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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 (Bmax) and not affinity (Kd). There was no significant change in [3H]HC-3 binding in the 6-OHDA-lesioned or transplanted animals, indicating that alterations in muscarinic receptors were not due to transynaptic degeneration of striatal cholinergic interneurons. The findings of downregulation of muscarinic receptors following long-term dopamine denervation and the subsequent normalization of muscarinic receptor density after fetal mesencephalic transplantation suggests that transplanted substantia nigra cells are able to restore inhibitory control on striatal cholinergic interneurons.

The effects of stimulation of guinea pig superior cervical ganglia (SCG) in vitro with 50 μM concentrations of dopamine, L -norepinephrine or L -isoproterenol in Eagle's Medium containing 5 mM theophylline were determined. Dopamine (50 μM) produced no stimulation of cyclic AMP levels, 50 μM norepinephrine produced a doubling of cyclic AMP levels, while 50 μM isoproterenol produced a 6-fold increase in cyclic AMP levels over control values. The increases were blocked by propranolol, indicating that they were due to stimulation of a β-adrenergic receptor-adenylate cyclase complex. In the rabbit SCG, by contrast, 50 μM dopamine produced an increase in cyclic AMP levels of 60% over control values. The possible involvement of the β-receptor complexin the modulation of ganglionic transmission was tested by subjecting ganglia to supramaximal stimulation for 8 min. No elevation of cyclic AMP levels occured in guinea pig SCG, but a doubling of cyclic AMP levels in rabbit SCG was noted. Current concepts of the function of cyclic AMP in neural transmission in the SCG involve a dopaminergic SIF cell, dopamine receptor-adenylate cyclase complex, and the generation of a slow inhibitory postsynaptic potential (s-IPSP). Previous research has indicated that the guinea pig SCG lacks a s-IPSP, and its SIF cells contain norepinephrine rather than dopamine. Since preganglionic stimulation of the guinea pig SCG has now been shown not to cause increased cyclic AMP levels, and since the cyclic AMP content of the guinea pig SCG is not increased following incubation with dopamine, we conclude that a dopamine receptor-adenylate cyclase complex is not involved in the modulation of neural transmission in the guinea pig SCG.

The distribution of specific [3H]hemicholinium-3 ( [3H]HC-3) binding sites sites throughout the rat forebrain was studied by means of quantitative light microscopic autoradiography. Tissue sections were labeled with 2.5 nM [3H]HC-3, apposed to tritium-sensitive film for 2 months and analyzed by computer-assisted densitometry. Regions of intense [3H]HC-3 labeling include the caudate-putamen, nucleus accumbens, olfactory tubercle, amygdala, habenula and the granule cell layer of the dentate gyrus. Little or no specific binding was detected in the corpus callosum, a white matter region. This distribution of specific [3H]HC-3 binding sites is compatible with a selective labeling of central cholinergic nerve terminals.

Muscarinic cholinergic receptors build up on the proximal side of a ligature placed on the rat sciatic and vagus nerves. These receptors appear to be flowing down the axons of at least a portion of the nerve fibers present in sciatic and vagus nerves. Most of the anterograde flowing muscarinic binding sites are displaceable with 10−4 M carbachol indicating that many are high affinity agonist binding sites. These receptors can also be localized in the ganglionic nerve cell bodies in the dorsal root ganglia which contribute fibers to the sciatic nerve. We hypothesize that muscarinic cholinergic receptors are synthesized in the ganglion cell bodies and transported distally in sensory nerve axons in the sciatic nerve.

Autoradiography was used to examine opioid receptor binding in the Mongolian gerbil, a genetic model of the epilepsies. Coronal brain sections of seizure-resistant (SR) and seizure-sensitive (SS) (both pre- and post-seizure conditions) gerbils were labeled with [3H]dihydromorphine. SS (pre-seizure) gerbils demonstrated overall greater brain opioid binding when compared to SR animals. The periaqueductal gray, substantia nigra and medial geniculate body were specific areas in SS (pre-seizure) gerbils which demonstrated highly significant increases in opioid binding compared to SR animals (% increase vs SR were 98%, 91.3% and 42.9%, respectively). Scatchard analysis demonstrated that the increase in opioid binding was due to an increase in the total number of receptors without a significant change in receptor affinity (i.e. periaqueductal gray area: total number of binding sites was 12.7 (SR) and 18.0 fmol/mg tissue (SS pre-seizure), while Kd values were 4.0 (SR) and 4.0 mM (SS pre-seizure). Opioid binding was also increased in the SS (post-seizure) animals when compared to SR animals, especially in the substantia nigra. However, when compared to SS (pre-seizure) gerbils, there was a general but not significant, decrease in opioid binding in SS post-seizure gerbils. The increased opioid binding in the SS (pre-seizure) gerbil compared to SR gerbils could reflect an up-regulation due to a deficit in endogenous ligand (e.g. a deficit in synthesis or decreased release) which could underlie the seizure diathesis in the gerbil.

The binding sites for a radiolabeled form of the potent activator of adenylate cyclase, forskolin, have been localized in the rat brain, pituitary and spinal cord. Using the quantitative technique of in vitro autoradiography, a high density of [3H]forskolin binding was detected in brain structures such as the caudate-putamen, nucleus accumbens, olfactory tubercle, globus pallidus, substantia nigra and the hilus of the area dentata. A comparison of the distribution of [3H] forskolin binding sites with those reported for several neurotransmitter receptor types indicated that forskolin identified adenylate cyclase was probably not linked to any single type of neurotransmitter receptor. These results also presented several new brain areas in which to investigate the neuronal role of adenylate cyclase.

The biochemical properties of the small, intensely fluorescent (SIF) cells of bovine and feline superior cervical ganglia were determined by studying the cyclic AMP responses to incubation in vitro with adrenergic agonists: the morphological properties were studied by fluorescence histochemistry and electron microscopy. Incubation with 50 micron dopamine elicited cyclic AMP levels 524% of control values in the bovine ganglion, but only 152% in the feline ganglion (a value which is not statistically significant). Incubation with 50 micron isoproterenol produced an increase of 356% of control values in the cow, but no increase in the cat. SIF cells were classified into two types by fluorescence histochemistry. The bovine ganglion contained 23.7% Type I (solitary) SIF cells, and 76.3% Type II (clustered) cells, whereas the feline ganglion contained 99.5% Type II cells. Thus the feline ganglion lacks both Type I SIF cells and a dopamine receptor-adenylate cyclase complex. On the basis of biochemical and morphological evidence, we hypothesize that the Type I SIF cell is an interneuron, and infer that the lack of a response to dopamine stimulation in the feline ganglion is caused by a scarcity of interneurons in this species.

The relative binding of the alpha 2 agonist [3H]para-aminoclonidine ([3H]PAC) was examined in discrete regions of the spontaneously hypertensive (SHR) and WKY normotensive rat brains by quantitative autoradiography. Substantially less binding was noted in various brain nuclei known to influence cardiovascular function when comparing the SHR to WKY rat brains. These areas included the nucleus of the solitary tract, dorsal motor nucleus of the vagus, locus coeruleus and paraventricular nucleus of the hypothalamus. This difference in binding was also noted in the bed nucleus of the stria terminalis and the nucleus reuniens of the thalamus. Other brain areas which are not believed to be directly involved in central cardiovascular control such as the cerebral cortex, dorsal lateral geniculate and hippocampus exhibited no significant difference in binding between the two strains. Scatchard analysis of [3H]PAC binding indicated that the difference in binding may be due to a selective loss of the high affinity alpha 2 agonist sites in the SHR. These results indicate that a selective loss of alpha 2 receptors in discrete brain cardiovascular control regions of the SHR may contribute to the elevated blood pressure seen in this strain.

Sertraline, a potent and selective inhibitor of serotonin uptake, was chronically administered to laboratory rats. Using in vitro receptor autoradiographic techniques, we found that the binding of [3H]dihydroalplrenolol ([3H]DHA) was reduced in cortex layers IV–VI. Results of a saturation experiment indicated that the reduction in cortex layer IV was due to a change in number but not affinity of β-adrenergic receptors.

The superior cervical ganglion (SCG) of the guinea pig has been investigated by a multidisciplinary approach. Dopamine (50 μM) produced no increase in cyclic AMP levels above control values of 27.9 pmole/mg protein, but 50 μM isoproterenol produced cyclic AMP levels of 210 pmole/mg protein, indicating the existence of a β-adrenergic receptor-adenylate cyclase complex. The SIF cells were studied by fluorescence histochemistry, which indicated that two morphological types were present. A few Type I cells of the guinea pig SCG were solitary, but most were present in clusters containing many Type II cells. Immunohistochemical localization of antibodies to dopamine-β-hydroxylase (DBH) andphenylethanolamine-N-methyl-transferase (PNMT) demonstrated that both types of SIF cell localize antibodies to DBH but not PNMT, providing strong evidence that norepinephrine is the neurotransmitter for all the SIF cells of the guinea pig SCG. Determination of the ratio of norepinephrine to dopamine confirmed that no other dopamine pools exist in the guinea pig SCG.

The distribution of the high affinity gamma-aminobutyric acid (GABA) receptor labeled by [3H]muscimol, has been studied in the rat brain by light microscopic autoradiography. Receptors in slide-mounted tissue sections were labeled in vitro with [3H]muscimol. Most of the gray matter areas presented grain densities significantly higher than background or white matter areas. Wide variations in receptor densities were found between different brain areas and nuclei. Areas with very high grain densities are the granule cell layer of the cerebellum, external plexiform layer of the olfactory bulb and nuclei of the thalamus, such as the ventral nucleus, lateral nucleus and dorsal geniculate body. The molecular layer of the hippocampus and the external (I–IV) layers of the cortex are also rich in GABA receptors. The basal ganglia have moderate concentrations of receptors, while the pons, medulla and brainstem have only low concentrations of autoradiographic grains. These distributions are discussed in correlation with the known distributions of GABAergic terminals and the presence of inhibitory GABAergic mechanisms.

Specific binding of [125I]angiotensin II ([125I]Ang II) to sections of dog brain was determined by in vitro receptor autoradiography. Highly discrete, dark images representing specific binding of [125I]Ang II were observed in areas corresponding to the nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, ventrolateral medulla, pineal, subfornical organ, nucleus medianus, septum, organum vasculosum of the lamina terminalis and the anterior pituitary. The specific binding was frequently present either as a narrow band or tiny spot within a small portion of the nuclei to which the binding corresponded. The location of these Ang II recognition sites in regions associated with regulation of autonomic and neuroendocrine function provides further evidence for a role of this peptide within the central nervous system.

High affinity gamma-aminobutyric acid, benzodiazepine, strychnine (glycine), dopamine, spirodecanone, alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic and muscarinic cholinergic binding sites were localized by semiquantitative autoradiography in rat and, in some instances, in monkey and human retinae using [3H]muscimol, [3H]flunitrazepam, [3H]strychnine, [3H]spiperone, [3H]prazosin, [3H]para-aminoclonidine, [3H]dihydroalprenolol and [3H]quinuclidinyl benzylate, respectively. In nearly every case, the inner plexiform layer (IP) contained a high receptor density. The distribution of alpha 1 sites was unusual in that binding was concentrated in the outer plexiform layer (OP). Dopaminergic and, to a lesser extent, beta-adrenergic binding was diffusely distributed in the outer nuclear layer, the OP, the inner nuclear layer and the IP. The ganglion cell layer displayed significant benzodiazepine binding. The intraretinal distribution of pre- and postsynaptic markers of these neurotransmitters is discussed.

In vitro opioid receptor binding in the dorsal hippocampal formation and parietal cortex was surveyed in ground squirrels (Citellus lateralis) in the contrasting physiological states of hibernation and euthermia (i.e. not hibernating). Computer-assisted autoradiographic analysis of coronal sections incubated with [3H]dihydromorphine (DMH; 4 nM) revealed statistically significant reductions in specific opioid binding associated with hibernation. In the dorsal hippocampal formation of hibernating animals, binding in the striatum radiatum of CA3, hilus of the dentate gyrus and molecular layer of the dentate gyrus exhibited decreases up to 34% compared to euthermic animals. The stratum radiatum of CA3 exhibited the smallest decrease overall. DHM binding in parietal cortex displayed significant hibernation-related reductions, although they were not uniformly observed across all lamine at the 3 different brain levels examined. These experiments present evidence of changes in brain opioid binding related to the mammalian state of hibernation. The results suggest that changes in opioid receptor binding during hibernation may contribute to the earlier reported5 apparent failure of morphine physical dependence to develop during hibernation.

Discrete localization of enkephalin immunoreactive sites in the rat forebrain was undertaken using animals with and without colchicine pretreatment. Both the immunofluorescence and unlabeled PAP antibody techniques were employed using antisera directed against methionine-enkephalin. We have found several areas of enkephalin fiber and cell localizations not previously described, as well as confirming the results of other researchers. Thus, we found large amounts of perikaryal enkephalin immunoreactivity in the lateral septal nucleus, bed nucleus of the stria terminalis, the preoptic nuclei, the ventral premammillary nucleus, prelateral mammillary nucleus, the medial mammillary nucleus, paraventricular nucleus, the periventricular, ventromedial, dorsomedial and posterior nuclei of the hypothalamus, the arcuate nucleus, and the ventral nucleus of the lateral geniculate body. High concentrations of immunoreactive neuronal fibers not immediately associated with enkephalin perikarya were seen in the nucleus accumbens, medial forebrain bundle, globus pallidus, mammillothalamic tract, the subthalamic nucleus, and the caudal portion of the zona incerta. Many of the areas presented in this study correspond with areas known to exert some regulation over neuroendocrine function as well as various motor and sensory functions. Nuclei with high immunoreactive fiber content also correspond in many instances with areas reported to have high concentrations of opiate receptors. Possible enkephalin-containing tracts and the relationships of enkephalin-containing cells in fiber bundles are also pointed out.

(+)-cis-[3H]Methyldioxolane ((+)-[3H]CD), a potent muscarinic agonist, was used to label high-affinity agonist states of muscarinic receptors in thin tissue sections of the rat central nervous system. Light microscopic autoradiography of atropine-sensitive (+)-[3H]CD binding sites revealed regions of dense labeling (superior colliculus, inferior colliculus, lateral geniculate body, hypoglossal (XII) nucleus, facial (VII) nucleus, tractus diagonalis) and regions of sparse labeling (hippocampus, dentate gyrus). The inverse regional correlation between high-affinity (+)-[3H]CD states and binding sites for the muscarinic antagonists [3H]pirenzepine (r = −0.79) and (-)-[3H]quinuclidinyl benzilate (r = −0.30) underscores potentially important differences between agonist and antagonist binding to CNS tissue slices.

We have reported that the ability of high doses of methamphetamine to impair dopamine and serotonin synthesis in the rat brain is attenuated when animals are pretreated with gradually increasing doses of methamphetamine. To examine the mechanism of this tolerance phenomenon, the effect of methamphetamine on several neurochemical parameters was determined in naive and methamphetamine-pretreated rats. The elevation of nigral substance P concentrations by methamphetamine was attenuated in pretreated compared to naive rats. The methamphetamine-induced reduction in [3H]sulpiride binding in the rat neostriatum and nucleus accumbens was similarly attenuated in animals pretreated with methamphetamine. Determination of brain concentrations of methamphetamine and amphetamine revealed significantly lower concentrations of both compounds in the brains of pretreated compared to naive animals. The results indicate a reduction in the ability of methamphetamine to increase dopamine transmission in the brains of methamphetamine-pretreated rats. Furthermore, this effect appears to be due, at least in part, to a change in the disposition of methamphetamine in pretreated animals.

By using kainic acid (KA) to perform chemical lesions in the guinea pig cerebellum, we have caused degeneration of Purkinje cells without affecting granule cell morphology. Near the injection site we found a large decrease in autoradiographically labeled histamine-H 1 and benzodiazepine receptors of the molecular layer while those receptors distant from the injection site were unaffected. GABA receptors in the granule cell layer remained uniformly constant even immediately adjacent to the lesion site. This evidence suggests that histamine-H 1 and benzodiazepine receptors are present on neuronal elements (possibly on Purkinje cell dendrites) in the molecular layer of the cerebellum and that GABA receptors are associated with the KA-resistant granule cells.

Ibotenic acid lesions of the caudate-putamen in rat brain resulted in dramatic reductions in [3H]SCH 23390 binding in both the ipsilateral caudate-putamen and substantia nigra reticulata as assessed by quantitative autoradiography. Nigral ibotenic acid and 6-hydroxydopamine lesions did not significantly alter the binding in either structure. This indicates that D1 receptors in the caudate-putamen are postsynaptic on striatal neurons, while those in the substantia nigra reticulata are presynaptic on nerve terminals originating in the caudate-putamen.

Previous studies have shown that muscarinic cholinergic agonists bind to 3 distinct receptor sites in brain, each being distinguished by their affinity for the agonists. Antagonists, on the other hand, bind to these sites with the same high affinity. The relative concentrations of the two high and the low affinity agonist sites are known to vary in different brain regions. The goal of this study was to localize separate populations of high and low affinity receptor sites in brain at the light microscopic level. All muscarinic receptors in slide-mounted intact tissue sections were labeled with [3H]N-methyl scopolamine ([3H]NMS), but in some experiments carbachol was added to selectively inhibit the binding of [3H]NMS to the high affinity sites. Autoradiograms of these tissue sections were generated by the apposition of emulsion-coated coverslips. Certain brain areas showed a relatively large displacement of [3H]NMS by carbachol indicating high concentrations of high affinity agonist binding sites. These areas included lamina IV of the cerebral cortex, nucleus tractus diagonalis, some thalamic nuclei, the zona incerta and the dorsal lateral geniculate body.