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OBJECTIVES Clinical high-frequency spinal cord stimulation (hfSCS) (>250 Hz) applied at subperception amplitudes reduces leg and low back pain. This study investigates, via labeling for c-fos-a marker of neural activation, whether 500 Hz hfSCS applied at amplitudes above and below the dorsal column (DC) compound action potential (CAP) threshold excites dorsal horn neurons. MATERIALS AND METHODS DC CAP thresholds in rats were determined by applying single biphasic pulses of SCS to T12 -T13 segments using pulse widths of 40 or 200 μsec via a ball electrode placed over the left DC and increasing amplitude until a short latency CAP was observed on the L5 DC and sciatic nerve. The result of this comparison allowed us to substitute sciatic nerve CAP for DC CAP. SCS at T12 -T13 was applied continuously for two hours using: sham or hfSCS at 500 Hz SCS, 40 μsec pulse width, and 50, 70, 90, or 140% CAP threshold. Spinal cord slices from T11 -L1 were immunolabeled for c-fos, and the number of c-fos-positive cells was quantified. RESULTS 500 Hz hfSCS applied at 90 and 140% CAP threshold produced substantial (≥6 c-fos + neurons on average per slice per segment) c-fos expression in more segments between T11 and L1 than did sham stimulation (p < 0.025, 90% CAP; p < 0.001, 140% CAP, Fisher's Exact Tests) and resulted in more c-fos-positive neurons on average per slice per segment ipsilateral to than contralateral to the SCS electrode at 70, 90, and 140% CAP threshold (p < 0.01, Wilcoxon Signed Rank Tests). CONCLUSIONS The finding of enhanced c-fos expression in the ipsilateral superficial dorsal horn provides evidence for activation/modulation of neuronal circuitry associated with subperception hfSCS.

Angina pectoris is cardiac pain that typically is manifested as referred pain to the chest and upper left arm. Atypical pain to describe localization of the perception, generally experienced more by women, is referred to the back, neck, and/or jaw. This article summarizes the neurophysiological and pharmacological mechanisms for referred cardiac pain. Spinal cardiac afferent fibers mediate typical anginal pain via pathways from the spinal cord to the thalamus and ultimately cerebral cortex. Spinal neurotransmission involves substance P, glutamate, and transient receptor potential vanilloid-1 (TRPV1) receptors; release of neurokinins such as nuclear factor kappa b (NF-kb) in the spinal cord can modulate neurotransmission. Vagal cardiac afferent fibers likely mediate atypical anginal pain and contribute to cardiac ischemia without accompanying pain via relays through the nucleus of the solitary tract and the C1-C2 spinal segments. The psychological state of an individual can modulate cardiac nociception via pathways involving the amygdala. Descending pathways originating from nucleus raphe magnus and the pons also can modulate cardiac nociception. Sensory input from other visceral organs can mimic cardiac pain due to convergence of this input with cardiac input onto spinothalamic tract neurons. Reduction of converging nociceptive input from the gallbladder and gastrointestinal tract can diminish cardiac pain. Much work remains to be performed to discern the interactions among complex neural pathways that ultimately produce or do not produce the sensations associated with cardiac pain.

Phenobarbital (PB) was administered to pregnant mice during days 9-21 of gestation. Forebrain and cerebellar [3H]flunitrazepam ([3H]FLU) binding was assayed in the offspring at birth and at 21 days of age. Prenatal treatment produced a decrease in the number (Bmax) of [3H]FLU receptors in both the forebrain and cerebellum at birth. A small decrease in the [3H]FLU dissociation constant (KD) values in the forebrain was also detected at birth, but no changes were seen in the [3H]FLU KD values in the cerebellum. No changes were observed in forebrain and cerebellar [3H]FLU Bmax or KD values at 21 days of age, indicating that the effects of prenatal exposure to PB on [3H]FLU binding are eliminated during the postnatal development of the forebrain and cerebellum. The receptor affinity for the triazolopyridazine CL 218,872, which distinguishes the type I and type II benzodiazepine (BDZ) receptors, was not altered by prenatal PB treatment. The coupling of the BDZ receptor to the gamma-aminobutyric acid and pentobarbital binding sites was unaffected by exposure to PB in utero.

The capsaicin receptor TRPV1 is a nonselective cation channel that is expressed in sensory neurons. In this study, we examined the role of the nonreceptor cellular tyrosine kinase c-Src kinase in the modulation of the rat TRPV1. Capsaicin-induced currents in identified colonic dorsal root ganglion neurons were blocked by the c-Src kinase inhibitor PP2 and enhanced by the tyrosine phosphatase inhibitor sodium orthovandate. PP2 also abolished currents in human embryonic kidney-293 cells transfected with rat TRPV1, whereas cotransfection of TRPV1 with v-Src resulted in fivefold increase in capsaicin-induced currents. In cells transfected with dominant-negative c-Src and TRPV1, capsaicin-induced currents were decreased by approximately fourfold. TRPV1 co-immunoprecipitated with Src kinase and was tyrosine phosphorylated. These studies demonstrate that TRPV1 is a potential target for cellular tyrosine kinase-dependent phosphorylation.

Kava has been used for centuries by Pacific Islanders for its tranquilizing and sedative effects. Recent clinical trials suggest that kava has therapeutic value for the treatment of anxiety. Demonstration of kava's anxiolytic effects in animals under controlled conditions would provide additional support for its clinical potential as an anxiolytic and would facilitate investigation of its mechanism(s) of action.This study systematically characterized the acute dosage-dependent anxiolytic and sedative effects of kava extract in well established quantitative murine behavioral assays and compared kava- and diazepam-induced behavioral changes.Various doses of an ethanolic extract of kava root or diazepam were administered intraperitoneally to BALB/cByJ inbred mice. Behavioral changes were measured in the mirrored chamber avoidance assay and elevated plus-maze assay. Reduced latency to enter and increased time spent in a normally avoided environment operationally defined anxiolysis. Sedation was defined by a significant decrease in locomotor activity in a circular arena.Kava extract produced statistically significant dose-dependent anxiolytic-like behavioral changes in both assays of anxiolysis. ED(50) values for kava-induced increases in time spent inside the mirrored chamber and on the open arms of the plus maze were 125 mg/kg and 88 mg/kg, respectively. Kava extract also caused a profound decrease in locomotor activity (ED(50) of 172 mg/kg). Flumazenil, a competitive benzodiazepine receptor antagonist, blocked both the anxiolytic and sedative effects of diazepam, but had no effect on kava's behavioral actions.Kava extracts produce significant murine anxiolytic-like behavioral changes and sedation that are not mediated through the benzodiazepine binding site on the GABA(A) receptor complex.

The ability of a selective 2-opiate receptor antagonist, naltriben, to modulate ethanol discrimination was investigated in a rat model using a drug discrimination procedure. Rats were trained to discriminate ethanol (1.25 g/kg, IP) from saline on a fixed-ratio schedule, FR10. Once rats had acquired the ethanol-saline discrimination, ethanol dose–response tests were conducted with 15-min pretest injections. Following the characterization of the ethanol dose-response curve, the effect of naltriben on ethanol's discriminative stimulus was assessed by administering naltriben (0.032–5.6 mg/kg, IP) 15 min before the ethanol administration. In the present study, naltriben did not have any modulatory effect on ethanol discrimination, suggesting that either Δ 2 -opiate receptors are not involved in the formation of ethanol's discriminative stimulus or the antagonism of Δ 2 -opiate receptors is not sufficient to alter ethanol's compound discriminative stimulus.

The role of genotype in susceptibility to the behavioral actions of benzodiazepines is not well characterized. To develop a model for such studies, we have characterized the anxiolytic and sedative activities of diazepam in C57BL/6J and A/J inbred mice. C57BL/6J mice were more responsive than A/J mice to diazepam-induced anxiolytic-like activity in the mirrored chamber aversion assay and the elevated plus-maze assay. Basal activity of the two strains did not differ in either assay. In contrast, the two strains were equally responsive to the anxiolytic effects of the 5-HT1Areceptor partial agonist, buspirone. C57BL/6J mice were also more susceptible to the sedative effects of diazepam than were A/J mice. Flumazenil blocked the effects of diazepam in these behavioral assays. Measurement of diazepam and nordiazepam in blood and brain suggested that the response differences are of a pharmacodynamic rather than a pharmacokinetic nature. Taken together, these findings indicate that C57BL/6J and A/J mice provide a valuable tool for behavioral genetic studies of the mechanisms underlying the pharmacological actions of benzodiazepines.

The present study employed neuropharmacological and receptor binding protocols to determine if diazepam-sensitive (DS) gamma-aminobutyric acid-A (GABA(A)) receptors in the nucleus tractus solitarius (NTS) participate in autonomic regulation of cardiovascular function. The first set of protocols was designed to determine if GABA(A) receptors in the NTS were functionally modulated by the benzodiazepine agonist, diazepam. Mean arterial pressure and heart rate responses to microinjection of GABAergic substances into the NTS were examined in urethane-anesthetized rats. Microinjection of the GABA(A) agonist isoguvacine into the NTS increased mean arterial pressure and heart rate, and these effects were blocked by the GABA(A) receptor antagonist, bicuculline. Preadministration of diazepam into the NTS potentiated the pressor actions of isoguvacine and had variable effects on heart rate changes. Flumazenil, a benzodiazepine antagonist, blocked the diazepam-induced potentiation of the pressor response to isoguvacine. The second protocol employed receptor autoradiography to examine the presence of DS and diazepam-insensitive (DI) GABA(A) receptors in the NTS. Autoradiography confirmed that DS GABA(A) receptors were present in the NTS; however, no measurable levels of DI GABA(A) receptors were detected. We conclude that GABA(A)-mediated integration of central autonomic control in the NTS is mediated solely by DS GABA(A) receptors.

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.

The ability of a selective 5-hydroxytryptamine (5-HT(3)) receptor antagonist, ICS 205-930 (3-tropanyl-indole-1-carboxylate, tropisetron), to block the discriminative stimulus effects of ethanol was investigated in rats that were trained to discriminate ethanol (1.25 g/kg ip) from saline with food as the reinforcement. Prior administration of ICS 205-930, at the dose of 0.01 mg/kg, significantly decreased ethanol's discriminative stimulus effect at ED(75) dose of ethanol, while higher doses of ICS 205-930 (10 and 17 mg/kg) showed enhancement of ethanol's discriminative effects at ED(0), ED(25), and ED(50) doses of ethanol. Under conditions where ICS 205-930 (10, 17 mg/kg) was tested alone, rats responded exclusively on the saline-appropriate lever. These effects occurred without significantly altering response rates or blood ethanol concentrations. The results suggest that the 5-HT(3) antagonist ICS 205-930 at lower concentration decreases, and at higher concentration enhances the discriminative stimulus effects associated with a lower to moderate dose of ethanol.

THIS study extends the pharmacological characterization of a novel quantitative murine behavioral method, the mirrored chamber aversion assay, which appears to be selectively sensitive to anxiolytic agents. Behavioral effects of acute diazepam administration were compared with those of the 5-HT 1A anxiolytic buspirone and those of ethanol in C57BL/6J mice. These known anxiolytics produced a dose-dependent reduction in avoidance behavior of large magnitude, as evidenced by statistically significant decreases in latency to enter and increases in time spent in the mirrored chamber. Anxiolytic-like effects of these compounds in the mirrored chamber assay differed from those observed by the elevated plusmaze method. The behavioral effects of the test compounds were not due to alteration of locomotor activity. These findings indicate that the murine mirrored chamber assay responds to several agents known to be anxiolytic in man but differs from the plusmaze in the pharmacological spectrum of the anxiolytics to which it is sensitive.

This paper reviews selected aspects of benzodiazepine binding site heterogeneity. These include receptor heterogeneity revealed by biochemical determinations of receptor numbers, autoradiographic localization in histological sections of brain, lesion studies, solubilization of receptors, and photoaffinity labelling. The data summarized support the concept of benzodiazepine receptor multiplicity. In addition, we have reviewed recent work on peripheral-type benzodiazepine binding sites and suggest that further study of these sites may increase our understanding of both the central and peripheral actions of benzodiazepines and other ligands.

The binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I-, followed by Br- less than Cl- less than F-. A similar rank order was observed for the concentration of ion required to produce half-maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I- than in Cl-. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.

Ca2+ transport activity in synaptosomal membranes has been identified as having two major components: Ca2+-stimulated ATP hydrolysis and ATP-dependent CA2+ uptake. Both processes exhibit similar affinities for Ca2+ and operate maximally under identical buffer conditions. Subcellular fractionation studies revealed the Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake activities to be highest in synaptic plasma membrane fractions 1 and 2, with lesser activity in synaptic vesicles and mitochondria. Progressive treatment with Triton X-100 activated, then decreased Ca2+/Mg2+ ATPase, Mg2+ ATPase and Ca2+ ATPase. ATP-dependent Ca2+ uptake was progressively decreased by similar treatment with Triton X-100. These studies illustrate that Ca2+ ATPase and ATP-dependent Ca2+ uptake may provide two important mechanisms for buffering of cytosolic Ca2+ at the nerve terminal. These systems may function to rapidly sequester cytosolic Ca2+ following a rise during depolarization and then extrude Ca2+ from the terminal against a concentration gradient. This regulation of cytosolic Ca2+, represented by two processes of the type seen in other plasma membranes, may play critical roles in calcium homeostasis in nerve cells.

A number of investigators have shown compelling evidence for multiplicity of benzodiazepine (BDZ) receptors. The present study addresses the query of BDZ receptor heterogeneity, in vitro, with respect to temperature. In competition studies involving rat cerebellar tissue, CL 218,872 produced Hill slopes near unity at both 0 degree C and 37 degrees C. In contrast, similar experiments utilizing cortical tissue from rats and mice produced Hill slopes of 0.69 and 0.66 at 0 degree C and 37 degrees C respectively. 3H-Flunitrazepam-photoaffinity labeling of cortical and cerebellar membranes was conducted at 0 degree C and 37 degrees C. SDS-PAGE fluorographic analyses of photolysed 3H-flunitrazepam (3H-Flu) revealed one intensely labeled 51K band in the cerebellum at both temperatures, which was specifically chased by diazepam. Similar experiments conducted in cortical tissue revealed photoaffinity labeling of at least three distinct macromolecules, one intense 51K and two less intense 55K and 59K bands. Labeling of each of these bands was chased specifically by diazepam. These data, taken together, indicate the existence of regional BDZ receptor heterogeneity under physiological conditions.

The postnatal development of benzodiazepine (BDZ) receptors was monitored in Heterogeneous Stock (HS) mice, and the BDZ receptors were characterized and categorized into Type I and Type II receptors. When the number of 3 H-Flu binding sites (B max ) was assessed at weekly intervals after the birth of the animal, the number of sites in both the cortex and cerebellum increased significantly if the data was expressed as fmol/mg tissue. On the other hand, no significant change in 3 H-Flu binding sites was evidenced in the cortex, and the number of 3 H-Flu binding sites in the cerebellum decreased during postnatal development if B max values were expressed as fmole/mg protein. When receptor binding data was analyzed for the presence of Type I and Type II BDZ receptors, the changes in K D values for 3 H-Flu binding during development could be accounted for by changes in relative proportions of Type I and Type II receptors present in the cortex and cerebellum during the maturation process. Type II receptors predominated in both cortex and cerebellum at birth, and Type I receptors proliferated primarily during the first two weeks of postnatal life. In the cortex of adult mice there were approximately equal numbers of Type I and Type II BDZ receptors. In the cerebellum of adult mice, computer assisted analysis of binding data could not distinguish the presence of two distinct BDZ binding sites. However, Hill coefficients and overall binding constants determined from data on CL-218,872 displacement of 3 H-Flu binding to cerebellar membranes indicated that cerebellar tissue from adult mice did contain a heterogeneous array of BDZ receptors.

Opiate agonists, morphine, levorphanol and β-endorphin increased calcium accumulation in rat peritoneal mast cells. This effect was dose dependent and β-endorphin was 10 times more potent than morphine. The stimulation was stereospecific and inhibited by naloxone. The site of the opiate action apears to be on the outer surface of the plasma membrane since lysis of the mast cell did not alter the the response to morphine. Tolerance to the opiate effect was not seen after chronic morphine administration. Morphine did not stimulate histamine release even at relatively high doses in vivo or high concentrations in vitro. It is reasoned that the enhancing effects on external calcium accumulation may reduce the critical cytosol calcium level for affecting histamine release.

1. Acute administration of ethanol (2.5 gm/kg, i.p.) to rats inhibits the cytosolic buffering of Ca2+ in nerve terminals. 2. Ca2+ ATPase and ATP-dependent Ca2+ uptake are both inhibited 30 min after a single dose of ethanol. 3. Chronic ethanol administration (6%, 14 days) did not inhibit Ca2+ ATPase but significantly stimulated ATP-dependent Ca2+ uptake. 4. Lubrol WX treatment of acute ethanolicmembranes reverses the inhibition of Ca2+ ATPase seen following ethanol. 5. Lubrol WX treatment of chronic ethanolic membranes prevents the increase in ATP-dependent Ca2+ uptake seen in ethanolic membranes. 6. Both acute and chronic ethanol-induced changes in Ca2+ transport within nerve teminals may involve lipid-dependent parameters of the membrane which may underlie neuronal adaptation.

1. Acute administration of ethanol (4 g/kg, i.p.) to mice inhibits the sequestration of calcium into endoplasmic reticulum-like organelles in synaptosomal membranes. 2. Ethanol administration inhibits both Ca2+-stimulated adenosine triphosphate hydrolysis and ATP-dependent calcium uptake in the vesicles at time of loss of righting reflex. 3. At recovery of righting reflex, the Ca2+-ATPase activity returns to normal levels, while the ATP-dependent uptake remains inhibited. 4. The effect of ethanol is specific for the sequestration (active transport) of calcium since calcium binding to synaptic membranes is not altered. 5. Alteration in mechanisms responsible for synaptosomal buffering of cytosolic Ca2+ levels by in vivo ethanol may contribute to altered transmitter release rates following ethanol adminstration.

The mechanisms by which ethanol produces its effects on the central nervous system are both diverse and complicated. An increasing amount of evidence, however, suggests that many of the primary and secondary effects of this chemical may be related to its interaction with the cell membrane (Hunt, 1975). During the initial phase of ethanol absorption, there is a depression of motor function, leading to overall depression of the central nervous system. Continued administration of ethanol leads to a tolerance of this sedative-hypnotic effect so that, over time, the animal or human no longer responds to the same concentration of ethanol with characteristic depression.

t-[35S]Butylbicyclophosphorothionate [( 35S]TBPS) has been shown to bind to the GABAA receptor complex. The binding is modulated allosterically by drugs that interact at components of the receptor complex. The present studies were designed to evaluate the influence of ionic environment and state of equilibrium on the allosteric modification of [35S]TBPS binding. In both I- and Cl- under nonequilibrium conditions, diazepam, gamma-aminobutyric acid (GABA), and pentobarbital (PB) stimulate and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibits [35S]TBPS binding. In addition, there is an inhibitory component to the effect of GABA and PB at higher drug concentrations. These effects are blocked by the appropriate antagonists for each drug. In Cl-, the stimulation of [35S]TBPS binding by drugs disappears at equilibrium, whereas the inhibition by GABA and PB persists. The inhibitory effect of DMCM in Cl- also disappears at equilibrium. When assayed in I- at equilibrium, however, DMCM stimulates [35S]TBPS binding. In addition, bicuculline, which is without effect under nonequilibrium conditions in either Cl- or I-, stimulates [35S]TBPS binding in I- at equilibrium. The persistent effects of DMCM, bicuculline, and GABA in I- are accompanied by alterations in the affinity of [35S]TBPS for its receptor. In addition, the stimulation of [35S]TBPS binding by GABA is associated with a decreased number of [35S]TBPS binding sites. These data demonstrate that receptor complex interactions with anions influence the responsiveness to drug binding.

Synaptosomal membranes accumulate 3–6 times more Ca2+ in the presence of ATP (50–1000 μM) than basal Ca2+ accumulation (-ATP). The location of this Ca2+ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca2+ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 μg/ml), sodium azide (100 μM) and dinitrophenol (100 μM) differentiate mitochondrial from nonmitochondrial Ca2+ accumulation under conditions that are [Ca2+]- and ATP-dependent. In the presence of low concentrations of ATP (200 μM), the Ca2+ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca free 2+ =2.5 μM. When Ca free 2+ is increased to 6.8 μM, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca2+ accumulating mechanisms in synaptosomal membranes depend on both [Ca2+] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca2+ uptake may be a viable mechanism for the regulation of synaptosomal Ca2+ levels.

A cDNA clone of an alpha subunit of the human GABA-A receptor has been isolated. The human clone (pCLL800) contains 1055 nucleotides in an open reading frame and 260 nucleotides in the 5′ non-coding region. The 351 amino acid sequence of this human alpha subunit shows 97% homology with its bovine counterpart. Hybridization of pCLL800 to Northern blots shows a 3.9/4.3 Kb RNA doublet in human cortex, rat whole brain, cortex, hippocampus, midbrain, olfacotry bulb and cerebellum. Developmental studies show that the levels of the rat alpha mRNA increase between one and three weeks of age in a manner similar to the development of the benzodiazepine binding sites.

The effect of the Type I benzodiazepine (BDZ) receptor agonist, CL 218,872, on convulsions generated by low doses of methyl β-carboline-3-carboxylic acid (MBCC), bicuculline, picrotoxin and pentylenetetrazole (PTZ) in mice was examined. Low doses of CL 218,872 enhanced the convulsions produced by all agents except PTZ. An anticonvulsant action of CL 218,872 was observed at higher doses. Since CL 218,872 exhibits proconvulsive effects at low doses, and a proconvulsant action is a characteristic of compounds classified as BDZ antagonists, it appears that CL 218,872 has some antagonist action.

Characteristics of [3H]flunitrazepam ([3H]FLU) binding to cortical and cerebellar membrane receptors were examined following chronic (six days) administration of phenobarbital (PB) to C57B1 mice. Following PB treatment, the number of [3H]FLU binding sites (Bmax) was significantly reduced in both cerebral cortex and in cerebellum. No change in the affinity (KD) of these binding sites was observed. Using 3-methyl-6-[3-(trifluormethyl)phenyl]-1,2,4,-triazolo [4,3-β]pyridazine (CL-218,872), further analysis revealed a significant decrease in the number of high-affinity CL-218,872 binding sites in cerebellar tissue. In the forebrain areas, however, a significant decrease in the number of low-affinity binding sites was found. Finally, the enhancement of [3H]FLU binding, produced by in vitro addition of pentobarbital, was significantly less pronounced in the cerebellum of PB-treated animals.