Chronic nicotine administration does not increase nicotinic receptors labeled by [125I]epibatidine in adrenal gland,superior cervical ganglia,pineal or retina

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in CNS and peripheral tissues following continuous exposure to saline or nicotine hydrogen tartrate (3.3 or 10 mg/kg/day) for 14 days via osmotic pumps. Initially, binding of [3H](-)nicotine, [3H]cytisine and [3H]epibatidine to nAChRs was compared to determine the suitability of each for these kinds of studies. The predominant nAChR labeled by agonists in the cerebral cortex is an alpha 4 beta 2 subtype, whereas the predominant nicotinic receptors in the adrenal gland, superior cervical ganglia and pineal gland contain an alpha 3 subunit, and they do not bind either [3H](-)nicotine or [3H]cytisine with high affinity. In retina some nAChRs bind all three ligands with high affinity, and others appear to bind only [3H]epibatidine. Thus, only [3H]epibatidine had high enough affinity to be useful for measuring the nAChRs in all of the tissues. The receptors from nicotine-treated rats were then measured using [125I]epibatidine, which has binding characteristics very similar to [3H]epibatidine. Treatment with the two doses of nicotine hydrogen tartrate increased binding sites in the cerebral cortex by 40% and 70%, respectively. In contrast, no significant changes in the density of receptor binding sites were found in the adrenal gland, superior cervical ganglia, pineal gland or retina. These data indicate that chronic administration of nicotine even at high doses does not increase all nicotinic receptor subtypes, and that receptors containing alpha 3 subunits may be particularly resistant to this nicotine-induced change.     

脊髓背角痛觉传递和调制的一些化学解剖学观察

本实验研究了脊髓背角内Ｃ纤维末梢的分布和突触学特征及其一些神经递质化学构筑;定量观察了急性痛引起背角的递质变化;显示了初级传入Ｃ纤维,抑制性中间神经元和背角伤害性感受神经元三者之间的突触关系,并探讨它们在痛觉信息传递和调制中的作用。     

Synaptic organization of substance P, glutamate and GABA-immunoreactive boutons on functionally identified neurons in cat spinal deeper dorsal horn

In order to determine how nociceptive input conveyed by the C-fibers terminating in superficial lam-inae of the spinal cord reaches the wide dynamic range (WDR) cells in deeper dorsal horn, which functions as ascend-ing projection pathway, the morphological features of some WDR cells in the deeper dorsal horn of the cat lumbar spinal cord were studied by intracellular injection of horseradish peroxidase and physiological characterization. One of the fully stained neurons with somata in lamina V and dendrites that entered lamina Ⅱ were examined by electron mi-croscopy. Immunogold staining of ultrathin sections through the labeled proximal dendrites in lamina Ⅱ revealed that these dendrites received numerous synapses from substance P and glutamate immunoreactive (IR) axons, which were considered originating from C-fibers. In addition, many GABA-IR terminals were found presynaptic to the labeled dendrites. The results, therefore, suggest that the information carried by primary afferent can be sent from t     

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5-[125I]iodo-3-(2-azetidinylmethoxy)pyridine ([125I]A-85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A-85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 receptors. Although the predominant subtype in rat brain was alpha4beta2, non-alpha4beta2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, alpha3beta4-like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in alpha3beta4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in alpha3beta2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.     

Binding and functional activity of nicotinic cholinergic receptors in selected rat brain regions are increased following long-term but not short-term nicotine treatment

Chronic nicotine exposure up-regulates neuronal nicotinic receptors, but the functional consequences for these receptors is less well understood. Following 2 weeks of nicotine or saline treatment by osmotic minipump, the functional activity of nicotinic receptors was measured by concentration-response curves for epibatidine-stimulated (86)Rb efflux. Nicotine-treated animals had a significantly higher maximal efflux in cerebral cortex and superior colliculus, but not in thalamus or interpeduncular nucleus plus medial habenula. This increase was confirmed in a separate experiment with stimulation by single concentrations of epibatidine (cortex, superior colliculus) or nicotine (cortex only). Chronic nicotine did not alter (86)Rb efflux stimulated by cytisine, an alpha3beta4-selective agonist, or by potassium chloride, in any region. Short-term (16 h) nicotine exposure caused no changes in either (86)Rb efflux or receptor binding measured with [(3)H]epibatidine. Binding was significantly increased after 2 weeks nicotine exposure in cortex, superior colliculus and thalamus, but not in interpeduncular nucleus plus medial habenula. The increases in epibatidine-stimulated (86)Rb efflux in the four regions tested was linearly correlated with the increases in [(3)H]epibatidine binding in these regions (R(2) = 0.91), suggesting that rat brain receptors up-regulated by chronic nicotine are active. These results have important consequences for understanding nicotinic receptor neurobiology in smokers and users of nicotine replacement therapy.     

Effects of sulfhydryl modification on adrenal nicotinic acetylcholine receptors: disulfide integrity is not essential for activation

The importance of disulfide bridges in muscle nicotinic receptors is well established; however, for neuronal nicotinic receptors, the effects of sulfhydryl modification are less definitive. In these studies the effects of treatment with the mild reducing agent, dithiothreitol, on adrenal nicotinic receptors are described. We have found that following dithiothreitol treatment, adrenal chromaffin cells retained the ability to be stimulated by a variety of nicotinic receptor agonists including nicotine, acetylcholine, cytisine, epibatidine, and bromoacetylcholine. However, with dithiothreitol treatment, changes in apparent affinities were seen with two agonists, epibatidine and bromoacetylcholine. These effects of dithiothreitol on apparent affinities were concentration-dependent and reversible upon treatment with an oxidizing agent. Dithiothreitol treatment also produced effects on secretion that were independent of nicotinic receptor activation. Our results are unlike those in other tissues containing nicotinic receptors and suggest that subunit composition of nicotinic receptors influences the functional outcome of sulfhydryl modification.     

Transduction and transmission properties of primary nociceptive afferents.

The prototypical primary nociceptive afferent is the polymodal C-fiber nociceptor, which responds to noxious thermal, mechanical, and chemical stimuli. C-fiber nociceptors are peripheral terminals of small neurons in the dorsal root ganglia (DRG). DRG neurons must therefore supply their peripheral terminals with the molecular machinery for the encoding of noxious stimuli into trains of action potentials. The following phenomena are known for this encoding process in vivo: 1) adaptation: for a constant stimulus intensity the action potential discharge decreases slowly within 2-3 seconds, 2) fatigue: recovery from adaptation may take ten minutes or more, 3) sensitization: preceding tissue damage enhances the response, particularly to heat stimuli. Recent studies in vitro have provided important clues about the molecular mechanisms underlying these phenomena. Several membrane receptors and channels are specifically expressed in small nociceptive neurons, such as vanilloid receptors (VR1), purinergic receptors (P2X3), acid sensing ion channels (ASIC), and TTX-resistant Na-channels. In the near future, we may therefore expect major advances in our understanding of the transduction of noxious stimuli into generator potentials and transformation into trains of action potentials. Along the axon that leads from the innervated tissue to the spinal cord, primary nociceptive afferents have a limited capacity to transmit high impulse rates, suggesting a different composition of voltage-gated channels than in other primary afferents (low-threshold mechanoreceptors and thermoreceptors). Finally, the DRG neuron also supplies its central terminals with the molecular machinery for synaptic transmission and its presynaptic modulation. Progress in understanding the cellular mechanisms at both ends of the primary nociceptive neuron promises to lead to new analgesic treatment modalities for both acute and chronic pain.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

Rigidified acetylcholine mimics: conformational requirements for binding to neuronal nicotinic receptors

Rigidified derivatives have been designed and synthesized assuming the g+t conformer of acetylcholine (N-C-C-O=+60 degrees, C-C-O-C=180 degrees ) as active conformation for binding to cytisine sensitive neuronal nicotinic receptors. The SAR of the compounds evaluated, along with those of more flexible analogues, support the g+t conformer hypothesis and highlight the stringent steric limitation of this nicotinic receptor sub-type. Compound 3e has low microM affinity for cytisine sensitive nicotinic receptor binding sites while being selective with regard to the alpha-bungarotoxin sensitive subclass. We also report few compounds with microM affinity for the alpha-bungarotoxin sensitive subclass.     

α3, β2, and β4 Form Heterotrimeric Neuronal Nicotinic Acetylcholine Receptors in Xenopus Oocytes

Abstract: One of the problems faced when using heterologous expression systems to study receptors is that the pharmacological and physiological properties of expressed receptors often differ from those of native receptors. In the case of neuronal nicotinic receptors, one or two subunit cDNAs are sufficient for expression of functional receptors in Xenopus oocytes. However, the stoichiometries of nicotinic receptors in neurons are not known and expression patterns of mRNA coding for different nicotinic receptor subunits often overlap. Consequently, one explanation for the discrepancy between properties of native versus heterologously expressed nicotinic receptors is that more than two types of subunit are necessary for correctly functioning receptors. The Xenopus oocyte expression system was used to test the hypothesis that more than two types of subunit can coassemble; specifically, can two different β subunits assemble with an α subunit forming a receptor with unique pharmacological properties? We expressed combinations of cDNA coding for α3, β2, and β4 subunits. β2 and β4, in pairwise combination with α3, are differentially sensitive to cytisine and neuronal bungarotoxin (nBTX). α3β4 receptors are activated by cytisine and are not blocked by low concentrations of nBTX; acetylcholine-evoked currents through α3β2 receptors are blocked by both cytisine and low concentrations of nBTX. Coinjection of cDNA coding for α3, β2, and β4 into oocytes resulted in receptors that were activated by cytisine and blocked by nBTX, thus demonstrating inclusion of both β2 and β4 subunits in functional receptors.     

Two subgroups of lung vagal C-fibers with different vulnerabilities to blockades by perivagal capsaicin and vagal cooling in dogs

Perivagal capsaicin treatment and vagal cooling are two techniques that have been widely used to study the respiratory reflexes mediated by lung vagal C-fibers because they can block the neural conduction of unmyelinated fibers. We hypothesized that there are two subgroups of lung vagal C-fibers which have different vulnerabilities to blockades by these two techniques. To test this hypothesis, afferent activity arising from lung vagal C-fibers was recorded in 29 anesthetized, paralyzed, and artificially ventilated dogs. Afferent C-fiber activity was recorded before and after various concentrations of perivagal capsaicin treatment or before and during various temperatures of vagal cooling. Of the 89 lung vagal C-fibers studied, 73 fibers were classified as the group of "low resistance" to capsaicin, while the other 16 were classified as the group of "high resistance". The former group differed from the latter due to their afferent activity being blocked at relatively low concentrations of perivagal capsaicin and at relatively low temperatures of vagal cooling. Our results suggest that lung vagal C-fibers can be categorized into two subgroups, based upon their different blocking thresholds for perivagal capsaicin and vagal cooling. Our data may provide information for researchers to further differentiate the respiratory reflexes originating from these two subgroups of lung vagal C-fibers.     

The mechanisms underlying long-term potentiation of C-fiber evoked field potentials in spinal dorsal horn

Long-term potentiation (LTP) of C-fiber evoked feld potentials in spinal dorsal horn is first reported in 1995. Since then, the mechanisms underlying the long-lasting enhancement in synaptic transmission between primary afferent C-fibers and neurons in spinal dorsal horn have been investigated by different laboratories. In this article, the related data were summarized and discussed.     

N-methylcytisine--a selective ligand of nicotinic receptors of acetylcholine in the CNS

The ability of cytisine and its N-methyl derivatives to bind to nicotinic acetylcholine receptors (nAChR) from different tissues was studied. Cytisine and N-methylcytisine have high affinity (KD = 50 nM) to nAChR from squid optical ganglia. N,N-dimethylcytisine did not show high affinity to this receptor. In the case of nAChR from T. marmorata, cytisine was the only effective inhibitor of 14C-tubocurarine specific binding (Ki = 700 nM). N-methyl- and N,N-dimethylcytisine did not displace 14C-tubocurarine at a concentration of 0.1 mM. The results obtained indicate that there are some differences in the structure of nAChR binding sites from squid and T. marmorata optical ganglia.     

Neurotoxic effect of capsaicin in mammals

Capsaicin is now widely used to explore and/or prove the role of peptide-containing primary afferent neurones in different somato- and viscerosensory functions. The present paper deals with the morphological effects of capsaicin administered according to currently used experimental paradigms. As it has been repeatedly confirmed in the recent literature, administration of capsaicin to newborn mammals results in a highly selective degeneration of a particular population of small sized, B-type primary afferent neurones located in spinal and cranial sensory ganglia. Chemosensitive i.e. capsaicin sensitive primary sensory neurones (CPSNs) correspond to primary sensory ganglion cells which contain neuropeptides. The permanent functional impairments and the decrease in the peptide contents of the sensory neurones observed after neonatal capsaicin treatment may be accounted for an irreversible loss of CPSNs. Direct application of capsaicin to peripheral nerves results in an apparently irreversible functional impairment of unmyelinated afferent fibres implicated in nociceptive, viscerosensory and neurogenic inflammatory mechanisms. Morphological observations indicate that perineural treatment with capsaicin initiates a selective but delayed degeneration process of unmyelinated afferent nerve fibres presumably due to an inhibition of intraneuronal transport mechanisms. In contrast with perineural capsaicin treatment affecting the chemistry and function of the whole sensory neurone, injection of capsaicin into the subarachnoid space results in an irreversible abolition of the "afferent" but not the "efferent" function of CPSNs. Accordingly, noxious thermal or chemical stimuli applied to the peripheral innervation areas of the trigeminal nucleus caudalis or the affected segments of the spinal cord fail to induce nociceptive reflexes because of the degeneration of the central terminals of CPSNs. However, in these same skin areas, application of chemical irritants invariably evoked the neurogenic inflammatory response, indicating that CPSNs deprived of their central terminals maintain their capacity to synthesize and release the peptide(s) responsible for the initiation of that response. In contrast with previous findings, our recent studies furnished evidence for a selective neurodegenerative action of systemically injected capsaicin in adult mammals, as well. Therefore, some of the irreversible functional impairments produced by capsaicin in adult animals may result from the degeneration of a particular subpopulation of CPSNs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nicotinic Modulation of [3H]Dopamine Release from Striatal Synaptosomes: Pharmacological Characterisation

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. We have compared the effects of a number of nicotinic agonists and antagonists on a perfused synaptosome preparation preloaded with [3H]dopamine. (-)-Nicotine, acetylcholine, and the nicotinic agonists cytisine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), at micromolar concentrations, stimulated the release of [3H]dopamine from striatal nerve terminals. Carbamylcholine was a much weaker agonist. The actions of (-)-nicotine, cytisine, and DMPP were inhibited by low concentrations of the nicotinic antagonists dihydro-beta-erythroidine, mecamylamine, pempidine, and neosurugatoxin; alpha-bungarotoxin was without effect, and extending the time of exposure to this toxin resulted in only very modest inhibition. This pharmacology points to a specific nicotinic receptor mechanism that is clearly distinct from that at the neuromuscular junction. Atropine failed to antagonise the effects of acetylcholine and carbamylcholine, suggesting that no muscarinic component is involved. The nicotinic receptor ligands (-)-[3H]nicotine and 125I-alpha-bungarotoxin bound to specific sites enriched in the synaptosome preparation. Drugs tested on the perfused synaptosomes were examined for their ability to interact with these two ligand binding sites in brain membranes. The differential sensitivity to the neurotoxins alpha-bungarotoxin and neosurugatoxin of the 125I-alpha-bungarotoxin and (-)-[3H]nicotine binding sites, respectively, leads to a tentative correlation of the (-)-[3H]nicotine site with the presynaptic nicotinic receptor on striatal nerve terminals.     

Role of vagal afferents in the control of abdominal expiratory muscle activity in the dog.

We examined the contribution of afferent vagal A- and C-fibers on abdominal expiratory muscle activity (EMA). In seven spontaneously breathing supine dogs anesthetized with alpha-chloralose we recorded the electromyogram of the external oblique muscle at various vagal temperatures before and after the induction of a pneumothorax. When myelinated fibers were blocked selectively by cooling the vagus nerves to 7 degrees C, EMA decreased to 40% of control (EMA at 39 degrees C). With further cooling to 0 degrees C, removing afferent vagal C-fiber activity, EMA returned to 72% of control. On rewarming the vagus nerves to 39 degrees C, we then induced a pneumothorax (27 ml/kg) that eliminated the EMA in all the dogs studied. Cooling the vagus nerves to 7 degrees C, during the pneumothorax, produced a slight though not significant increase in EMA. However, further cooling of the vagus nerves to 0 degrees C caused the EMA to return vigorously to 116% of control. In three dogs, intravenous infusion of a constant incrementally increasing dose of capsaicin, a C-fiber stimulant, decreased EMA in proportion to the dose delivered. These results suggest that EMA is modulated by a balance between excitatory vagal A-fiber activity, most likely from slowly adapting pulmonary stretch receptors, and inhibitory C-fiber activity, most likely from lung C-fibers.     

Neurotrophin and GDNF family ligand receptor expression in vagal sensory nerve subtypes innervating the adult guinea pig respiratory tract

We combined retrograde tracing techniques with single-neuron RT-PCR to compare the expression of neurotrophic factor receptors in nodose vs. jugular vagal sensory neurons. The neurons were further categorized based on location of their terminals (tracheal or lungs) and based on expression of the ionotropic capsaicin receptor TRPV1. Consistent with functional studies, nearly all jugular neurons innervating the trachea and lungs expressed TRPV1. With respect to the neurotrophin receptors, the TRPV1-expressing jugular C-fiber neurons innervating both the trachea and lung compartments preferentially expressed tropomyosin-receptor kinase A (TrkA), with only a minority of neurons expressing TrkB or TrkC. The nodose neurons that express TRPV1 (presumed nodose C-fibers) innervate mainly intrapulmonary structures. These neurons preferentially expressed TrkB, with only a minority expressing TrkA or TrkC. The expression pattern in tracheal TRPV1-negative neurons, nodose tracheal presumed Aδ-fiber neurons as well as the intrapulmonary TRPV1-negative presumed Aβ-fiber neurons, was similar to that observed in the nodose C-fiber neurons. We also evaluated the expression of GFRα receptors and RET (receptors for the GDNF family ligands). Virtually all vagal sensory neurons innervating the respiratory tract expressed RET and GFRα1. The jugular neurons also categorically expressed GFRα3, as well as ～50% of the nodose neurons. GFRα2 was expressed in ～50% of the neurons irrespective of subtype. The results reveal that Trk receptor expression in vagal afferent neurons innervating the adult respiratory tract depends more on the location of the cell bodies (jugular vs. nodose ganglion) than either the location of the terminals or the functional phenotype of the nerve. The data also reveal that in addition to neurotrophins, the GDNF family ligands may be important neuromodulators of vagal afferent nerves innervating the adult respiratory tract.     

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.     

Cortical Presynaptic Control of Dorsal Horn C–Afferents in the Rat

Lamina 5 sensorimotor cortex pyramidal neurons project to the spinal cord, participating in the modulation of several modalities of information transmission. A well-studied mechanism by which the corticospinal projection modulates sensory information is primary afferent depolarization, which has been characterized in fast muscular and cutaneous, but not in slow-conducting nociceptive skin afferents. Here we investigated whether the inhibition of nociceptive sensory information, produced by activation of the sensorimotor cortex, involves a direct presynaptic modulation of C primary afferents. In anaesthetized male Wistar rats, we analyzed the effects of sensorimotor cortex activation on post tetanic potentiation (PTP) and the paired pulse ratio (PPR) of dorsal horn field potentials evoked by C–fiber stimulation in the sural (SU) and sciatic (SC) nerves. We also explored the time course of the excitability changes in nociceptive afferents produced by cortical stimulation. We observed that the development of PTP was completely blocked when C-fiber tetanic stimulation was paired with cortex stimulation. In addition, sensorimotor cortex activation by topical administration of bicuculline (BIC) produced a reduction in the amplitude of C–fiber responses, as well as an increase in the PPR. Furthermore, increases in the intraspinal excitability of slow-conducting fiber terminals, produced by sensorimotor cortex stimulation, were indicative of primary afferent depolarization. Topical administration of BIC in the spinal cord blocked the inhibition of C–fiber neuronal responses produced by cortical stimulation. Dorsal horn neurons responding to sensorimotor cortex stimulation also exhibited a peripheral receptive field and responded to stimulation of fast cutaneous myelinated fibers. Our results suggest that corticospinal inhibition of nociceptive responses is due in part to a modulation of the excitability of primary C–fibers by means of GABAergic inhibitory interneurons.     

Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 subunits

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.