The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

Metrifonate and dichlorvos: Effects of a single oral administration on cholinesterase activity in rat brain and blood

Cholinesterase activities in rat forebrain, erythrocytes, and plasma were assessed after a single oral administration of metrifonate or dichlorvos. In 3-month-old rats, the dichlorvos (10 mg/kg p.o.)-induced inhibition of cholinesterase reached its peak in brain after 15–45 min and after 10–30 min in erythrocytes and plasma. Cholinesterase activity recovered rapidly after the peak of inhibition, but did not reach control values in brain and erythrocytes within 24 h after drug administration. The recovery of plasma cholinesterase activity, in contrast, was already complete 12 h after dichlorvos treatment. Metrifonate (100 mg/kg p.o.) had qualitatively similar inhibition kinetics as dichlorvos, albeit with a slightly delayed onset. Peak values were attained 45–60 min (brain) and 20–45 min (blood), after drug administration. Apparently complete recovery of cholinesterase activity was noted in both tissues 24 h after treatment. The dose-dependence of drug-induced inhibition of cholinesterase in rat blood and brain was determined at the time of maximal inhibition, i.e., 30 min after dichlorvos treatment and 45 min after metrifonate treatment. The oral ED₅₀ values obtained for dichlorvos were 8 mg/kg for brain and 6 mg/kg for both erythrocyte and plasma cholinesterase. The corresponding oral ED₅₀ values for metrifonate were 10 to 15 times higher, i.e., 90 mg/kg in brain and 80 mg/kg in erythrocytes and plasma. In rats deprived of food for 18 h before drug treatment, the corresponding ED₅₀ values for metrifonate were 60 and 45 mg/kg, respectively, indicating an about two-fold higher sensitivity of fasted rats to metrifonate-induced cholinesterase inhibition compared to non-fasted rats. Compared to 3-month-old rats, 19-month-old rats showed a higher sensitivity towards metrifonate and dichlorvos. At the time of maximal inhibition, there was a strong correlation between the degree of cholinesterase inhibition in brain and blood. These results demonstrate that single oral administration of metrifonate and dichlorvos induces an inhibition of blood and brain cholinesterase in the conscious rat in a dose-dependent and apparently fully reversible manner. While the efficiency of a given dose of inhibitor may vary with the satiety status or age of the animal, the extent of brain ChE inhibition can be estimated from the level of blood ChE activity.     

A proenkephalin A-derived peptide analogous to bovine adrenal peptide E from frog brain: Purification, synthesis, and behavioral effects

A peptide derived from the posttranslational processing of proenkephalin A was isolated from an extract of the brain of the European green frog Rana ridibunda and its primary structure established as: Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg¹⁰-Pro-Glu-Trp-Trp-Gln-Asp-Tyr-Gln-Lys-Arg²⁰-Tyr-Gly-Gly-Phe-Met. The structure was confirmed by chemical synthesis. The peptide represents an amphibian equivalent of bovine adrenal peptide E [preproenkephalin A (206–230)-peptide] but the sequence contains two amino acid substitutions (Met¹⁵ → Gln and Leu²⁵ → Met) compared with the mammalian peptide. The data support previous hypotheses that the Leu-enkephalin sequence is not present in preproenkephalin A of amphibians. Intracerebroventricular injections of frog peptide E (10 and 100 ng) in mice had no significant effect on horizontal locomotor activity. The peptide, in doses up to 1 μg, had no effect on latency of escape jumping in the hot plate test and the peptide (100 ng) did not modify responses (paw licking, rearing, and escape jumping) in morphine-treated mice.     

Membrane receptors for estrogen,progesterone, and testosterone in the rat brain: Fantasy or reality

Summary 1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes.2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17-E-6-[¹²⁵I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimatedK _d of about 3±0.7 nM (X ± SE;n=3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with aK _d of 34±7 nM and, aB _max of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (K _d=1–2 nM) and one low affinity (K _d=62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[¹²⁵I]BSA indicate that the estimatedK _d of 30±2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement withK _d values computed from Scatchard-derived data using the LIGAND algorithm.3. 17-E-6-[¹²⁵I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[¹²⁵I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22.4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase.5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the reality of membrane steroid receptors.     

Anatomical distribution of NADPH-cytochrome P450 reductase and cytochrome P4502D forms in rat brain: Effects of xenobiotics and sex steroids

Since the brain is not a homogeneous organ, but one dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monoxygenase system in brain, as well as the regulation of its expression, is important in elucidating its function in that organ. In order to study these issues, female rats-both ovariectomized and not-were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and P450 reductase determined using polymerase chain reaction. Results of this investigation indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR was utilized to demonstrate the effects of xenobiotics (phenobarbital, β-naphthoflavone, imipramine) and sex hormones (testosterone, estrogen) on the levels of these messages in the female rat brain. Significant induction of message for P4502D forms was noted with testosterone in the absence of estrogen. The level of mRNA for reductase was not significantly influenced by any of the treatments, however. These results raise the issue of a sexual dimorphism in the rat regarding P4502D expression in brain.     

Neurological disorder and excessive accumulation of calcium in brain of clinically vitamin A-deficient rats

Three groups of rats were fed two types of synthetic diets for 52 d. The—A group was allowed free access to a vitamin A-deficient diet and showed classical signs of vitamin A deficiency. The brain was the only organ in our experiment where no significant weight difference was present among the three groups. In the brain, calcium concentration was significantly higher in the—A group when compared with the PF (Pair-fed; allowed restricted amount of control diet) and +A groups (allowed free access to control diet). In the tibia, calcium and magnesium concentrations were significantly lower in the—A group when compared with other two groups. Excessive accumulation of calcium in brain and apparently similar unbalance in bone, mineral concentration were observed in central nervous system (CNS) degenerative diseases. Our results suggest that abnormal metabolism of calcium and magnesium in some tissues and excessive accumulation of calcium in brain may be responsible for the development of neurological disorders in vitamin A-deficient rats.     

A direct comparison of the masculinizing effects of testosterone,androstenedione, estrogen,and progesterone on the development of the zebra finch song system

To assess which hormones are capable of masculinizing the neural song system of zebra finch hatchlings, we implanted female hatchlings with estrogen (estradiol [E2], 75 μg, n = 9), testosterone (T, 75–88 μg, n = 13), androstenedione (AE, 75 μg, n = 7), progesterone (P, 117 μg, n = 10), or nothing (Blanks, n = 10) and compared these to unimplanted males (n = 7). Implants, consisting of a hormone and Silastic mixture encased in polyethylene tubing, were placed under the skin of the breast on the day of hatching. Birds were killed when they were subadult (58 to 68 days old). We measured volumes of area X, the higher vocal center (HVC), and the robust nucleus of the archistriatum (RA); measured soma sizes in the lateral magnocellular nucleus of the neostriatum (IMAN), HVC, and RA: and counted RA neurons. E2 masculinized all measures in the song system and nearly sex-reversed the size of RA neurons. T masculinized volumes of nuclei and soma sizes but not the number or spacing of RA neurons. E2 was always at least as effective as T in masculinizing measures of the song system and was usually more effective. AE and P did not significantly masculinize any measure. These data suggest that E2 is more potent than aromatizable androgens or P in masculinizing the female song system in development and that the action of E2 alone may be sufficient to masculinize the volume of song control nuclei and the size and number of neurons. © 1995 John Wiley & Sons, Inc.     

猪脑组织水解液及其益智食品的研制

本文初步探讨了多脂质物质（脑组织）水解条件的优化过程及其益智产品的研制。经过多项对比实验得出了以双酶分段水解以及水化法除脂质提取脑组织水解液的初步工艺,并通过微震化工艺达到了掩蔽不良气味,稳定有效成分的效果。