Benzodiazepine Receptor Binding in Cerebellar Cortex: Observations in Olivopontocerebellar Atrophy

Benzodiazepine receptor binding was measured in cerebellar cortex of 15 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The majority of these patients had a moderate to marked Purkinje cell loss, as judged by the lowered levels of dentate nucleus gamma-aminobutyric acid (GABA), a marker of Purkinje cells. Despite the reduction in Purkinje cell number cerebellar cortical benzodiazepine receptor density was either normal or slightly elevated in the OPCA patients. These results are in contrast to the findings in a mutant strain of mice deficient in Purkinje cells in which the concentration of benzodiazepine receptors in cerebellum is greatly reduced. Our data indicate that in the human, cerebellar cortical benzodiazepine receptors are either not significantly associated with Purkinje cells or that in OPCA Purkinje cell loss triggers a de novo synthesis of extra benzodiazepine binding sites. It is concluded that, in contrast with the rodent, in the human benzodiazepine receptor binding may not serve as a marker for cerebellar Purkinje cells.     

Neuronal [3H]Benzodiazepine Binding and Levels of GABA, Glutamate, and Taurine Are Normal in Huntington''s Disease Cerebellum

Alterations in one subunit of the proposed GABA receptor complex, namely, the GABA receptor, have been observed in Huntington's disease cerebellum. We measured binding to a second subunit, the benzodiazepine binding site, in the autopsied cerebellum of 12 patients dying with adult-onset Huntington's disease. Neuronal benzodiazepine ([3H]flunitrazepam) binding density (Bmax) and affinity in cerebellar cortex of the Huntington's disease patients were not significantly different from control values. Similarly, maximal GABA stimulation of benzodiazepine binding was normal in the Huntington's disease cerebellum. In addition, no significant changes were observed in the concentrations of GABA, glutamate, and taurine in cerebellar cortex, nor of GABA in the dentate nucleus.     

Differential supersensitivity of beta-receptor subtypes in rat cortex and cerebellum after central noradrenergic denervation

Inhibition of ³H-dihydroalprenolol binding to rat cortex and cerebellum β-receptors by the selective β₁-antagonist practolol, and the selective β₂-agonist salbutamol, was shallow and biphasic, with log-logit slopes less than 1.0. The relative affinities of these inhibitors suggested that the predominant β-adrenergic receptor population in cortex and cerebellum was β₁ and β₂ respectively. specific lesion of the ascending dorsal norepinephrine bundle, in addition to increasing β-receptor number in the cortex, significantly increased the affinity of practolol, but did not change the affinity of salbutamol, at cortex β receptor sites. Similar lesions decreased cerebellar β-receptor binding and reduced the affinity of salbutamol but not of practolol for those same sites. Iterative computer analysis of the inhibition data showed mixed populations of β₁- and β₂- receptors in both cortex and cerebellum. Dorsal NE bundle lesion doubled the number of cortical β₁-receptors, but did not alter the number of β₂-receptors. In contrast, these lesions induced a selective decrease in cerebellar β₂-receptors. It is concluded that the relevant neuronal β-receptors which are postsynaptic to central NE nerve terminals are β₁ in the cerebral cortex and β₂ in the cerebellum.     

GABA receptor binding in the parkinsonian brain

The binding of GABA to postsynaptic receptors was studied in the cerebral and cerebellar cortex, caudate nucleus, putamen, pallidum and substantia nigra from autopsy brains of 12 parkinsonian patients and 11 controls. GABA receptor binding in the substantia nigra was significantly decreased in the parkinsonian brain. In the other brain regions, however, GABA binding was unchanged. There was no correlation between GABA binding and sex, age, duration or severity of the disease. The results suggest the involvement of nigral GABA receptor in Parkinson's disease.     

COMPARISON OF THE BINDING’ OF β-ALANINE AND y-AMINOBUTYRIC ACID IN SYNAPTOSOMAL-MITOCHONDRIAL FRACTIONS OF RAT BRAIN1

Abstract— Na⁺-dependent ‘binding’ of β-alanine and GABA was examined with synaptosomal-mitochondrial fractions of rat brain incubated for 10 min at 0°C. GABA was bound to a much greater extent than β-alanine to particles of cerebral cortex, whole cerebellum and brain stem. For cerebral cortex, the binding capacity (B_max) for GABA was about 18 limes greater than that for β-alanine. and the affinity of the particles for GABA was about 2′ times greater than for β-alanine. The order of potency of GABA binding to brain regions was cerebral cortex > cerebellum > brain stem, whereas that for β-alanine was the reverse. If the binding of β-alanine is taken to indicate the glial component of the Na⁺-dependent binding process for GABA, then most of the GABA was bound to neuronal elements under the conditions employed.     

Loss of rat ventricular alpha 1-adrenergic receptors during aging

Ventricular alpha 1-adrenergic receptor concentration, measured by specific binding of [3H]-prazosin, decreases by 33% as rats age from 3 to 24 months. No age changes occur in binding affinity for [3H]-prazosin or potency of various alpha-adrenergic agonists and antagonists to displace [3H]-prazosin. The ratio of membrane protein to ventricular wet weight also does not change significantly with age. These results suggest a possible mechanism for loss of cardiovascular alpha-adrenergic responsiveness during aging.     

Contents of several amino acids in the cerebellum, brain stem and cerebrum of the 'staggerer', 'weaver' and 'nervous' neurologically mutant mice.

The content of glutamate, GABA, aspartate, glycine and alanine was determined in the cerebellum, brain stem and cerebrum of three different mutant mice which have been named ‘staggerer’, ‘weaver’ and ‘nervous’ on the basis of neurological symptoms. In the ‘staggerer’ and ‘weaver’ mutants there is an almost complete absence of granule cells in the cerebellar cortex while in the ‘nervous’ mutant there is a loss of Purkinje cells (and to a lesser extent a loss of granule cells) in the cerebellar cortex. In the cerebellum of the ‘weaver’ mutant, the content of glutamate was signficantly lower (P < 0.025) than control values (8.77 ± 0.76 vs 12.0 ± 1.3 μmol/g tissue wet wt) and the contents of GABA and glycine were significantly greater than normal levels. In the cerebellum of the ‘staggerer’ mutant, the content of glutamate was significantly lower (6.62 ± 0.70 μmol/g) and the contents of glycine and alanine significantly higher than control values. In the cerebrum and brain stem regions of the staggerer mutant, weaver mutant and the normals the contents of the five amino acids were the same. The contents of glycine and alanine in the cerebellum, GARA and glycine in the brain stem and GABA and alanine in the cerebrum of the nervous mutants were higher than control values. The data are discussed in terms of a possible role for glutamate functioning as an excitatory transmitter when released from the cerebellar granule cells.     

Modulation of glutamate release from parallel fibers by mGlu4 and pre-synaptic GABA(A) receptors

The regulation of pre-synaptic glutamate release is important in the maintenance and fidelity of excitatory transmission in the nervous system. In this study, we report a novel interaction between a ligand-gated ion channel and a G-protein coupled receptor which regulates glutamate release from parallel fiber axon terminals. Immunocytochemical analysis revealed that GABA(A) receptors and the high affinity group III metabotropic glutamate receptor subtype 4 (mGlu4) are co-localized on glutamatergic parallel fiber axon terminals in the cerebellum. GABA(A) and mGlu4 receptors were also found to co-immunoprecipitate from cerebellar membranes. Independently, these two receptors have opposing roles on glutamate release: pre-synaptic GABA(A) receptors promote, while mGlu4 receptors inhibit, glutamate release. However, coincident activation of GABA(A) receptors with muscimol and mGlu4 with the agonist (2S)-S-2-amino-4-phosphonobutanoic acid , increased glutamate release from [(3) H]glutamate-loaded cerebellar synaptosomes above that observed with muscimol alone. Further support for an interaction between GABA(A) and mGlu4 receptors was obtained in the mGlu4 knockout mouse which displayed reduced binding of the GABA(A) ligand [(35) S]tert-butylbicyclophosphorothionate, and decreased expression of the α1, α6, β2 GABA(A) receptor subunits in the cerebellum. Taken together, our data suggest a new role for mGlu4 whereby simultaneous activation with GABA(A) receptors acts to amplify glutamate release at parallel fiber-Purkinje cell synapses.     

Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells

GABA(A) receptors are chloride channels composed of five subunits. Cerebellar granule cells express abundantly six subunits belonging to four subunit classes. These are assembled into a number of distinct receptors, but the regulation of their relative proportions is yet unknown. Here, we studied the composition of cerebellar GABA(A) receptors after targeted disruption of the delta subunit gene. In membranes and extracts of delta-/- cerebellum, [(3)H]muscimol binding was not significantly changed, whereas [(3)H]Ro15-4513 binding was increased by 52% due to an increase in diazepam-insensitive binding. Immunocytochemical and Western blot analysis revealed no change in alpha(6) subunits but an increased expression of gamma(2) subunits in delta-/- cerebellum. Immunoaffinity chromatography of cerebellar extracts indicated there was an increased coassembly of alpha(6) and gamma(2) subunits and that 24% of all receptors in delta-/- cerebellum did not contain a gamma subunit. Because 97% of delta subunits are coassembled with alpha(6) subunits in the cerebellum of wild-type mice, these results indicated that, in delta-/- mice, alpha(6)betagamma(2) and alphabeta receptors replaced delta subunit-containing receptors. The availability of the delta subunit, thus, influences the level of expression or the extent of assembly of the gamma(2) subunit, although these two subunits do not occur in the same receptor.     

GABAA alpha6-containing receptors are selectively compromised in cerebellar granule cells of the ataxic mouse, stargazer

Stargazer mice fail to express the gamma2 isoform of transmembrane alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptor regulatory proteins that has been shown to be absolutely required for the trafficking and synaptic targeting of excitatory AMPA receptors in adult murine cerebellar granule cells. Here we show that 30 +/- 6% fewer inhibitory gamma-aminobutyric acid, type A (GABA(A)), receptors were expressed in adult stargazer cerebellum compared with controls because of a specific loss of GABA(A) receptor expression in the cerebellar granule cell layer. Radioligand binding assays allied to in situ immunogold-EM analysis and furosemide-sensitive tonic current estimates revealed that expression of the extrasynaptic (alpha6betaxdelta) alpha6-containing GABA(A) receptor were markedly and selectively reduced in stargazer. These observations were compatible with a marked reduction in expression of GABA(A) receptor alpha6, delta (mature cerebellar granule cell-specific proteins), and beta3 subunit expression in stargazer. The subunit composition of the residual alpha6-containing GABA(A) receptors was unaffected by the stargazer mutation. However, we did find evidence of an approximately 4-fold up-regulation of alpha1betadelta receptors that may compensate for the loss of alpha6-containing GABA(A) receptors. PCR analysis identified a dramatic reduction in the steady-state level of alpha6 mRNA, compatible with alpha6 being the primary target of the stargazer mutation-mediated GABA(A) receptor abnormalities. We propose that some aspects of assembly, trafficking, targeting, and/or expression of extrasynaptic alpha6-containing GABA(A) receptors in cerebellar granule cells are selectively regulated by AMPA receptor-mediated signaling.     

β-Endorphin: Characteristics of binding sites in the rabbit cerebellar and brain membranes

Specific binding of human β-endorphin to rabbit cerebellar and brain membranes was measured using $\text{[}{}^3\text{H}{}_2\text{-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the primary ligand. In both tissues binding was time dependent and saturable, with apparent equilibrium dissociation constants of 0.275 nM and 0.449 nM in the cerebellum and brain, respectively. The binding capacity of cerebellum is greater than that of brain. Kinetic studies showed that the association rate constants were 2.7 × 10⁷ M^?1min^?1 for cerebellum and 2.4 × 10⁷ M^?1min^?1 for brain. Dissociation of tritiated β_h-endorphin from both cerebellum and brain is not consistent with a first order decay from a single site. In the cerebellum, these is a time-dependent increase in slowly dissociating complex. The potency of several opioid peptides and opiates to inhibit the binding of tritiated β_h-endorphin was determined. Ligands with preference for μ, δ, and κ opiate receptor (morphine, Metenkephalin and ethylketocyclazocine) all have similar affinities toward β_h-endorphin sites in both brain and cerebellar membranes.     

Electroconvulsive Shock and Reserpine: Effects on β-Adrenergic Receptors in Rat Brain

Abstract Electroconvulsive shock (ECS) administered once daily for up to 14 days decreases β-adrenergic receptor binding in the cortex and hippocampus in a time-dependent manner. The decrease in binding in the cortex lasts at least 1 week after the last shock. In the striatum, hypothalamus, or cerebellum, 14 days of ECS did not produce significant changes in β-adrenergic receptor binding. The brain regional pattern of β-adrenergic receptor changes suggests that repeated ECS affects β-adrenergic receptors in brain regions that receive a noradrenergic innervation activated by ECS. The effects of ECS on neurotransmitter receptor binding appear to be highly selective. Of five receptors in the cortex and three receptors in the hippocampus measured, only β-adrenergic receptor binding is decreased. Chronic footshock stress does not alter β-adrenergic receptor binding sites in the cortex, indicating that the effects of ECS are not due to stress alone. The effects of ECS on reserpine-induced alterations in β-adrenergic receptor binding sites were also examined. Ten days of ECS following chronic reserpine injections reverses the increased binding of β-adrenergic receptors     

Synaptic Chemistry Associated with Aberrant Neuronal Development in the Reeler Mouse

Abstract: Pre- and postsynaptic neurochemical markers for several afferent and intrinsic neuronal systems were measured in the mouse mutant, reeler. In the neocortex of the reeler, the relative positions of the polymorphic and pyramidal cells were inverted but this was not associated with alterations in the content/mg protein of synaptic markers for noradrenergic [tyrosine hydroxylase (TH), norepinephrine (NE), NE uptake], cholinergic [choline acetyltransferase (ChAT), quinuclidinyl benzilate (QNB) binding], γ-aminobutyric acid (GABA)ergic (glutamate decarboxylase, GABA uptake, GABA receptors, GABA) or glutamatergic (glutamate uptake, receptors, glutamate) neurons. The laminar distributions of the hippocampal neurons were disrupted and associated with mild hypoplasia; consistent with this alteration, the content/mg protein of some GABAergic (GABA uptake) and glutamatergic (glutamate receptors) markers were slightly increased. The reeler cerebellum was characterized not only by misalignment of neurons but also by a marked loss of granule cells. Commensurate with the degree of cerebellar hypoplasia, the total amount of glutamate content, [³H]l-glutamate uptake activity, [³H]muscimol, and [³H]QNB ligand binding were reduced in the reeler cerebellum. In contrast, presynaptic markers for the noradrenergic (TH, NE) climbing fibers and the cholinergic (ChAT) mossy fibers were significantly increased/mg protein but their total content/cerebellum was near normal. Our data support suggestions that cerebellar granule cells use glutamate as their neurotransmitter and contain GABA and cholinergic receptors. The findings also suggest that misplaced cortical and cerebellar neurons retain normal neurochemical characteristics and that the morphologic alterations do not markedly affect the quantitative development of aminergic afferent systems.     

Effects of x-irradiation on the levels of glutamate,aspartate and GABA in different regions of the cerebellum of the rat

The effect of the x-irradiation-induced loss of cerebellar granule and stellate cells on the levels of glutamate, aspartate and GABA in regions of the rat cerebellum was determined. The level of glutamate was significantly lower in the neuron-depleted cerebellar cortex while GABA levels were higher than control values in the cerebellar cortex and white matter of the x-irradiated rats. Aspartate levels were not changed by x-irradiation in any cerebellar region. The data is discussed in terms of the proposed role of glutamate as the excitatory neurotransmitter released from granule cells.     

Cerebellar GABA(A) receptor alterations in hypoxic neonatal rats: Role of glucose, oxygen and epinephrine supplementation

Hypoxia in neonates causes dysfunction of excitatory and inhibitory neurotransmission resulting in permanent brain damage. The present study is to understand the cerebellar GABA(A) receptor alterations and neuroprotective effect of glucose supplementation prior to current sequence of resuscitation - oxygen and epinephrine supplementation in hypoxic neonatal rats. Hypoxic insult caused a significant decrease in GABA(A) receptor number along with down regulated expression of GABA(Aα1,) GABA(Aα5), GABA(Aδ) and GABA(Aγ3) receptor subunits in the cerebellum which accounts for the respiratory inhibition. Hypoxic rats supplemented with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GABA(A) receptor subunits expression to near control. Glucose can reduce ATP-depletion-induced alterations in GABA receptors, thereby assisting in overcoming the neuronal damage caused by hypoxia. Resuscitation with oxygen alone and epinephrine was less effective in reversing the receptor alterations. The reduction in the GABA(A) receptors functional regulation during hypoxia plays an important role in cerebellar damage. Resuscitation with glucose alone and glucose with oxygenation to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

Inhibition of GABA system involved in cyclosporine-induced convulsions.

In this study, we attempted to clarify the mechanisms mediating cyclosporine-evoked convulsions. Cyclosporine (50 mg/kg, i.p.) significantly enhanced the intensity of convulsions induced by bicuculline (GABA receptor antagonist), but not those induced by strychnine (glycine receptor antagonist), N-methyl-D-aspartic acid, quisqualic acid or kainic acid (glutamate receptor agonists). Bicuculline plus cyclosporine-induced convulsions were significantly suppressed by an activation of GABAergic transmission with diazepam, phenobarbital and valproate. The GABA turnover estimated by measuring aminooxyacetic acid-induced GABA accumulation in the mouse brain was significantly inhibited by cyclosporine (50 mg/kg, i.p.). When cultured rat cerebellar granule cells were exposed to 1 microM cyclosporine for 24 hr, the specific [3H]muscimol (10 nM) binding to intact granule cells decreased to 53% of vehicle controls. The present study provides the first evidence suggesting that cyclosporine inhibits GABAergic neural activity and binding properties of the GABAA receptor. These events are closely related to the occurrence of adverse central effects including tremors, convulsions, coma and encephalopathy under cyclosporine therapy.     

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA_A-receptor ligand [³H]TBOB to rat forebrain and cerebellar membrane preparations with IC₅₀ (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [³H]flunitrazepam and [³H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [³H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [³H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC₅₀ values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA_A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.     

Changes in β-Adrenergic Receptor Subtypes in Alzheimer-Type Dementia

Using ligand binding techniques, we studied beta-adrenergic receptor subtypes in brains obtained at autopsy from seven histologically normal controls and seven histopathologically verified cases with Alzheimer-type dementia (ATD). Inhibition of [3H]dihydroalprenolol [( 3H]DHA) binding by the selective beta 1 antagonist, metoprolol, results in nonlinear Hofstee plots, suggesting the presence of the two receptor subtypes in the human brain. The calculated ratios of beta 1/beta 2-adrenergic receptors in control brains are as follows: frontal cortex, 49:51; temporal cortex, 31:69; hippocampus, 66:34; thalamus, 23:77; putamen, 70:30; caudate, 48:52; nucleus basalis of Meynert (NbM), 43:57; cerebellar hemisphere, 25:75. Compared with the controls, total concentrations of beta-adrenergic receptors were significantly reduced only in the thalamus of the ATD brains. beta 1-Adrenergic receptor concentrations were significantly reduced in the hippocampus and increased in the NbM and cerebellar hemisphere, whereas beta 2-adrenergic receptor concentrations were significantly reduced in the thalamus, NbM, and cerebellar hemisphere and increased in the hippocampus and putamen of the ATD brains. These results suggest that beta 1- and beta 2-adrenergic receptors are present in the human brain and that there are significant changes in both receptor subtypes in selected brain regions in patients with ATD.     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.