Age-dependent changes in second messenger and rolipram receptor systems in the gerbil brain

Summary Age-related alterations in binding sites of major second messengers and a selective adenosine 3,5-cyclic monophosphate (cyclic-AMP) phospho-diesterase (PDE) in the gerbil brain were analysed by receptor autoradiography. [³H]Phorbol 12,13-dibutyrate (PDBu), [³H]inositol 1,4,5-trisphosphate (IP₃), [³H]forskolin, [³H]cyclic-AMP, and [³H]rolipram were used to label protein kinase C (PKC), IP₃ receptor, adenylate cyclase, cyclic-AMP dependent protein kinase (PKA), and Ca²⁺/calmodulin-mdependent cyclic-AMP PDE, respectively. In middle-aged gerbils (16 months old), [³H]PDBu binding was significantly reduced in the hippocampal CA 1 sector, thalamus, substantia nigra, and cerebellum, compared with young animals (1 month old). [³H]IP₃ binding revealed significant elevations in the nucleus accumbens, hippocampal CA 1 sector, dentate gyrus, and a significant reduction in cerebellum of middle-aged gerbils. [³H]Forskolin binding in middle-aged animals was significantly increased in the nucleus accumbens and hilus of dentate gyrus, but was diminished in the substantia nigra and cerebellum. On the other hand, in middle-aged animals, [³H]cyclic-AMP binding revealed a significant elevation only in the hippocampal CA3 sector, whereas [³H] rolipram binding showed a significant reduction in the thalamus and cerebellum. Thus, the age-related alteration in these binding sites showed different patterns among various brain regions in middle-aged gerbils indicating that the binding sites of PKC, IP₃, and adenylate cyclase are more markedly affected by aging than those of PKA and cyclicAMP PDE and that the hippocampus and cerebellum are more susceptible to these aging processes than other brain regions. The findings suggest that in-tracellular signal transduction is affected at an early stage of senescence and this may lead to neurological deficits.     

Withdrawal from butorphanol dependence alters binding of [H]phorbol dibutyrate to protein kinase C, but not of [H]forskolin to adenylate cyclase

The time course of autoradiographic binding of major second messengers in the rat brain was studied at 2, 7, and 24 h after withdrawal from butorphanol infusion. [³H]Forskolin and [³H]phorbol 12,13-dibutyrate (PDBu) were used to label adenylate cyclase and protein kinase C (PKC), respectively. Rats were rendered dependent by intracerebroventricular infusion of butorphanol (26 nmol μl⁻¹ h⁻¹) via osmotic minipumps for 3 days. Withdrawal was initiated by abrupt cessation of the butorphanol infusion. The levels of [³H]forskolin binding were not changed at any time or in any brain area, except for an increase following 7 h of withdrawal in the brainstem only. The levels of [³H]PDBu binding were significantly increased (13–47%) in multiple areas of the rat brain following 7 h of withdrawal from butorphanol infusion. These findings suggest that the phosphoinositide cycle system is more susceptible to alteration during butorphanol dependence than is the adenylate cyclase system in the rat brain.     

Autoradiographic analysis of second-messenger systems in the gerbil brain

Quantitative in-vitro autoradiographic study was performed to localize two prominent second-messenger systems (the adenylate cyclase and phosphoinositide systems) in the normal gerbil brain. [3H] Forskolin and [3H] phorbol 12, 13-dibutyrate (PDBu) were used to identify the regional distribution of adenylate cyclase and protein kinase C, respectively. The localization of the forskolin binding was not uniform, being particularly concentrated in the striatum, the accumbens nucleus, the olfactory tubercle, the substantia nigra, the CA3 region of the hippocampus and the molecular layer of the cerebellum. On the other hand, the PDBu binding was rather uniform, although the superficial layer of the cerebral neocortices, the strata oriens of the CA1 region of the hippocampus and the molecular layer of the cerebellum showed relatively dense binding. Quantitative autoradiography of the second-messenger systems in the brain is expected to provide important information concerning the role of neurotransmitters in the pathophysiology of various conditions.     

Age-related changes of sodium-dependentd-[3H]aspartate and [3H]FK506 binding in rat brain

Summary We investigated age-related changes in excitatory amino acid transport sites and FK506 binding protein (FKBP) in 3-week-, and 6-, 12-, 18- and 24-month-old Fischer 344 rat brains using receptor autoradiography. Sodium-dependentd-[³H]aspartate and [³H]FK506 were used to label excitatory amino acid transport sites and immunophilin (FKBP), respectively. In immature rats (3-week-old), sodium-dependentd-[³H]aspartate binding was lower in the frontal cortex, parietal cortex, striatum, nucleus accumbens, whole hippocampus, thalamus and cerebellum as compared to adult animals (6-month-old), whereas [³H]FK506 binding was significantly lower only in the hippocampus, thalamus and cerebellum. [³H]FK506 binding exhibited no significant change in the brain regions examined during aging. However, sodium-dependentd-[³H]aspartate binding showed a conspicuous reduction in the substantia nigra in 18-month-old rats. Thereafter, a significant reduction in sodium-dependentd-[³H]aspartate binding was found in the thalamus, substantia nigra and cerebellum in 24-month-old rats. Other regions also showed about 10–25% reduction in sodium-dependentd-[³H]aspartate binding. The results indicate that excitatory amino acid transport sites are more susceptible to aging process than immunophilin. Further, our findings demonstrate the conspicuous differences in the developmental pattern between excitatory amino acid transport sites and immunophilin in immature rat brain.     

Withdrawal from butorphanol dependence alters binding of [3H]phorbol dibutyrate to protein kinase C, but not of [3H]forskolin to adenylate cyclase

The time course of autoradiographic binding of major second messengers in the rat brain was studied at 2, 7, and 24 h after withdrawal from butorphanol infusion. [3H]Forskolin and [3H]phorbol 12,13-dibutyrate (PDBu) were used to label adenylate cyclase and protein kinase C (PKC), respectively. Rats were rendered dependent by intracerebroventricular infusion of butorphanol (26 nmol microliter-1 h-1) via osmotic minipumps for 3 days. Withdrawal was initiated by abrupt cessation of the butorphanol infusion. The levels of [3H]forskolin binding were not changed at any time or in any brain area, except for an increase following 7 h of withdrawal in the brainstem only. The levels of [3H]PDBu binding were significantly increased (13-47%) in multiple areas of the rat brain following 7 h of withdrawal from butorphanol infusion. These findings suggest that the phosphoinositide cycle system is more susceptible to alteration during butorphanol dependence than is the adenylate cyclase system in the rat brain.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.     

Effects of naftidrofuryl oxalate, a 5HT2 antagonist, on neurotransmission and transduction systems in the gerbil hippocampus

We investigated the effects of age and naftidrofuryl oxalate (Naftidrofuryl), a 5-HT₂ antagonist, on neurotransmission and transduction systems in the gerbil hippocampus using quantitative autoradiography. [³H]Quinuclidinyl benzilate (QNB), [³H]cyclohexyl-adenosine (CHA), [³H]MK-801, and [³H]muscimol were used to label muscarinic acetylcholine, adenosine A1, N-methyl-d-aspartate (NMDA), and γ-aminobutyric acid-A (GABA_A) receptors, respectively. [³H]PN200-110 labeled L-type Ca²⁺ channels. [³H]Forskolin, [³H]cyclic adenosine monophosphate (cAMP), [³H]phorbol 12,13-dibutyrate (PDBu), and [³H]inositol 1,4,5-triphosphate (IP₃) were used to label adenylate cyclase, cAMP-dependent protein kinase, protein kinase C (PKC), and IP₃ receptors, respectively. Approximately 20% reductions in [³H]QNB, [³H]forskolin, and [³H]PDBu binding were observed in the hippocampus of 9-month-old gerbils in comparison with 5-week-old gerbils. Treatment with Naftidrofuryl (10 mg/kg, i.p., once a day for 7 days) ameliorated these reductions. No changes were found in [³H]CHA, [³H]MK-801, [³H]muscimol, [³H]PN200-110, [³H]cAMP, and [³H]IP₃ binding. The results suggest that Naftidrofuryl may have beneficial effects on the age-related alterations in signal transmission and transduction systems in the brain. Because the acetylcholine system, adenylate cyclase, and PKC are considered to be involved in learning and memory processes, the result may have clinical implications.     

Mapping of second messenger and rolipram receptors in mammalian brain

Autoradiographic localizations of major second messengers and a selective cyclic adenosine monophosphate (cyclic-AMP) phosphodiesterase in the brain were visualized in the gerbil and the rat using receptor autoradiography. [3H]Phorbol 12,13-dibutyrate (PDBu), [3H]inositol 1,4,5-trisphosphate (IP3), [3H]forskolin, [3H]cyclic-AMP, and [3H]rolipram were used to label protein kinase C, IP3 receptor, adenylate cyclase, cyclic-AMP-dependent protein kinase (cyclic-AMP-DPK), and Ca2+/calmodulin-independent cyclic-AMP phosphodiesterase (PDE), respectively. Most second messengers and rolipram binding activities were especially found in the limbic system, basal ganglia, and cerebellum. Marked differences were noted in the hippocampus, where cyclic-AMP and rolipram binding activities were very low in gerbils but high in rats. In contrast, regional localization in the binding sites of PDBu, IP3, and forskolin in gerbil brain was relatively similar to that in rat brain. Further, alteration of the cyclic-AMP and rolipram binding sites was studied in the gerbil hippocampus 7 days after 10-min cerebral ischemia. The results suggest that the gerbil differs from the rat with respect to the characteristic neurons or interneurons, especially in the hippocampal formation. This finding may help further elucidate the relationship or difference between gerbils and rats for brain function and behavioral pharmacology. Furthermore, our results suggest that cyclic-AMP and rolipram binding sites are predominantly distributed on the pyramidal cell layer of the hippocampal CA1 sector and that transient cerebral ischemia can cause marked reduction in these binding sites in the hippocampus.     

α2-adrenoceptor-GTP binding regulatory protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats

The characterization of [³H]clonidine binding and effects of GTP, forskolin, islet-activating protein (IAP) and cholera toxin on adenylate cyclase activity were investigated in cerebral cortical membranes from 70-day-old and 2-year-old rats. Neither K_d nor B_max values in [³H]clonidine binding were changed between day 70 and year 2. The activation of adenylate cyclase by forskolin was significantly higher in senescent than in adult animals. The inhibitory effect of adrenaline, which was completely abolished by the pretreatment with IAP/NAD on forskolin/GTP-stimulated cyclase activity, was low in senescent rats compared to that in adult ones. The stimulatory effect of cholera toxin/NAD was also low at the senescent stage compared to that at the adult stage. It is suggested that ligand binding affinity and the density in α₂-adrenoceptors do not change between day 70 and year 2 but that GTP binding and/or coupling activity of inhibitory as well as stimulatory GTP binding regulatory protein to catylytic unit decrease in synaptic membranes of 2-year-old compared to those of 70-day-old rat brain.     

Post- and presynaptic lesions in the CA1 region of hippocampus: Effect on [3H]forskolin and [3H]phorboldibutyrate ester binding

Summary The effect of transient cerebral ischemia and intraventricular injection of kainic acid on adenylate cyclase and protein kinase C as labeled by [³H]forskolin ([³H]FOR) and [³H]phorboldibutyrate ester ([³H]PDBU) in several rat brain microregions was investigated in a quantitative autoradiographic study. Four days after transient four vessel occlusion a 80% loss of [³H]FOR and a 35% loss of [³H]PDBU binding could be measured in the CA1 stratum radiatum of operated Wistar rats as compared to control rats. Four days after intraventricular injection of kainic acid only a marginal loss of [³H]FOR and a 30% increase of [³H]PDBU binding was seen in the CA1 stratum radiatum while in the CA3 stratum lucidum and radiatum respectively a 30% loss of [³H]FOR and no significant change in [³H]PDBU binding was observed. As transient cerebral ischemia and intraventricular kainic acid injection are depleting the hippocampal CA1 region of CA1 pyramidal cells and axons of CA3 pyramidal cells respectively in rat brain, these findings strongly suggest that both adenylate cyclase and protein kinase C are localized in CA1 pyramidal cells of rat hippocampus.Part of this study has been presented at the 16th C.I.N.P. Congress, Munich, August 15–19, 1988.     

Neuronal localization of cannabinoid receptors and second messengers in mutant mouse cerebellum

Four lines of mutant mice were used to investigate (1) the neuronal localization of cannabinoid receptors in the cerebellar molecular layer and (2) the anatomical association of these receptors with elements of the two second messenger systems in the brain. Two of the mutant lines--Purkinje cell degeneration and nervous--are selectively deficient in Purkinje cells; the other two--weaver and reeler--are deficient in granule cells. In the heterozygous mice, [3H]CP 55,940 binding to cannabinoid receptors was discretely and densely localized to the molecular layer, as was [3H]forskolin binding to adenylate cyclase and [3H]phorbol 12,13-dibutyrate binding to protein kinase C, a component of the phosphoinositide cycle. [3H]CP 55,940 and [3H]forskolin binding was selectively reduced in weaver and reeler homozygous mice but unchanged in Purkinje cell deficient and nervous homozygotes. No decreases in [3H]phorbol 12,13-dibutyrate binding were found in any of the homozygous mutants relative to the heterozygous littermates. The results suggest that cannabinoid receptors and adenylate cyclase are localized to granule cell axons in the molecular layer, whereas protein kinase C is equally distributed in parallel fibers and Purkinje cell dendrites.     

Upregulation of Adenosine Al Receptors and Forskolin Binding Sites Following Chronic Treatment with Caffeine or Carbamazepine: A Quantitative Autoradiographic Study

The effects of feeding a diet enriched in caffeine or carbamazepine (CBZ) were investigated in rats in a quantitative autoradiographic study of adenosine A1 receptors (labeled by [3H]cyclohexyladenosine, [3H]CHA) and adenylate cyclase (labeled by [3H]forskolin). Although regional distribution of [3H]CHA and [3H]forskolin binding sites differed in some areas, chronic CBZ as well as chronic caffeine upregulated both of them. The changes in receptor densities occurred in the same brain microregions, suggesting that caffeine and CBZ act as antagonists at similar subpopulations of adenosine A1 receptors and [3H]forskolin binding sites. Therefore, a selective interaction of these two drugs with distinct adenosine A1 receptors (and adenylate cyclase) probably does not explain the differential effects of caffeine and CBZ on neuronal activity.     

Quantitative autoradiography of [H]forskolin binding sites in the rat brain

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.     

Dopamine receptor sensitivity following nigrostriatal lesion in the aged rat

This investigation sought to determine whether the ability to regulate dopamine receptor sensitivity following removal of dopaminergic innervation is altered during aging. Aged (24–26 months old) Fischer 344 rats compared with young (6 months old) rats had lower levels of dopamine and dopamine receptor binding ([³H]ADTN), but no change of dopamine-stimulated adenylate cyclase activity. Unilateral lesion of the nigrostriatal pathway produced equivalent dopaminergic denervation in rats of both age groups. The denervated striata of young rats had greatly enhanced dopaminergic sensitivity as evidenced by apomorphine induced rotational behavior and increased dopamine stimulated adenylate cyclase activity and [³H]ADTN binding. Old rats responded similarly with a very high degree of increased dopaminergic sensitivity in both the behavioral and biochemical parameters, demonstrating that the ability to regulate dopamine receptors remains basically intact. However, deficits of supersensitivity occured in apomorphine induced rotational behavior and [³H]ADTN binding and there was a large deficit in the guanine nucleotide sensitive subcomponent of [³H]ADTN binding. Supersensitivity of dopamine stimulated adenylate cyclase was not altered. The diminished ability to develop supersensitivity to [³H]ADTN binding could contribute to decreased [³H]ADTN binding in unlesioned rats.     

Brain forskolin binding in mice dependent on and tolerant to ethanol

Chronic ethanol ingestion by mice was previously shown to result in decreased activation of adenylate cyclase by guanine nucleotides and beta-adrenergic agonists, and in the loss of the high affinity beta-adrenergic agonist binding site in frontal cortex and hippocampus but not in cerebellum. These results indicate a regional specificity of ethanol's actions on beta-adrenergic receptors, the guanine nucleotide binding protein (Gs) and/or adenylate cyclase. To further detail the anatomical specificity of the effects of ethanol ingestion on receptor-coupled adenylate cyclase (AC) systems we have quantified the binding of [3H]forskolin to brain sections of control and ethanol-fed mice. High-affinity forskolin binding, thought to represent the complex of the alpha-subunit of Gs (as) and AC, was decreased in several brain areas including frontal cortex and hippocampus, but not in cerebellum, nucleus accumbens and certain other brain areas of ethanol-fed mice. Guanine nucleotides, such as Gpp(NH)p, generally enhanced forskolin binding in control animals. In ethanol-fed mice, however, Gpp(NH)p failed to enhance forskolin binding in most brain regions. These findings suggest that chronic ethanol ingestion may decrease the amount or function of as-AC in certain brain regions. Moreover, the regulation of the formation of this complex in different brain regions may affect responses to ethanol ingestion in mice.     

Effect of seizures kindled by subconvulsant doses of pentylenetetrazol on dopamine receptor binding and dopamine-sensitive adenylate cyclase in the rat

Possible alterations in dopamine receptor regulation were assessed in pentylenetetrazol (PTZ)-kindled rats by examination of dopamine (DA)-sensitive adenylate cyclase activity and the specific binding of [³H]spiroperidol in various brain regions. A reduction in [³H]spiroperidol-labeled high-affinity binding sites in the amygdaloid-pyriform cortex followed the kindling of seizures by chronic administration of PTZ. Similarly, a reduction of low-affinity binding sites was observed in the frontal cortex of PTZ-kindled rats. No changes in the apparent affinity of both high- and low-affinity binding sites were found after kindling except in the amygdala-pyriform cortex. The affinity of the low-affinity binding site was significantly reduced in this brain region. The lack of significant changes in the number and affinity of binding sites after acute seizures induced by electroconvulsive shock or a convulsant dose of PTZ suggests that the changes in receptor binding after PTZ kindling were not related to seizure sequelae. Basal and DA-sensitive adenylate cyclase activities remained unchanged after PTZ kindling. In conjunction with our previous observations of significant alterations in [³H]spiroperidol binding after amygdaloid kindling, the present findings suggest that altered DA receptor regulation may be a general mechanism involved in the development of increased seizure susceptibility.     

Inhibition of [3H]forskolin binding by Gpp[NH]p may reflect adenylate cyclase coupling to Gi.

The effect of the GTP analogue guanylyl-5'-imidodiphosphate (Gpp[NH]p) on [3H]forskolin binding was studied in rat brain using autoradiography. In the striatum the presence of 100 microM Gpp[NH]p produced a 40% increase in binding, whereas a decrease of about 30% was observed with low Gpp[NH]p concentrations (0.1 microM). In the molecular layer of the cerebellum all concentrations of Gpp[NH]p decreased [3H]forskolin binding. The decrease in binding disappeared in both striatum and the molecular layer of cerebellum in sections pretreated with 100 microM N-ethylmaleimide (NEM) for 10 min. NEM pretreatment did not significantly affect the stimulation of [3H]forskolin binding by micromolar concentrations of Gpp[NH]p in the striatum, but reversed the decrease observed in the molecular layer of the cerebellum, to an increase. Based on these data we suggest that the effects of Gpp[NH]p on [3H]forskolin binding may involve both Gs and Gi, where stimulation produces an increase and decrease in binding, respectively. The regional effects of Gpp[NH]p may reflect differences in the responsiveness of adenylate cyclase to Gs- and Gi-mediated effects.     

Distribution of protein kinase C in the brain of Apteronotus leptorhynchus as revealed by phorbol ester binding.

An antibody to the mammalian protein kinase C alpha (PKCalpha) subunit and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 80 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in gymnotiform brain. The 80-kDa band was enriched in fish forebrain and cerebellum compared with hypothalamus and brainstem areas. [3H]Phorbol 12,13-dibutyrate ([3H]PDBu) binding was used as a marker for the distribution of protein kinase C (PKC). [3H]PDBu binding was nearly completely displaced by excess cold PDBu; specific [3H]PDBu binding sites were heterogenously distributed with high densities in some gray matter regions and negligible densities in fiber tracts. A very high density of [3H]PDBu binding sites were found in the dorsal forebrain with far lower densities in most ventral forebrain nuclei. Low binding densities were observed in preoptic and hypothalamic areas with the exception of the nucleus diffusus and nucleus tuberis anterior. The thalamus and midbrain also had only low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was negligible. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal molecular layer, which contains cerebellar parallel fibers; the other layers of the ELL had far lower binding densities.     

Alpha 2-adrenoceptor-GTP binding regulatory protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats

The characterization of [3H]clonidine binding and effects of GTP, forskolin, islet-activating protein (IAP) and cholera toxin on adenylate cyclase activity were investigated in cerebral cortical membranes from 70-day-old and 2-year-old rats. Neither Kd nor Bmax values in [3H]clonidine binding were changed between day 70 and year 2. The activation of adenylate cyclase by forskolin was significantly higher in senescent than in adult animals. The inhibitory effect of adrenaline, which was completely abolished by the pretreatment with IAP/NAD on forskolin/GTP-stimulated cyclase activity, was low in senescent rats compared to that in adult ones. The stimulatory effect of cholera toxin/NAD was also low at the senescent stage compared to that at the adult stage. It is suggested that ligand binding affinity and the density in alpha 2-adrenoceptors do not change between day 70 and year 2 but that GTP binding and/or coupling activity of inhibitory as well as stimulatory GTP binding regulatory protein to catalytic units decrease in synaptic membranes of 2-year-old compared to those of 70-day-old rat brain.     

Activation of protein kinase C by phorbol dibutyrate modulates GABAA receptor binding in rat brain slices

Effects of protein kinase C (PKC) activation on the function of the GABA/benzodiazepine receptor-chloride complex were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam and [³⁵S]TBPS in rat brain slices. The density of [³H]muscimol binding was highest in cerebellar granular layers and high in both the frontal cortex and thalamus, but binding levels in the hippocampus were low. After activation of PKC by 100 nM phorbol-12,13-dibutyrate (PDBu), [³H]muscimol binding was decreased in the frontal cortex, striatum and thalamus, but binding levels were not changed in the hippocampus or cerebellum. The density of [³H]flunitrazepam binding was high in the cortex, hippocampus and molecular layers of cerebellum but was low in thalamus. PDBu increased the [³H]flunitrazepam binding only in the striatum and in part of the cortex and thalamus after activation of PKC. After activation of PKC by PDBu, [³⁵S]TBPS binding was increased in most areas, but binding levels were not changed in the brainstem or cerebellum. The receptor binding was markedly decreased in almost all areas by the addition of 2.5 mM Mg²⁺. Elevated [³⁵S]TBPS binding produced by PDBu was significantly inhibited by the addition of Mg²⁺. These results suggest that the activation of PKC potentiates benzodiazepine and TBPS binding, but decreases muscimol binding in a region-specific manner in the rat brain.