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.     

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.     

Autoradiographic studies in animal models of hemi-parkinsonism reveal dopamine D2 but not D1 receptor supersensitivity. I. 6-OHDA lesions of ascending mesencephalic dopaminergic pathways in the rat

The selective dopaminergic antagonist ligands [3H]SCH 23390 and [3H]sulpiride were used to reveal autoradiographically dopamine D1 and D2 receptors, respectively, in brain sections from rats which had received unilateral 6-hydroxydopamine (6-OHDA) injections destroying ascending nigrostriatal neurones. The binding of both ligands to striatal sections was first shown to be saturable, reversible and of high affinity and specificity [( 3H]SCH 23390: Bmax 2.16 pmol/mg protein, Kd 1.4 nM; [3H]sulpiride; Bmax 0.67 pmol/mg protein, Kd 10.7 nM). After unilateral stereotaxic 6-OHDA injections, rats rotated contralaterally when challenged with apomorphine (0.5 mg/kg), or specific D1 or D2 agonists, SKF 38393 (1.0-5.0 mg/kg) and LY 171555 (0.05-0.5 mg/kg), respectively. Loss of forebrain dopaminergic terminals was assessed autoradiographically using [3H]mazindol to label dopamine uptake sites. A loss of approximately 90-95% of uptake sites was reproducibly accompanied by an enhanced density of binding ipsilaterally for the D2 ligand, [3H]sulpiride, in all areas of the striatum, but most markedly in the lateral areas. An increase in the D2 binding site density was also seen in the ipsilateral nucleus accumbens and the olfactory tubercle. In contrast, in the same animals, the striatal D1 receptors were far less affected by dopaminergic denervation, with no consistent changes seen in the binding of [3H]SCH 23390. These results suggest that dopamine D2 receptors are more susceptible than D1 receptors to changes after dopaminergic denervation, which is expressed as an increase in the density of binding sites revealed here with [3H]sulpiride.     

Selective cortical infarction reduces [3H]sulpiride binding in rat caudate-putamen: autoradiographic evidence for presynaptic D2 receptors on corticostriate terminals

Although the existence of presynaptic D2 dopamine receptors on corticostriate terminals has been supported by numerous receptor-binding studies, recent autoradiographic data has failed to demonstrate loss of striatal D2 receptors following cortical lesions. In the present study, Long-Evans rats were subjected to unilateral middle cerebral artery (MCA) infarction in order to produce reproducible lesions of the neocortex without damaging subcortical structures. Animals were sacrificed 2 and 4 wk following lesion and brains were prepared for receptor autoradiography. D2 receptors were studied using the selective ligand [3H]sulpiride, while D1 dopamine receptors were examined using [3H]SCH 23390. Sodium-dependent, high-affinity choline uptake sites were labeled with [3H]hemicholinium-3, thereby providing a quantitative measure of cholinergic neuronal integrity. Unilateral cortical infarction resulted in approximately a 20% reduction in [3H]sulpiride binding in several discrete regions of the ipsilateral caudate-putamen (CPu), but not in the nucleus accumbens. D2 receptor binding was also reduced significantly in some areas of the contralateral CPu when compared with [3H]sulpiride binding in sham-operated, control animals. In contrast, D1 receptors (as identified by [3H]SCH 23390 and high-affinity choline uptake sites (labeled with [3H]-HC-3) were not affected by the cortical lesion. The results provide autoradiographic confirmation of the existence of presynaptic D2 receptors on corticostriate terminals.     

Facilitation of amphetamine-induced rotation by muscarinic antagonists is correlated with M2 receptor affinity

This study examined the relationship between the affinity of cholinergic drugs for muscarinic receptor subtypes and their potency in potentiating or inhibiting amphetamine-induced rotation. The ascending nigrostriatal dopaminergic pathway was unilaterally lesioned in male Wistar rats using 6-hydroxydopamine. In these rats, ipsiversive rotation induced by amphetamine sulphate (1 mg/kg, s.c.) was dose-dependently inhibited by the cholinergic agonists oxotremorine, RS86 and pilocarpine and by the acetylcholinesterase inhibitor physostigmine. In contrast the cholinergic antagonists scopolamine, secoverine and dicyclomine facilitated amphetamine-induced rotation. Agonist and antagonist potencies were then compared with M1 and M2 binding site affinities estimated by displacing [3H]pirenzepine from forebrain and [3H]QNB from brainstem homogenates. The data suggest a relationship between antagonist potency and M2 binding site affinity.     

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°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.     

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.     

Effects of serotonergic denervation on the density and plasticity of brain muscarinic receptors in the rat.

This study investigated whether serotonergic lesion may affect density, sensitivity, and plasticity of muscarinic receptors in hippocampus and cerebral cortex. Intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT) in rats produced a 90% reduction in cortical and hippocampal 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents. In these brain areas, the 5,7-DHT lesion did not affect the overall density of muscarinic receptors or those of M1 and non-M1 muscarinic receptor subtypes as assayed using [3H]N-methylscopolamine ([3H]NMS), [3H]pirenzepine, and [3H]NMS in the presence of pirenzepine, respectively. In addition, the binding of the muscarinic agonist [3H]oxotremorine-M (OXO-M), taken as an indirect index of coupling efficiency of non-M1 receptors with G-proteins, did not change significantly in cortex and hippocampus of 5,7-DHT-lesioned rats. Similarly, carbachol-induced accumulation of [3H]inositol phosphates (InPs) in hippocampal miniprisms showed no significant differences between tissues from 5,7-DHT-lesioned and sham-operated rats. In sham-operated rats, an intraperitoneal (i.p.) injection of scopolamine (10 mg/kg once daily) during 21 days caused an increased density of [3H]NMS binding sites in cortex (+20%) and hippocampus (+26%). This up-regulation was restricted to non-M1 receptors subtypes. In 5,7-DHT-lesioned rats, chronic scopolamine failed to modify significantly the density of cortical or hippocampal M1 or non-M1 receptors. These results suggest 1) that 5-HT denervation did not affect the density and sensitivity of muscarinic receptors and 2) that the ability of cortical and hippocampal non-M1 receptors to up-regulate following repeated injection of scopolamine requires the integrity of 5-HT neurons terminating in these brain structures.     

Effects of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin on the dopaminergic and cholinergic receptors as evaluated by positron emission tomography in the Rhesus monkey

Summary The effects of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (R-THBP) on the central cholinergic and dopaminergic systems in the Rhesus monkey brain were investigated by positron emission tomography (PET) with the muscarinic cholinergic receptor ligands (N-[¹¹C]methyl-benztropine) and dopaminergic receptor ligands selective for D₁ D₂, and D₃ subtypes ([¹¹C]SCH23390, N-[¹¹C]methyl-spiperone, and (+)[¹¹C]UH232, respectively). None of the doses (3, 10, and 30 mg/kg i.v.) of R-THBP used significantly affected the regional cerebral blood flow (rCBF as determined by Raichle's H₂ ¹⁵O method), and 10 mg/kg of R-THBP had little effect on the regional cerebral metabolic rate of glucose (rCMRglc) in the Rhesus monkey brain, as assessed by the graphical [¹⁸F]fluoro-deoxyglucose method. The effect of R-THBP on the muscarinic cholinergic system was dose dependent; while 3 mg/kg of R-THBP did not significantly alter the uptake ratio of N-[¹¹C]methyl-benztropine in several brain regions to that in the cerebellum, 10 and 30 mg/kg of R-THBP significantly reduced the uptake ratio in the thalamus, as well as in the frontal and temporal cortices. None of the doses (3, 10, and 30 mg/kg i.v.) of R-THBP tested affected [¹¹C]SCH23390 (dopamine D₁ receptor) binding. However, the k3 value for N-[¹¹C]methyl-spiperone (dopamine D₂ receptor) binding, which represents the association rate × B_max value, was significantly decreased in the striatum. The uptake ratio of (+)[¹¹C]UH232 (dopamine D₃ receptor) in the striatum to that in the cerebellum was also decreased by administration of R-THBP (3 and 30 mg/kg i.v.). These findings suggest that R-THBP acts on dopamine D₂ and D₃ receptors selectively without markedly affecting dopamine D₁ receptor binding. Furthermore, the changes in cholinergic and dopamine D₂ and D₃ receptors in vivo can not be attributed to a change in rCBF but may depend on the action of R-THBP.Abbreviations R-THBP 6R-L-erythro-5,6,7,8-tetrahydrobiopterin - PET positron emission tomography - rCBF regional cerebral blood flow - rCMRglc regional cerebral metabolic rate of glucose     

Sequential changes of cholinergic and dopaminergic receptors in brains after 6-hydroxydopamine lesions of the medial forebrain bundle in rats

Summary. We studied sequential changes in muscarinic cholinergic receptors, high-affinity choline uptake sites and dopamine D₂ receptors in the brain after 6-hydroxydopamine lesions of the medial forebrain bundle in rats. The animals were unilaterally lesioned in the medial forebrain bundle and the brains were analyzed at 1, 2, 4 and 8 weeks postlesion. [³H]Quinuclidinylbenzilate (QNB), [³H]hemicholinum-3 (HC-3) and [³H]raclopride were used to label muscarinic cholinergic receptors, high-affinity choline uptake sites and dopamine D₂ receptors, respectively. The degeneration of nigrostriatal pathway produced a transient decrease in [³H]QNB binding in the parietal cortex of both ipislateral and contralateral sides at 2 and 8 weeks postlesion. [³H] QNB binding also showed a mild but insignificant decrease in the ipsilateral striatum throughout the postlesion periods. No significant change was observed in the substantia nigra (SN) of both ipsilateral and contralateral sides throughout the postlesion periods. In contrast, [³H]HC-3 binding showed no significant change in the parietal cortex of both ipsilateral and contralateral sides during the postlesion. However, [³H]HC-3 binding was upregulated in the ipsilateral dorsolateral striatum throughout the postlesion periods. The ventromedial striatum also showed a significant increase in [³H]HC-3 binding at 1 week and 2 weeks postlesion. On the other hand, no significant change in [³H]raclopride binding was found in the parietal cortex of both ipsilateral and contralateral sides during the postlesion. [³H]Raclopride binding showed a conspicuous increase in the ipsilateral striatum (35–52% of the sham-operated values in the lateral part and 39–54% in the medial part) throughout the postlesion periods. In the contralateral side, a mild increase in [³H]raclopride binding was also found in the striatum (10–15% of the sham-operated values in the lateral part and 22% in the medial part) after lesioning. However, a significant decline in [³H]raclopride binding was observed in the ipsilateral SN and ventral tegmental area during the postlesion. The present study indicates that 6-hydroxydopamine injection of medial forebrain bundle in rats can cause functional changes in high-affinity choline uptake site in the striatum, as compared with muscarinic cholinergic receptors. Furthermore, our studies demonstrate an upregulation in dopamine D₂ receptors in the striatum and a decrease in the receptors in the SN and ventral tegmental area after the 6-hydroxydopamine injection. Thus, these findings provide further support for neurodegeneration of the nigrostriatal pathway that occurs in Parkinson's disease. Received April 26, 1999; accepted November 12, 1999     

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.     

Normalization of subtype-specific muscarinic receptor binding in the denervated hippocampus by septodiagonal band grafts

Unilateral fimbria-fornix lesions were made by aspiration in female Sprague-Dawley rats. In a group of these rats, fetal septal tissue was transplanted into the lesion cavity. Lesion of the fimbria-fornix resulted in a reduction of cholinergic input to the hippocampal formation as indicated by the loss of acetylcholinesterase (AChE)-positive staining in all ipsilateral hippocampal laminae and a loss of [3H]hemicholinium-3 binding to cholinergic terminals in the strata oriens (82% reduction) and radiatum (77% reduction) of areas CA2 and CA3 and in the molecular layer of the dentate gyrus (83% reduction). In contrast, the density of muscarinic receptor binding ([3H]QNB) increased in the strata oriens (80% increase) and radiatum (70% increase) in areas CA2-CA4. This was shown to be due to an actual increase in receptor number (Bmax) and not to a change in affinity (KD). Analysis of muscarinic receptor subtypes indicated that the increase in receptor binding in the stratum radiatum was of the M-1 subtype ([3H]-pirenzepine) and in the stratum oriens was of the M-2 subtype ([3H]QNB + 100 nM pirenzepine). In the host hippocampus after fetal septal graft, the staining for AChE, the binding of [3H]hemicholinium-3, and the binding of muscarinic receptors (both the M-1 and M-2 receptor subtypes) were all comparable to nonlesioned control values. These data indicate that the fetal septal grafts have reinnervated the host hippocampus and have made synaptic contact with host cells in a manner capable of regulating postsynaptic muscarinic receptors.     

Differential vulnerability of dopamine receptors in the mouse brain treated with MPTP

We investigated the chronological changes of dopamine D1 and D2 receptors and dopamine uptake sites in the striatum and substantia nigra of mouse brain treated with 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) by quantitative autoradiography using [3H]SCH23390, [3H]raclopride and [3H]mazindol, respectively. The mice received i.p. injections of MPTP (10 mg/kg) four times at intervals of 60 min, the brains were analyzed at 6 h and 1, 3, 7 and 21 days after the last the injection. Dopamine D2 receptor binding activity was significantly decreased in the substantia nigra from 7 to 21 days after MPTP administration, whereas such binding activity was significantly increased in the medial part of the striatum at 21 days. There was no alteration of dopamine D1 receptor binding activity in either the striatum or the substantia nigra for the 21 days. The number of dopamine uptake sites gradually decreased in the striatum and the substantia nigra, starting at 6 h after MPTP administration, and the lowest levels of binding activity were observed at 3 and 7 days in the striatum (18% of the control values in the medial part and 30% in the lateral part) and at 1 day in the substantia nigra (20% of the control values). These results indicate that severe functional damage to the dopamine uptake sites occurs in the striatum and the substantia nigra, starting at an early stage after MPTP treatment. Our findings also demonstrate the compensatory up-regulation in dopamine D2 receptors, but not dopamine D1 receptors, in the striatum after MPTP treatment. Furthermore, our results support the existence of dopamine D2 receptors, but not dopamine D1 receptors, on the nigral neurons. The present findings suggest that there are differential vulnerabilities to MPTP toxicity in the nigrostriatal dopaminergic receptor systems of mouse brain.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

Developmental profiles of various cholinergic markers in the rat main olfactory bulb using quantitative autoradiography

The existence of possible relationships among the developmental profile of various cholinergic markers in the main olfactory bulb (OB) was assessed by using in vitro quantitative autoradiography. Muscarinic receptors were visualized with [³H]pirenzepine (muscarinic M1-like sites) and [³H]AF-DX 384 (muscarinic M2-like sites); nicotinic receptors by using [³H]cytisine (nicotinic 42-like subtype) and [¹²⁵I]α-bungarotoxin (nicotinic 7-like subtype); cholinergic nerve terminals by using [³H]vesamicol (vesicular acetylcholine transport sites) and [³H]hemicholinium-3 (high-affinity choline uptake sites). These various cholinergic markers exhibited their lowest levels at birth and reached adult values by the end of the 4–5 postnatal weeks. However, the density of presynaptic cholinergic markers and nicotinic receptors at postnatal day 2 represented a large proportion of the levels observed in adulthood, and displays a transient overexpression around postnatal day 20. In contrast, the postnatal development of cholinergic muscarinic M1-like and M2-like receptors is apparently regulated independently of the presynaptic cholinergic markers and nicotinic receptors. Two neurochemically and anatomically separate olfactory glomeruli subsets were observed in the posterior OB of the developing rat. These atypical glomeruli expressed large amounts of [³H]vesamicol- and [³H]hemicholinium binding sites without significant amounts of muscarinic M1, M2, or nicotinic α4β2 receptor binding sites. A significant density of [¹²⁵I]α-bungarotoxin binding sites could be detected only at early postnatal ages. A few olfactory glomeruli specifically restricted to the dorsal posterior OB expressed a high density of [³H]cytisine binding sites but lacked significant binding of the two presynaptic cholinergic markers used here, suggesting their noncholinergic but cholinoceptive nature. © 1996 Wiley-Liss, Inc.     

Modulation of hippocampal norepinephrine release by cholinergic agonists is altered by AF64A lesion

The effect of lesioning hippocampal cholinergic neurons with the neurotoxin AF64A on the ability of cholinergic agonists to modulate stimulation-induced release of ³H-norepinephrine (NE) from rat hippocampal slices was studied. Rats received intracerebroventricular injections of either AF64A (ethylcholine mustard aziridinium, 2 nmol) or vehicle (sham operated). Six weeks after treatment, release of ³H-NE evoked by electrical stimulation (2 Hz, 2 min) in the presence or absence of cholinergic agonists and/or antagonists was measured. Activation of M₂ receptors with oxotremorine (in the presence of the M₁ antagonist pirenzepine) caused a small inhibition of NE release, which was abolished in hippocampi from AF64A-treated rats. The K_d for high-affinity binding of the selective M₂ ligand [³H] AF-DX 384 was increased 10-fold in lesioned tissues. The M₁ selective agonist McN-A-343 produced a significant enhancement of NE release, which was unchanged by AF64A lesion. Binding studies with [³H] pirenzepine showed no change in the affinity or number of M₁ receptors. Nicotine also caused a significant enhancement of evoked NE release, but this effect was markedly reduced in tissues from AF64A-treated rats. AF64A treatment caused a twofold decrease in the number of [³H] nicotine binding sites. This study suggests that long-term lesion of hippocampal cholinergic neurons with AF64A alters the function of postsynaptic muscarinic M₂ and nicotinic cholinergic receptors that modulate the release of NE in the hippocampus.     

Reduction in striatal D2 dopamine receptor mRNA and binding following AF64A lesions

Unilateral lesions by a cholinotoxin, receptor autoradiography, andin situ hybridization techniques were employed to determine if dopaminergic receptors are located on cholinergic interneurons in the caudate-putamen (CPu). Lesion of the CPu with small amounts of the cholinotoxin AF64A resulted in a significant decrease in D₂ receptor mRNA and D₂ receptor binding. The loss was more pronounced in lateral and central portions of the CPu. Results obtained using [³H] SCH23390 binding to D₁ receptors indicated that there was no change in this dopamine receptor subtype in the AF64A-lesioned CPu. A decrease in D₂ receptor mRNA and receptor binding in AF64A-lesioned animals indicates that a population of postsynaptic D₂ receptors is associated with the cholinergic interneurons. Lack of any change in [³H]SCH23390 binding in the AF64A-lesioned animals suggests that D₁ receptors are not located on cholinergic neurons. These results provide evidence to support the selectivity of the lesion when used as indicated.     

Muscarinic receptor localization and function in rabbit carotid body

Acetylcholine and muscarinic agonists inhibit chemosensory activity in the rabbit carotid sinus nerve (CSN). Because the mechanism of this inhibition is poorly understood, we have investigated the kinetics and distribution of muscarinic receptors in the rabbit carotid body with the specific muscarinic antagonist [3H]quinuclidinylbenzilate ([3H]QNB). Equilibrium binding experiments identified displaceable binding sites (1 microM atropine) with a Kd = 71.46 pM and a Bmax = 9.23 pmol/g tissue. These binding parameters and the pharmacology of the displaceable [3H]QNB binding sites are similar to specific muscarinic receptors identified in numerous other nervous, muscular and glandular tissues. Comparisons of specific binding in normal and chronic CSN-denervated carotid bodies suggest that muscarinic receptors are absent on afferent terminals in the carotid body; however, nearly 50% of the specific [3H]QNB binding is lost following chronic sympathectomy, suggesting the presence of presynaptic muscarinic receptors on the sympathetic innervation supplying the carotid body vasculature. Autoradiographic studies have localized the remainder of [3H]QNB binding sites to lobules of type I and type II parenchymal cells. In separate experiments, the muscarinic agonists, oxotremorine (100 microM) stimulation of the in vitro carotid body. Our data suggest that muscarinic inhibition in the rabbit carotid body is mediated by receptors located on type I cells which are able to modulate the excitatory actions of acetylcholine at nicotinic sites.     

Genetic control of dopamine and serotonin receptors in brain regions of inbred mice

In this report the genetic determinants of dopamine and serotonin receptors are investigated. We have used two types of radioreceptor binding assays to identify and quantify these neurotransmitter receptors in various brain regions of inbred mice. In the first method dopamine and serotonin sites are quantified using [3H]spiperone in the presence of appropriate blanking agents. These results are compared with those obtained by the use of [3H]domperidone and [3H]mianserin to label D2 and S2 sites, respectively. Both methods yield nearly identical results. Strain differences in D2 sites are found in the striatum, olfactory tubercle and pituitary. The density of dopaminergic sites is uncorrelated in the 3 brain regions in all mouse strains studied, suggesting that genetic determination of receptor density is independently regulated in each region. Similar observations have been made for S2 receptors in the striatum, hypothalamus, olfactory tubercle and frontal cortex. Analysis of D3 and D2 binding sites in recombinant inbred lines suggests that each site may be determined monogenically.     

Reduced D2 dopamine and muscarinic cholinergic receptor densities in caudate specimens from fluctuating parkinsonian patients.

Binding of spiperone and 3-quinuclidinyl benzilate (QNB), both labeled with hydrogen 3 (3H), were measured in caudate tissue obtained from 8 living parkinsonian patients at the time of cerebral transplantation. This was clinically homogeneous group of patients. All remained predominantly responsive to levodopa, although with marked disability secondary to clinical fluctuations (short-duration responses) and medication-induced dyskinesias; all were receiving substantial doses of levodopa and 6 of the 8 patients were additionally receiving bromocriptine or pergolide. Binding densities of dopamine D2 receptors, as measured by [3H]spiperone binding, were reduced in this group of patients, compared to caudate specimens from autopsy control subjects. This findings may reflect medication-induced receptor downregulation. Parallel changes occurred with muscarinic cholinergic receptors; [3H]QNB binding was significantly reduced, compared to autopsy control values. This reduction of muscarinic receptors might be due to loss of nigrostriatal terminals that are known to contain muscarinic receptors. Alternatively, muscarinic receptors may have been downregulated by increased corticostriatal glutamatergic input to cholinergic cells, inferred to be present based on the prominent levodopa-induced dyskinesias. Finally, receptor deficits could have been a reflection of more widespread degenerative cerebral disease, although levodopa-refractory symptoms were generally not pronounced in these patients.