Diphenylhydantoin: pre- and postnatal administration alters diazepam binding in developing rat cerebral cortex.

Close correlations between the development of the anticonvulsant effects of diphenylhydantoin and increases in tritiated diazepam binding were observed in rats from fetal day 16 to maturation. In contrast, significant decreases in tritiated diazepam binding were observed in 2- and 3-week-old rats that were exposed in utero to diphenylhydantoin. These changes can be correlated with reported increases in seizure susceptibility after prenatal exposure to diphenylhydantoin.

Receptors for the age of anxiety: pharmacology of the benzodiazepines.

Investigation of the actions of the benzodiazepines has provided insights into the neurochemical mechanisms underlying anxiety, seizures, muscle relaxation, and sedation. Behavioral, electrophysical, pharmacological, and biochemical evidence indicates that the benzodiazepines exert their therapeutic effects by interacting with a high-affinity binding site (receptor) in the brain. The benzodiazepine receptor interacts with a receptor for gamma-aminobutyric acid, a major inhibitory neurotransmitter, and enhances its inhibitory effects. The benzodiazepine receptor may also interact with endogenous substances and several naturally occurring compounds, including the purines and nicotinamide, are candidates for this role. Both the purines and nicotinamide possess some benzodiazepine-like properties in vivo, although further work will be required to confirm their possible roles as endogenous benzodiazepines.

Regional differences in the distribution of muscarinic cholinergic receptors in the macaque cerebral cortex.

The in vitro autoradiographic technique was used to characterize the density and laminar distribution of muscarinic cholinergic receptors in 12 cytoarchitectonic areas in the frontal, parietal, and occipital lobes of the rhesus monkey. The entire population of muscarinic receptors was labeled with [3H]quinuclidinyl-benzilate; the M1 receptor subtype was labeled with [3H]pirenzepine; and the density of the M2 receptor subtype was estimated by subtracting the density of M1 receptors from the total population. The overall density of M1 and M2 receptor subtypes was similar throughout the cerebral cortex. However, their laminar distribution varied regionally. In cortical regions of the parietal and occipital lobes and in the primary motor cortex of the frontal lobe, both M1 and M2 receptor subtypes were concentrated in the supragranular layers. By contrast, in prefrontal cortical areas, the combined population of M1 and M2 receptors was evenly distributed across the cortical layers, though M1 receptors were most dense and M2 receptors least dense in layer IV. The difference in the distribution of cholinergic receptors in the prefrontal cortex compared to other neocortical areas reveals a degree of chemoarchitectural specificity of this region with respect to cholinergic markers that has escaped immunohistochemical and other anatomical and functional techniques.

Quantitative autoradiographic mapping of serotonin 5-HT1 and 5-HT2 receptors and uptake sites in the neocortex of the rhesus monkey.

The in vitro autoradiographic technique was used to characterize the distribution of serotonin 5-HT1 and 5-HT2 receptors and uptake sites in 11 cortical areas of frontal, parietal, and occipital lobes in the rhesus monkey; 5-HT1 receptors were labeled with [3H]5-HT; 5-HT2 receptors were labeled with [3H]ketanserin; and 5-HT uptake sites were labeled with [3H]citalopram. Five-HT1 and 5-HT2 receptors and 5-HT uptake sites were found in every cortical area examined with the absolute concentration of 5-HT1 receptors higher than that of 5-HT2 receptors in all areas. In eight regions of prefrontal and parietal as well as in prestriate cortex, 5-HT1 and 5-HT2 receptors had complementary distribution profiles: 5-HT1 receptors were concentrated in layers I and II and the upper strata of layer III, while 5-HT2 receptors had their highest concentration throughout layers III and IV. Only the primary motor and visual cortex had receptor distributions different from that described above. Thus, in the primary visual cortex, both 5-HT1 and 5-HT2 receptors were found in high concentration in sublayer IVc beta, though the density of 5-HT1 receptor was also high in other subdivisions of layer IV and in layers III, V, and VI. In the primary motor cortex, both receptor subtypes were concentrated in layers I and II and the upper strata of layer III. The pattern of distribution of serotonin uptake sites did not match the patterns of distribution of either 5-HT1 or 5-HT2 receptors alone; rather it approximated the combined patterns of distribution of both receptor subtypes. The complementary patterns of distribution of 5-HT1 and 5-HT2 receptors in most areas of the monkey cerebral cortex suggest that these two receptor subtypes may make differential contributions to cortical functions.

Autoradiographic localization of CRF1 and CRF2 binding sites in adult rat brain.

The regional distribution of corticotropin-releasing factor1 (CRF1) and CRF2 binding sites was assessed autoradiographically in adult rat brain. The differential pharmacological profiles of the CRF1 and CRF2 receptor subtypes were used for the discrimination of the CRF1 and CRF2 receptor subtypes in rat brain. Pharmacological characterization at the human CRF1 receptor subtype, expressed in baculovirus-infected Sf9 cells, showed high affinity binding (Ki < or = 10.0 nM) for the peptide agonists sauvagine, urotensin I, rat/human CRF, and ovine CRF. Pharmacological characterization at the rat CRF2 receptor subtype expressed in CHO cells showed a rank order affinity for the peptide agonists such that sauvagine, urotensin I and rat/human CRF showed high affinity binding whereas ovine CRF had a Ki value of 300 nM. Based on this differential binding affinity for ovine CRF, [125I]sauvagine binding in the presence of increasing concentrations of ovine CRF was used to discriminate CRF1 from CRF2 receptor subtypes in rat brain. The CRF1 receptor subtype was found to be localized to various regions of the cerebellum, as well as to several cortical areas. The CRF2 receptor subtype was shown to be localized to the lateral septal nucleus, entorhinal cortex, and to amygdaloid and hypothalamic regions. The present autoradiographic findings provide evidence that each subtype has a distinct regional distribution, thus strengthening the suggestion that CRF1 and CRF2 receptors serve different roles in mediating CRF function. Such data suggest that the development of CRF receptor subtype selective antagonists should help to delineate the role of CRF1 and CRF2 receptor subtypes in central nervous system function.

Consequences of benzodiazepine receptor occupancy.

The pharmacological consequences of the occupancy of benzodiazepine receptors have been a recent area of active research. There is good agreement between the electrophysiological effects of benzodiazepines and their binding to benzodiazepine receptors when both are studied in vitro under identical conditions. Compounds of different structure from the benzodiazepines can occupy the receptor in a way, which produces little overt effect (imidazodiazepines) or actually causes actions opposite to the benzodiazepines (beta-carbolines, inverse agonists). Several biochemical tests (GABA-shift, photo-shift) for distinguishing these different behavioral properties are described. A model is described for the interactions at membranes of agonists, antagonists and inverse agonists with benzodiazepine receptors in the GABA receptor complex.

Altered gamma-aminobutyric acid/benzodiazepine interaction after chronic diazepam exposure.

Binding to components of the GABA/benzodiazepine receptor complex was examined in cortical membranes from rats treated for 3 weeks with continuously releasing diazepam pellet implants. Chronic diazepam treatment resulted in a decrease in the ability of GABA to inhibit benzodiazepine inverse agonist binding. The amount of binding of benzodiazepine agonist, antagonist, and inverse agonist to benzodiazepine recognition sites was unaltered by the chronic treatment, as were the potencies of these benzodiazepine ligands in inhibiting agonist (3H-flunitrazepam) binding. Chloride channel binding (35S-TBPS) was also unchanged by chronic diazepam treatment. A change in GABA/benzodiazepine coupling and/or a decreased effectiveness of GABA may be responsible for the desensitization of GABA/benzodiazepine interaction.

Chronic administration of beta-carboline-3-carboxylic acid methylamide by continuous intraventricular infusion increases GABAergic function.

The repeated, intraperitoneal administration of the benzodiazepine receptor inverse agonist, FG 7142 (beta-carboline-3-carboxylic acid methylamide), leads to pharmacological kindling and an associated decrease in GABA-stimulated influx of 36Cl- into cortical membrane preparations. The chronic administration of benzodiazepine agonists results in the development of tolerance and also results in a decrease in GABA-stimulated uptake of 36Cl-. The present study was designed to evaluate further the paradoxical reports that both chronic treatment with benzodiazepine receptor agonists and inverse agonists results in a decreased ability of GABA to stimulate uptake of 36Cl- into cortical membrane preparations. The effects of continuous administration of FG 7142 on GABA-stimulated uptake of 36Cl-, the threshold for bicuculline-induced seizures and the proconvulsant actions of acute administration FG 7142 were evaluated. The continuous administration of FG 7142 resulted in an increased capacity of GABA to stimulate the uptake of 36Cl- into cortical membrane preparations and a significant increase in the seizure threshold for bicuculline following the acute administration of FG 7142. These data, therefore, indicate that changes in GABAergic function following chronic administration of GF 7142 are dependent on the regimen of administration of drug. The results also suggest that the GABA receptor homeostatically responds to continuous occupation by inverse agonists by an upregulation of its functional response to GABA.

Distribution of dopaminergic receptors in the primate cerebral cortex: quantitative autoradiographic analysis using [3H]raclopride, [3H]spiperone and [3H]SCH23390.

A widespread distribution of dopamine D1 receptors in the neocortex is well recognized. However, the presence of dopamine D2 receptors in this structure has only recently been established [Martres et al. (1985) Eur. J. Pharmac. 118, 211-219; Lidow et al. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 6412-6416]. In the present paper, a highly specific antagonist, [3H]raclopride, was used for autoradiographic determination of the distribution of D2 receptors in 12 cytoarchitectonic areas of the frontal, parietal, and occipital lobes of the rhesus monkey. A low density of D2-specific [3H]raclopride binding (1.5-4.0 fmol/mg tissue) was detected in all layers of all cortical areas studied. Throughout the entire cortex, the highest density of binding was consistently found in layer V. This is a unique distribution not observed so far for any other neurotransmitter receptor subtype in monkey cerebral cortex, including D1 receptor. In addition, a comparison was made of the distribution of [3H]raclopride and [3H]spiperone, which has been commonly used in previous attempts to label cortical D2 receptors. We found marked differences in the distribution of these two radioligands. In the prefrontal cortex, the pattern of [3H]spiperone binding in the presence of ketanserin resembled the combined distribution of 5-HT1C serotoninergic and alpha 2-adrenergic sites as well as D2 receptors. Thus, [3H]raclopride provides a better estimation of the D2 receptor distribution than does [3H]spiperone. The distribution of D2-specific binding of [3H]raclopride was also compared with the D1-specific binding of [3H]SCH23390 in the presence of mianserin to block labeling to 5-HT2 and 5-HT1C sites. The density of D1-specific [3H]SCH23390 binding was 10-20 times higher than that of D2-specific [3H]raclopride binding throughout the cortex. The densities of both [3H]raclopride and [3H]SCH23390 binding sites display a rostral-caudal gradient with the highest concentrations in prefrontal and the lowest concentrations in the occipital cortex. However, the binding sites of these two ligands had different laminar distributions in all areas examined. In contrast to preferential [3H]raclopride binding in layer V, a bilaminar pattern of [3H]SCH23390 labeling was observed in most cytoarchitectonic areas, with the highest concentrations in supragranular layers I, II and IIIa and infragranular layers V and VI. Whereas [3H]raclopride binding was similar in all cytoarchitectonic areas, [3H]SCH23390 exhibited some region-specific variations in the primary visual and motor cortex. The different regional and laminar distributions of D1 and D2 dopaminergic receptors indicates that they may subserve different aspects of dopamine function in the cerebral cortex.

Distribution of major neurotransmitter receptors in the motor and somatosensory cortex of the rhesus monkey.

The in vitro quantitative autoradiographic technique was used to characterize the distributions of alpha 1, alpha 2, beta 1 and beta 2 adrenergic, D1 and D2 dopaminergic, 5-HT1 and 5-HT2 serotonergic, M1 and M2 cholinergic, GABAA and benzodiazepine receptors in the motor (Brodmann's area 4) and somatosensory (Brodmann's areas 3, 1 and 2) cortex of the adult rhesus monkey. All receptor subtypes studied were present throughout all layers of both areas. In the somatosensory cortex, each receptor had its own laminar distribution. Some subtypes of the same receptor (5-HT1 and 5-HT2; alpha 1 and alpha 2) had complementary distributions while others (beta 1 and beta 2; D1 and D2; M1 and M2) had largely overlapping distributions. In contrast, different receptors had remarkably coincidental distributions in the motor cortex. In this area, they all tended to concentrate in layers I, II and the upper part of layer III. However, such coextensive distribution of many types of neurotransmitter receptors is not observed in motor cortex of rats and humans and therefore may be a distinctive feature of motor cortex in the rhesus monkey. The findings described in this paper indicate that somatosensory and motor areas are distinct in their receptor architecture and that receptor autoradiography provides a useful complement to classical histological techniques in elucidating areal differences in the cortex.

Effects of continuous diazepam administration on GABAA subunit mRNA in rat brain.

Rats treated chronically with diazepam develop tolerance to diazepam effects and show changes in sensitivity of GABAergic systems. In order to investigate possible molecular mechanisms associated with these changes, we have evaluated the effects of acute and chronic diazepam treatment on levels of mRNA for the alpha 1 and beta 1 subunits of the GABAA receptor. Northern blots were hybridized with 32P-labeled GABA alpha 1 and beta 1 cDNA probes, and resulting bands were quantified by autoradiography and densitometry. Levels of alpha 1 mRNA were significantly decreased in cerebral cortex but not in cerebellum or hippocampus of chronic diazepam-treated rats. Acute diazepam treatment did not change levels of alpha 1 mRNA in any of the brain regions. Levels of beta 1 mRNA were examined by Northern blot analysis and also by solution hybridization analysis using a 32P-labeled riboprobe. Both methods showed that beta 1 mRNA was not significantly changed by chronic diazepam treatment. These results demonstrate a specific change in alpha 1 subunit that is associated with a state of altered GABA sensitivity and provide further support for the regional heterogeneity of chronic diazepam effects.

In vitro autoradiography of GTPgamma[35S] binding at activated NPY receptor subtypes in adult rat brain.

Guanylyl 5'-[gamma[35S]thio]-triphosphate (GTPgamma[35S]) binding to NPY receptor-activated G-proteins was measured in adult rat brain sections in order to determine the neuroanatomical distribution of NPY receptor subtypes. Using the pharmacological specificity of the NPY receptor subtypes, differential stimulation of GTPgamma[] binding by subtype-specific agonists was used to demonstrate the differential distribution of these subtypes in rat brain. Treatment of rat brain slices with selective agonists for the NPY receptor subtypes in the presence of 2000 microM GDP was used to discriminate populations of NPY receptor subtypes. Activation of a NPY Y1 receptor subtype by human [Leu31Pro34]NPY stimulated GTPgamma[35S] binding in the rank order: frontal cortex>dentate gyrus>inferior colliculus>/=thalamus>hypothalamus. In contrast, NPY Y2/Y5 peptide agonist, human PYY(3-36), stimulated GTPgamma[35S] binding in the rank order: hypothalamus>substantia nigra>hippocampus>frontal cortex>/=inferior colliculus. Stimulation of NPY Y5 receptor subtypes by a NPY Y5 selective agonist, rat/human D-Trp, was shown to stimulate GTPgamma[35S] binding in the hypothalamus and discrete nuclei of the thalamus. Little GTPgamma[35S] binding in the dentate gyrus, frontal cortex, or inferior colliculus was measured following stimulation with D-Trp. Stimulation of GTPgamma[35S] binding by [Leu31Pro34]NPY, but not by the other NPY receptor agonists, was blocked by the selective NPY Y1 receptor antagonist, BIBP 3226. In conclusion, functional coupling at NPY receptor subtypes can be shown in rat brain and populations of NPY receptor subtypes can be anatomically discriminated by NPY agonist stimulation of GTPgamma[35S] binding in rat brain.

Developmental profile of polyadenylated and non-polyadenylated GABAA receptor subunit mRNAs.

The ratio of mRNA not selected for polyadenylation (non-poly(A)+ selected) to mRNA selected for polyadenylation (poly(A)+) for the beta 1, alpha 1 and gamma 2 subunits of the GABAA receptor complex was examined in rats as a function of age. RNA was extracted from whole brain of rats that were either 0, 1, 3, 5 or over 60 days of postnatal age. Poly(A)+ mRNA was purified by oligo(dT)-cellulose chromatography. Non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor beta 1, alpha 1 and gamma 2 subunits were examined by Northern blot analysis using cDNA probes specific for these subunits. Levels of GABAA receptor beta 1 subunit mRNA were also examined by solution hybridization analysis with a beta 1 riboprobe. Analysis of Northern blots revealed that levels of poly(A)+ beta 1 subunit mRNA were highest at 0 days of age, but decreased and reached adult levels by 5 days of postnatal age. However, levels of the beta 1 subunit message extracted from non-poly(A)+ selected mRNA were not significantly different at any of the ages examined, suggesting the existence of a population of beta 1 subunit mRNA that is not polyadenylated. The age-related discrepancy between beta 1 subunit levels measured in non-poly(A)+ selected mRNA and poly(A)+ mRNA was also observed using solution hybridization analysis. In contrast, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the alpha 1 subunit of the GABAA complex increased from 0 days of age to adulthood. Similarly, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor gamma 2 subunit increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)

Development changes in pharmacological responsivity of the acoustic startle reflex: effects of picrotoxin.

Using the acoustic startle reflex as the behavioral measure, qualitatively different responses to the GABA antagonist picrotoxin were obtained in developing rats before and after 21 days postnatal (PN) age. Dose-dependent increases in acoustic startle were seen following picrotoxin in PN day 15-16 rat pups. In contrast, dose-dependent decreases in startle following picrotoxin were observed in adult rats. The switch from excitation to inhibition of startle was found to occur abruptly on PN day 21. Excitatory responses to picrotoxin were also found in adult rats following localized infusions of picrotoxin into lumbar spinal cord regions, but not into the forebrain. These results give evidence that picrotoxin-sensitive sites that modulate increases in startle reflex behavior mature first and are analogous to sites in the adult spinal cord, whereas picrotoxin-sensitive sites that modulate decreases in startle reflex behavior mature later (greater than or equal to PN day 21) and are localized in more rostral brain areas.

Raphe origin of serotonergic nerves terminating in the cerebral ventricles.

The dorsal and median raphe nuclei of the midbrain are known to contain the perikarya of origin of the major serotonergic (indoleamine) neurons projecting to the parenchyma of the forebrain. Lesions were placed in these nuclei to determine whether serotonin-containing nerve terminals in the cerebral ventricular system are also derived from the raphe nuclei. Brain tissue from control rats and rats 2-7 days after placement of raphe lesions was examined by fluorescence and electron microscopy. By the third day after lesion there was a marked reduction in the formaldehyde-induced fluorescence of supra-ependymal terminals. By the same time virtually all supra-ependymal terminals showed advanced degenerative changes as visualized by electron microscopy. There was a degeneration of supra-ependymal terminals in all parts of the cerebral ventricular system examined, including the epithalamic region (e.g., habenula and pineal recess; serotonin-containing terminals in the latter areas had previously been thought to arise from modified pinealocytes in the pineal recess). We conclude that most, if not all, supra-ependymal nerve terminals are derived from serotonergic cells of origin in the raphe nuclei.

Afferents to brain stem nuclei (brain stem raphe, nucleus reticularis pontis caudalis and nucleus gigantocellularis) in the rat as demonstrated by microiontophoretically applied horseradish peroxidase.

Using a retrograde tracer technique with microiontophoretically applied horseradish peroxidase (HRP), afferent projections to the brain stem raphe nuclei (BR, raphe magnus, pallidus and obscurus) and to two adjacent reticular nuclei, nucleus reticularis pontis caudalis (nRPC) and nucleus gigantocellularis (nGC) were identified. The most striking difference between the afferent projections to the BR and the adjacent nuclei as determined by this method is that afferents to the BR originate primarily from structures rostral to the pons, especially the mesencephalic central gray and the dorsal and ventral tegmentum. In contrast, the two reticular nuclei studied (nGC and nRPC) received afferent projections within or caudal to the pons-medulla. For example, the nGC receives prominent afferent projections from the gray matter of the spinal cord. In addition, evidence for interconnections between all of the adjacent nuclei (BR, nGC and nRPC) was found. Such afferent projections are compatible with the notion that the brain stem raphe nuclei may serve as connections within the brain stem for a descending system, while the nGC may be a relay in a feedback loop between the spinal cord and the reticular formation.

Time course for development of anticonvulsant tolerance and GABAergic subsensitivity after chronic diazepam.

The time courses for development of neuronal and behavioral tolerance to diazepam (DZ) were estimated in rats continuously exposed to low levels of DZ for 3, 7, 14 or 21 days. Microiontophoretic sensitivity of dorsal raphe neurons to gamma-aminobutyric acid (GABA) was initially facilitated after short-term exposure to DZ released from implanted capsules for up to 3 days but returned to control levels by 7 days postimplantation and continued to decrease thereafter. GABAergic sensitivity remained depressed for a minimum of 5 days following removal of DZ capsules. To obtain a behavioral measure of tolerance, the anticonvulsant activity of DZ against bicuculline-induced seizures was also assessed. Rats studied 3 days after capsule implantation showed a significant elevation in seizure threshold. Seizure liability returned to control levels ca. 7 days after chronic treatment was initiated. These results indicate that tolerance to anticonvulsant efficacy against bicuculline seizures are temporally related to the onset of reduced GABA sensitivity on dorsal raphe neurons during prolonged exposure to DZ.

GABAergic subsensitivity of dorsal raphe neurons in vitro after chronic benzodiazepine treatment in vivo.

Previous studies have shown that chronic benzodiazepine treatment reduces the in vivo sensitivity of dorsal raphe neurons (DRN) to microiontophoretically applied gamma-aminobutyric acid (GABA). We have examined sensitivity of DRN in vitro using a modified midbrain slice technique which allows side-by-side analysis of slices from control and chronic diazepam-treated rats. GABA sensitivity of raphe neurons was reduced in slices from rats treated for 3 weeks with diazepam, compared to control sensitivity. Thus, GABA subsensitivity following chronic diazepam treatment appears to be dependent on changes intrinsic to the midbrain area.

Development of long-term subsensitivity to GABA in dorsal raphe neurons of amygdala-kindled rats.

Previously we reported a long-term change in neuronal sensitivity to GABA following amygdala kindling. Dorsal raphe neurons of amygdala-kindled rats exhibited significant subsensitivity to GABA 4 weeks after the last fully generalized (Stage 5) seizure. We hypothesized that this alteration in GABA sensitivity might reflect neuronal changes corresponding to kindled seizure susceptibility and subsequent experiments have investigated this hypothesis. The progression towards neuronal subsensitivity to GABA during amygdala kindling can be correlated with the Stage to which an animal has been kindled. That is, when measured 4 weeks after the last kindled seizure, dorsal raphe neurons are supersensitive to GABA following a Stage 2 seizure, not different from controls following a Stage 3 seizure and subsensitive to GABA following a Stage 5 seizure. In addition, subsensitivity to GABA appears to be permanent in that it is still measurable 3 months after the last Stage 5 seizure. Thus, amygdala kindling produces long-term, perhaps permanent, changes in neuronal sensitivity to GABA and these changes reflect the Stage to which an animal has been kindled.

Interaction of diphenylhydantoin and benzodiazepines in the CNS.

Using extracellular unit recording and microiontophoretic techniques, the anticonvulsant diphenylhydantoin (DPH) was found to increase the physiological efficacy of the benzodiazepines. This increased biological effect could be correlated with an enhanced specific binding of benzodiazepines measured in vivo following pretreatment of rats with DPH. The increased binding of benzodiazepines is due to an increase in the total number of benzodiazepine binding sites without an alteration in the affinity of these sites for [3H]diazepam. The data show that the effects of DPH on benzodiazepine binding are qualitatively different and independent from the effects of gamma-amino-butyric acid. Based on the dose-responsive relationship between benzodiazepine binding effects and the anticonvulsant activity of DPH and reports of other convulsant, anticonvulsant compounds which alter benzodiazepine binding, it is suggested that the benzodiazepine binding site may be relevant to convulsant-anticonvulsant activity.