Hippocampal muscarinic supersensitivity after AF64A medial septal lesion excludes M1 receptors

Stereotaxic injection of AF64A, into the medial septum of the rat, resulted in significant loss of presynaptic cholinergic markers in this structure. No significant change was observed for the presynaptic neuronal markers for dopamine- and serotonin-containing neurons in either the medial septum or hippocampus. The AF64A lesion also resulted in a significant reduction of muscarinic receptors as demonstrated by a loss of [3H]QNB binding in the medial septum. Subtype analysis showed the decrease of receptor binding in the medial septum to be due to a loss of M1 receptors as well as other muscarinic receptor subtypes. In the hippocampal formation, [3H]hemicholinium-3 binding was significantly reduced in the molecular layer of the dentate gyrus, and in the stratum oriens and stratum radiatum of the hippocampus. AF64A lesion resulted in a significant increase (Bmax) in non-M1 muscarinic receptors in hippocampal stratum oriens, in areas CA2, CA3, and CA4. AF64A lesion of the medial septum did not result in muscarinic receptor alterations in any other region of the hippocampal formation examined. These results indicate that postsynaptic muscarinic receptors in the stratum oriens of the CA2 to CA4 region of the hippocampus mediate primarily the function of the cholinergic cell bodies of the medial septum. These receptors are not of the M1 subtype.     

Cholinergic denervation-like changes in rat hippocampus following developmental lead exposure

We investigated the effects of developmental lead exposure from embryonic day 16 (E16) through postnatal day 28 (PN28), on cholinergic and catecholaminergic markers in the septohippocampal pathway in rats through fourth month of age. Lead exposure resulted in a persistent 30–40% reduction of [³H]hemicholinium-3 ([³H]HC-3) binding in the hippocampus through PN120, and 20–30% reduction of septal and hippocampul choline acetyltransferase (ChAT) activity which persisted through PN84 but returned to control levels in both septum and hippocampus at PN112. The muscarinic ligand [³H]quinuclidinyl benzylate ([³H]QNB) binding was reduced in the septum at PN28 but did not differ significantly from controls at PN56–PN112. Neither short- nor long-term effects of Pb exposure on [³H]QNB binding were seen in the hippocampus. Similar to the effects of fimbria-fornix transection, Pb exposure resulted in a long-term 50–90% increase of tyrosine hydroxylase(TH) activity in the hippocampus, although neither treatment affected TH activity in the septum. The lead-induced increase in hippocampul TH was significantly attenuated by superior cervical ganglionectomy. It is concluded that the effects of perinatal lead exposure resemble in several respects those seen following surgical disruption of the septohippocampal pathway in adult animals. The denervation-like effects in the hippocampus may be an important factor in long-term learning and cognitive impairments following developmental exposure to low-levels of lead.     

Alterations in [3H]kainate and N-methyl-D-aspartate-sensitive L-[3H]-glutamate binding in the rat hippocampal formation following fimbria-fornix lesions.

Following lesions of the fimbria-fornix, there is a time-dependent increase in interictal spikes and seizure susceptibility. This may result from sprouting of local excitatory and inhibitory circuits in response to the loss of subcortical and commissural innervation of the hippocampal formation. We used receptor autoradiography to examine the density of N-methyl-D-aspartate (NMDA)-sensitive L-[3H]glutamate and [3H]-kainate (KA) binding sites in the hippocampal formation at 5 days, 3 months, and 1 year following bilateral aspiration lesions of the fimbria-fornix. At 5 days post-lesion, the CA3 and CA1 strata radiatum and oriens displayed a decrease (20-42%, P less than 0.01) in NMDA-sensitive L-[3H]glutamate binding. The initial decrease was followed by a moderate recovery at later time points but was still evident at 1 year postlesion. This may reflect a lesion-induced turnover of synaptic complexes, down-regulation of postsynaptic receptors, or loss of presynaptic receptors. Five days following fimbria-fornix lesion there was also a decrease (13-15%, P less than 0.05) in [3H]KA binding in CA3 strata radiatum and pyramidale. However, at 3 months postlesion KA receptor density was elevated by 29-33% (P less than 0.01) in the outer molecular layer of the dentate gyrus with no significant change in binding to the inner molecular layer. By 1 year postlesion, the density of [3H]KA binding sites was not significantly different from that observed in control animals of the same age. The increase in KA receptor density in the outer molecular layer 3 months after fimbria-fornix lesion may reflect sprouting of the perforant path input or mossy fibers to this region and contribute to the increase in interictal spikes and seizures susceptibility.     

Early septal lesion: Effect on the development of the cholinergic system in rat hippocampus

The development of the cholinergic system in the rat hippocampal formation was studied following lesion of the septal region at an age of 2–4 days postnatal (i.e. the lesion was performed prior to the establishment of the septohippocampal connections). The steep increase in acetylcholinesterase (AChE) level, that under normal conditions take place during the second and the third week postnatal, was not observed in early lesioned animals, and AChE level at maturity was about 30% of control. AChE level of adult-lesioned animals was about 15% of control, suggesting an age-dependent plasticity in response to the lesion. Early deafferentation did not seem to alter the pattern of development of muscarinic binding sites as measured by specific binding of [³H]quinuclidinyl benzilate ([³H]QNB). Total [³H]QNB bound per hippocampus of adult, early-lesioned animals was about 70% of control, but this reduction could be accounted for by the atrophy observed in the hippocampal formation following early lesion. Binding of [³H]QNB per protein in early lesioned animals did not differ from normal. Thus the development and the level of muscarinic binding sites in the hippocampal formation do not seem to depend upon normal establishment of presynaptic contacts.     

Characterization of muscarinic cholinergic receptors in the submandibular gland of the rat

The specific muscarinic ligand [3H]quinuclidinyl benzilate ([3H]QNB) was used to label acetylcholine receptors in the submandibular gland of the rat. Specific binding of [3H]QNB increased linearly with tissue concentration in the range of 0.02-0.3 mg of protein/ml. Kinetic analysis of [3H]QNB binding revealed the presence of a single population of high affinity binding sites, with a dissociation constant of 87.2 pM and a Hill coefficient of 0.95. The binding was saturable and the receptor density was 214 fmol/mg of protein. The rate constants at 37 degrees C for association and dissociation of the [3H]QNB-receptor complex were 5.98 X 10(-8) M-1 X min-1 and 6.6 X 10(-3) X min-1, respectively. The ratio k-1/k+1 gave a Kd value of 11.1 pM, similar to the Kd value (13.1 pM) determined by kinetic parameters when extrapolated at infinitely low receptor concentration. Muscarinic antagonists displaced [3H]QNB from muscarinic receptors with a Hill coefficient near to 1.0. Displacement curves for muscarinic agonists and for the atypical antagonist pirenzepine had Hill values significantly less than one. In the presence of 0.1 mM GPP(NH)P, the potency of agonists but not antagonists in displacing [3H]QNB binding decreased 2 to 3-fold. The [3H]QNB binding site was sensitive to the inhibitory effect of various sulfhydryl reagents. Repeated treatments of rats with an acetylcholinesterase inhibitor led to a decreased density of muscarinic receptors in the submandibular gland. This alteration was specific for the muscarinic recognition site and was paralleled by a reduced sensitivity to carbachol.     

Autoradiographic analysis of muscarinic receptors and choline-uptake mechanisms within foetal basal forebrain transplants

The pharmacological characteristics of both muscarinic receptors and high-affinity choline uptake sites were examined within intracerebral implants of foetal basal forebrain cell suspensions. Approximately 12 weeks after implantation, the transplants were identified by acetylcholinesterase histochemistry. Muscarinic receptors were labelled by [3H]quinuclidinyl benzylate (QNB) autoradiography. The M1 and M2 receptor components of QNB binding were differentiated by pirenzepine competition. The distribution of the high-affinity choline uptake site was examined using [3H]hemicholinium-3 (HC3) autoradiography. Unilateral lesion of the nucleus basalis reduced [3H]QNB (8-25%) and [3H]HC3 (19-43%) binding throughout host frontoparietal cortex ipsilateral to the lesion but did not significantly alter these cholinergic markers within cingulate cortex, subcortical white matter, striatum or septum. Saturation analysis of the implanted tissue revealed the presence of a single population of [3H]QNB and [3H]HC3 binding sites with affinities similar to those of the host tissue (KD = 0.43 nM and 20.4 nM respectively). However, the receptor profile which developed appeared to be intrinsic to the implant; it was unaffected by the site of implantation and was dissimilar to that which ultimately developed in the donor tissue when left in situ.     

Calcium antagonist, adenosine A1, and muscarinic bindings in rat hippocampus after transient ischemia

The protective roles of Ca2+ channel blockers against ischemic hippocampal damage are still debated. We used autoradiography to study postischemic L-type Ca2+ channels (1,4-dihydropyridine Ca2+ channel blocker binding), adenosine A1 receptors, and muscarinic cholinergic receptors in the rat hippocampus using [3H]PN200-110 (PN), [3H]cyclohexyladenosine (CHA), and [3H]quinuclidinyl benzilate (QNB), respectively, in 49 rats subjected to 20 minutes of forebrain ischemia. The rats were decapitated after 1 (n = 7), 3 (n = 7), 6 (n = 8), 12 (n = 7), 24 (n = 6), 48 (n = 6), or 168 (n = 8) hours of recirculation; eight control rats were sham-operated but experienced no cerebral ischemia. Reduced receptor binding preceding the delayed death of CA1 pyramidal cells was first observed in the stratum oriens of the CA1 subfield. Significant reductions in [3H]PN, [3H]CHA, and [3H]QNB bindings of this stratum compared with control were noticed after 3 (35%, p less than 0.01), 12 (31%, p less than 0.01), and 1 (10%, p less than 0.05) hours of recirculation, respectively. By 168 hours after ischemia (when the populations of CA1 pyramidal cells were depleted) all strata in the CA1 subfield had lost most of their receptor sites, and [3H]PN, [3H]CHA, and [3H]QNB bindings in the stratum oriens were decreased to 23%, 30%, and 63% of control (p less than 0.01). Although [3H]PN binding in the CA3 subfield did not change significantly during 168 hours after ischemia, the histologically intact dentate gyrus exhibited a 31% loss of binding sites compared with control (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative autoradiographic analysis of muscarinic cholinergic and adenosine A1 binding sites after transient forebrain ischemia in the gerbil

The influence of transient forebrain ischemia on adenosine A₁ and muscarinic cholinergic receptors in the gerbil brain 1–27 days after recirculation was studied. The topographical distribution and the alteration in the adenosine A₁ and muscarinic receptor sites were analyzed by means of quantitative receptor autoradiography using [³H]cyclohexyladenosine ([³H]CHA) and [³H]quinuclidinyl benzilate ([³H]QNB), respectively. In most regions examined, the temporal profiles of the alteration of the receptor density were in accordance with the histopathological findings. [³H]CHA binding activity decreased suddenly after neuronal damage, while [³H]QNB grain density showed a gradual decrease in the dorsolateral caudate-putamen and in the CA₁ subfield of the hippocampus. In the caudate-putamen, [³H]CHA and [³H]QNB binding activity in the dorsal aspect was markedly reduced 1–27 days after ischemia. [³H]CHA binding activity in the ventromedial region of the caudate-putamen also decreased 1–3 days after ischemia, though neuronal damage was restricted to the dorsolateral aspect. Neuronal death in CA₁ was preceded by the decrease in [³H]QNB binding activity in the stratum radiatum 1 and 2 days after ischemia. Marked decrease in [³H]QNB and [³H]CHA binding activity was noted in the CA₁ subfield 3–27 days after recirculation. Three to 27 days after ischemia, the A₁ binding activities in the CA₃ subfield of the hippocampus and int he dentate gyrus were reduced despite the normal appearance of these areas throughout the reperfusion period. Muscarinic binding sites in the CA₃ subfield were also reduced 27 days after ischemia. Despite minimal neuronal damage in the lateral septal nucleus and in the substantia nigra, the A₁ binding activity in these regions was reduced by 70% and 50%, respectively. These results provide further evidence that the muscarinic receptors in the dorsolateral region of the caudate-putamen are localized postsynaptically on small and medium-sized neurons and that those in the CA₁ subfield of the hippocampus are localized on the CA₁ pyramidal cells.     

Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland.

Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]diphenyl-acetoxy-N-methyl-piperidine ([3H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25 degrees C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [3H]QNB and [3H]4-DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M1, M3 subtypes selective antagonist) > atropine (general) > pirenzepine (M1). Methoctramine (M2) didn't antagonise the [3H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca2+ level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M1 and M3 muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined.     

Lack of [3H]quinuclidinyl benzylate binding to biologically relevant binding sites on mononuclear cells.

We analyzed the binding characteristics of [3H]quinuclidinyl benzylate ([3H]QNB), a muscarinic cholinergic ligand, to rat and human mononuclear cells (MNC). Under various assay conditions, atropine-sensitive, saturable binding occurred with an apparent Kd of 10 nM. Conditions which disrupted the MNC membrane reduced total binding and eliminated specific binding. Muscarinic agonists were unable to inhibit [3H]QNB binding to MNC at concentrations up to 10(-2) M. Stereoisomers dexetimide and levetimide were equipotent inhibitors of binding (IC50 2 x 10(-5) M). We conclude that, although atropine-sensitive binding of [3H]QNB to MNC occurs, the binding is not consistent with the presence of a biologically relevant muscarinic cholinergic receptor.     

Postischemic alteration of muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in selectively vulnerable areas: an autoradiographic study of gerbil brain.

We performed receptor autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of a voltage dependent L-type calcium channel blocker 1 h-1 month after transient cerebral ischemia in the gerbil brain. [3H]Quinuclidinyl benzilate (QNB), [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Transient ischemia was induced for 10 min. [3H]QNB and [3H]CHA binding showed no significant alteration in selectively vulnerable areas at an early stage (1-24 h) of recirculation. However, the dentate molecular layer which was resistant to ischemia revealed a significant decrease in the [3H]CHA binding sites 24 h after ischemia. Thereafter, the [3H]QNB and [3H]CHA binding showed significant reduction in most of selectively vulnerable areas. Marked reduction was especially found in the dorsolateral part of striatum and the hippocampal CA1 sector which was the most vulnerable to ischemia. In contrast, [3H]PN200-110 binding showed a transient elevation in the hippocampal CA1 sector, the dentate molecular layer and the thalamus 1 h of recirculation. However, the striatum and neocortex revealed no alteration in the [3H]PN200-110 binding. Thereafter, the reduction in the [3H]PN200-110 binding was seen only in the dorsolateral part of the striatum and the hippocampal CA1 sector. The results suggest that transient cerebral ischemia can cause the alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of L-type calcium channel blocker in most of selectively vulnerable areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic acetylcholine receptors are localized on striatal cyclic GMP-containing neurons

The localization of muscarinic acetylcholine receptors has been determined using in vitro binding of radiolabeled quinuclidinyl benzilate ([3H]QNB), a specific reversible muscarinic receptor antagonist. All cyclic GMP-immunoreactive neurons in the rat striatum show clustering of [3H]QNB silver grains overlying their somata following autoradiographic analysis. The autoradiography of total binding of silver grains over the cyclic GMP-containing neurons was approximately 800 times as dense as the surrounding neuropil localization of radioligand binding sites. Incubation of striatal tissue slices in the presence of micromolar atropine, to determine non-specific binding of [3H]QNB, decreased the autoradiographic silver grain density of the neuropil about 2.5 times, and lessened the number of receptor sites detectable on cyclic GMP-positive neurons at least 5-fold. Biochemical examination of [3H]QNB binding on tissue sections demonstrated that the ligand binding is saturable and dependent on section thickness for the muscarine receptor subtype of acetylcholine.     

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.     

Downregulation of muscarinic receptors in the rat caudate-putamen after lesioning of the ipsilateral nigrostriatal dopamine pathway with 6-hydroxydopamine (6-OHDA): normalization by fetal mesencephalic transplants

The autoradiographic distribution and density of muscarinic receptors was studied in the neostriatum of rats with long-term unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic pathway and in lesioned rats who had additionally received embryonic substantia nigra grafts in the dopamine denervated striatum. Muscarinic receptors were labeled with [³H]quinuclidinyl benzilate (QNB), M₁ receptors were directly labeled with [³H]pirenzepine (PZ) and non-M₁ receptors were labeled by the competition of 100 nM PZ with [³H]QNB. The density and distribution of muscarinic receptors were directly compared to the sodium-dependent, high-affinity, choline uptake sites as labeled with [³H]hemicholinium-3 (HC-3). In the 6-OHDA-lesioned animals, there was a 25% reduction in muscarinic receptors labeled with [³H]QNB. Subtype analysis showed that there was a reduction of both M₁ (−26%) and non-M₁ (−33%) receptors. A normal density of both muscarinic receptor populations was found in animals with successful transplants. Saturation analysis demonstrated that the changes, in muscarinic receptor density, were due to a change in receptor number (B_max) and not affinity (K_d). There was no significant change in [³H]HC-3 binding in the 6-OHDA-lesioned or transplanted animals, indicating that alterations in muscarinic receptors were not due to transynaptic degeneration of striatal cholinergic interneurons. The findings of downregulation of muscarinic receptors following long-term dopamine denervation and the subsequent normalization of muscarinic receptor density after fetal mesencephalic transplantation suggests that transplanted substantia nigra cells are able to restore inhibitory control on striatal cholinergic interneurons.     

The effect of time following exposure to trimethyltin (TMT) on cholinergic muscarinic receptor binding in rat hippocampus

Adult male Long-Evans rats were given 6 mg/kg trimethyltin (TMT). Rats were killed 1, 3, 7, 14, 21, 35 or 60 d later. An untreated control group was included. Brain sections were processed using film autoradiography to visualize in the hippocampus either total muscarinic receptor binding ([³H]quinuclidiny] benzilate: [³H]QNB), or M₁ receptors ([³H]pirenzepine; [³H]PZ), or M₂ receptors ([³H]oxotremorine-M; [³H]OXO-M). A reduction in [³H]QNB binding was found in CA1 and CA3c 7 d after TMT, but not in CA3a,b, or the dentate gyrus. [³H]PZ binding was decreased throughout Ammon’s horn by 14 d after treatment. [³H]OXO-M binding decreased 1 d after exposure in CA1 and in all subfields of Ammon’s horn by d 3. Neither [³H]PZ or [³H]OXO-M binding decreased in the dentate gyrus of TMT-treated rat at any time point. The temporal patterns of receptor loss may be explicable by reference to timing of fiber and cell body degeneration reported in previous studies and the regional differences may account for discrepancies between reports of either substantial decreases or no loss in hippocampal muscarinic receptors after TMT exposure.     

Development of the cholinergic system in control and intra-uterine growth retarded rat brain

The activity of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and muscarinic receptors was studied in control rats and in rats growth-retarded in utero because of reduction of the blood supply 5 days before birth. The different markers of the cholinergic system were estimated at P (postnatal day) 6, 9, 12, 15, 22 and 60 in cerebellum, hypothalamus, septum, striatum and CA1, CA3 and fascia dentata of the hippocampus. In control rats, there was a transient increase in ChAT activity in the septum during the second week of postnatal development. In the intrauterine growth retarded rats there was a marked delay in this developmental rise in CA1, CA3 at P6 and P9 and in the fascia dentata at P14 respectively. This delayed rise enzyme activity was associated with a significant reduction of muscarinic binding sites [( 3H]QNB) in the hippocampus. AChE staining showed a similar development in both groups. Therefore, the undernutrition produced by a reduction of the blood supply 5 days before birth is associated with a delayed maturation of cholinergic functions.     

Characterization of muscarinic acetylcholine receptors in cultured adult skin fibroblasts: Effects of the swedish Alzheimer's disease APP 670/671 mutation on binding levels

Summary We have characterised the muscarinic receptor subtypes found in human skin fibroblasts and compared binding levels in cell lines from members of the Alzheimer's disease family with the Swedish amyloid precursor protein (APP) 670/671 mutation. Binding studies with [³H] quinuclidinyl benzilate ([³H]QNB) and the M2/M4 selective antagonist [³H](±)-5,11-dihydro-11-{[(2-[(di-propylamino)methyl]-1-piperidinyl}ethyl)amino]carbonyl}-6H-pyrido(2,3-b)(1,4) benzodiazepine-6-one ([³H]AF-DX 384) revealed the presence of a single population of muscarinic receptors on lysed fibroblast membranes. [³H]QNB binding was displaced by a number of selective muscarinic ligands with a rank order of potency: atropine>himbacine>methoctramine>(±)-p-fluoro-hexahydro-sila-difenidol hydrochloride>pirenzepine>muscarinic-toxin-3. APP 670/671 mutation carrying cell lines showed 25–35% lower levels of muscarinic receptors labelled with [³H]QNB, [³H]N-methyl scopolamine and [³H]AF-DX 384, compared to controls. This difference was not statistically significant due to large individual variation. It is concluded that muscarinic receptors on adult skin fibroblasts are predominantly of the M2 subtype. Since these cells do not possess M1 and M3 receptor subtypes, they are unlikely to provide a good model for studying muscarinic receptor regulation of APP processing.     

Muscarinic receptors in preoptic area and hypothalamus: effects of cyclicity, sex and estrogen treatment

Cholinergic muscarinic receptor binding was measured in the preoptic area (POA) and whole hypothalamus (HTH) of adult Sprague-Dawley rats using the tritiated antagonist quinuclidinyl benzilate ([3H]QNB) as the ligand. Binding of [3H]QNB expressed as fmol/mg protein was 30% higher in POA than in HTH from gonadectomized rats. Cyclic changes were observed in the POA with the highest binding at proestrus and the lowest binding at diestrus. In HTH, no significant changes occurred over the estrous cycle. Estrogen treatment (10 micrograms of estradiol benzoate (EB)/120 g b. wt./48 and 24 h before sacrifice) increased [3H]QNB binding by 42% in the POA and 17% in HTH, relative to the ovariectomized controls. The enhancement of [3H]QNB binding in POA as compared with controls was evident with both the filtration and the centrifugation methods, although binding levels were higher when centrifugation assay was used. A lower estrogen dose (2 micrograms EB/rat/48 and 24 h before sacrifice) which is routinely used to activate lordotic behavior in female rats increased muscarinic binding by 26% in the POA but had no appreciable effect in HTH. A significant sex difference was found in the ability of estrogen to induce [3H]QNB binding in the central nervous system (CNS). Estrogen was ineffective in altering [3H]QNB binding in either brain region of castrated males, although the level and pattern of cholinergic binding between untreated gonadectomized males and females were similar.2+ These data suggest that physiological changes in estrogen secretion over the estrous cycle are capable of modulating cholinergic muscarinic binding in the POA and these changes may be of physiological relevance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional distribution of cholinergic muscarinic receptors in spinal cord

Quinuclidinylbenzilate ([³H]QNB) binding sites are present in the rat spinal cord. The binding sites are muscarinic in character based on displacement of [³H]QNB by cholinoceptive drugs. They are distributed rather uniformly along the cord, although the receptor density is greater in gray matters than in white matter. Binding to white matter may be associated with glial cells. Within the gray matter, the receptor density is higher in the ventral born than in the dorsal horn. In the thoracic region receptor density is about equal in the intermediate zone and ventral horn. Midthoracic transection of the cord does not change the receptor density or the dissociation constant of [³H]QNB in the lumbar cord. In contrast, treatment with the neurotoxin, 6-aminonicotinamide, which produces lesions of the cord, loss of motor control and paralysis, reduces the receptor density and affinity of [³H]QNB for lumbar gray matter but not white matter. The presence of [³H]QNB binding sites throughout the spinal cord as well as the documented presence of acetylcholine-containing neurons, suggest that muscarinic receptors play a role in all phases of spinal cord physiology.     

Sequential changes in muscarinic acetylcholine, adenosine A1 and calcium antagonist binding sites in the gerbil hippocampus following repeated brief ischemia.

We performed quantitative autoradiography to determine sequential alterations in the binding of muscarinic cholinergic and adenosine A1 receptors and of an L-type calcium channel blocker in the gerbil hippocampus following repeated brief ischemic insults. [3H]Quinuclidinyl benzilate (QNB). [3H]cyclohexyladenosine (CHA) and [3H]PN200-110 were used to label muscarinic and adenosine A1 receptors and L-type calcium channels, respectively. Changes at 1 h, 6 h, 1 day, 4 days and 1 month after three 2-min ischemic insults were compared with changes after single 2- or 6-min ischemia. Two-minute ischemia, which causes no histopathological neuronal damage, produced no persistent alterations in binding sites. We observed a transient and mild increase in binding activities, especially in [3H]CHA binding, at 1 h of recirculation. Following 6-min ischemia and three 2-min ischemic insults. [3H]QNB and [3H]PN200-110 binding decreased by more than 50% in the CA1 subfield by 1 month, but [3H]CHA binding decreased transiently by 20-30% at 4 days when delayed neuronal death of hippocampal CA1 pyramidal cells took place. Reductions in binding, especially in [3H]QNB binding, following three 2-min ischemic insults were greater and appeared earlier than those after 6-min ischemia. Furthermore, alterations extended to the CA3 subfield and the dentate gyrus following repeated insults. Thus, alterations in receptor binding after repeated ischemic insults were greater than those after equivalent single period of ischemia.