Age-Related Alterations In Pre-Synaptic and Receptor-Mediated Cholinergic Functions In Rat Brain

Fractional [³H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [³H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [³H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 M) significantly decreased stimulation-induced fractional [³H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [³H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [³H]ACh release from striatum of both age groups is modulated mainly by M₃ muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-³H]inositol decreased with aging, carbachol-stimulated [³H]myo inositol-1-fosfat (IP₁) accumulation was found similar in striatal, cortical and hippocampal slices.     

STEREOSPECIFIC IN VlTRO BINDING OF [3H]DEXETIMIDE TO BRAIN MUSCARINIC RECEPTORS

A binding assay for muscarinic cholinergic receptors has been developed using labelled dexetimide as ligand and a filtration technique. The main features of this assay are its stereospecific nature, the very high affinity of the ligand for the specific receptors sitcs and its very low affinity for non-specific binding sites. The latter point was further investigated using labelled levitimide, the inactive enantiomer. The binding was found to be neither stereospecific nor saturable and displacement by both enantiomers revealed a particular curve with a very flattened course. Kinetic experiments with [³H]dexetimide suggest the occurrence of a heterogenous population of muscarinic receptors in the rat striatum. A study of the regional distribution of muscarinic receptors in rat brain showed a high concentration in the dopaminergic areas, the cortex and the hippocampus, but practically none in the cerebellum. The subcellular distribution pattern revealed a marked enrichment of [³H]dexetimide stereospecific binding sites in the microsomal fraction of rat striatum and hippocampus. Such a distribution was not found with [³H]levitimide. All the characteristics of this binding assay make dexetimide a very appropriate ligand for labelling muscarinic receptors in vitro as well as in vivo.     

A unique regulatory profile and regional distribution of [3H]pirenzepine binding in the rat provide evidence for distinct M1 and M2 muscarinic receptor subtypes

We recently demonstrated that the non-classical muscarinic receptor antagonist [³H]pirenzepine ([³H]PZ) identifies a high affinity population of muscarinic sites in the rat cerebral cortex. We now report that cortical muscarinic sites to which [³H]PZ binds with high affinity are modulated by ions but not guanine nucleotides. We also have examined equilibrium [³H]PZ binding in homogenates of various rat tissues using a new rapid filtration assay. All regional saturation isotherms yielded a similar high affinity dissociation constant (K_d = 2 ? 8 nM) in 10 mM sodium-potassium phosphate buffer. Receptor density (B_max in fmol/mg tissue) varied as follows: corpus striatum = 154.5, cerebral cortex = 94.6, hippocampus = 94.3, ileum = 1.3, cerebellum = 1.0, and heart = 0.45. The cerebral cortex and hippocampus possess 61 percent of striatal binding sites, while the ileum, cerebellum and heart contain only 0.84 percent, 0.65 percent and 0.29 percent of striatal sites respectively. The [³H]PZ sites in heart, ileum, and cerebellum represent 3.1 percent, 9.6 percent, and 10.4 percent of the sites obtained by using [³H](?)quinuclidinyl benzilate. Thus, [³H]PZ labels high affinity muscarinic receptor binding sites with a tissue distribution compatible with the concept of distinct M₁ and M₂ receptor subtypes. Accordingly, regions such as heart, cerebellum, and ileum would be termed M₂, though each have an extremely small population of the M₁ high affinity [³H]PZ site. [³H]PZ therefore appears to be a useful ligand for M₁ receptor identification. Furthermore, the inability to demonstrate a significant effect of guanine nucleotides upon high affinity [³H]PZ binding to putative M₁ receptors suggests that M₁ sites may be independent of a guanine regulatory protein.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

Quantitative assessment of heterogeneous 3H-spiperone binding to rat neostriatum and frontal cortex

Anomalies of the binding of ³Hspiperone to rat cerebral membranes have been examined. By employing a very low ligand concentration ($\text{～ 25}\text{pM}{}^3\text{Hspiperone}$) we have demonstrated that even within the corpus striatum, ³Hspiperone appears to bind to multiple sites and that dopaminergic and serotonergic agents can selectively inhibit from these sites. In the corpus striatum, 75–80% of the ³Hspiperone specific binding can be inhibited with high affinity by dopaminergic drugs while some 20–30% is inhibited with high affinity by serotonergic compounds. The two ³Hspiperone sites, which we have shown to have affinities of 31 and 325 pM, may therefore represent dopaminergic and serotonergic sites. At higher concentrations of ³Hspiperone, however, the picture may be complicated by a further low affinity site. The great selectivity shown by dopaminergic agonists for the two ³Hspiperone sites explains the ‘flattened’ displacement curves reported for ³Hspiperone/agonist interactions. As dopaminergic agents show the greater affinity for the high affinity ³Hspiperone site, it is tempting to speculate that this site has the greatest association with the dopamine receptor.     

Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [³H] cis methyldioxolane ([³H] CD). Characterization studies on rat forebrain homogenates showed that [³H] CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [³H] CD binding in the rat brain correlated generally with [³H] (?)3-quinuclidinyl benzilate ([³H] (?)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 μM, the GTP analogue, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], caused a 43–77% inhibition of [³H] CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [³H] CD binding was greater in the heart than in the ileum. In contrast to its effects on [³H] CD binding, Gpp(NH)p caused an increase in [³H] (?)QNB binding in the heart heart and ileum and no change in [³H] (?)QNB binding in the corpus striatum. When measured by competitive inhibition of [³H] (?)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100 μM) caused an increase in the IC₅₀ values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies on NEM treated forebrain homogenates revealed an enhancement of [³H] CD binding by NEM.     

Effect of phencyclidine on [3H]QNB binding

Intraperitoneal injection of phencyclidine before intravenous injection of [³H] Quinuclidinyl benzilate (QNE, 1.6 μg/kg) significantly increased the amount of radioactivity found in the brains of female C57BL/6J mice one hour after the ³H-QNB administration. This effect was found in hypothalamus, cortex, hippocampus and striatum and was decreased by pretreatment of the animal with atropine. The magnitude of the enhancement varied as a function of dose but did not change across the time span studied. These data are in contrast to our findings and those of others of inhibition of the specific binding of ³H-QNB to muscarinic cholinergic receptors by PCP $\text{in vitro}$. When atropine or PCP was administered $\text{in vivo}$ and the tissue later analyzed $\text{in vitro}$, no effects of the drugs were observed on ³H-QNB binding. The reasons for the differences remain a matter of speculation.     

[3H] pirenzepine selectively identifies a high affinity population of muscarinic cholinergic receptors in the rat cerebral cortex

The specific binding of [³H] pirenzepine was investigated in homogenates of rat cerebral cortex, cerebellar cortex, and heart. Specific binding of [³H] pirenzepine in the cerebral cortex as defined by displacement with atropine sulfate (1μM) was of high affinity (K_d = 4–10 nM, receptor density = 1.06 pmoles/mg protein), stereoselective, and competitive with drugs specific for the muscarinic receptor. In contrast, few [³H] pirenzepine binding sites were demonstrated in cerebellar and heart homogenates.     

The alteration of serotonin binding sites in aged human brain

The $\text{in}$$\text{vitro}$ binding of [³H]serotonin ([³H]5-HT) to cerebral cortex from young and old adult humans was studied. With cortex from human males 23–29 years old, the binding of [³H]5-HT was a saturable process, and bound [³H]5-HT could be displaced by unlabeled 5-HT or by lysergic acid diethylamide (LSD). Two separate binding sites were discernible by Scatchard analysis. The dissociation constants were 7 nM (Kd₁) and 52 nM (Kd₂), and the total number of binding sites were 0.65 (n₁) and 2.06 (n₂) pmoles/mg protein, respectively. In cerebral cortex from aged humans (61–70 years old), the dissociation constant for [³H]5-HT binding was 198 nM, and the total number of binding sites were 4.78 pmoles/mg protein. The alteration of serotonin binding sites may be related to cerebral malfunction in old people, particularly to mental depression and sleep disturbances that commonly occur.     

Determination of acetylcholine by inhibition of the specific binding of tritiated cis-methyldioxolane: application to the study of the effects of acetylcholine accumulation in a murine neuroblastoma culture

The radioreceptor assay for acetylcholine (ACh) is based on the ability of the ACh to compete with the specific binding of [3H] cis-methyldioxolane to muscarinic receptors of rat cerebral cortex membranes. The technique described was used to measure ACh levels in NS 20 cells treated with an organophosphorus compound. The down regulation of muscarinic acetylcholine receptors of neuroblastoma cells observed is probably related to ACh accumulation.     

Time course of decline of radiolabeled acetylcholine formed following intracerebroventricular administration of tritiated choline: Effects of oxotremorine and scopolamine

Rats were injected intracerebroventricularly with 5 Ci of [methyl-³H]choline. The time course of decline of the rediolabeled acetylcholine (ACh) formed was estimated in the ispilateral cerebral cortex and striatum. The [³H]ACh levels declined biphasically from the cerebral tissue. The initial decline proceeded rapidly, after which labeled ACh declined more slowly. Scopolamine (1 mg/kg, i.v.) caused a significant increase in the rat of [³H]ACh disappearance, which can be interpreted as an enhancement of ACh release. By contrast, oxotremorine (0.8 mg/kg, i.v.) markedly reduced the [³H]ACh disappearance. The results show that drug-induced changes in cholinergic neuronal activities can be estimated from the disappearance of radioactive ACh after labeling the endogenous transmitter through intracerebroventricular administration of labeled choline.     

Characterization of N-[3H]Methylcarbamylcholine Binding Sites and Effect of N-Methylcarbamylcholine on Acetylcholine Release in Rat Brain

The present experiments show that N-[3H]-methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro-beta-erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro-beta-erythroidine and d-tubocurarine, but not by alpha-bungarotoxin or by the muscarinic antagonist atropine. The MCC-induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

Effect of lithium and sodium ions on opiate-and dopamine-receptor binding

The effects of lithium and sodium were studied in the corpus striatum and cerebral cortex of rats. Lithium was inhibitory at low concentrations but at 20 mM it increased the binding of [G-³H]naloxone (specific activity 15.6 Ci/mmol). Sodium stimulated the high-affinity binding of this compound. Membranes obtained from the rats treated with lithium showed lower specific binding of both [³H]naloxone and [³H]DHM. Binding of [³H]d-alanine Leu-enkephalin was not changed in the brains of lithium-treated rats, but that of [³H]-spiroperidol was lowered. Cerebral cortex and striatum of lithium-treated rats had a decreased apparent dissociation constant and a lower receptor concentration of naloxone binding sites.     

The activation of phosphatidylinositol turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors

In the rat cerebral cortex, the comparative effects of various muscarinic agonists on the release of [³H]dopamine ([³H]DA), [³H]acetylcholine ([³H]ACh), and [³H]5-hydroxytryptamine ([³H]5-HT) from superfused nerve endings and on phosphatidylinositol (PI) turnover were studied. Acetylcholine (ACh) was found to be the most potent among the agonists tested on all three release systems examined, and also on the activation of PI turnover. Oxotremorine and bethanechol were very weak agonists when tested as stimulators of PI turnover. However, oxotremorine was very effective as a release modulator, while bethanechol was completely ineffective. Our data suggest that the activation of PI turnover is not directly involved in the modulation of neurotransmitter release mediated by presynaptic muscarinic receptors.     

Specific invivo binding of 77Br-brombenperidol in rat brain

The $\text{in vivo}$ binding of the radiobrominated neuroleptic brombenperidol in rat brain was studied. The accumulation of the radiolabeled neuroleptic was high in the striatum and relatively low in the cerebellum, cortex, and blood. Striatal binding of brombenperidol was saturable and displaced by subsequent administration of benperidol. The rationale for the development of ⁷⁵Br-brombenperidol as a radiopharmaceutical for the non-invasive imaging of cerebral dopamine receptor areas is presented.     

Muscarinic receptors and responses in intact embryonic chick atrial and ventricular heart cells

We have studied muscarinic acetylcholine (ACh) receptors in intact atrial and ventricular heart cells dissociated from 8-day chick embryos and maintained in sparse cell cultures. Two specific antagonists, [³H]quinuclidinyl benzilate (QNB) and [³H]N-methyl scopolamine (NMS), bind to surface sites with affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{s}\text{? 40}\text{and}\text{400}\text{p}\text{M}$, respectively). The concentration of [³H]QNB sites in ventricular cell cultures (460 fmole/mg protein) was comparable to the concentration of sites in atrial cultures (420 fmole/mg protein). The same result was obtained with [³H]NMS. Autoradiography following incubation in saturating concentrations of [³H]QNB shows that nearly all of the atrial and ventricular myocytes were labeled and that the distribution of grains over individual cells was uniform. The mean binding site density was 109/μm² for atrial cells 117/μm² for ventricular cells. In contrast to the antagonist binding results, microelectrode recordings from individual myocytes or from small clusters of cells showed that many more atrial myocytes (89%) were sensitive to 10^?4M carbachol than were ventricular myocytes (26%). Saline extract of embryonic brain tissue added to the culture medium did not alter the number or distribution of ligand binding sites but it produced a 2.6-fold increase in the number of carbachol-sensitive ventricular cells.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

Differential decrease of the central beta-adrenergic receptor in the rat after subchronic infusion of desipramine and clenbuterol

The effect of subchronic infusion of desipramine, a norepinephrine uptake inhibitor, and clenbuterol, a beta-adrenergic agonist, on the central beta receptor of the rat was determined using in vitro [³H]dihydroalprenolol binding. Desipramine produced significant decreases of the receptor in neocortex and hippocampal formation, and clenbuterol effected such decreases in corpus striatum and cerebellum. Both drugs caused a marked decrease in the activity of isoproterenol-sensitive adenylate cyclase in neocortex. The alpha₂ receptor of neocortex and cerebellum was unchanged by either drug as assessed by in vitro$\text{[}{}^3\text{H}\text{]p-}\text{aminoclonidine}$ binding. The results are discussed in terms of the different mechanisms of action of desipramine and clenbuterol, and the efficacy of these two drugs in the treatment of depression.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.