Epibatidine binding sites and activity in the spinal cord

Epibatidine has been shown to be the most potent nicotinic agonist in several neuronal nicotinic receptor preparations. Similar to other nicotinic agonists, intrathecal (−)-epibatidine elicits dose-dependent increases in pressor, heart rate and pain responses in rats, as well as an increase in latency to withdraw from a noxious thermal stimulus. The latter response requires higher doses and is of shorter duration, suggesting interaction with multiple subtypes of spinal nicotinic receptors. In the present study, we relate the binding properties of (±)-[³H]epibatidine in spinal cord membrane preparations to the cardiovascular and behavioral responses. Unlike (−)-[³H]cytisine or (−)-[³H]nicotine, (±)-[³H]epibatidine reveals two sites; the ratio of high affinity to low affinity sites is 2:1. The rank ordering of potencies of the nicotinic agonists in displacing (±)-[³H]epibatidine binding from spinal cord membranes correlates with the potencies in eliciting cardiovascular and behavioral responses upon spinal administration. The nicotinic receptor antagonists, α-lobeline, dihydro-β-erythroidine and methyllycaconitine, also displayed similar rank ordering of potencies in displacing (±)-[³H]epibatidine, (−)-[³H]cytisine or (−)-[³H]nicotine binding to spinal nicotinic receptors. Virtually all the nicotinic analogs exhibited a Hill coefficient of less than one in competing with (±)-[³H]epibatidine to spinal cord membranes indicating their interaction with at least two classes of binding sites. Intrathecal (−)-epibatidine, in addition to eliciting an initial and subsequently a sustained pressor and tachycardic response, also exhibited a transient intervening bradycardia which coincided temporally with the duration of the analgesia. Repeated administration of (−)-epibatidine desensitized its responses as well as the cardiovascular and behavioral responses to spinal nicotine and cytisine. Intrathecal α-lobeline showed selectivity for blocking the analgesic response, whereas methyllycaconitine exhibited selectivity for the pressor and irritation responses. The NMDA receptor antagonist, AP-5, inhibited the pressor, tachycardic and irritation responses elicited by intrathecal (−)-epibatidine, confirming a role for spinal excitatory amino acids released by the nicotinic agonist. © 1997 Elsevier Science B.V. All rights reserved.     

Presence of an endogenous factor which inhibits binding of α-bungarotoxin 2.2 to its receptor

Cerebral cortical membranes and supernatant from rat were prepared by centrifugation of tissue homogenates at 45,000 g for 10 min. The supernatant fraction thus obtained was found to significantly inhibit α-bungarotoxin binding to the membrane preparation. After a 3 min incubation period, the supernatant inhibited toxin binding by approximately 65%, while the inhibition declined to about 40% after 30 min of incubation, presumably due to the slow reversility of α-bungarotoxin binding. The choice of buffer was found to be an important determinant of the degree of inhibition observed, with 10 mM Tris pH 7.4 providing the most effective condition. This inhibition of toxin binding to cortical membranes by the 45,000 g supernatant was shown not to be due to adsorption of the radiolabeled compound to soluble or residual particulate material in the supernatant fraction. Specificity of the supernatant for the α-bungarotoxin site was demonstrated; a supernatant fr action could be prepared which inhibited α-bungarotoxin binding by 50% but had no effect on [³H]spiroperidol (DA₂ and 5-HT₂), [³H]prazosin, (α₁-adrenergic), [³H]5-hydroxytryptamine (5-HT₁) and [³H]quinuclidinylbenzilate (muscarinic cholinergic) binding. The inhibition of toxin binding also occurred in several other CNS regions including hippocampus, brainstem, spinal cord and cerebellum with an 80 to 90% inhibition of binding occuring in the latter two regions. In addition, the 45,000 g cortical supernatant completely prevented the binding of α-bungarotoxin to extrajunctional neuromuscular receptors and inhibited the binding to junctional receptors by 50%. Supernatants prepared from heart, liver and kidney or bovine serum albumin, at a concentration similar to the supernatant fraction, did not alter radiolabeled toxin binding to cortical membranes, while supernatant prepared from striated muscle tissue was effective. These results suggest there may be an endogenous ligand for the α-bungarotoxin 2.2 binding site in tissues which receive nicotinic cholinergic innervation.     

Localization of M1 Muscarinic Receptors in Rat Brain Using Selective Muscarinic Toxin-1

Mambas, African snakes of the genus Dendroaspis, produce several types of toxins that are of pharmacological interest. The novel muscarinic toxin-1 (MT-1), from the green mamba Dendroaspis angusticeps, binds specifically to muscarinic M₁ receptors in homogenates of rat cerebral cortex. Iodination of the toxin, ¹²⁵I-muscarinic toxin-1 (¹²⁵I-MT-1), renders the toxin selective for M₁ muscarinic receptors. Quantitative measurement of ¹²⁵I-MT-1 autoradiography in rat brain sections indicated highest labeling in the nucleus accumbens, striatum, and dentate gyrus. High densities of ¹²⁵I-MT-1 binding sites were located in the CA1 region of the hippocampus, frontal, and parietal cortices. Moderate densities of binding sites were seen in temporal cortex, and hippocampal subregions CA2, CA3, and CA4, whereas low labeling was observed in the cerebellum and spinal cord.     

Nicotinic cholinergic receptors in brain synaptosomes

In order to elucidate pharmacological characteristics of nicotinic receptors in the brain, competitive binding of nicotine analogues or cholinergic agents to brain particles was studied utilizing [³H]nicotine. The binding of [³H]nicotine to brain crude mitochondrial or synaptosomal fraction was progressively inhibited by the addition of increasing amounts of nicotine or nornicotine, but cotinine had little effect. Of the myelin, synaptosomal and mitochondrial subfractions of the crude mitochondrial fraction, [³H]nicotine binding was almost exclusively confined to synaptosomes. In this binding, the affinity constant was 1.97 × 10⁹/M and the maximum binding capacity was 7.30 × 10⁻⁸ M/g of protein. This binding was reduced by 50% in the presence of 4 × 10⁻⁷ MD-tubocurarine and 10⁻⁶ M carbamylcholine, while atropine was the least effective of the drugs tested. These findings suggest the possible existence of nicotinic cholinergic receptors in the synaptic region. A study of regional differences in nicotinic cholinergic receptors showed that higher specific bindings of nicotine to synaptosomes occured in the hypothalamus, hippocampus and thalamus. These findings suggest that the nicotinic cholinergic mechanism plays an important role in these regions of the central nervous system.     

The action of chlorpromazine at an isolated cholinergic synapse

The effects of chlorpromazine (CPZ) on cholinergic transmission were studied at the isolated neuromuscular synapse of the frog. It was found that 5 × 10⁻⁶ M CPZ produces the following effects: (1) a reduction in end-plate potential amplitude, mainly through inhibition of transmitter release at presynaptic nerve terminals; (2) a reduction in amplitude of focally recorded end-plate current without detectable change in nerve terminal potential: (3) a decrease in amplitude of miniature end-plate potentials; and (4) an increase in the frequency of spontaneous liberation of transmitter both in normal and calcium-free Ringer's solution. It is concluded that CPZ inhibits cholinergic transmission by a complex action on presynaptic and postsynaptic elements. The relation of these findings to central cholinergic activities of CPZ is discussed.     

Evidence for two types of nicotinic receptors in the cat carotid body chemoreceptor cells

Current concepts on the location and functional significance of nicotinic receptors in the carotid body rest on α-bungarotoxin binding and autoradiographic studies. Using an in vitro preparation of the cat carotid body whose catecholamine deposits have been labeled by prior incubation with the tritiated natural precursor [³H]tyrosine, we have found that nicotine induces release of [³H]catecholamines in a dose-dependent manner (IC₅₀=9.81 μM). We also found that mecamylamine (50 μM) completely abolished the nicotine-induced release, while α-bungarotoxin (100 nM; ≈20 times its binding K_d) only reduced the release by 56%. These findings indicate that chemoreceptor cells, and perhaps other carotid body structures, contain nicotinic receptors that are not sensitive to α-bungarotoxin and force a revision of the current concepts on cholinergic mechanisms in the carotid body chemoreception.     

Regional distribution of cholinergic neurons in human spinal cord transections in the patients with and without motor neurons disease

Regional distribution of enzymic activities in acetylcholine (ACh) metabolism was examined on thinly-sectioned transverse slices of human spinal cords obtained during autopsy of 5 motor neuron disease (MND) and 5 control patients without MND. Choline acetyltransferase (ChAT) activity was highly concentrated in the ventral horn regions (gray and white matters) of cervical, thoracic and lumbar spinal cord of non-MND patients. This enzyme activity was found to be remarkably low in the ventral gray and white matter of MND patients compared with that of the controls. Although the distribution of acetylcholinesterase (AChE) activity was found to be high in both ventral and dorsal gray matter of the spinal cord, little difference was observed between each corresponding region of MND and control patients, except relatively low enzyme activity in the cervical ventral horn region of MND patients. Muscarinic cholinergic receptors, examined as specific [³H]quinuclidinylbenzilate ([³H]QNB) binding, was also highly concentrated in the ventral and dorsal gray matter of the control spinal cord, and was strongly reduced in the ventral horn region of MND patients, indicating a quite similar distribution pattern of ChAT activity. These biochemical changes of cholinergic transmission system may be paralleled to the morphological degeneration of the spinal lower motor neurons in MND patients. Activity of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase), a marker enzyme of central myelin structure, was evenly distributed throughout the whole spinal cord section, without regard to the gray and white matter, of both MND and control patients.     

Immunologic similarities between the hypothalamic α-bungarotoxin receptor and theTorpedo californica nicotinic cholinergic receptor

The solubilized rat central nervous system (hypothalamic) nicotinic cholinergic receptor and the Torpedo nicotinic cholinergic receptors are immunologically similar technique. Antibodies to the Electrophorus and Torpedo receptors also decrease the rate of α-bungarotoxin binding to these membraneous receptors. It is concluded that the Torpedo and hypothalamic nicotinic receptors are immunologically similar and that receptor binding sites for α-bungarotoxin and antibodies are physically close. These studies indicate that α-bungarotoxin can be used to study the nicotinic cholinergic receptor of the rat hypothalamus.     

Up-regulation of spinal μ-opioid receptor function to activate G-protein by chronic naloxone treatment

The effects of repeated s.c. administrations of an μ-opioid receptor antagonist naloxone on the G-protein activation induced by μ-opioid receptor agonists [ -Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAMGO), endomorphin-1 and endomorphin-2 in the mouse spinal cord was studied, monitoring guanosine-5′-o-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding. All μ-opioid receptor agonists concentration-dependently increased the [³⁵S]GTPγS binding. The increases of [³⁵S]GTPγS binding induced by agonists were significantly enhanced in mice pretreated with naloxone. Under the present condition, chronic treatment with naloxone significantly increased the density of [³H]DAMGO binding sites with an increase in K_d values in spinal cord membranes, indicating an increase in μ-opioid receptors on the membrane surface. These findings suggest that chronic treatment with an μ-opioid receptor antagonist naloxone leads to the supersensitivity to activate G-protein by μ-opioid receptor agonists with an increase in μ-opioid receptors in membranes of the mouse spinal cord.     

Neuronal bungarotoxin displaces [I]α-bungarotoxin binding at the neuromuscular junction as well as to the spinal cord during embryogenesis

α-Bungarotoxin (αBTX) administration in ovo prevents motoneuron apoptosis during development. This process may be mediated by α BTX-sensitive nicotinic cholinoceptors in the spinal cord, at the neuromuscular junction or at both sites. In order to differentiate between these possibilities, neuronal bungarotoxin binding (NBTX) binding to embryonic muscle and spinal cord was investigated in the chick.     

The filum terminale of the frog spinal cord, a non transformed glial preparation: I. Morphology and uptake of γ-aminobutyric acid

The filum terminale of the frog spinal cord is a rather pure glial cell preparation, largely devoid of neuronal elements.γ-Aminobutyric acid (GABA) is taken up by the frog filum terminale (FT) via a Na⁺-dependent, ouabain-inhibited, saturable high affinity transport system with a K_m of 2.7 × 10⁻⁵ M. The rate of the FT GABA uptake is significantly greater than the velocities observed in the spinal cord. In fact, the V_max increases caudally beyond the level of the last root, and is maximal in the FT per se. β-Alanine is a competitive inhibitor of the FT high affinity transport system for GABA (K_i 11.1 × 10⁻⁵ M). In addition to GABA, the FT also takes up β-alanine, glycine, glutamate and aspartate at rates significantly higher than those shown by the spinal cord of the frog. Light and electron microscope level radioautography clearly shows that GABA uptake occurs primarily in the glial cells and also in ependymal cells present in the FT. In that the FT contains few ependymal cells and a large number of glia, it is fair to state that most of the GABA accumulated by the FT reflects the glial transport of this amino acid.Unlike the adult frog, the spinal cord of the tadpole does not show any regional differences in the rate of GABA transport during early development. However, during later developmental stages, the rates of GABA transport increase in the caudal portion of the tadpole cord as compared to the more rostral areas. Close to metamorphosis, the terminal portion of the tadpole cord, which is destined to become the filum terminale of the frog, accumulates GABA at rates not greatly different from those observed in the FT of the adult frog. Therefore, the tadpole spinal cord is a useful preparation in which to study the dynamic properties of normal non-transformed glia as influenced by a changing neuronal population, whereas the frog FT is a unique preparation for the study of some properties of normal glia largely in the absence of neurons.     

A postmortem study of brain nicotinic receptors in Parkinson's and Alzheimer's disease

Brain nicotinic receptors were studied in the frontal cortex, temporal cortex, hippocampus and caudate nucleus in patients with Parkinson's disease (PD), Alzheimer's disease (AD) and controls. The B_max and K_d values of (−)-[³H]nicotine binding were determined with a Scatchard analysis. The number of nicotinic receptors declined both in PD and in AD patients in all brain areas examined. The K_d values were unchanged. There was a negative correlation between the degree of dementia in PD patients and the number of nicotinic receptors in the frontal cortex. A similar correlation was seen between the muscarinic/nicotinic receptor ratio in the frontal cortex and the degree of dementia in PD patients. The present findings indicate that nicotinic receptors are affected not only in AD, but also in PD and that dysfunction of the cholinergic system in the frontal cortex is involved in the dementia process in PD.     

α-Bungarotoxin-acetylcholine receptors in the chick ciliary ganglion: Effects of deafferentation and axotomy

α-Bungarotoxin (α-BuTX) binds in a saturable and practically irreversible fashion to membrane-associated receptors in the ciliary ganglion of the adult chick. The binding of toxin to receptors is competitively inhibited by nicotinic cholinergic ligands, and for these properties the receptors are regarded as acetylcholine receptors of the nicotinic type (α-BuTX-AChRs). The rate constant of association (K₁) and dissociation (K₋₁) of the toxin-receptor reaction has been estimated to be K₁ = 7.4 × 10⁴ M⁻¹sec⁻¹ and K₋₁ = 9.6 × 10⁻⁶sec⁻¹, respectively. Light autoradiography shows that most, if not all, the receptors are related to surface membrane, probably to synaptic areas of both choroid and ciliary neurons. The choroid neurons contain more receptors that the ciliary ones. A single chick ciliary ganglion binds specically 47 fmole of α-BuTX in situ corresponding to 2.83 × 10¹⁰ α-BuTX-AChRs/ganglion.No changes in number and distribution of the toxin receptors occur following preganglionic denervation. Conversely, postganglionic axotomy causes a rapid disappearance of the receptors in situ. Since binding experiments in vitro revealed a partial, instead of a total, loss of the receptors, it is suggested that the disappearance of the receptors in situ includes both a partial loss of the original receptors and the masking of the residual ones.     

Acetylcholine receptor availability and transmission efficacy

Recovery of cholinergic transmission after in vivo blockade with α-bungarotoxin (α-BTX), and the relationship of recovery to availability of unbound acetylcholine receptors (AChR) were studied in rat diaphragm. When 83% of endplate acetylcholine receptor binding sites were blocked, transmission was absent. A barely detectable recovery of the blocked receptors (25 h after exposure to α-bungarotoxin) restored transmission. In fact, 25% of the endplate receptor ACh binding sites were just sufficient for action potential generation. As discussed, slow turnover (t₁₂ = 11 days) of junctional receptors would be sufficient to provide the observed recovery of transmission. Good agreement was observed between the minimum fraction of total receptor sites required for transmission and the computed fraction of maximum quantal release of acetylcholine required to reach threshold. In addition, the data are consistent with the hypothesis that at the neuromuscular junction, AChR exists in considerable excess.     

Analysis of NMDA Receptors in the Human Spinal Cord

NMDA receptors in postmortem human spinal cord were analyzed using [³H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The averageK_Dfor [³H]MK-801 binding was 1.77 nM with aB_maxof 0.103 pmol/mg. The EC₅₀for stimulation of [³H]MK-801 binding withl-glutamate was 0.34 μM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.     

Identified spinal motoneurons of young rats possess nicotinic acetylcholine receptors of the heteromeric family

The aim of the present study was to determine whether, in young rats, spinal motoneurons possess functional nicotinic acetylcholine receptors. Motoneurons were identified either by retrograde labelling or by choline acetyltransferase immunohistochemistry. Whole-cell recordings were performed in spinal cord slices cut at the lumbar level. In voltage clamp, acetylcholine evoked a rapidly activating inward current. In current clamp, it depolarized the motoneuron membrane and induced action potential firing. The acetylcholine-evoked current was strongly reduced by d-tubocurarine or dihydro-beta-erythroidine, broad spectrum nicotinic antagonists, but was almost insensitive to methyllycaconitine, a nicotinic antagonist selective for receptors containing the alpha7 subunit. Moreover, exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, an alpha7-specific agonist, was without effect. In young animals, light-microscopic autoradiography showed that in the central grey matter all laminae were intensely and equally labelled by [3H]epibatidine. A dense [125I]-alpha-bungarotoxin binding was also found in all laminae, with slightly lower levels in the superficial layers of the dorsal horns and in the ventral part of the grey matter. In adults, the density of [3H]epibatidine binding sites was much lower in the entire grey matter, except in layer 2 of the dorsal horn, and [125I]-alpha-bungarotoxin binding sites were present only in some selected areas. Our data indicate that spinal motoneurons possess functional nicotinic receptors of the heteromeric type and suggest that nicotinic cholinergic transmission may play a significant role in the developing spinal cord.     

Behavior and binding: Correlations between α1-adrenergic stimulation of acoustic startle and α1-adrenoceptor occupancy and number in rat lumbar spinal cord

The relationship between alterations in α₁-adrenoceptors and behavioral effects of α₁-adrenergic agonists were investigated in a localized region of the rat central nervous system. Direct infusion of the α₁-adrenergic agonists,d-amphetamine or phenylephrine. into the subarachnoid space of the lumbar cord (intrathecal administration) increased the amplitude of the acoustic startle reflex, The magnitude of this behavioral facilitation correlated highly with the degree of α₁-adrenoceptor occupation measured by [³H]prazosin binding in lumbar spinal tissue. Using an in vitro estimate of receptor occupation, maximal potentiation of startle occurred following approximately 30% occupation of the receptors, using eitherd-amphetamine or phenylephrine. Intrathecal administration of 6-OHDA produced a 95% decrease in spinal norepinephrine and markedly enhanced the behavioral response to intrathecal phenylephrine as well as the number of α₁-adrenoceptors. The correlation between the time course of the behavioral and binding changes was 0.99. No change in receptor affinity (K_D) or receptor occupation by phenylephrine was found after 6-OHDA. The data indicate that receptor binding parameters do have predictive value for behavior, especially if localized regions of the nervous system, critical to the behavior, are analyzed.     

3-[2,4-Dimethoxybenzylidene]anabaseine (DMXB) selectively activates rat α7 receptors and improves memory-related behaviors in a mecamylamine-sensitive manner

The α7 nicotinic receptor agonist 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB; GTS-21) was investigated for its ability to: (1) activate a variety of nicotinic receptor subtypes in Xenopus oocytes; (2) improve passive avoidance and spatial Morris water task performances in mecamylamine-sensitive manners in bilaterally nucleus basalis lesioned rats; and (3) elevate high-affinity [³H]acetylcholine (ACh) and high-affinity α-[¹²⁵I]bungarotoxin binding in rat neocortex following 2 weeks of daily injections. DMXB (100 μM) activated α7 homo-oligomeric receptors, without significant activity at α2-, α3- and α4-containing subtypes. Mecamylamine blocked rat α7 receptors weakly if co-administered with agonist, but much more potently when pre-applied. Bilateral ibotenic acid lesions of the nucleus basalis interfered with passive avoidance and spatial memory-related behaviors. DMXB (0.5 mg/kg, i.p.) improved passive avoidance behavior in lesioned animals in a mecamylamine-sensitive manner. DMXB (0.5 mg/kg 15 min before each session) also improved performance in the training and probe components of the Morris water task. DMXB-induced improvement in the probe component but not the training phase was mecamylamine-sensitive. [³H]ACh binding was elevated after 14 days of daily i.p. injections with 0.2 mg/kg nicotine but not after 1 mg/kg DMXB. Neither drug elevated high-affinity α-[¹²⁵I]bungarorotoxin binding over this interval.     

Effects of chronic treatment with (+)- and (−)-nicotine on nicotinic acetylcholine receptors and N-methyl-d-aspartate receptors in rat brain

In this study, the effects of chronic treatment with (+)-nicotine on brain nicotinic acetylcholine receptors (nAChRs) andN-methyl-d-aspartate (NMDA) receptors, as well as on animal body weight were compared with those of chronic treatment with (−)-nicotine. Male Sprague-Dawley rats were s.c. injected with saline, (+)-nicotine (2.0 mg free base/kg b.w.) or (−)-nicotine (0.45 mg free base/kg b.w.) for 18 days. Brain nAChRs were investigated by (-)-[³H]nicotine binding. A significant increase in the high-affinity (−)-[³H]nicotine (5 nM)-binding sites was observed the cortex, hippocampus, midbrain and striatum but not in the cerebellum of the rats treated with either (+)- or (−)-nicotine. The displacement curves of (−)-[³H]nicotine/(−)-nicotine in the cortices of rats treated with either (+)- or (−)-nicotine showed only one population of high-affinity binding sites, whereas both high- and low-affinity binding sites were observed in the cortices of control animals. Brain NMDA receptors were studied by [³H]MK-801, which binds to the NMDA receptor-ion channel complex. A significant decrease in theB_max, but not in theK_D for [³H]MK-801 binding in the cortices of rats treated with either (+)- or (−)-nicotine was only detected under certain experimental conditions where the NMDA receptors seem not to be maximally activated. The body weight of the animals treated with (−)-nicotine was significantly lower than that of the control animals, whereas there was no difference in body weight between (+)-nicotine- and saline-treated animals. These results show that (+)-nicotine treatment has an effect on nAChRs in rat brains which is similar to that of (−)-nicotine treatment, but an effect on body weight which differs from (−)-nicotine, and also suggest that nicotinic agonists may interact with brain NMDA receptors in vivo.     

Muscarinic cholinergic receptors in the human spinal cord: differential localization of [H]pirenzepine and [H]quinuclidinylbenzilate binding sites

The localization of muscarinic cholinergic receptor subtypes was studied in the human spinal cord using in vitro labelling of cryostat sections with [³H]quinuclidinylbenzilate (QNB) and [³H]pirenzepine (PZ) followed by autoradiography. The highest densities of [³H]QNB binding were localized in laminae II (substantia gelatinosa) and IX (motor neurons); in contrast, the highest density of [³H]PZ binding was localized to lamina II where the binding density was 22—32% higher than in lamina IX. These results suggest that the M₁ and M₂ muscarinic cholinergic receptor subtypes may be differentially localized in sensory and motor regions of the human spinal cord.