimulant drugs and other striatal-based behavior.

Patterns of Nicotinic Receptor Antagonism: Nicotine Discrimination Studies

Evaluation of the discriminative stimulus effects of drugs is a useful procedure for identification of receptor mediation of in vivo drug effects. This assay can be enhanced when the stimulus effects of different doses of agonist are evaluated. In the present study, rats were trained to discriminate small or large doses of nicotine from saline, and interactions of these effects with nicotinic receptor antagonists and partial agonists were determined. The insurmountable nicotine antagonist mecamylamine blocked both the discriminative stimulus and response rate-reducing effects of nicotine but was less effective against the large dose of nicotine. The α4β2*-selective, competitive antagonist dihydro-β-erythrodine (DHβE) antagonized the discriminative stimulus effects of both doses but was less effective against the larger training dose of nicotine. Schild analyses of DHβE suggested that different nicotinic receptor populations may be mediating the stimulus effects of large and small doses of nicotine. This suggestion was supported by observations that the discriminative stimulus effects of the partial agonist cytisine were more like those of the large dose than of the small dose of nicotine and that cytisine antagonized the effects of only the small nicotine dose. Varenicline produced nicotine-like effects in both training dose groups but reduced the discriminative stimulus effects of intermediate doses of nicotine in the group trained to the small dose of nicotine. Overall, these results suggest that small doses of nicotine produce their stimulus effects via α4β2* nicotine receptors, whereas larger doses of nicotine recruit additional nicotine receptor subtypes, as revealed by drug discrimination assays in rats.

Nicotine decreases DNA methyltransferase 1 expression and glutamic acid decarboxylase 67 promoter methylation in GABAergic interneurons

Tobacco smoking is frequently abused by schizophrenia patients (SZP). The major synaptically active component inhaled from cigarettes is nicotine, hence the smoking habit of SZP may represent an attempt to use nicotine self-medication to correct (i) a central nervous system nicotinic acetylcholine receptor (nAChR) dysfunction, (ii) DNA-methyltransferase 1 (DMT1) overexpression in GABAergic neurons, and (iii) the down-regulation of reelin and GAD(67) expression caused by the increase of DNMT1-mediated hypermethylation of promoters in GABAergic interneurons of the telencephalon. Nicotine (4.5-22 micromol/kg s.c., 4 injections during the 12-h light cycle for 4 days) decreases DNMT1 mRNA and protein and increases GAD(67) expression in the mouse frontal cortex (FC). This nicotine-induced decrease of DNMT1 mRNA expression is greater (80%) in laser microdissected FC layer I GABAergic neurons than in the whole FC (40%), suggesting selectivity differences for the specific nicotinic receptor populations expressed in GABAergic neurons of different cortical layers. The down-regulation of DNMT1 expression induced by nicotine in the FC is also observed in the hippocampus but not in striatal GABAergic neurons. Furthermore, these data show that in the FC, the same doses of nicotine that decrease DNMT1 expression also (i) diminished the level of cytosine-5-methylation in the GAD(67) promoter and (ii) prevented the methionine-induced hypermethylation of the same promoter. Pretreatment with mecamylamine (6 micromol/kg s.c.), an nAChR blocker that penetrates the blood-brain barrier, prevents the nicotine-induced decrease of FC DNMT1 expression. Taken together, these results suggest that nicotine, by activating nAChRs located on cortical or hippocampal GABAergic interneurons, can up-regulate GAD(67) expression via an epigenetic mechanism. Nicotine is not effective in striatal medium spiny GABAergic neurons that primarily express muscarinic receptors.

The nicotinic activation of the denervated sympathetic neuron of the rat

Nicotinic responses to endogenous acetylcholine and to exogenously applied agonists have been studied in the intact or denervated rat sympathetic neuron in vitro, by using the two-microelectrode voltage-clamp technique. Preganglionic denervation resulted in progressive decrease of the synaptic current (excitatory postsynaptic current, EPSC) amplitude, which disappeared within 24 h. These effects were accompanied by changes in ion selectivity of the nicotinic channel (nAChR). The extrapolated EPSC null potential (equilibrium potential for acetylcholine action, ESyn) shifted from a mean value of −15.9±0.7 mV, in control, to −7.4±1.6 mV, in denervated neurons, indicating a decrease of the permeability ratio for the main components of the synaptic current (PK/PNa) from 1.56 to 1.07. The overall properties of AChRs were investigated by applying dimethylphenylpiperazinium or cytisine and by examining the effects of endogenous ACh, diffusing within the ganglion after preganglionic tetanization in the presence of neostigmine. The null potentials of these macrocurrents (equilibrium potential for dimethylphenylpiperazinium action, EDMPP; and equilibrium potential for diffusing acetylcholine, EACh, respectively) were evaluated by applying voltage ramps and from current–voltage plots. In normal neurons, ESyn (−15.9±0.7 mV) was significantly different from EDMPP (−26.1±1.0) and EACh (−31.1±3.3); following denervation, nerve-evoked currents displayed marked shifts in their null potentials (ESyn=−7.4±1.6 mV), whereas the amplitude and null potential of the agonist-evoked macrocurrents were unaffected by denervation and its duration (EDMPP=−26.6±1.2 mV). It is suggested that two populations of nicotinic receptors, synaptic and extrasynaptic, are present on the neuron surface, and that only the synaptic type displays sensitivity to denervation.

Declines in different beta2* nicotinic receptor populations in monkey striatum after nigrostriatal damage.

In the present study we used the nicotinic ligand 5-iodo-A-85380 [5-iodo-3(2(S)-azetidinylmethoxy)pyridine], which selectively binds to beta2-containing nicotinic acetylcholine receptors, to elucidate the nicotinic receptor subtypes affected by nigrostriatal damage in the monkey. Autoradiographic studies in control monkeys showed that 5-[(125)I]A-85380 ([(125)I]A-85380) binds throughout the brain with the characteristics of a nicotinic receptor ligand. Competition experiments with cytisine and nicotine yielded K(i) values of approximately 1 and 10 nM, respectively, with complete inhibition of [(125)I]A-85380 binding at a 10(-6) M concentration of these ligands. In contrast, alpha-conotoxin MII blocked radioligand binding in the striatum by 30% at the highest concentrations, suggesting that a subset of striatal [(125)I]A-85380 sites are alpha-conotoxin MII-sensitive. Monkeys treated with the nigrostriatal neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed a selective decrease in striatal [(125)I]A-85380 sites, with a 42% reduction in the caudate and putamen of animals with moderate nigrostriatal lesioning and a 53% decline in the striatum of severely lesioned animals. Our previous work had demonstrated that there were two populations of nicotinic receptors eliminated after nigrostriatal damage, an alpha-conotoxin MII-sensitive and an alpha-conotoxin MII- resistant subtype. Analysis of both striatal [(125)I]A-85380 and [(125)I]epibatidine competition studies extend our earlier studies by demonstrating that the alpha-conotoxin MII-sensitive sites eliminated after moderate nigrostriatal lesioning appear to be composed of two nicotinic receptor subtypes. The data may be important for potential therapeutic approaches because they suggest that there are at least three populations of nicotinic receptors in monkey striatum, of which two are selectively vulnerable to nigrostriatal damage, while the third is more resistant.

Differential nicotinic receptor expression in monkey basal ganglia: Effects of nigrostriatal damage

Our previous work showed that there were marked declines in 125I-α-conotoxin MII labeled nicotinic receptors in monkey basal ganglia after nigrostriatal damage, findings that suggest α3/α6 containing nicotinic receptors sites may be of relevance to Parkinson’s disease. We now investigate whether there are differential changes in the distribution pattern of nicotinic receptor subtypes in the basal ganglia in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned animals compared to controls to better understand the changes occurring with nigrostriatal damage. To approach this we used 125I-α-conotoxin MII, a marker for α3/α6 nicotinic receptors, and 125I-epibatidine, a ligand that labels multiple nicotinic subtypes. The results demonstrate that there were medial to lateral gradients in nicotinic receptor distribution in control striatum, as well as ventromedial to dorsolateral gradients in the substantia nigra, which resembled those of the dopamine transporter in these same brain regions. Treatment with MPTP, a neurotoxin that selectively destroys dopaminergic nigrostriatal neurons, led to a relatively uniform decrease in nicotinic receptor sites in the striatum, but a differential effect in the substantia nigra with significantly greater declines in the ventrolateral portion. Competition analysis in the striatum showed that α-conotoxin MII sensitive sites were primarily affected after lesioning, whereas multiple nicotinic receptor populations were decreased in the substantia nigra. From these data we suggest that in the striatum α3/α6 nicotinic receptors are primarily localized on dopaminergic nerve terminals, while multiple nicotinic receptor subtypes are present on dopaminergic cell bodies in the substantia nigra. Thus, if activation of striatal nicotinic receptors is key in the regulation of basal ganglia function, α3/α6-directed nicotinic receptor ligands may be more relevant for Parkinson’s disease therapy. However, nicotinic receptor ligands with a broader specificity may be more important if receptors in the substantia nigra play a dominant role in controlling nigrostriatal activity.

Nicotine and Nicotinic Receptors; Relevance to Parkinson’s Disease

The development of nicotinic agonists for therapy in neurodegenerative disorders such as Parkinson’s disease is an area currently receiving considerable attention. The rationale for such work stems from findings that reveal a loss of nicotinic receptors in Parkinson’s disease brains. These results, coupled with reports that nicotine treatment relieves some of the symptoms of this disorder, provides support for the contention that nicotine and/or nicotinic agonists may be beneficial for acute symptomatic treatment. Moreover, the observation that there is a decreased incidence of Parkinson’s disease with tobacco use, possibly due to the nicotine in tobacco products, may imply that such drugs are useful for long-term neuroprotection. However, there are multiple nicotinic receptor populations in the brain with different functional properties. Identification of the subtypes involved in nigrostriatal dopaminergic activity is therefore critical for the rational use of selective therapeutic agents for symptomatic treatment and/or neuroprotection. Accumulating evidence, both in rodents and nonhuman primates now indicate that α6 ∗ 1 Asterisk denotes receptors containing a specific α and/or β nicotinic subunit. 1 nicotinic receptors are present on nigrostriatal dopaminergic neurons, and furthermore, that receptors containing this subunit may be most vulnerable to nigrostriatal damage, at least in nonhuman primates. These data suggest that nicotinic receptor ligands directed to α6 ∗ nicotinic receptors might be particularly relevant for Parkinson’s disease therapeutics.

Stimulation of non-α7 nicotinic receptors partially protects dopaminergic neurons from 1-methyl-4-phenylpyridinium-induced toxicity in culture

Previous work has shown that nicotine treatment protects against nigrostriatal degeneration in rodents, findings that may be of relevance to the decreased incidence of Parkinson’s disease in cigarette smokers. In the present studies, we investigated the effect of nicotine against 1-methyl-4-phenylpyridinium-induced toxicity in dopaminergic ventral mesencephalic cultures to identify the nicotinic receptor population that may be involved. [ 3H]Epibatidine, a ligand that binds to receptors containing α2–α6 subunits, bound to at least two populations of sites that were up-regulated by nicotine in a time and dose dependent manner. We next examined the effect of nicotine on cultures treated with 1-methyl-4-phenylpyridinium, a neurotoxin that selectively damages nigrostriatal dopaminergic neurons. Pre-treatment with nicotine, at 10 −7–10 −4 M, partially prevented the toxin-induced decline in dopaminergic cells. Pre-exposure to nicotine for 24 h resulted in optimal protection, suggesting that receptor up-regulation may contribute to the observed neuroprotective effect. Nicotine-mediated protection was blocked by pre-incubation with the nicotinic receptor antagonist d-tubocurarine (10 −4 M), but not the α7 receptor-selective antagonist α-bungarotoxin (10 −7 M). Our results show that nicotinic receptor activation partially protects nigral dopaminergic neurons from 1-methyl-4-phenylpyridinium-induced toxicity in culture and that this appears to occur through an interaction at non-α7 containing receptors.

Tonic nicotinic modulation of serotoninergic transmission in the spinal cord.

The spinal serotoninergic projection from the raphe magnus has been shown to modulate nociceptive inputs, and activation of this projection mediates nicotine-elicited analgesia. Here, we investigate the interactions between cholinergic and serotoninergic systems in the spinal cord, by conducting serotonin [5-hydroxytryptamine (5-HT)] efflux experiments on mouse spinal slices. At least three spinal populations of nicotinic receptors are distinguished that affect 5-HT release. The first could be directly located on serotoninergic terminals, is insensitive to nanomolar concentrations of methyllicaconitine (MLA), and may be subjected to a basal (not maximal) cholinergic tone. The second is tonically and maximally activated by endogenous acetylcholine, insensitive to nanomolar concentrations of MLA, and present on inhibitory neurons. The last is also present on inhibitory neurons but is sensitive to nanomolar concentrations of MLA and not tonically activated by acetylcholine. Multiple nicotinic acetylcholine receptor populations thus differentially exert tonic or not tonic control on 5-HT transmission in the spinal cord. These receptors may be major targets for nicotine effects on antinociception. In addition, the presence of a tonic nicotinic modulation of 5-HT release indicates that endogenous acetylcholine plays a role in the physiological regulation of descending 5-HT pathways to the spinal cord.

Effect of neostigmine on the hippocampal noradrenaline release: role of cholinergic receptors.

The effect of the cholinesterase inhibitor neostigmine on hippocampal noradrenaline (NA) release was studied using in vivo microdialysis. Local application of neostigmine significantly increased the release of NA. The effect was potentiated by coperfusion of the nicotinic antagonist mecamylamine but was completely blocked by the muscarinic antagonist atropine. The neostigmine-evoked NA release was not affected by the M2-selective muscarinic antagonist gallamine but was completely blocked by the M1-selective muscarinic antagonist pirenzepine. While muscarinic antagonists had no effect on the resting release of NA, mecamylamine increased it. Our data indicate that acetylcholine can stimulate the hippocampal NA release via M1 muscarinic receptors and that a population of nicotinic receptors mediate inhibitory tone on hippocampal NA release. The fact that neostigmine is able to enhance both cholinergic and noradrenergic neurotransmission may help to understand the beneficial effect of cholinesterase inhibitors in Alzheimer's disease.

Effects of α-erabutoxin, α-bungarotoxin, α-cobratoxin and fasciculin on the nicotine-evoked release of dopamine in the rat striatum in vivo

Snake neurotoxins (NTX) have proven to be valuable tools for the characterisation of muscular nicotinic acetylcholine receptor structure and function. It is very likely that they could also be utilised to identify subtypes of neuronal nicotinic receptors controlling specific functions within the central nervous system. In this study we examined the effects of long alpha NTX (α-bungarotoxin, α-Bgt, and α-cobratoxin, α-Cbt) and short alpha NTX (α-erabutoxin a, α-Ebt) as well as the anticholinesterase toxin fasciculin-2 (FAS), on the nicotine-evoked release of dopamine (DA) in the striatum, using the in vivo push-pull technique. The short toxins α-Ebt and FAS blocked the extracellular increase of DA evoked by nicotine at 4.2 μM concentrations and α-Ebt was more potent, as reflected by the blockade at the lower dose of 0.42 μM. In contrast, the long toxins showed a different profile of action. α-Cbt did not show any blockade of the nicotine-evoked release of DA at the doses studied while α-Bgt did block it only at the higher dose (4.2 μM) These results indicate that short neurotoxins show a stronger interaction with striatal nicotinic receptors subtypes controlling DA release when compared to the long ones. This interaction of short neurotoxin polypeptides and presynaptic receptors may permit the further elucidation of the particular nicotinic receptor populations responsible for the modulation of striatal DA release.

A population of nicotinic receptors is associated with thalamocortical afferents in the adult rat: laminal and areal analysis.

In the adult rat brain, a prominent population of nicotinic cholinoceptors binds 3H-nicotine with nanomolar affinity. These receptors are abundant in most thalamic nuclei and in neocortical layers 3/4, which receive a major thalamic input. To test whether cortical nicotinic receptors are associated with thalamocortical afferents, unilateral excitotoxic (N-methyl-D-aspartate) lesions were made in one of four thalamic nuclear groups (anterior, ventral, medial geniculate, or dorsal lateral geniculate) or in temporal cortex. After 1 or 4 weeks of survival, cortical 3H-nicotine binding was quantified via autoradiography. Thalamic lesions resulted in a partial loss of 3H-nicotine binding in ipsilateral cerebral cortex. In each thalamic lesion group, the greatest decrease (35-45%) occurred within the cortical layers and area (i.e., cingulate, parietal, temporal, or occipital cortex) receiving the densest thalamocortical innervation. Binding of 3H-nicotine was also reduced within the thalamus local to the lesion, particularly at the longer survival time. Saturation analysis, performed in frontoparietal cortical tissue homogenates following ventral thalamic lesions, revealed a significant (34%) reduction in receptor density but not affinity. Direct excitotoxic lesions of the neocortex (temporal cortex) tended to preserve 3H-nicotine binding in layers 3/4, despite local neuronal loss. These results, taken with other published findings, suggest that some nicotinic cholinoceptors in adult rat cerebral cortex are located on thalamocortical terminals. This organizing principle appears to apply not only to sensory and motor relay projections but also to association nuclei that project to allocortical areas. These receptors may provide a local mechanism for nicotinic cholinergic modulation of thalamocortical input.

Locomotor activation and dopamine release produced by nicotine and isoarecolone in rats

1. Isoarecolone was approximately 250 times less potent than nicotine as an inhibitor of [3H]-nicotine binding to rat brain membranes. Isoarecolone failed to inhibit the binding of the nicotinic ligand [125I]-alpha-bungarotoxin or of the muscarinic ligand [3H]-QNB. 2. Nicotine (0.01-30 microM) evoked the release of [3H]-dopamine from striatal and frontal cortex synaptosomes, with EC50 values of approximately 0.5 microM in each case. This release was largely mecamylamine-sensitive. 3. Isoarecolone (1-200 microM) evoked predominantly mecamylamine-sensitive dopamine release from both striatal and cortical synaptosomes, with a potency at least 20 times less than that of nicotine. The maximum effect of isoarecolone was less than that of nicotine, particularly in the frontal cortex preparation. 4. In control rats treated chronically with saline, neither nicotine nor isoarecolone had clear effects on locomotor activity at the doses tested. Chronic treatment with nicotine clearly sensitized rats to the locomotor activating effect of isoarecolone was seen at a dose about 40 times larger than that of nicotine. 5. The low potency and efficacy of isoarecolone in facilitating sensitized locomotor activity resembled its lower potency and efficacy, compared with nicotine, in evoking dopamine release in vitro. The agonist profile of the nicotinic receptor population mediating dopamine release may determine the pharmacological characteristics of consequent locomotor behaviour.

Neural mechanisms underlying nicotine dependence

There is little doubt that many habitual smokers find it difficult to quit the habit because they have become addicted to the nicotine present in the smoke. This paper addresses some of the pharmacological mechanisms underlying this addiction and discusses how an understanding of these mechanisms may contribute to the more effective use of nicotine replacement therapy during smoking cessation. It considers critically the evidence that the "rewarding" properties of nicotine, which serve to reinforce drug-seeking behaviour, are related to stimulation of the mesolimbic dopamine system of the brain. The critique focuses specifically on the evidence that many central nicotinic receptors, including those which mediate the effects of the drug on dopamine secretion, are readily desensitized by chronic exposure to agonist and that hypotheses which assume that nicotine inhaled from tobacco smoke invariably results in stimulation of the receptors must be treated with caution. Nicotinic receptors in the brain are, however, heterogeneous in nature with different molecular structures and pharmacologies. It is concluded that the reinforcing properties of nicotine sought by smokers may reflect both stimulation and desensitization of the different nicotinic receptor populations, and that smokers may adjust their smoking habits to achieve the balance of receptor stimulation and desensitization which they find most reinforcing. It seems likely that the efficacy of the different nicotine formulations during the treatment of smoking cessation may also reflect their ability to stimulate or desensitize brain nicotinic receptors.

Properties of α-bungarotoxin binding cholinergic receptors in pyrethroid susceptible and resistant tobacco budworm moths

1. In membrane preparations from whole head homogenates of tobacco budworm, Heliothis virescens (F.), moths from a pyrethroid susceptible (S) strain and from a pyrethroid resistant (R) strain with multiple resistance mechanisms including nerve insensitivity, the nicotinic acetylcholine receptor antagonist [ 125I]-α-bungarotoxin (BGT) was found to bind reversibly with high affinity to a single class of noninteracting high density sites. Although no significant interstrain differences were found in saturation experiments with regard to radioligand affinity and number of binding sites, half-times for [ 125I]BGT dissociation were about 3 min longer in S than in R moth preparations. 2. Nicotinic drugs generally were more potent than muscarinic drugs in displacing bound [ 125I]BGT from membranes of both strains. There were no significant interstrain differences with d-tubocurarine, decamethonium, (±)-quinuclidinyl benzilate, and (−)-scopolamine; however, (−)-nicotine (by 10×) and atropine (by 2×) were more potent in displacing [ 125I]BGT bound to membrane from S than from R moths. 3. Five pyrethroids, four formamidines, and one carbamate were without significant effect on [ 125I]BGT binding in membranes from both strains when tested at concentrations of 1 × 10 −5 and 1 × 10 −7 M. However, DDT at 1 × 10 −5 M gave about 20% stimulation of [ 125I]BGT binding in preparations from S moths. 4. It was concluded that [ 125I]BGT was binding to populations of nicotinic receptors in membranes from S and R moths. Although some differences may exist in the nature of the receptors between the two strains, studies with insecticides did not provide any basis for associating nerve insensitivity resistance to pyrethroids with tobacco budworm moth nicotinic receptors as probed with [ 125I]BGT.

Thymopoietin, a thymic polypeptide, potently interacts at muscle and neuronal nicotinic alpha-bungarotoxin receptors.

Current studies suggest that several distinct populations of nicotinic acetylcholine (ACh) receptors exist. One of these is the muscle-type nicotinic receptors with which neuromuscular nicotinic receptor ligands and the snake toxin alpha-bungarotoxin interact. alpha-Bungarotoxin potently binds to these nicotinic receptors and blocks their function, two characteristics that have made the alpha-toxin a very useful probe for the characterization of these sites. In neuronal tissues, several populations of nicotinic receptors have been identified which, although they share a nicotinic pharmacology, have unique characteristics. The alpha-bungarotoxin-insensitive neuronal nicotinic receptors, which may be involved in mediating neuronal excitability, bind nicotinic agonists with high affinity but do not interact with alpha-bungarotoxin. Subtypes of these alpha-toxin-insensitive receptors appear to exist, as evidenced by findings that some are inhibited by neuronal bungarotoxin whereas others are not. In addition to the alpha-bungarotoxin-insensitive sites, alpha-bungarotoxin-sensitive neuronal nicotinic receptors are also present in neuronal tissues. These latter receptors bind alpha-bungarotoxin with high affinity and nicotinic agonists with an affinity in the microM range. The function of the nicotinic alpha-bungarotoxin receptors are as yet uncertain. Thymopoietin, a polypeptide linked to immune function, appears to interact specifically with nicotinic receptor populations that bind alpha-bungarotoxin. Thus, in muscle tissue where alpha-bungarotoxin both binds to the receptor and blocks activity, thymopoietin also potently binds to the receptor and inhibits nicotinic receptors-mediated function. In neuronal tissues, thymopoietin interacts only with the nicotinic alpha-bungarotoxin site and not the alpha-bungarotoxin-insensitive neuronal nicotinic receptor population. These observations that thymopoietin potently and specifically interacts with nicotinic alpha-bungarotoxin-sensitive receptors in neuronal and muscle tissue, together with findings that thymopoietin is an endogenously occurring agent, could suggest that this immune-related polypeptide represents a ligand for the alpha-bungarotoxin receptors. The function of thymopoietin at the alpha-bungarotoxin receptor is as yet uncertain; however, a potential trophic, as well as other roles are suggested.

Thymopoietin, a thymic polypeptide, specifically interacts at neuronal nicotinic alpha-bungarotoxin receptors.

alpha-Bungarotoxin (alpha-BGT), a snake venom polypeptide, interacts potently and specifically with a nicotinic receptor population in neuronal tissue. However, the identity of this site is unclear, because, unlike at the neuromuscular junction and in electroplax, in nervous tissue the toxin does not block nicotinic cholinergic responses. Therefore, we sought endogenous compounds other than acetylcholine that could interact with the neuronal alpha-BGT site. In the present experiments, thymopoietin, a polypeptide isolated from the thymus, is shown to inhibit potently alpha-BGT binding to brain membranes in a dose-dependent manner (IC50 = 3.1 nM). This effect was not shared by a wide variety of other peptides, including thysplenin, a closely related polypeptide. Thymopoietin did not inhibit the binding of other radioligands known to interact with different populations of cholinergic receptors, such as [3H]nicotine and [3H]methylcarbachol, which bind to nicotinic receptors, or [3H]quinuclidinylbenzilate, which binds to muscarinic receptors. These results show that thymopoietin potently and specifically affects 125I-alpha-BGT binding to brain membranes and suggest that thymopoietin might be an endogenous ligand for alpha-BGT receptors in neuronal tissue.

Distribution and density of cholinergic receptors at the motor endplates of a denervated mouse muscle

The localization and quantitation of the acetylcholine receptor at the normal and 5-day denervated mouse diaphragm endplate has been evaluated by means of labeling with tritiated α-bungarotoxin followed by electron microscope autoradiography. The toxin binds specifically and irreversibly to the acetylcholine receptor molecules of skeletal muscle. The distribution of silver grains over denervated endplates labeled to saturation with this toxin is consistent with a receptor location on the postjunctional membrane at a mean density similar to that of the innervated endplate, 8,500/μm 2. As in the innervated endplate, the receptors are not located uniformly along the membrane, but are concentrated at the fold crests, i.e., that area nearest to the axonal membrane when it is present, at a density near to 22,000/μm 2. Since morphometric measurements show that the total postjunctional membrane at the denervated endplate is unchanged at 5 days, we deduce from the mean receptor site density that the total receptor population at the endplate remains the same. On the basis of these findings, we conclude that only a very small population of nicotinic receptors could be associated with the terminal axon, and perhaps that none at all are present there. Further, the receptors on the postjunctional membrane are unaffected in either density or distribution following the removal of neural influences by denervation even though these same influences have a profound effect on extrajunctional receptors at later periods of denervation were not possible due to degenerative tissue changes associated with denervation.