Regional changes in the cholinergic system in mice lacking monoamine oxidase A

Elevated brain monoamine concentrations resulting from monoamine oxidase A genetic ablation (MAOA knock-out mice) lead to changes in other neurotransmitter systems. To investigate the consequences of MAOA deficiency on the cholinergic system, we measured ligand binding to the high-affinity choline transporter (CHT1) and to muscarinic and nicotinic receptors in brain sections of MAOA knock-out (KO) and wild-type mice. A twofold increase in [³H]-hemicholinium-3 ([³H]-HC-3) binding to CHT1 was observed in the caudate putamen, nucleus accumbens, and motor cortex in MAOA KO mice as compared with wild-type (WT) mice. There was no difference in [³H]-HC-3 labeling in the hippocampus (dentate gyrus) between the two genotypes. Binding of [¹²⁵I]-epibatidine ([¹²⁵I]-Epi), [¹²⁵I]-α-bungarotoxin ([¹²⁵I]-BGT), [³H]-pirenzepine ([³H]-PZR), and [³H]-AFDX-384 ([³H]-AFX), which respectively label high- and low-affinity nicotinic receptors, M1 and M2 muscarinic cholinergic receptors, was not modified in the caudate putamen, nucleus accumbens, and motor cortex. A small but significant decrease of 19% in M1 binding densities was observed in the hippocampus (CA1 field) of KO mice. Next, we tested acetylcholinesterase activity and found that it was decreased by 25% in the striatum of KO mice as compared with WT mice. Our data suggest that genetic deficiency in MAOA enzyme is associated with changes in cholinergic activity, which may account for some of the behavioral alterations observed in mice and humans lacking MAOA.     

Differential regulation of muscarinic and nicotinic receptors by cholinergic stimulation in cultured avian retina cells

Sustained cholinergic stimulation of retina cells grown in primary aggregate and monolayer cultures regulated the concentration of muscarinic but not nicotinic receptors. Muscarinic receptor sites, quantified by the binding of [³H]quinuclidinyl benzilate to membranes and the binding of [³H]N-methyl-scopolamine to intact cells, decreased up to 84% following long-term incubation of cultures in muscarinic agonists. This decrease was blocked by atropine and was not induced by chronic nicotine treatment. The rate of the muscarinic response was biphasic. A rapid binding decrease of 30% occurred within 15 min. The slower phase was half-maximal by 6 h and was complete by 24 h. Neither the fast nor the slow receptor loss was reversed by the guanine nucleotide GppNp. Three different depolarizing agents (gramicidin D, protoveratrine, and ouabain) blocked the cholinergic-induced receptor loss, but the hyperpolarizing ionophore valinomycin had no effect. In contrast to the muscarinic response, nicotinic receptor binding was not altered by chronic receptor stimulation. Exposure to receptor-saturating doses of carbamylcholine or nicotine for 48 h did not change [¹²⁵I]α-bungarotoxin or [³H]bromoacetylcholine binding. Differential regulation of acetylcholine receptors is discussed in relation to the possible physiological role of receptor regulation by receptor activity.     

Comparative ontogenic profile of cholinergic markers,including nicotinic and muscarinic receptors,in the rat brain

The ontogenic profiles of several cholinergic markers were assessed in the rat brain by using quantitative in vitro receptor autoradiography. Brain sections from animals at different stages of development were processed with [³H]AH5183 (vesamicol; vesicular acetylcholine transport sites), [³H]N-methylcarbamylcholine (α₄β₂ nicotinic receptor sites), [³H]hemicholinium-3 (high-affinity choline uptake sites), [³H]3-quinuclidinyl benzilate (total population of muscarinic receptor sites), [³H]4-DAMP (muscarinic M₁/M₃ receptor sites), [³H]pirenzepine (muscarinic M₁ receptor sites), and [³H]AF-DX 116 and [³H]AF-DX 384 (muscarinic M₂ receptor sites) as radiolabeled probes. The results revealed that, by the end of the prenatal period (embryonic day 20), the densities of nicotinic receptor and vesicular acetylcholine transport sites already represented a considerable proportion of those observed in adulthood (postnatal day 60) in different laminae of the frontal, parietal, and occipital cortices, in the layers of Ammon's horn fields and the dentate gyrus of the hippocampal formation, as well as in the amygdaloid body, the olfactory tubercle, and the striatum. In contrast, at that stage, the densities of total muscarinic, M₁/M₃, M₁, and possibly M₂ receptor and high-affinity choline uptake sites represent only a small proportion of levels seen in the adult. Differences were also observed in the postnatal ontogenic profiles of nicotinic, muscarinic, vesamicol, and high-affinity choline uptake sites. For example, between postnatal weeks 3 and 5, the levels of M₁/M₃ and M₁ sites were at least as high as in the adult, whereas M₂ and high-affinity choline uptake site densities appeared to be delayed and to reach adult values only after postnatal week 5. With regard to cholinergic innervation in the developing rat brain, the present findings suggest a temporal establishment of several components of the cholinergic systems. The first components are the vesicular acetylcholine transporter and nicotinic sites; these are followed by M₁/M₃ and M₁ sites and, finally, by M₂ and high-affinity choline uptake sites. © 1996 Wiley-Liss, Inc.     

Muscarinic and nicotinic cholinergic binding sites in alzheimer's disease cerebral cortex

The total population of muscarinic receptors and a subpopulation of muscarinic receptors with high affinity for agonists were measured with [³H]quinuclidinyl benzilate and [³H]acetylcholine, respectively, in homogenates of cerebral cortex from control and Alzheimer's disease brains. No significant differences between control and Alzheimer's diseased cortex were found in either the total population of receptors or the subpopulation with high affinity for agonists in either the frontal or temporal poles. Nicotinic cholinergic receptors labeled by [³H]acetylcholine were measured in homogenates and by autoradiography in the same brain areas. In contrast to muscarinic sites, binding to nicotinic sites was markedly decreased in Alzheimer's disease cortex. Autoradiography of [³H]acetylcholine binding to nicotinic sites indicated that in control cortex these sites are more concentrated in laminae IV–VI than in the superficial laminae, and that in Alzheimer's disease there is loss of these sites in all cortical laminae.     

α-Bungarotoxin blocks the nicotinic receptor mediated increase in cell number in a neuroendocrine cell line

Exposure of H69 small cell lung carcinoma cells to nicotinic agonists resulted in a significant increase (up to 100%) in cell number after 6 to 12 days. The effect of nicotine (10⁻⁸ M to 10⁻⁴ M) was both dose and time dependent as was that of another nicotinic agonist cytisine (10⁻⁶ M to 10⁻⁴ M). Interstingly, both the nicotine and cytisine induced increases in H69 cell number were blocked by α-bungarotoxin, as well as d-tubocurarine a nicotinic blocker which appears to interact with most nicotinic receptors. These results suggest that the nicotine induced increase in cell number is mediated through an interaction at the nicotinic α-bungarotoxin receptor. This idea is further supported by experiments which show (1) that H69 cells possess high affinity α-bungarotoxin sites (K_d = 25 nM, B_max = 10.4 fmol/10⁶ cells) with the characteristics of a nicotinic α-bungarotoxin receptor and (2) that the potencies of nicotinic receptor ligands in the α-bungarotoxin binding assay were similar to those observed in the functional studies. Northern analysis showed that mRNA for α7, a putative nicotinic α-bungarotoxin binding subunit, and for α5 were present in H69 cells. The present data provide further evidence that nicotine increases cell number in small cell lung carcinoma and are the first to show that this effect is mediated through an interaction at the nicotinic α-bungarotoxin receptor population. These results suggest that the α-bungarotoxin site may be involved in modulating proliferative responses in neuroendocrine derived SCLC cells.     

Muscarinic and dopaminergic receptor subtypes on striatal cholinergic interneurons

Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A, caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers [3H]hemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-containing neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D2 receptors as evidenced by a decrease in [3H]sulpiride binding (42% reduction) and decrease of muscarinic non-M1 receptors as shown by a reduction in [3H]QNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in [3H]sulpiride and [3H]QNB binding was due to a change in Bmax not Kd. Intrastriatal injection of AF64A failed to alter dopamine D1 or muscarinic M1 receptors labeled with [3H]SCH23390 and [3H]pirenzepine, respectively. In addition, no change in [3H]forskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M1) are presynaptic on cholinergic interneurons (hence, autoreceptors), and a subpopulation of dopamine D2 receptors are postsynaptic on cholinergic interneurons. Furthermore, dopamine D1, muscarinic M1 and [3H]forskolin-labeled adenylate cyclase are not localized to striatal cholinergic interneurons.     

Autoradiographic comparison of muscarinic M1 and M2 binding sites in the CNS of spontaneously hypertensive and normotensive rats

Spontaneously hypertensive rats (SHR) respond with exaggerated pressor responses of central origin in response to pharmacologic stimulation of brain muscarinic receptors when compared with those to normotensive Wistar Kyoto (WKY) rats. At least part of the enhanced response to central muscarinic stimulation may be due to alterations in the expression of one or more of the five subtypes of muscarinic receptors. SHR are also known to exhibit regional alterations in the levels of mRNA encoding the M1, M2 and M4 receptors. In this study, we estimated the number of specific muscarinic receptor binding sites in 12-week-old SHR and WKY by measuring the binding of M1- and M2-selective ligands. Using standard autoradiographic techniques, coronal sections obtained from 12-week-old SHR and WKY were incubated with [³H]pirenzepine or [³H]AFDX 384 to label M1 and M2 receptors, respectively. Although both strains exhibited similar distribution patterns for both binding sites, sections derived from SHR expressed a significant increase in the number of [³H]pirenzepine binding sites compared to normotensive WKY in caudate putamen, CA3 region of the hippocampus, cingulate cortex, substantia nigra, posterior hypothalamic area and tuberomammillary nucleus. An increased number of [³H]AFDX 384 binding sites in SHR were observed in the olfactory tubercle, nucleus accumbens, basolateral amygdaloid nucleus, rostroventrolateral medulla and nucleus paragigantocellularis. Decreases in the number of [³H]AFDX 384 binding sites in SHR were also observed in the parietal cortex, medial geniculate, and lateral hypothalamic area. Statistically significant site-selective differences in binding densities between strains ranged from 4.0% to 35.5% of WKY means. These alterations in the expression of M1 and M2 binding sites in cardiovascular regions may contribute to the strain's hyper-responsiveness to cholinergic drugs and possibly to the appearance of other autonomic or behavioral phenotypes exhibited by SHR, including the hypertensive state itself.     

Distribution,of [3H]quinuclidinyl benzilate, [3H]nicotine,and [125I]alpha-bungarotoxin binding sites in the nucleus tractus solitarii of the cat

The distribution of muscarinic and nicotinic cholinergic binding sites in the cat nucleus tractus solitarii was studied by the technique of in vitro autoradiography. Using the antagonist [³H]quinuclidinyl benzilate, muscarinic binding sites were differentially located in subdivisions of the nucleus tractus solitarii. The majority of muscarinic binding sites were located predominantly in the caudal half of the nucleus, reaching their greatest amounts at the mid levels of the nucleus tractus solitarii. The medial, dorsolateral, intermediate, and interstitial subdivisions contained the highest densities of quinuclidinyl benzilate binding sites. Nicotinic cholinergic binding sites, using [³H]nicotine and [¹²⁵I]alpha-bungarotoxin, had unique patterns of distribution. With [³H]nicotine the majority of binding sites were located in rostral levels of the nucleus with very few binding sites present in the caudal half. In contrast, [¹²⁵I]alpha-bungarotoxin binding sites were present mainly in subdivisions located in the caudal half of the nucleus, i.e., commissural, ventrolateral, dorsolateral, medial, and intermediate subdivisions, and dropped off precipitously at more rostral levels. The differential distribution of [³H]nicotine and [¹²⁵I]alpha-bungarotoxin suggests the two ligands may be labeling different types of nicotinic binding sites in the nucleus tractus solitarii. The unique distribution of muscarinic and nicotinic cholinergic binding sites in the various subdivisions of the nucleus solitarii suggests that muscarinic and nicotine mechanisms may play an active role in the regulation of the diverse autonomic functions at the level of the nucleus tractus solitarii. © 1993 Wiley-Liss, Inc.     

Spontaneously hypertensive rats: further evaluation of age-related memory performance and cholinergic marker expression

OBJECTIVE: The spontaneously hypertensive rat (SHR), often used to study cardiovascular disease processes, may also be utilized to model certain central nervous system changes associated with memory disorders. Previous work in our laboratory indicated that central nicotinic acetylcholine receptors are markedly diminished and that memory-related task performance is impaired in this rodent phenotype. Due to the well-documented importance of the central cholinergic system to memory processes and its vulnerability to the effects of aging, it was of interest to measure other cholinergic markers and to further evaluate memory function in older SHRs. METHOD: Radial arm maze performance was used to assess working memory, quantitative receptor autoradiography with [3H]-pirenzipine, [3H]-AFDX-384 and [3H]-epibatidine (combined with cytisine) was used to determine the densities of muscarinic-M1 and -M2 and nicotinic cholinergic alpha3 receptors, respectively. Immunoblotting experiments were also used to determine the expression of the presynaptic cholinergic markers, choline acetyltransferase and the vesicular acetylcholine transporter. RESULTS: Radial arm maze performance was impaired in hypertensive (compared with normotensive Wistar and Wistar-Kyoto) rats, regardless of age. M1 binding was increased in frontal and prefrontal cortical areas in SHR (p < 0.05), whereas M2 densities were higher in the hypertensive phenotype in the caudate putamen. A lower expression of alpha3-containing nicotinic receptors was observed in the superior colliculus in SHRs. Age-related differences in the expression of the vesicular acetylcholine transporter were noted in the hippocampus. CONCLUSION: The SHR may be useful to model some aspects (particularly hypertension-related) of memory disorders, especially those in which cholinergic function is altered.     

An autoradiographic analysis of cholinergic receptors in mouse brain

Autoradiographic techniques were used to localize cholinergic receptors in the central nervous system of female DBA mice. Nicotinic receptors were identified using [3H]-L-nicotine and alpha-[125I]-bungarotoxin (BTX); [3H]-quinuclidinyl benzilate (QNB) was used to examine muscarinic receptor binding. There was little overlap between the regional distribution of binding sites for these ligands. Nicotine binding was highest in thalamic nuclei, the superior colliculus and the interpeduncular nucleus. For BTX binding, high density receptor populations were identified in the hippocampus, caudate putamen, colliculi (superior and inferior) and various nuclei in the hypothalamus and hindbrain. Muscarinic receptors were distributed more uniformly than nicotinic receptors; the colliculi, hippocampus and cerebral cortex had the highest level of QNB binding. Species differences between rats and mice in terms of cholinergic receptor binding are discussed.     

Cholinergic receptor binding and autoradiography in brains of non-neurological and senile dementia of Alzheimer-type patients

Muscarinic and nicotinic cholinergic receptor distribution was studied by dry-mount autoradiography in brains obtained postmortem from patients with senile dementia of Alzheimer-type (SDAT) and non-neurological controls. Sections were incubated with either [N-methyl-³H]scopolamine, ([³H]NMS) or [¹²⁵I]α-bungarotoxin, ([¹²⁵I]α-BTX). No significant difference in the affinity and number of muscarinic and nicotinic receptors was found in hippocampus, frontal, temporal and cingulate cortex between SDAT patients and non-neurological controls. However, some SDAT cases showed diffuse instead of laminar [³H]NMS labeling in cortical regions. The labeling pattern was not affected by the presence of neuritic plaques and neurofibrillary tangles.     

Nicotinic acetylcholine binding sites in Alzheimer''s disease

In Alzheimer's disease (AD), there is a loss of presynaptic cholinergic markers in the cerebral cortex, but the nature of cholinergic receptor changes is unclear. In this study, [3H]acetylcholine and [3H]nicotine were used to label nicotinic cholinergic binding sites in cerebral cortical tissues obtained at autopsy from patients with AD and from matched controls. A consistent and severe loss of nicotinic receptors was found in AD.     

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.     

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.     

Effects of chronic sleep deprivation on central cholinergic receptors in rat brain

Rats subjected to chronic total sleep deprivation (TSD) by the disk-over-water method have shown very large, sustained rebounds in paradoxical sleep (PS) (also known as REM sleep). Other studies have indicated that cholinergic mechanisms are involved in the instigation and maintenance of PS. Hypothetically, the large PS rebounds could be mediated by an upregulation of cholinergic receptors during TSD. To evaluate this hypothesis, regional brain cholinergic receptors were compared in rats subjected to 10-day TSD by the disk-over-water method (TSD rats), yoked control (TSC) rats which received the same physical stimulation but with much smaller reductions in sleep, and home cage control (HCC) rats. l-[³H]nicotine and [³H]quinuclidinyl benzilate were used as specific cholinergic radioligands for nicotinic and muscarinic receptor binding assays, respectively. Nicotinic receptor binding was not significantly different among groups for any of the brain regions assayed, including frontal cortex, parietal cortex, thalamus, amygdala, hippocampus, anterior hypothalamus, posterior hypothalamus, caudate, limbic system (including septal area, olfactory tubercle, and nucleus accumbens), midbrain, pons, and medulla. Thus, there was no evidence that changes in nicotinic receptors mediate the PS rebounds. For muscarinic receptor binding, TSD rats differed significantly from control rats only in showing a higher binding affinity than TSC rats in the limbic system and a lower binding density than HCC rats in the hippocampus. On the other hand, significant differences in muscarinic receptor binding sites between rats selectively deprived of PS and their yoked controls were found only for the septal area. Although chronic sleep deprivation resulted in a few regionally specific changes in muscarinic receptor binding, compared to the large PS rebounds during recovery from chronic sleep deprivation, the changes in receptor binding were very small and were not apparent in the region most intimately related to the cholinergic instigation of PS, i.e., the pons. Thus, there was no substantial evidence that PS rebounds are mediated via cholinergic receptor upregulation.     

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.     

Regulation of α7 nicotinic acetylcholine receptors in mouse somatosensory cortex following whisker removal at birth

Previous studies in postnatal mouse demonstrating high levels of α7 nicotinic acetylcholine receptors on layer IV somatosensory cortical neurons coincident with the onset of functional synaptic transmission led us to investigate whether the number and/or the localization of these receptors could be regulated by activity. Accordingly, we examined α-bungarotoxin binding in mouse somatosensory cortex following removal of all of the vibrissae on one side of the face, either by vibrissal follicle cauterization or daily plucking beginning on the day of birth. Following vibrissa plucking, the levels of [¹²⁵I]α-bungarotoxin binding on postnatal day 6 were significantly higher (23 ± 7%) in the denervated cortex (contralateral to the peripheral manipulation) than the intact cortex. Cauterization also resulted in significantly higher (14 ± 3%) [¹²⁵I]α-bungarotoxin binding in the contralateral vs. the ipsilateral cortex. In contrast, there was no difference in [¹²⁵I]α-bungarotoxin binding in the left and right cortices of unoperated control animals. At postnatal day 14, levels of [¹²⁵I]α-bungarotoxin binding in layer IV were very low in control animals as well as in animals subjected to whisker plucking or cautery. These findings suggest that reducing activity in the somatosensory pathway regulates the density of α7 nicotinic acetylcholine receptors during the first postnatal week. However, the normal decrease in receptor density that is seen during the second postnatal week of development proceeds despite altered sensory activity. J. Comp. Neurol. 397:1–9, 1998. © 1998 Wiley-Liss, Inc.     

Compartment-specific changes in the density of choline and dopamine uptake sites and muscarinic and dopaminergic receptors during the development of the baboon striatum: a quantitative receptor autoradiographic study

In the fetal and young primate neostriatum, cholinergic and dopaminergic markers show patches of high density surrounded by a lower-density matrix. In the adult, the same markers display the opposite pattern, a lower density in striosomes, surrounded by a higher-density matrix. In order to understand the developmental sequences leading to the adult compartmental organization of the primate neostriatum, a quantitative technique was used to study the ontogeny of pre- and postsynaptic components of cholinergic and dopaminergic neurons in baboon caudate nucleus and putamen. The development of specific uptake mechanisms for choline and dopamine and receptors was studied by means of quantitative autoradiography of the specific binding of [3H]-hemicholinium-3 [( 3H]-HC3) and [3H]-mazindol [( 3H]-MAZ) to the choline and dopamine uptake systems, respectively. [3H]-pirenzepine [( 3H]-PIR) was used to label M1 muscarinic receptors and [3H]-spiroperidol [( 3H]-SPI) was used to label striatal dopamine D2 receptors. Serial sections were used for each ligand to determine the precise anatomical relationships between the binding patterns of the different markers. Our aim was to determine whether the adult striosomal distribution of the binding sites studied was due to 1) a selective decrease in patch/striosomal binding density or 2) a selective increase in matrix binding density. Our studies show that a postnatal decrease in the density of [3H]-HC3 sites in the patch/striosomes and an increase in the matrix density of [3H]-MAZ sites are the primary, but not the sole, changes in the compartmental distribution of these sites leading to the adult striosomal organization of the striatal cholinergic and dopaminergic innervation. D2 receptors follow the general developmental pattern of [3H]-MAZ and [3H]-HC3, changing their density of distribution in both compartments during the developmental period examined. In addition, M1 muscarinic receptors already display their adult pattern in the newborn baboon striatum, and therefore represent one of the first neurochemical makers to adopt its mature organization.     

Nicotine-induced release of noradrenaline from hypothalamic synaptosomes

In order to elucidate the functional role of nicotinic receptors in the hypothalamus, the drug-induced release of noradrenaline from hypothalamic synaptosomes was studied utilizing [³H] noradrenaline ([³H]NA). The release of [³H]NA from synaptosomes was significantly increased with an increase of the dose of nicotine, carbamylcholine chloride, reserpine or tyramine hydrochloride added to the medium, whereas arecoline, atropine sulfate or mecamylamine hydrochloride had no significant effect. Mecamylamine hydrochloride completely inhibited the nicotine- or carbamylcholine-induced release of [³H]NA at the concentration of 10⁻⁴ M, but had no effect on the reserpine- or tyramine-induced release of [³H]NA. A high concentration of potassium in the medium which depolarizes the synaptosome membrane significantly enhanced the release of [³H]NA. These results strongly suggest that there exist nicotinic cholinergic receptors in brain synaptic regions which play an important role in the function of hypothalamus by releasing noradrenaline and that the release mechanism of noradrenaline induced by nicotine is different from that induced by reserpine and tyramine. Although the existence of postsynaptic nicotinic receptor sites could not be ruled out, the present studies indicate the importance of presynaptic cholinergic receptors in the brain.     

High-affinity binding of [3H]acetylcholine to muscarinic cholinergic receptors

High-affinity binding of [3H]acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the [3H]acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity [3H]acetylcholine binding sites to total muscarinic binding sites labeled by [3H]quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, [3H] acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that [3H]acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.