Identification of putative high affinity nicotinic receptors on cultured cortical neurons

Primary cell cultures containing predominantly neurons or glia were prepared from fetal rat cerebral cortex. The presence of nicotinic receptor sites in neuronal cultures was indicated by the specific binding of L-[3H]nicotine to cell membrane preparations. No binding was observed with membrane preparations derived from glial cell cultures. Binding to neuronal membranes was saturable, reversible and stereoselective. Scatchard analysis revealed a single class of high affinity sites with a Kd of 3 nM and an average maximum number of binding sites of 25 fmol/mg of protein. The affinity of the sites was the same as that in adult cortical tissue, but the maximum number of sites was 25% of adult levels. The time course of binding exhibited complex kinetics that were consistent with the conversion of the sites to a high affinity state. The apparent equilibrium dissociation constant calculated from the kinetic rate constants for association (0.014 min-1 nM-1) and dissociation (0.03 min-1) was 2 nM, in good agreement with the results of equilibrium binding studies. In general, the pharmacological specificity of the sites, as judged from inhibition binding studies, was similar to that in adult brain. Nicotinic agonists were the most potent competitive inhibitors of [3H]nicotine binding and antagonists were the least effective. The D-isomer of nicotine was about 30-fold less potent than the L-isomer. The results show that cortical neurons contain high affinity nicotinic binding sites and that the properties of these sites are similar to those attributed to putative nicotinic cholinergic receptors in adult rat brain tissue.     

Classical genetic analysis of nicotine-induced seizures and nicotinic receptors

C3H/2lbg mice are more sensitive to nicotine-induced seizures than are DBA/2lbg mice. There are also differences in alpha-bungarotoxin (alpha-BTX) binding in the hippocampus and midbrain of these two strains, with the C3H mice having greater binding. Because alpha-BTX and nicotine appear to bind to nicotinic receptors in the central nervous system, it is possible that there may be a relationship between seizure sensitivity after a nicotine dose and nicotinic receptor concentration. To examine this relationship, a classical cross producing F1, F2 and backcross (F1 X C3H and F1 X DBA) generations from these two strains was utilized. Dose-response curves for nicotine-induced seizures were constructed for both parental strains and all crosses derived from them. Nicotinic receptors were also measured in three brain regions: cortex, midbrain and hippocampus. Both DL-[3H]nicotine and alpha-[125I]BTX were used to measure nicotinic receptors. The pattern of results for the six generations for alpha-BTX binding in the hippocampus paralleled that for seizure sensitivity. These results suggest that strain differences for both seizure sensitivity and receptor concentration in the hippocampus may be due to allelic differences at a single autosomal locus, with dominance for low seizure susceptibility and fewer alpha-BTX receptors.     

An autoradiographic analysis of cholinergic receptors in mouse brain after chronic nicotine treatment

Quantitative autoradiographic procedures were used to examine the effects of chronic nicotine infusion on the number of central nervous system nicotinic cholinergic receptors. Female DBA mice were implanted with jugular cannulas and infused with saline or various doses of nicotine (0.25, 0.5, 1.0 or 2.0 mg/kg/hr) for 10 days. The animals were then sacrificed and the brains were removed and frozen in isopentane. Cryostat sections were collected and prepared for autoradiographic procedures as previously described. Nicotinic cholinergic receptors were labeled with L-[3H]nicotine or alpha-[125I]bungarotoxin; [3H]quinuclidinyl benzilate was used to measure muscarinic cholinergic receptor binding. Chronic nicotine infusion increased the number of sites labeled by [3H]nicotine in most brain areas. However, the extent of the increase in binding as well as the dose-response curves for the increase were widely different among brain regions. After the highest treatment dose, binding was increased in 67 of 86 regions measured. Septal and thalamic regions were most resistant to change. Nicotinic binding measured by alpha-[125I]bungarotoxin also increased after chronic treatment, but in a less robust fashion. At the highest treatment dose, only 26 of 80 regions were significantly changes. Muscarinic binding was not altered after chronic nicotine treatment. These data suggest that brain regions are not equivalent in the mechanisms that regulate alterations in nicotinic cholinergic receptor binding after chronic nicotine treatment.     

Antidepressant binding sites in brain: autoradiographic comparison of [3H]paroxetine and [3H]imipramine localization and relationship to serotonin transporter

Binding of two different antidepressant drugs, [3H]paroxetine and [3H]imipramine in 30 rat brain regions was visualized, compared and quantified by means of autoradiography and densitometry. Specific binding of [3H]paroxetine to coronal sections of diencephalon represented 85% of total binding and was saturable and of high affinity (KD, 0.36 +/- 0.07 nM) with a maximum number of binding sites of 276 +/- 41 fmol/mg protein. The autoradiograms showed a heterogenous distribution of [3H]paroxetine in brain with selective accumulation of label in brain regions known to contain serotonergic terminals, axons and cell bodies (amygdaloid and raphe nuclei, superior colliculus, substantia nigra and medial forebrain bundle). Binding was displaced selectively with other serotonin uptake inhibitors (clomipramine and fluoxetine) and almost abolished by lesioning the serotonergic neurons with p-chloroamphetamine. The desipramine-sensitive [3H]imipramine binding was more diffuse with relatively high density in cerebral cortex and hippocampus and was only decreased partially in animals treated with p-chloroamphetamine. The results indicate that [3H]paroxetine, but not [3H]imipramine, is a ligand of choice to selectively label serotonergic structures in brain.     

Neuroanatomically selective down-regulation of beta adrenergic receptors by chronic imipramine treatment: relationships to the topography of [3H]imipramine and [3H] desipramine binding sites

The down-regulation of beta adrenergic receptors by chronic imipramine treatment was investigated with high resolution autoradiography of [125I]pindolol binding to brain sections. Neuroanatomically selective down-regulation of [125I]pindolol binding was found after chronic imipramine treatment. Subdivisions of the amygdala and hippocampus and discrete cortical regions were differentially affected. In the hippocampus, reduction of [125I]pindolol binding was observed in imipramine-treated rats in the CA-1 stratum radiatum and dentate molecular layer, but not in the CA-3 stratum radiatum. In the amygdala, the basolateral nucleus exhibited reduced [125I]pindolol binding after imipramine treatment but the central and medial nuclei were not affected. Chronic imipramine treatment was also associated with reduced [125I]pindolol binding in layer 1 of the cingulate cortex and layer 3 of the piriform cortex. In contrast, no effect on [125I]pindolol binding was apparent in the ventrolateral thalamic nucleus, caudate-putamen, lateral hypothalamus or layers 2 and 3 of the somatosensory cortex. In order to determine if regional variation in binding sites for imipramine, or its pharmacologically active metabolite desipramine, was responsible for the observed neuroanatomically selective reduction in [125I]pindolol binding, the binding of [3H]imipramine and [3H]desipramine was investigated. In some brain regions that exhibited high densities of [3H]imipramine and [3H]desipramine binding sites, [125I]pindolol binding was reduced after chronic treatment with imipramine. However, other regions that contained high densities of binding sites for antidepressant drugs did not show a reduction in [125I]pindolol binding after chronic imipramine treatment. Thus, regional binding of [3H]imipramine or [3H]desipramine cannot fully explain the neuroanatomical specificity of imipramine-induced beta adrenergic receptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological differences between the high-affinity muscarinic agonist binding states of the rat heart and cerebral cortex labeled with (+)-[3H]cismethyldioxolane

The high-affinity agonist binding state of muscarinic receptors in the rat heart and cerebral cortex has been pharmacologically characterized in parallel studies. Muscarinic sites were labeled and studied with the aid of a highly specific, rapid filtration binding assay using the potent muscarinic agonist (+)-[3H]CD. Homogenates of both tissues were found to contain a saturable high-affinity (Kd = 1-2 nM), low capacity (6-17% of (-)-[3H]QNB sites) (+)-[3H]CD binding state which demonstrated stereoselectivity and drug specificity typical of a muscarinic site. However, comparative studies of drug potency profiles in competition for myocardial and cerebral cortical (+)-[3H]CD-labeled membranes revealed several major pharmacological differences between muscarinic sites in these tissues. Whereas the muscarinic agonists pilocarpine and McN-A-343, the nonclassical antagonist pirenzepine, and the acetylcholinesterase inhibitor physostigmine reduced (+)-[3H]CD binding in both tissues, their inhibitory effects were more potent (4- to 77-fold) in cerebral cortical membranes. Conversely, gallamine, a nicotinic cholinergic antagonist, demonstrated a 36-fold greater potency at the high-affinity (+)-[3H]CD binding state in myocardial membranes. By comparison, other classical muscarinic agonists and antagonists were nearly equipotent as inhibitors of high-affinity (+)-[3H]CD binding in these two tissues. Thus, these studies for the first time demonstrate that muscarinic receptors in the heart and cerebral cortex can be distinguished pharmacologically by certain drugs which interfere with the high-affinity agonist binding state of the muscarinic recognition site and provide support for the subclassification of these receptors.     

3H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes as a measure of nicotine-induced changes in membrane potential.

The effect of nicotine on synaptosomal membrane potential in P2 preparations of rat cerebral cortex was investigated using a membrane permeant, lipophilic cation, [3H]tetraphenylphosphonium ([3H]TPP+). [3H]TPP+ accumulated in synaptosomes in a time-dependent manner and its accumulation was decreased when the extracellular potassium concentration was increased and in the presence of the sodium channel toxin, veratridine. Nicotine (1-1000 microM) decreased the accumulation of [3H]TPP+ in both P2 synaptosomal preparations and in synaptosomes purified using Percoll gradients. This effect of nicotine was mimicked by other nicotinic agonists (1,1-dimethyl-4-phenylpiperazinium iodide, cytisine, suberyldicholine and acetylcholine) and was partially blocked by 10 microM mecamylamine and 30 microM hexamethonium. Atropine (1 microM) and the removal of calcium from the incubation mixture both enhanced the effect of nicotine while the addition of physostigmine (10 microM) reduced the nicotine-induced decrease in [3H]TPP+ accumulation, evidence that acetylcholine released from the synaptosomes by nicotine may produce hyperpolarization of synaptosomes via stimulation of presynaptic muscarinic receptors. It is concluded that the effect of nicotine on [3H]TPP+ accumulation is mediated by nicotine stimulation of a ganglionic-type nicotinic cholinergic receptor and that this method of determining synaptosomal membrane potential will provide a functional measure of presynaptic nicotinic receptor activation.     

The binding of [3H]diazepam to rat brain homogenates

A technique is described for the measurement of specific [3H]diazepam binding to subcellular fractions of rat brain. Binding occurred in fresh or hypotonically shocked, frozen and thawed preparations; was independent of the buffer used; and was concentrated in the synaptosomal fraction. The total binding capacity of crude brain homogenate was entirely recovered in the particulate fractions (P1, P2 and P3). Binding was saturable and reached 34 pmol/g of tissue; half-maximal binding (KD) occurred at 3.2 nM. Hill and Scatchard analysis indicated that the binding was noncooperative and to a single class of sites. Binding was time dependent and reversible; the bimolecular association constant (K1) was 1.13 . 10(6) sec-1 M-1 and the first order dissociation constant (K-1) was 2.69 . 10(-3) sec-1. Binding was highest in cerebral cortex, hippocampus and cerebellum; intermediate in midbrain, hypothalamus, corpus striatum and medulla oblongata/pons; and lowest in spinal cord. Benzodiazepines inhibited binding of [3H]diazepam in a manner correlated with pharmacological activity in vivo, and binding was not inhibited by non-benzodiazepine anxiolytics, muscle relaxants, anticonvulsants or by strychnine and glycine. Distribution of [3H]diazepam binding in several regions of the rat central nervous system correlated with Na+-independent binding of gamma-aminobutyric acid in the same regions. The results may be in accord with the possible involvement of gamma-aminobutyric acid in the mechanism of action of the benzodiazepines but provide no support for a mechanism based on the interaction of benzodiazepines with central glycine receptors.     

Selective changes in sensitivity to cholinergic agonists and receptor changes elicited by continuous physostigmine infusion

A reduction in nicotinic receptors has been observed in brains of patients with Alzheimer's disease, and physostigmine, an indirect acting cholinergic agonist, has been suggested as a treatment of this disease. However, the effects of physostigmine treatment have not been thoroughly characterized. The central and peripheral effects of physostigmine were evaluated in C57BL/6 mice using a battery of behavioral and physiological tests that included respiratory rate, Y maze crossing and rearing activities, heart rate and body temperature. With the exception of the heart rate test, which was blocked by a peripheral muscarinic antagonist, all responses to physostigmine measured in the test battery seem to be mediated centrally. Sensitivity to physostigmine and to nicotine and oxotremorine were examined as were nicotinic and muscarinic receptors after continuous infusion of physostigmine. Mice (C57BL/6) continuously infused with physostigmine (0.1 mg/kg/h) for 10 days developed marked tolerance to physostigmine and exhibited a slight decrease in sensitivity to nicotine; sensitivity to oxotremorine was largely unaltered. Chronic physostigmine treatment resulted in a 62% inhibition of AChE activity and elicited a significant increase in the amount of L-[3H] nicotine binding in midbrain, hippocampus, striatum and colliculi; alpha-[125I]bungarotoxin and [3H]quinuclidinyl benzilatl binding were not changed. Although physostigmine treatment might partially reverse the reduction of brain nicotinic receptors seen with Alzheimer's disease, it is questionable whether it would be effective for a prolonged period since the up-regulated nicotinic receptors may not have normal function.     

Characterization of nicotinic receptors on cultured cortical neurons using anti-idiotypic antibodies and ligand binding

Monoclonal anti-idiotypic antibodies that represent the internal image of nicotine's natural isomer, L-nicotine, were used in conjunction with L-[3H]nicotine binding to characterize nicotinic receptors on neurons cultured from fetal rat cortex. Of the antibodies tested, two (422F11 and 420G11) were found that recognized a class of high affinity [3H]nicotine binding sites present on neuronal cells, but not on glia. The binding properties and pharmacological specificity of these sites compared well with those determined previously for putative nicotinic cholinergic receptors in adult rat brain. The binding of [3H]nicotine to neuronal receptors was effectively inhibited by both antibodies. Receptor-bearing cells were identified using indirect immunofluorescence. Approximately 20 to 30% of the cells were labeled specifically by the anti-idiotypes. Labeling was blocked by L-nicotine and other nicotinic agonists, but not by antagonists or by alpha and neuronal bungarotoxins. The majority of cells which were labeled had either bipolar or pyramidal morphology. Fluorescent labeling was associated with cell bodies as well as with axonal and dendritic processes, consistent with the proposed roles of neuronal nicotinic receptors in neuromodulation and synaptic transmission. The results suggest that anti-idiotypic antibodies may provide a new tool suitable for studying the locations, structure and functional significance of high affinity neuronal nicotinic receptors at the cellular level.     

Nicotine-induced tolerance and receptor changes in four mouse strains

The effects of chronic infusion of 3.0 mg/kg/hr of nicotine on the behavioral responses of four mouse strains (C3H, C57BL, BALB and DBA) to challenge doses of nicotine and on the levels of cholinergic receptors and enzymes were determined. Mice of the C57BL, BALB and DBA strains developed tolerance to the effects of nicotine on Y-maze activity, Y-maze rearing and body temperature. The C3H mice did not exhibit any evidence of tolerance to the effects of nicotine on these measures, but this strain did show tolerance to the effects of nicotine on acoustic startle response. None of the strains developed tolerance to the effects of nicotine on respiration rate, and only the BALB mice demonstrated tolerance to nicotine-induced bradycardia. Chronic nicotine treatment also elicited changes in putative brain nicotinic receptors. Increased binding of L-[3H]nicotine was seen in six brain regions in all four mouse strains. The changes in binding were similar for all the strains. The binding of alpha-[125I]bungarotoxin was also elevated in the four mouse strains, and modest differences among the strains were evident in this measure, with the C3H strain showing the smallest increases in four of the six brain regions. Chronic nicotine treatment did not alter brain muscarinic receptors, as measured with L-[3H]quinuclidinyl benzilate binding, nor were the activities of acetylcholinesterase or choline acetyltransferase altered, except that a small change in hippocampal choline acetyltransferase activity may have occurred. These results indicate that tolerance to nicotine is regulated by genetic factors and that changes in brain nicotinic receptor numbers are not sufficient to explain the development of tolerance.     

3H]tryptamine autoradiography in rat brain and choroid plexus reveals two distinct sites

In vitro autoradiographic techniques were used to examine the distribution of [3H]tryptamine binding sites in rat brain. The gross distribution and pharmacological characteristics of binding to brain sections resembled those seen in homogenate studies. Binding sites were found throughout the brain, with a preponderance of sites in the forebrain and limbic structures; highest levels were seen in the choroid plexus and the interpeduncular nucleus. Other regions exhibiting high levels of [3H]tryptamine binding include the cortex (especially lamina I), caudate putamen, hippocampus, anterior olfactory nucleus, olfactory tubercle, nucleus accumbens, amygdala, superior colliculus (superficial gray layer), locus ceruleus, the nucleus of the solitary tract and the pineal body. Although there were similarities in this distribution to that for binding sites of [3H]5-hydroxytryptamine ([3H]serotonin), the overall patterns were distinct. The binding site for [3H] tryptamine in the choroid plexus (termed T-2) was pharmacologically distinct from that in the rest of the brain (termed T-1); several compounds, including kynuramine, were potent inhibitors of [3H]tryptamine binding at the brain site, but not at the choroid plexus site. [3H]Serotonin also labels a site in the rat choroid plexus; this site was different from both [3H]tryptamine sites. Knowledge of the distribution of tryptamine binding sites in the brain will aid in efforts to ascertain the function of these sites.     

Comparison of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain

The properties of [3H]quinuclidinylbenzilate ( [3H]QNB) binding and [3H]pirenzepine ( [3H]PZ) binding to various regions of rat brain were compared. [3H]PZ appeared to bind with high affinity to a single site, with a Kd value of approximately 15 nM in the cerebral cortex. The rank order of potencies of muscarinic drugs to inhibit binding of either [3H]QNB or [3H]PZ was QNB greater than atropine = scopolamine greater than pirenzepine greater than oxotremorine greater than bethanechol. Muscarinic antagonists (except PZ) inhibited both [3H]PZ and [3H]QNB binding with Hill coefficients of approximately 1. PZ inhibited [3H]QNB binding in cortex with a Hill coefficient of 0.7, but inhibited [3H]PZ binding with a Hill coefficient of 1.0. Hill coefficients for agonists were less than 1. The density of [3H]PZ binding sites was approximately half the density of [3H]QNB binding sites in cortex, striatum and hippocampus. In pons-medulla and cerebellum, the densities of [3H]PZ binding sites were 20 and 0%, respectively, relative to the densities of [3H]QNB binding sites. When unlabeled PZ was used to compete for [3H]QNB binding, the relative number of high-affinity PZ binding sites in cortex, pons and cerebellum agreed with the relative number of [3H]PZ binding sites in those regions. The binding of [3H]PZ and [3H]QNB was nonadditive in cortex. GTP inhibited high-affinity oxotremorine binding, but not PZ binding. Together, these data suggest that [3H]PZ binds to a subset of [3H]QNB binding sites. Whether this subset reflects the existence of subtypes of muscarinic receptors or is a consequence of coupling to another membrane protein remains to be seen.     

Acute pentylenetetrazol injection reduces rat GABAA receptor mRNA levels and GABA stimulation of benzodiazepine binding with No effect on benzodiazepine binding site density.

The effects of a single convulsive dose of pentylenetetrazol (PTZ, 45 mg/kg i.p.) on rat brain gamma-aminobutyric acid type A (GABAA) receptors were studied. Selected GABAA receptor subunit mRNAs were measured by Northern blot analysis (with beta-actin mRNA as a standard). Four hours after PTZ, the GABAA receptor gamma2-mRNA was decreased in hippocampus, cerebral cortex, and cerebellum; alpha1-mRNA was decreased in cerebellum; and beta2 subunit mRNA was decreased in cortex and cerebellum. The alpha5 subunit mRNA level was not altered. Those mRNAs that had been reduced were increased in some brain regions at the 24-h time point, and these changes reverted to control levels by 48 h. PTZ effect on GABAA receptors was also studied by autoradiographic binding assay with the benzodiazepine agonist [3H]flunitrazepam (FNP), the GABAA agonist [3H]muscimol, and the benzodiazepine antagonist [3H]flumazenil. There was an overall decrease in [3H]FNP binding 12 but not 24 h after PTZ treatment. In contrast, [3H]muscimol binding was minimally affected, and [3H]flumazenil binding was unchanged after PTZ treatment. Additional binding studies were performed with well-washed cerebral cortical homogenates to minimize the amount of endogenous GABA. There was no PTZ effect on specific [3H]FNP binding. However, there was a significant reduction in the stimulation of [3H]FNP binding by GABA. The results showed that an acute injection of PTZ caused transient changes in GABAA receptor mRNA levels without altering receptor number but affected the coupling mechanism between the GABA and benzodiazepine sites of the GABAA receptor.     

Differential ontogeny of putative M1 and M2 muscarinic receptor binding sites in the murine cerebral cortex and heart

Studies with [3H]pirenzepine [( 3H]PZ) suggest that this nonclassical muscarinic antagonist selectively identifies putative M1 muscarinic receptors. We now compare the ontogeny of these putative M1 sites, identified by high-affinity [3H]PZ binding, with sites identified by the classical antagonist (-)-[3H]quinuclidinyl benzilate ((-)-[3H]QNB) in murine cerebral cortical and cardiac homogenates. Dissociation constants (Kd) for [3H]PZ (2.1-6 nM in the cortex and 2.0-21 nM in the heart) and for (-)-[3H]QNB (10-28 pM in the cortex and 10-39 pM in the heart) are similar in adult and neonatal tissues, whereas receptor density (maximum binding, femtomoles per milligram of protein) varies significantly. Cerebral cortical [3H]PZ binding rises from 14% at birth, to 88% of adult levels by day 14, peaks at 128% at day 28 and falls to the mean adult level of 606 fmol/mg of protein. Cerebral cortical (-)-[3H]QNB binding parallels [3H]PZ binding. Conversely, parallel studies show cardiac (-)-[3H]QNB density is 3- to 17-fold greater than the comparable density of high-affinity [3H]PZ binding sites throughout ontogeny. We conclude that: 1) the high ratio of [3H]PZ binding to (-)-[3H]QNB binding identifies the murine cerebral cortex as a tissue which contains predominantly putative M1 muscarinic binding sites; 2) the relatively low ratio of [3H]PZ binding to (-)-[3H]QNB binding throughout ontogeny identifies the murine heart as a tissue which contains primarily the putative M2 muscarinic binding site; and 3) M1 and M2 receptor binding sites show distinct developmental curves in the cerebral cortex and heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of aging on alpha-1 adrenergic stimulation of phosphoinositide hydrolysis in various regions of rat brain

The effects of aging were examined on the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis in three brain regions. Tissue minces of thalamus, cerebral cortex and hippocampus from 3-, 18- and 28-month-old male Fischer 344 rats were prelabeled with [3H]myoinositol. Exposure of these prelabeled minces to phenylephrine and (-)-norepinephrine revealed that accumulation of [3H]inositol phosphates was selectively reduced by 20 to 30% in the thalamus and cerebral cortex of the oldest age group. Analysis of concentration-response and competition binding curves indicated that this decrease was due to diminished agonist efficacy rather than diminished receptor affinity. The reduction in responsiveness to phenylephrine and (-)-norepinephrine in the cerebral cortex and the lack of any changes in the hippocampus parallel previously reported changes in the density of alpha-1 adrenergic receptors with aging. These data indicate that the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis is reduced in some, but not all, brain regions of aged Fischer 344 rats.     

Effects of chronic administration of nicotine on prolactin release in the rat: inactivation of prolactin response by repeated injections of nicotine

The effects of chronic injections of nicotine on nicotine-induced prolactin release in the rat were measured and compared to the effects of this treatment on [3H]acetylcholine binding to nicotinic cholinergic sites in the hypothalamus. Treatment with nicotine for 10 days (s.c. injections twice daily) abolished prolactin release in response to an acute i.v. injection of nicotine given 2, 6 or 8 days after the last of the chronic injections of nicotine. At each of these time points, the binding of [3H]acetylcholine in the hypothalamus from rats treated chronically with nicotine was significantly higher than in the hypothalamus from control rats. By 14 days after the last chronic injection of nicotine, the prolactin response to an acute injection of nicotine was restored. Coinciding with the return of the nicotine-induced prolactin response, the binding of [3H]acetylcholine had returned to control values. These results are consistent with the hypothesis that nicotine inactivates nicotinic cholinergic receptors in brain by an allosteric mechanism, and that prolonged inactivation of nicotinic cholinergic receptors leads to their increased number.     

Regional distribution of M1, M2 and non-M1, non-M2 subtypes of muscarinic binding sites in rat brain

The distribution of subtypes of the muscarinic receptor in homogenates of the rat brain was investigated by measuring the competitive inhibition of the binding [3H]N-methylscopolamine by pirenzepine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one). In most brain regions, the competitive binding curves for AF-DX 116 and pirenzepine were consistent with a two-site model. The dissociation constant of pirenzepine for its high-affinity site (M1 receptor) was approximately 10(-8) M, whereas the dissociation constant of AF-DX 116 for its high affinity site (M2 receptor) was approximately 10(-7) M. In many regions, particularly those in the forebrain, the sum of the densities of the M1 and M2 binding sites was substantially less than 100% of the total sites, indicating the existence of a third population of sites lacking high affinity for both pirenzepine and AF-DX 116. We have designated these latter sites as non-M1, non-M2 muscarinic receptors. In general, the densities of the M1 and non-M1, non-M2 binding sites were highest in cerebral cortex, corpus striatum and hippocampus, intermediate in thalamus and hypothalamus, and lowest in midbrain, medulla-pons and cerebellum, whereas the M2 binding site had a relatively low, uniform density throughout the brain. The binding capacity of [3H]N-methylquinuclidinyl benzilate was estimated to be 20 to 30% lower than that of [3H]quinuclidinyl benzilate in various regions of the forebrain, but not in more caudal regions of the brain where the two radioligands had approximately the same binding capacities. Treatment of homogenates of the cerebral cortex with benzilylcholine mustard caused a selective loss of the majority of the [3H]N-methylscopolamine binding sites but spared 25% of the sites labeled by [3H]quinuclidinyl benzilate The results of pirenzepine/[3H]quinuclinyl benzilate competitive binding experiments on cerebral cortex treated with benzilylcholine mustard showed that the residual binding sites for [3H] quinuclidinyl benzilate were enriched in M1 muscarinic receptors.     

Differences in d-[3H]lysergic acid diethylamide binding in mouse cortex and hippocampus in vivo and in vitro revealed by radioautography and rapid filtration studies

The localization of d-[3H]lysergic acid diethylamide ([3H]LSD) binding sites in mouse brain was compared in vivo and in vitro. Radioautography of brain sections incubated with 6 nM [3H]LSD in vitro revealed substantial specific binding in cortex (CTX), especially in layers III to IV and anterior cingulate gyrus, and in areas CA1 and dentate gyrus of hippocampus (HIP). In sections of brains from mice that received 100 nmol of [3H]LSD per kg and were killed 10, 15 or 30 min later, specific [3H]LSD binding in CTX had a pattern of distribution similar to that observed in vitro. In contrast, the pattern of specific [3H]LSD binding in HIP in vivo differed from the results obtained in vitro, in that it was sparse and lacked differential subregional distribution. The low specific [3H]LSD binding in vivo in HIP but not in CTX was confirmed by homogenate filtration studies of brain areas from mice that received 100 nmol of [3H]LSD per kg. The levels of free [3H]LSD, obtained after correction for time-dependent metabolism of [3H]LSD, did not vary among regions, but [3H]LSD specifically bound in HIP was 30 to 50% of that in CTX. In contrast, steady-state binding studies in vitro in membrane preparations from CTX and HIP demonstrated a similar density and affinity of [3H]LSD binding sites in the two regions. Comparison of [3H]LSD binding characteristics in vivo and in vitro suggests possible mechanisms causing the lower specific binding in HIP in vivo, including modulation of the binding sites that differ in CTX and HIP.     

Subtype-selective up-regulation by chronic nicotine of high-affinity nicotinic receptors in rat brain demonstrated by receptor autoradiography

Subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) are differentially sensitive to up-regulation by chronic nicotine exposure in vitro. To determine whether this occurs in animals, rats were implanted with minipumps containing saline +/- nicotine (6.0 mg/kg/rat/day) for 14 days. Autoradiography with [125I]epibatidine using 3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride (A-85380) or cytisine as selective competitors allowed quantitative measurement in 33 regions of 3 families of nAChR binding, with properties of alpha4beta2, alpha3beta4, and alpha3/alpha6beta2. Chronic nicotine exposure caused increases of 20 to 100% for alpha4beta2-like binding in most regions surveyed. However, binding to this subtype was not increased in some regions, including habenulopeduncular structures, certain thalamic nuclei, and several brainstem regions. In 9 of 33 regions, including catecholaminergic areas and visual structures, alpha3/alpha6beta2-like binding represented >10% of total binding. Binding to this subtype was up-regulated by nicotine in only two of these nine regions: the nucleus accumbens and superior colliculus. alpha3beta4-Like binding represented >10% of total in 15 of the 33 regions surveyed. Binding to this subtype was increased by nicotine in only 1 of these 15 regions, and actually decreased in subiculum and cerebellum. These studies yielded two principal findings. First, chronic nicotine exposure selectively up-regulates alpha4beta2-like binding, with relatively little effect on alpha3/alpha6beta2-like and alpha3beta4-like binding in vivo. Second, up-regulation by chronic nicotine exposure shows considerable regional variation. Differential subtype sensitivity to chronic nicotine exposure may contribute to altered pharmacological response in individuals who smoke or use nicotine replacement therapy.