QNB Binding Research Articles

Ontogeny of muscarinic receptors in the rat brain with emphasis on the differentiation of M 1- and M 2-subtypes— semi-quantitative in vitro autoradiography

The ontogeny of muscarinic acetylcholine receptors (mAChR) in the rat brain was studied with emphasis on the differentiation of M 1- and M 2-receptor subtypes through semi-quantitative in vitro autoradiography. [ 3Quinuclidinyl benzilate] (QNB) and [ 3H]pirenzepine (PZ) were used for labeling total mAChR and M 1-receptors, respectively. In the cerebral cortex of adult rats, [ 3H]QNB binding sites were more richly present in the superficial and deeper layers than in the middle layer, while M 1-receptors were diffusely observed in all the layers. This means that M 2-receptors are highly concentrated in the superficial and deeper layers. The ontogenetical differentiation of the laminar distribution between M 1- and M 2-receptor first appeared at 14 days of postnatal age. In the hippocampus and striatum whose mAChR were predominantly of the M 1-type in the adult rat brain, ontogenic patterns of M 1-receptors were almost identical to those of total mAChR. On the other hand, mAChR in the cerebellar cortex and lower brainstem of the adult rat were mainly of the M 2-subtype. In these areas, the ontogeny of total mAChR was apparently observed. However, M 1-receptors were not observed at any stage of the ontogeny. The above-mentioned results indicate that M 1- and M 2-receptors show distinct developmental behaviors in the rat brain.

Effect of Postmortem Factors on Muscarinic Receptor Subtypes in Rat Brain

Although prior studies have supported the validity of measuring total muscarinic receptor binding in postmortem brain, there has not been a study of postmortem effects on muscarinic receptor subtypes, M1 and M2, defined by high and low affinity for pirenzepine, respectively. We have examined in rat brain the effect of postmortem delay at room temperature, storage at 4 degrees C and -20 degrees C, and multiple freeze/thaw cycles on total muscarinic binding, measured with [3H]quinuclidinylbenzilate ([3H]QNB) and on M1 muscarinic binding, measured with [3H]pirenzepine ([3H]Pir). We found that delay at room temperature up to 4 h, or storage at 4 degrees C for 24 h or at -20 degrees C for 4 weeks, or 3 freeze/thaw cycles had no effect on [3H]QNB or [3H]Pir binding. Exposure of brain to room temperature for 15 h, however, led to an increase in [3H]QNB binding, without change in [3H]Pir. Scatchard analysis showed an increase in binding sites without a change in affinity. We conclude that [3H]QNB and [3H]Pir are valid measures of total and M1 muscarinic binding, respectively, under these circumstances, but that caution must be used in the interpretation of indirect measures of M2 binding.

Quantitative autoradiographic analysis of muscarinic cholinergic and adenosine A 1 binding sites after transient forebrain ischemia in the gerbil

The influence of transient forebrain ischemia on adenosine A 1 and muscarinic cholinergic receptors in the gerbil brain 1–27 days after recirculation was studied. The topographical distribution and the alteration in the adenosine A 1 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 CA 1 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 CA 1 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 CA 1 subfield 3–27 days after recirculation. Three to 27 days after ischemia, the A 1 binding activities in the CA 3 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 CA 3 subfield were also reduced 27 days after ischemia. Despite minimal neuronal damage in the lateral septal nucleus and in the substantia nigra, the A 1 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 CA 1 subfield of the hippocampus are localized on the CA 1 pyramidal cells.

Muscarinic receptors in human SH-SY5Y neuroblastoma cell line: regulation by phorbol ester and retinoic acid-induced differentiation

The specific binding of the muscarinic ligand [ 3H](-)quinuclidinyl benzilate ([ 3H]QNB) to cell membranes of human SH-SY5Y neuroblastoma cells was studied. Saturation isotherms yielded a K d = 0.28 ± 0.06 nM and a B max of 337 ± 47 pmol/g protein. Pirenzepine inhibited [ 3H]QNB binding; inhibition data showed best fit to a 2-site binding model revealing both a high affinity pirenzepine site (34%, K H = 10 nM) and a low affinity site (66%, K L = 1 μM). These results indicate that muscarinic receptors on SH-SY5Y cells may be subclassified as M 1/M 2 subtypes. Morphological and biochemical differentiation of these cells after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid (RA) resulted in a decrease and an increase in the number of muscarinic binding sites, respectively. Furthermore, TPA- and RA-treated cells showed a significant increase in acetylcholinesterase activity compared with non-treated cells. However, only RA-treated cells showed significant increase in choline acetyltransferase activity compared to nontreated cells. These findings demonstrate that TPA and RA can regulate both the number of muscarinic receptors and the acetylcholinesterase activity in human SH-SY5Y neuroblastoma cells.

Effect of chronic administration of haloperidol (intermittently) and haloperidol-decanoate (continuously) on D2 dopamine and muscarinic cholinergic receptors and on carbachol-stimulated phosphoinositide hydrolysis in the rat striatum.

It has been reported that apomorphine-induced stereotypy is sensitized after a chronic intermittent administration of haloperidol (HPD), but not after a chronic continuous exposure to haloperidol-decanoate (HPD-D). The present study was undertaken to investigate changes in the D2 dopamine and muscarinic receptors in the rat striatum after the administration of HPD intermittently and HPD-D continuously. The number of striatal [3H]spiperone binding sites increased significantly after HPD-D, but did not change after HPD. Neither the number of [3H](-)QNB binding sites nor carbachol-stimulated phosphoinositide hydrolysis changed after either HPD or HPD-D. These results indicate that the increase in striatal D2 receptors in rats administered HPD-D represents behavioral and biochemical tolerance, and that neither the D2 dopamine receptor supersensitivity nor muscarinic receptor hyposensitivity underlies sensitization of apomorphine-induced stereotypy.

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirerenzepine ([3H]PZ) and [3H] quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.

Selective interaction of tricyclic antidepressants with a subclass of rat brain cholinergic muscarinic receptors

Experiments were undertaken to determine whether the anticholinergic actions of tricyclic antidepressants are mediated by a selective interaction with a subclass of muscarinic receptors. To this end, the potencies of these antidepressants to inhibit [ 3H]-QNB binding to rat brain cerebral cortical membranes was compared to their potencies as antagonists of carbochol-stimulated inositol phosphate accumulation in cerebral cortical slices and carbachol-induced inhibition of GTP-stimulated adenylate cyclase in striatal membranes. Whereas amitriptyline was more potent than pirenzepine, a selective muscarinic M 1 receptor antagonist, in competing for [ 3H]-QNB binding sites and as an antagonist of carbachol-induced inhibition of adenylate cyclase, pirenzepine was substantially more active (ten-fold) than amitriptyline in blocking carbachol-stimulated phosphatidyl inositol turnover. Atropine was more potent than all other agents in these assays, failing to display any significant degree of selectivity. The results suggest that the tricyclic antidepressants, in particular amitriptyline, appear to be selective antagonists for muscarinic receptors associated with adenylate cyclase in striatal membranes. Given the current classification of cholinergic receptors, these findings indicate that the tricyclic antidepressants may be useful for defining the properties of M 2 receptors in brain.

A comparative study of the affinities of some tricyclic antidepressants for the muscarinic cholinergic receptor in human and guinea-pig bladder, ileum and brain in relation to differential drug potency

Following a report that nortriptyline was found useful in the control of enuresis in adults, presumably as an anticholinergic, its likely mechanism of action and apparent bladder specificity have now been investigated in vitro. The ratios of anticholinergic potencies (reciprocal of dissociation contants, Ki) for four different tricyclic antidepressants, derived from competitive binding assays with (−)[ 3H]QNB in tissue homogenates, in the order (human) detrusor muscle / ileal longitudinal muscle / caudate, are as follows: Nortriptyline, 5/4/7; desipramine, 2/1/5/; clomipramine, 4/3/27; amitriptyline, 25/14/56. The apparent selective effect of nortriptyline on the bladder cannot be ascribed to its higher affinity to bladder receptors. Still, this drug is the least discriminatory of the four. Hence, at a given concentration, it is expected to affect tissue embodying a low density receptor pool sooner than tissue having a large receptor reserve. The ratios of the densities of (−)[ 3H]QNB binding sites in the order detrusor muscle / ileal muscle / cortex is 1/3/5, supporting the present contention. In the guinea-pig, the ratios of the anticholinergic potency in the order bladder / proximal ileum / distal ileum / cortex are as follows: Nortriptyline, 25/5/6/33; desipramine, 8/2/2/14; amitriptyline, 100/14/20/100; clomipramine, 17/3/5/33. Also, the ratios of the densities of binding sites are 1/6/5/2. Hence, data derived from assays in the guinea-pig are not representative of those derived from human tissue

Characterization of cholinergic muscarinic receptors in cow tracheal muscle membranes: Effect of maturation

The parasympathetic nervous system is important in the control of basal airway muscle tone and caliber. We characterized muscarinic cholinergic receptors in isolated trachea! membranes from cows of three age groups (immature, < 2 weeks; transition, 3–5 months; and mature, > 5 years) using l-[ 3 H] quinuclidinyl benzilate (l-[ 3 H]QNB) as the radioligand. There were significant decreases in the densities of l[ 3 H]QNB binding sites with maturation ( B max: 2344 ± 169 vs 1381 ± 85 vs 1116 ± 80 mol/ mg protein for tissues from immature, transition and mature cows respectively). No change in the dissociation constant was observed with maturation ( K d : 0.38 ± 0.09 vs 0.55 ± 0.06 vs 0.50 ± 0.07 nM for tissues from immature, transition and mature animals respectively). The association and dissociation rate constants did not vary between tissues from immature and mature animals. The specific activity of the enzyme, acetylcholinesterase, was correlated with the density of l-[ 3 H]QNB binding sites present in the tracheal homogenates; that is, with maturation, there were significant decreases in acetyl-cholinesterase activity [0.28 ± 0.01 vs 0.16 ± 0.02 vs 0.08 ± 0.01 mol·1 −1·min −1·(mg protein) −1 for tissues from immature, transition and mature animals respectively]. All competition binding studies using muscarinic antagonists exhibited single site binding and did not show any differences in drug affinities between the age groups. In contrast, multiple binding sites were observed with carbachol, methacholine and muscarine, and there were significant decreases in receptor affinities for the muscarinic agonists. No changes in the proportion of high and low affinity sites were found. These results indicate that with maturation there are alterations in the properties of muscarinic receptors in tracheal smooth muscle.

Influence of veratridine on [ 3H]- l-quinuclidinyl benzilate ([ 3H]QNB) binding in mouse hindbrain

The neurotoxin veratridine is well known for its ability to open sodium channels in neuronal and muscle tissues in micromolar concentrations. It has also been shown that veratridine is an inhibitor of the potent muscarinic receptor antagonist l-quinuclidinyl benzilate (QNB) at these concentrations. These findings prompted us to examine the relationships between action potential sodium channels and muscarinic receptors in a glass-fiber filtration assay for [ 3H]QNB binding to mouse hindbrain membranes using agents known to affect interconversion of the affinity states in some muscarinic receptor populations, i.e. guanosine triphosphate (GTP) and magnesium (Mg 2+). The actions of the sodium channel antagonist tetrodotoxin (TTX) were also examined. Veratridine inhibited [ 3H]QNB binding with a K i , value of approximately 2.5 μM. This inhibition exhibited a competitive mechanism at higher concentrations (5–10 μM), while showing an apparent non-competitive action at low concentrations (1 μM). Magnesium caused a parallel shift to the right in the inhibition curve with a 32% increase in the veratridine K i . GTP caused a non-parallel shift to the left with the greatest displacement occurring at lower veratridine concentrations (2–5 μM). The addition of magnesium to GTP did not alter the action of GTP significantly. TTX (5 μM) caused a parallel shift of the veratridine inhibition curve to the right. In addition, TTX alone inhibited the binding of [ 3H]QNB. Therefore, it appears that there may be more than one binding site for veratridine which may be linked to the muscarinic system and that these may be action potential sodium channels.

The interaction of pancuronium and vecuronium with cardiac muscarinic receptors

The interaction of vecuronium, a monoquaternary analogue of pancuronium, with the cardiac muscarinic receptors in canine hearts was investigated in vitro by [3H] QNB binding assay and compared with that of pancuronium. Both pancuronium and vecuronium, which are nicotinic antagonists of competitive types, inhibited the binding of [3H] QNB to cardiac muscarinic receptors with values of IC50 of 5.41 X 10(-7) mol/l and 3.97 X 10(-6) mol/l respectively. According to the Kd and Bmax values on the Scatchard plot, pancuronium, while not influencing the number of receptors, had a 3-fold greater inhibitory effect than vecuronium on the affinity of the muscarinic receptors. The KI values of vecuronium and pancuronium showed that pancuronium had a 7.3-fold greater affinity with the receptors than vecuronium. We concluded that vecuronium had a direct inhibitory action on the binding of [3H] QNB to the canine heart muscarinic receptors, but this action was much weaker than pancuronium.

Binding studies with [3H]cis-methyldioxolane in different tissues. Under certain conditions [3H]cis-methyldioxolane labels preferentially but not exclusively agonist high affinity states of muscarinic M2 receptors.

Special conditions--tricine buffer containing Ca2+ and Mg2+, 22 degrees C (TCM)--allow to label a much higher proportion of muscarinic receptors by [3H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984a). Taking the maximum number of binding sites, Bmax, of [3H]QNB as 100%, Bmax of [3H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [3H]CD and [3H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [3H]CD system, affinities of antagonists are slightly higher in the [3H]QNB system. In both assay systems receptors of heart and pons/medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [3H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [3H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in Bmax and not in the dissociation constant KD. Competition of unlabelled pirenzepine with [3H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M2-receptors) are labelled by [3H]CD.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in cholinergic markers following kainic acid lesion of the ventral globus pallidus in rat.

Choline acetyltransferase (CAT) and glutamate decarboxylase (GAD) activities and [3H] quinuclidinyl benzilate ([3H]QNB) binding were determined in the rat frontal cortex following damage to the basal forebrain cholinergic system. Pre- and postsynaptic changes in the cholinergic system with the passage of time were also studied. After a unilateral injection of kainic acid into the right ventral globus pallidus, the GAD levels remained unaffected, but the CAT levels decreased to 63.4% after 7 days. After 12 weeks, the CAT levels had returned to 87% of the control value. The Bmax of [3H]QNB binding for the muscarinic receptor was higher in the ipsilateral cortex up to 4 weeks. On the other hand, the KD value at 12 weeks was higher without a change in the Bmax of the [3H]QNB binding. These findings might indicate an ongoing compensatory receptor mechanism of denervation supersensitivity as a response to early changes in presynaptic cholinergic activity and the production of postsynaptic effect with presynaptic cholinergic damage over a long period of time.

Cross-tolerance between muscarinic agonists: Role of muscarinic receptors

In order to explore the relationship between response to muscarinic agonists and brain muscarinic receptors, two mouse strains that differ in acute sensitivity (DBA and C3H) were injected chronically with DFP or infused with oxotremorine. Chronic DFP-treated DBA mice were not tolerant to DFP's effects on any measure, but they were cross-tolerant to the effects of oxotremorine on heart rate and body temperature. DFP-treated C3H mice were not tolerant to DFP or cross-tolerant to oxotremorine on any measure. Oxotremorine infusion resulted in tolerance to oxotremorine in both mouse strains, and chronically infused DBA mice were cross-tolerant to DFP on five of the six measures. Oxotremorine-infused C3H mice were cross-tolerant to DFP on two of the measures. These results suggest that genetic factors influence the development of tolerance or cross-tolerance. These genetic factors do not seem to be related to changes in brain QNB binding. Both mouse strains showed comparable changes in QNB binding following chronic DFP and oxotremorine with DFP eliciting reductions in QNB binding in striatum and hippocampus and oxotermorine eliciting reductions in nearly every brain region. However, tolerance and cross-tolerance did not seem to correlate with changes in binding which suggests that the relationship between receptor changes and responses to muscarinic agonists must be examined further.

Dissociation of decreased numbers of muscarinic receptors from tolerance to DFP

Several studies have demonstrated that chronic treatment with organophosphates, such as DFP, elicits a decreased number of brain muscarinic receptors (measured by the binding of QNB) which has been presented as an explanation for tolerance to the organophosphates. The purpose of the studies presented here was to assess whether graded changes in QNB binding could be attained following different methods of chronic DFP treatment, and whether tolerance to DFP paralleled these changes. Male DBA mice were injected with DFP every 4 days or 2 days for 30 days or daily for 14 days. The animals were subsequently challenged with DFP or the muscarinic agonist, oxotremorine, and respiratory rate, heart rate, body temperature, Y-maze activity and rearing were recorded. Chronic DFP-treated animals were supersensitive to the effects of DFP on respiratory rate, heart rate, and body temperature whereas a modest tolerance to the effects of oxotremorine on respiratory rate, heart rate, and body temperature was seen. Neither tolerance nor supersensitivity were observed for the effects of DFP and oxotremorine on the Y-maze measures. Chronic DFP treatment elicited reduced binding of QNB in striatum, cortex, and hippocampus with the group that had been treated every other day exhibiting the greetest changes. The changes in drug response did not parallel changes in QNB binding which raises questions as to the cause of the reduction in binding.

Decreased binding of the muscarinic antagonist [ 3H]N-methylscopolamine in mouse brain following acute treatment with an organophosphate

The effect of the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP) on mouse brain muscarinic acetylcholine receptors was assessed using the muscarinic antagonists [ 3H]N-methylscopolamine ([ 3H]NMS) and [ 3H]quinuclidinyl benzilate ([ 3H]QNB). No alteration in the maximal binding capacity (B max) or equilibrium dissociation constant (K D) was observed in brain homogenates obtained from mice 12 h after a single injection of DFP when [ 3H]NMS was employed as the ligand. However, one administration of DFP produced a 24 and 33% decrease in B max as measured by [ 3H]NMS binding after 18 and 24 h, respectively. A similar decrease in B max was found after two (31%) and three (29%) days of daily DFP treatment. On the other hand, Scatchard analysis using [ 3H]QNB binding in the brain revealed no difference in K D or B max between untreated and 24 h DFP-treated mice. Thus, there is a differential alteration in mouse brain muscarinic acetylcholine receptors using these two ligands which suggests that [ 3H]NMS binding sites are more sensitive to regulation following acute organophosphate administration.

Dissociation of contraction and muscarinic receptor binding to isolated smooth muscle cells

We examined changes in [3H]QNB binding and cell length induced by muscarinic ligands in a suspension of single smooth muscle cells isolated from the canine stomach. Cells contracted following a brief (30 s) exposure to picomolar concentrations of muscarinic agonists and yielded ED50 values of 1.0 +/- 0.7 pM for oxotremorine, 12.5 +/- 1.8 pM for carbachol, and 16.0 +/- 2.9 pM for metacholine. Contraction was inhibited by atropine with a pA2 value of 10.2 +/- 1.1. The binding of [3H]QNB was rapid and reversible and was stereospecific and pharmacologically appropriate. Specific binding of [3H]QNB was saturable and bound with high affinity (KD 1.04 +/- 0.23 nM) to a single class of sites, of which there were approximately 200,000/cell. In competition experiments antagonist binding was generally homogeneous, whereas that of agonists was heterogeneous and subpopulations of binding sites with different affinities for agonists were identified. The Ki value of 8.1 +/- 1.1 nM for inhibition of QNB binding by atropine was greater than the pA2 of 10.2 +/- 1.1 derived from contraction studies. Furthermore, whereas picomolar concentrations of agonists induced cell contraction, substantially higher concentrations (10 nM to 10 mM) were required to inhibit [3H]QNB binding to the isolated cells.

Calmodulin regulation of the cholinergic receptor in the rat heart during ontogeny and senescence

The contents of calmodulin and cholinergic muscarinic receptor binding sites in the hearts of fetal, adult and aged rats have been examined. A biphasic pattern of calmodulin development was observed. A relatively high level of calmodulin appeared on gestational days 14–15 followed by a steady but significant decrease at birth and during the first week of postnatal life. The level of calmodulin then increased significantly during the first month followed by a decrease at 6 and 26 months of age. Calmodulin contents were significantly higher in the atrium than in the ventricle in the age groups of 1–26 months. The number of [ 3H] QNB binding sites showed a steady increase during the gestational periods studied, reaching a peak at 9 days after birth and followed by a significant ( P < 0.05) decline at 6 and 26 months of age. A good correlation between the levels of [ 3H]. QNB binding and calmodulin was observed from day 9 of the postnatal period to 26 months of age. In the presence of calcium, calmodulin induced a dose-dependent receptor binding loss in the hearts of postnatal, young adult and aged rats under phosphorylating conditions. These findings support the suggestion that calmodulin may regulate cholinergic functions during ontogeny and senescence.

Behavioral and physiological effects associated with changes in muscarinic receptors following administration of an irreversible cholinergic agonist (BM 123).

Previous work in our laboratory has shown that the aziridinium ion of BM 123 (N-[4(2-chloroethylmethylamino)-2-butynyl]-2 pyrrolidone) is a potent and selective muscarinic agonist and binds irreversibly to muscarinic receptors (mAChR). The present series of experiments was designed to study the effects of BM 123 on behavioral and physiological variables known to be sensitive to manipulations of the cholinergic neurotransmitter system. BM 123 was injected into the tail vein of Sprague-Dawley rats, reducing mAChR to approximately 10% of normal as judged by [3H](-)QNB binding. Oxotremorine was injected IV for purposes of comparison. Behavioral and physiological variables were measured daily for 26 days. Physiological variables (e.g., tremor, chromodacryorrhea, salivation, and temperature) showed effects in less than 5 min after injection and returned to their pretreatment baselines within minutes. Nociceptive thresholds, dependent on sensory-perceptual processes, showed peak changes of approximately +230% and returned to normal within hours. Motoric responses, i.e., drinking and general activity, recovered in 3-4 days. Learned responses and those requiring temporal discrimination took 8-11 days to recover and were the only responses paralleling the return of the mAChRs to their normal levels. Changes elicited by oxotremorine recovered more rapidly than those elicited by BM 123. The results suggest that the different variables measured are dependent on different densities of functional receptors. Implications for a theoretical model are discussed.

Effects of aging and cholinergic deafferentation on putative muscarinic cholinergic receptor subtypes in rat cerebral cortex

Despite a 34% decrease in the activity of choline acetyltransferase (ChAT) in the rat cerebral cortex following lesions of the nucleus basalis, there were no changes in the B max of the antagonist ligands [ 3H]quinuclidinyl benzilate ((−)-[ 3H]QNB) or (−)-[ 3H]N-methylscopolamine ((−)-[ 3H]NMS). Furthermore, this treatment produced no significant change in the proportions or affinities of muscarinic receptors having high and low affinity for pirenzepine or (−)-NMS. These data indicate that putative M 2 muscarinic receptors are not restricted to ChAT-containing neurons in rat cerebral cortex. In senescent compared to mature rats there was no significant loss of ChAT activity although a significant reduction in the B max of both (−)-[ 3H]QNB and (−)-[ 3H]NMS binding was observed. However, no changes in the competition of pirenzepine or (−)-NMS for the remaining (−)-[ 3H]QNB binding sites were observed. Therefore, there is no evidence for any differential regulation of either putative muscarinic receptor subtype in response to cholinergic deafferentation or as a function of the natural aging process.