A splice variant of E2-2 basic helix-loop-helix protein represses the brain-specific fibroblast growth factor 1 promoter through the binding to an imperfect E-box.

We previously demonstrated that a cis-element (-489 to -467) in the brain-specific fibroblast growth factor (FGF)-1 promoter (FGF-1.B) binds multiple nuclear factors, and this binding enhances transcriptional activity of this promoter. Here we report the isolation of three cDNA clones, VL1, VL2 and VL3, from a human brain stem cDNA expression library using four tandem repeats of the 26-base pair sequence (-492 to -467) as the probe. These cDNA clones represent the variant of bHLH protein E2-2/SEF2-1 in having 12 additional nucleotides encoding the amino acids RSRS. The glutathione S-transferase (GST) fusion proteins of VLl, VL2, and VL3 immunologically react with anti-E2-2 antibody and anti-GST-VL2 antibody. Electrophoretic mobility shift assay and methylation interference assay revealed that the GST fusion proteins specifically bind to an imperfect E-box sequence (GACCTG) present in the 26-base pair sequence. Transient expression of the full-length E2-2 without RSRS in U1240MG glioblastoma cells resulted in repression of FGF-1.B promoter activity. We further showed a significant repression of promoter activity (>40 fold) by E2-2 (lacking the amino acid sequence RSRS) when the E47 reporter construct, containing a hexameric E-box site, was used. In contrast, the E2-2 variant containing the RSRS sequence has no significant effect on either the FGF-1 promoter or E47 promoter. These results suggest that the relative abundance of the two splice variants of E2-2 in brain could be an important determinant for the expression of FGF-1.

Uncoupling of GABAA/benzodiazepine receptor alpha 1, beta 2, and gamma 2 subunit mRNA expression in cerebellar Purkinje cells of staggerer mutant mice.

The mammalian GABAA/benzodiazepine (GABAA/BZ) receptor is comprised of several subunit isoforms: alpha 1-6, beta 1-13, gamma 1-3 and delta. In the present studies, the expression of alpha 1, beta 2, and gamma 2 subunit mRNAs was examined in cerebellar Purkinje cells and deep cerebellar neurons of staggerer mutant mice during postnatal development. In control animals, the three subunit mRNAs were present at high density in Purkinje cells which, in adult animals, form a monolayer at the interface of the granule cell and molecular layers. The number of Purkinje cells in the staggerer cerebellar cortex is reduced; the majority of those that remain are retained within the granule cell layer and are unable to receive normal afferent synapses from granule cells. The three subunit mRNAs were ex pressed at similar levels in both staggerer and control Purkinje cells until postnatal day 9. After this time, although the alpha 1 subunit mRNA was maintained at control levels in staggerer Purkinje cells, the expression of beta 2 and gamma 2 subunit mRNAs decreased, and was largely absent by postnatal day 20. The loss of beta 2 and gamma 2 mRNA expression in staggerer was specific to Purkinje cells, since all three mRNAs were present throughout postnatal development in other brain regions, including the deep cerebellar nuclei. The present studies indicate that in cerebellar Purkinje cells, the GABAA/BZ receptor alpha 1, and beta 2, and gamma 2, subunit mRNAs are regulated by distinct mechanisms which are differentially affected by the staggerer mutation.

Developmental changes in the expression of gamma-aminobutyric acidA/benzodiazepine receptor subunit mRNAs in the murine inferior olivary complex.

The pharmacological and physiological properties of ligand-gated ion channels are dependent on their subunit composition; spontaneously occurring changes in subunit composition during neuronal development may result in dramatic functional differences between embryonic and adult forms of the receptor complex. In the present study, in situ hybridization with antisense cRNA probes was used to examine the subunit composition of the gamma-aminobutyric acidA/benzodiazepine (GABAA/BZ) receptor in the developing inferior olivary complex. This receptor is thought to be a pentameric chloride channel comprised of selected alpha, beta, gamma, delta, and rho subunits, the majority of which have several isoforms: alpha 1-6, beta 1-4, gamma 1-4, and rho 1,2. Among the 13 subunit variants present in the mammalian central nervous system, alpha 2-5, beta 3, and gamma 1,2 mRNAs are expressed at significant levels in the inferior olivary complex. Two clearly different temporal patterns of GABAA/BZ receptor subunit mRNA expression were observed: The expression of alpha 3, alpha 5, beta 3, and gamma 2 mRNAs was at a peak during embryonic and early postnatal development followed by rapid down-regulation thereafter. Conversely, alpha 2, alpha 4, and gamma 1 mRNA expression was very low or absent during early development, and a pronounced increase was observed at the end of postnatal week 1. These studies suggest that there are developmental changes in the subunit composition of the GABAA/BZ receptor in inferior olivary neurons. These changes in subunit expression, which occur during a period of major alterations in afferent and efferent synaptic connections, may subserve a change in the role of GABA from its function as a neurotrophic factor to that of an inhibitory neurotransmitter.

GABAA/benzodiazepine receptor gamma 2 subunit gene expression in developing normal and mutant mouse cerebellum.

Recent studies have identified several subunits (alpha, beta, gamma and delta) of the gamma-aminobutyric acidA/benzodiazepine receptor; each consists of several variants. The gamma 2 subunit appears to mediate the interaction of the alpha and beta subunits making the receptor capable of modulation by benzodiazepines. In the present studies, the expression of mRNA encoding the gamma 2 subunit was examined in the cerebellum during development and in adult Purkinje cell degeneration, lurcher and reeler mutant mice. In the normal adult cerebellum, in situ hybridization with [35S]cRNA probes revealed a strong signal over the Purkinje cell layer and deep cerebellar nuclei, and a weaker signal over basket, stellate and granule cells. Labeling over Purkinje cells was detectable at birth, gradually becoming stronger and more punctate during postnatal weeks 1 and 2, as Purkinje cells formed a monolayer between the molecular and granule cell layers. Adult levels of grain density were reached by P20. The external germinal layer, which contained proliferating granule cells, was unlabeled throughout development; however, weak labeling was detected over the internal granular layer at the end of postnatal week 1, as granule cells began their migration across the molecular layer. During the second postnatal week, punctate labeling became visible over the molecular layer in a distribution indicative of basket and stellate cells. In adult Purkinje cell degeneration and lurcher mutants, in which Purkinje cells have degenerated, no punctate labeling characteristic of mature Purkinje cells was detected. In adult and developing reeler mutants, where all classes of cells are malpositioned throughout the cerebellum, the punctate hybridization signal was present and clearly associated with Purkinje cells in all cortical regions. Our results suggest that developing Purkinje cells express the gamma 2 gene at a time prior to receiving GABAergic inhibitory input, and that the continued expression in the adult is not affected by the absence of afferents.

Auditory sensory gating in the rat hippocampus: modulation by brainstem activity.

Auditory stimuli repeated at short intervals result in diminished evoked responses recorded from the skull surface and from the hippocampus in the rat. The rat has been used to model diminished responses to repeated auditory stimuli--a phenomenon seen in normal human subjects, but often absent in schizophrenics. In this study, we examined the neural circuitry involved in the processing and gating of auditory responses recorded from the hippocampus of the rat. Evoked potentials and single neuron activity with diminished responses to the second of paired tones were recorded in the brainstem reticular formation in the paragigantocellular region at the caudal level of the pons, but diminished responses were not observed in the primary auditory relay nuclei. Electrical stimulation of this region of the brainstem reticular formation was able to substitute for the first, or conditioning, auditory tone to produce sensory gating of the response to the second, or test, tone when recording from the hippocampus. Stimulation of the auditory nuclei up to the level of the lateral lemniscus, but not the superior colliculus, was also able to substitute for an auditory stimulus to produce sensory gating in the hippocampus. The gating of hippocampal responses to auditory stimuli may thus involve pathways which branch from the lemniscal auditory pathway at the level of the lateral lemniscus and ascend to the hippocampus via the brainstem reticular formation.

Differential expression of GABAA/benzodiazepine receptor beta 1, beta 2, and beta 3 subunit mRNAs in the developing mouse cerebellum.

Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian cerebellum. Cerebellar granule, Purkinje, and deep nuclear neurons are known to receive GABAergic afferents. Since GABA exerts its inhibitory effects via GABA receptors, it is of interest to determine the temporal relationship between the formation of GABAergic synapses and the expression of genes coding for the GABA receptor. In a previous study, we have examined the developmental expression of binding sites for [3H]muscimol, which binds with high affinity to the beta subunits of the GABAA/benzodiazepine (GABAA/BZ) receptor. In the present study, [35S]cRNA probes were used to examine the appearance and distribution of GABAA/BZ beta 1, beta 2, and beta 3 subunit mRNAs in the developing C57BL/6 mouse cerebellum by in situ hybridization. In the adult cerebellum, the distribution of the three subunit mRNAs was clearly different, despite considerable overlap, and their temporal expression differed throughout postnatal development. The beta 1 hybridization signal appeared within the cerebellar cortex during the second postnatal week as a discrete band at the interface of the molecular and granule cell layers. Grains were distributed diffusely over small densely staining cells surrounding the Purkinje cells; relatively few grains were visible over Purkinje cell bodies themselves. This distribution may reflect an association with Bergmann glia or basket cells. The beta 2 and beta 3 hybridization signals were present considerably earlier than that of the beta 1 mRNA. The beta 2 signal was present at birth in the molecular/Purkinje cell layer; as development progressed, the signal became increasingly intense over both granule and Purkinje cells. At birth, the beta 3 subunit mRNA was present in the external germinal and molecular layers, later becoming largely localized within the granule cell layer. Dense beta 2 and beta 3 cRNA probe labeling was present over the adult granule cell layer. Moderate levels of beta 2 signal were seen over Purkinje cell bodies; considerably less labeling was observed with the beta 3 probe. The adult distribution of beta 2 and beta 3 cRNA probes showed good spatial correspondence with the known GABAA receptor beta subunit markers, [3H]-muscimol and the mAb 62-3G1 antibody, each being present within the granule cell layer. Our results indicate that the temporal expression of GABAA/BZ receptor beta subunit messages within a given cell type may be independently regulated, and that acquisition of the beta 2 and beta 3 mRNAs occurs before these cells become integrated into mature synaptic circuits.

Ontogeny of GABAA/benzodiazepine receptor subunit mRNAs in the murine inferior olive: transient appearance of beta 3 subunit mRNA and [3H]muscimol binding sites.

The GABAA/benzodiazepine receptor consists of at least four subunits, alpha, beta, gamma and delta, each comprised of several variants. The developmental expression of the alpha 1, beta 1-3, gamma 2 and delta subunits was studied in the murine inferior olivary nucleus by in situ hybridization with antisense cRNA probes. The postnatal appearance and distribution of [3H]flunitrazepam and [3H]muscimol binding sites, alpha and beta subunit-specific ligands respectively, were also studied autoradiographically. The beta 3 subunit was transiently expressed in each of the subnuclei of the inferior olive: The signal was strong at birth, increased throughout postnatal week 1 and rapidly declined thereafter to low adult levels. A similar pattern of labeling was observed with [3H]muscimol. Detectable levels of alpha 1 subunit mRNA hybridization signal and [3H]flunitrazepam binding sites were also present in the inferior olive at birth, decreasing thereafter. Low to moderate levels of beta 1, beta 2, and gamma 2 subunit mRNAs were present in olivary neurons throughout postnatal development, while delta mRNAs were largely absent. It has been reported previously that, during the 2nd postnatal week, the ratio of climbing fiber terminals to Purkinje cells is reduced from 3:1, as observed in neonates, to the 1:1 relationship observed in the adult cerebellar cortex. Our results raise the possibility that the subunit composition of the GABAA/benzodiazepine receptor in inferior olivary neurons undergoes changes during development, and that this process may be related to the elimination of multiple climbing fiber innervation of cerebellar Purkinje cells.

Phencyclidine and auditory sensory gating in the hippocampus of the rat.

The psychotomimetic drug 1-(1-phenylcyclohexyl) piperidine (PCP, phencyclidine) was found to cause a deficit in the gating of the response of the hippocampal neuron to repeated auditory stimuli, which is similar to a particular physiological feature observed in human psychosis. Other drugs, with sigma agonist and/or N-methyl-D-aspartate (NMDA) antagonist effects, were administered and their ability to cause a loss of auditory gating was compared to that of PCP. The rank order of effectiveness was levoxodrol > PCP and (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801) > N-allylnormetazocine (SKF 10047) > dexoxodrol > 3-(+/-)2-carboxypiperazine-4-yl) propyl-1-phosphonate (CPP). Further studies of two of the drugs, PCP and MK-801, showed that selective lesioning of the noradrenergic input with the neurotoxin DSP4, as well as less selective depletion of monoamines with reserpine, blocked the loss of gating. Phencyclidine, and other drugs with the same spectrum of action, most likely disrupt gating by increasing noradrenergic activity through a sigma mechanism.

Cholinergic gating of response to auditory stimuli in rat hippocampus.

Rapid decrement of response to repeated stimuli is a characteristic of hippocampal neurons. To assess the possible role in this process of cholinergic afferents from the medial septal nucleus, a series of cholinergic antagonists were administered intraventricularly to chloral hydrate-anesthetized rats. Auditory stimuli were delivered in pairs to the rats, and the evoked response was recorded from an electrode in the CA3 layer of the hippocampus. The most prominent component of the auditory evoked potential recorded in this region (N40) showed over 60% decrement in the amplitude of the response to the second stimulus when the two stimuli were delivered 0.5 s apart. Only neuromuscular-type nicotinic antagonists, alpha-bungarotoxin and (+)-tubocurarine, disrupted this decrement of response to repeated auditory stimuli. The muscarinic antagonist, scopolamine, and the ganglionic-type nicotinic antagonists, kappa-bungarotoxin and mecamylamine, were without effect. The results suggest that a subset of nicotinic receptors mediate the gating of response to auditory stimuli in the hippocampus.

Nicotine interferes with GABA-mediated inhibitory processes in mouse hippocampus.

Previous studies indicated that the excitatory effects of nicotine may be mediated via interference with GABAergic transmission. Here, several variants of the paired-pulse paradigm were employed to ascertain whether nicotine interferes with endogenous inhibitory circuits in the hippocampus. Nicotine attenuated the inhibition evoked by antidromic (alvear) stimulation in the CA1 region in a concentration-dependent manner (EC50 = 60-75 microM). This same phenomenon was also observed for the GABAA receptor antagonist bicuculline (0.1 microM). Orthodromic-orthodromic paired-pulse paradigms were found to be unsuitable for investigating the effects of epileptogenic agents such as nicotine and bicuculline on endogenous inhibition.

5 alpha-Pregnan-3 alpha-ol-20-one blocks nicotine-induced seizures and enhances paired-pulse inhibition.

5 alpha-Pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) blocked seizures induced by nicotine (4 mg/kg, i.p.) in C3H male mice with an ID50 of 2.37 +/- 0.66 mg/kg (average +/- 95% confidence limit). This steroid (1 microM) also increased paired-pulse inhibition in the hippocampus approximately 40% after 50 min exposure; nicotine (200 microM) partially reversed this effect. Since nicotine and 3 alpha-OH-DHP may have opposite effects on endogenous inhibitory systems, it is proposed that nicotine-induced seizures may involve a disinhibitory mechanism and that 3 alpha-OH-DHP protects against seizures by preventing disinhibition.