Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor.

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.

Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases.

Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c-fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.

Midbrain microinfusions of prolactin increase the estrogen-dependent behavior, lordosis.

Microinfusions of rat prolactin into the dorsal midbrain of estrogen-treated, ovariectomized rats increased lordosis behavior. Midbrain microinfusions of antiserum to prolactin into rats displaying maximum lordosis had the opposite effect. The distribution of a prolactin-like substance in the brain was studied immunocytochemically. The results suggest that a hypothalamic neuronal system projecting to the midbrain contains a prolactin-like substance that plays a role in facilitating this behavior and therefore may mediate some of the effects of estrogen on the brain. These data, together with others from studies of the prolactin gene and its regulation, indicate that it may be possible to analyze a sequence of molecular events in the brain that facilitate a behavioral response.

Two novel GABAA receptor subunits exist in distinct neuronal subpopulations.

Two cDNAs encoding novel GABAA receptor subunits were isolated from a rat brain library. These subunits, gamma 2 and delta, share approximately 35% sequence identity with alpha and beta subunits and form functional GABA-gated chloride channels when expressed alone in vitro. The gamma 2 subunit is the rat homolog of the human gamma 2 subunit recently shown to be important for benzodiazepine pharmacology. Cellular localization of the mRNAs encoding the gamma 2 and delta subunits in rat brain revealed that largely distinct neuronal subpopulations express the two subunits. The delta subunit distribution resembles that of the high affinity GABAA receptor labeled with [3H]muscimol; the gamma 2 subunit distribution resembles that of GABAA/benzodiazepine receptors labeled with [3H]flunitrazepam. These findings have implications for the composition of two different GABAA receptor subtypes and for information processing in networks using GABA for signaling.

The role of receptor subtype diversity in the CNS.

The molecular characterization of neuroreceptors and voltage-gated ion channels has revealed that receptor subtype heterogeneity is a common feature of chemical and electrical signal reception. The use of distinct genes encoding receptor subtypes is a favoured mechanism for generation of this diversity. We propose that the significance of the multiplicity and diversity of signal reception proteins is to increase the information-handling capacity of neurons. This may contribute to neural plasticity.

Estrogen increases proenkephalin messenger ribonucleic acid levels in the ventromedial hypothalamus of the rat.

The effects of estrogen on proenkephalin (PE) gene expression were measured in neurons of the ventromedial hypothalamus. Slot blot hybridization analysis indicates that the levels of PE mRNA in the ventromedial hypothalamus of ovariectomized rats increase 3.1-fold after 2 weeks of estrogen replacement. In situ hybridization reveals that the estrogen-inducible enkephalinergic neurons are located in the ventrolateral aspect of the ventromedial nucleus, a subnucleus known to contain many estrogen-concentrating neurons. The increase in PE mRNA levels is due to both a 63% increase in the number of detectable PE mRNA-containing neurons and a 2.0-fold increase in the levels of PE mRNA per enkephalinergic neuron (1.63 x 2.0 = 3.3-fold overall induction). This estrogen-regulated enkephalinergic cell group may represent part of the neural network mediating estrogen's effects on reproductive behavior and/or other neuroendocrine processes.

Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions.

Two bioactive products of pituitary adenylate cyclase-activating polypeptide (PACAP) prohormone have been isolated from ovine hypothalamus: PACAP38 with 38 residues and PACAP27 corresponding to the N-terminal 27 residues of PACAP38. Immunocytochemical and RIA results support the existence of PACAP in the rat brain, posterior pituitary, and various peripheral tissues. Furthermore, high affinity PACAP-binding sites have been detected in the rat brain, anterior pituitary, and cultured astrocytes which differ from those in lung, liver, and cultured mouse splenocytes. In the present study additional rat tissues were examined to elucidate the location and characteristics of PACAP-binding sites using [125I] PACAP27 with conventional methods of receptor autoradiography and RRA. Binding specificity was established by displacement with unlabeled PACAP27 or a related peptide, vasoactive intestinal polypeptide (VIP). PACAP27-binding sites were localized autoradiographically in the testis, epididymis, adrenal gland, lung, liver, prostate gland, and seminal vesicle; binding sites were not detected in the heart, kidney, or thymus. In the testis and epididymis, a PACAP27-binding site was localized on germinal cells and in the adrenal gland on medullary chromaffin cells. Excess VIP did not displace PACAP27 binding localized in these three tissues. A site with a greater affinity for PACAP27 than for VIP was detected in adrenal gland and epididymis, characteristic of a site recognized previously in hypothalamus, anterior pituitary, and cultured astrocytes. The PACAP-specific site was more abundant in these tissues than a second site to which PACAP27 and VIP bound with similar affinities. Accordingly, the first site has been named type I. In lung, liver, prostate, and seminal vesicle, VIP displaced PACAP27 binding localized autoradiographically. Lung and liver contained an abundant site to which PACAP27 and VIP bound with similar affinities. This binding site, measured previously in lung, liver, and cultured splenocytes, may be shared by PACAP and VIP and has been named type II. Taken together, these data support the existence of two high affinity binding sites for PACAP with different tissue distribution.

Localization of cells containing LHRH-like mRNA in rat forebrain using in situ hybridization.

Using in situ hybridization, we localized cells in the rat forebrain which contain mRNA that hybridizes with a radiolabeled, synthetic oligodeoxyribonucleotide (59-mer) complementary to human LHRH mRNA in the region which includes the coding sequence for the decapeptide. These brain areas have been shown previously to contain immunoreactive LHRH cell bodies.

Prolactin mRNA exists in rat hypothalamus.

Using radiolabeled DNA complementary to rat pituitary prolactin mRNA, we probed RNA gel blots from three rat tissues: pituitary, hypothalamus, and liver. Poly (A)-enriched RNA from male and female hypothalami contained a hybridizable RNA which was the same size as, though less abundant than, mature pituitary PRL mRNA. These results support the proposal that the rat brain synthesizes PRL.

Reduced ischemic brain injury in interleukin-1 beta converting enzyme-deficient mice.

A variety of recent studies suggest a role for both inflammatory cytokines such as interleukin-1 beta (IL-1 beta), and apoptosis in ischemic brain injury. Because IL-1 beta converting enzyme (ICE) is required for the conversion of proIL-1 beta to its biologically active form, and has homology with proteins that regulate apoptosis in invertebrates, we studied the effect of cerebral ischemia on brain injury in mutant mice deficient in the ICE gene (ICE knockout [KO] mice). Focal cerebral ischemia, produced by occlusion of the middle cerebral artery, resulted in brain edema (increased water and sodium content) at 4 hours and a histologically defined brain lesion at 24 hours. Both of these markers of brain injury were significantly reduced in the ICE KO mice as compared to wild-type C57BL/6 mice. Regional cerebral blood flow, determined using the flow tracer, N-isopropyl [methyl 1,3-(14)C] p-iodoamphetamine (14C-IMP), was similar in the two strains of mice, indicating that the reduced brain injury in the KO mice was not a result of a lesser degree of ischemia. These data show that ICE contributes to the development of ischemic brain damage, and that it plays a role at an early time in the pathologic process. Although the mechanism of this effect is uncertain, our results suggest that pharmacologic inhibition of ICE may be a useful treatment for stroke.

Interleukin-1 beta dissociates beta-amyloid precursor protein and beta-amyloid peptide secretion.

A heightened production of interleukin 1 beta (IL-1 beta) has been reported in microglial-associated amyloid deposits in Alzheimer's disease (AD) brains. These plaques are composed predominantly of beta/A4 peptide derived from beta-amyloid precursor protein (beta APP). We demonstrate that short-term (1 h) IL-1 beta-treatment increases beta APPs secretion and concomitantly decreases cell-associated beta APP in human H4 neuroglioma cells. Long-term (5 h) IL-1 beta treatment did not alter secreted or cell-associated beta APP content. In contrast, the secretion of beta/A4-containing epitope was not affected by short-term IL-1 beta stimulation; however, long-term IL-1 beta treatment decreased the amount of beta/A4-containing epitope secreted from the cells. These results show that IL-1 beta modifies the processing and secretion of beta APP to exacerbate perhaps the neuropathology of AD.

The Adhesion Molecule on Glia (AMOG) Is Widely Expressed by Astrocytes in Developing and Adult Mouse Brain.

Adhesion molecule on glia (AMOG) is a 45 - 50 kD cell surface glycoprotein structurally similar to the Na, K-ATPase beta-subunit and associated with the catalytic subunit of this enzyme. Previous immunofluorescence results had suggested that AMOG is transiently expressed on Bergmann glia during mouse cerebellar development, and antibody-inhibition results have implicated it in the migration of granule neurons. We report that, while AMOG mRNA is detected in Bergmann glia during the migratory period, this astrocyte derivative continues to express AMOG mRNA at similar levels in adult mice suggesting a functional role for AMOG in adulthood. Evidence from RNA and protein blot analyses that AMOG is present before birth, increasing about ten fold in adult mouse brain and cerebellum is also provided. RNA blot analysis of astrocyte-enriched cell populations and in situ hybridization results show that astrocytes synthesize AMOG mRNA in all regions of the developing and adult brain. In the adult, AMOG mRNA is more abundant in grey than white matter and, among grey matter regions, highest in cerebellar cortex. These results indicate a relationship between density of neuronal elements and AMOG expression. It is further speculated that AMOG is part of a Na,K-ATPase complex expressed preferentially by astrocytes in mouse brain.

Localization of preproenkephalin mRNA in the rat brain and spinal cord by in situ hybridization.

To determine the localization in rat brain and spinal cord of individual neurons that contain the messenger RNA coding for the opioid peptide precursor preproenkephalin, we performed in situ hybridization with a tritiated cDNA probe complementary to a protion of preproenkephalin mRNA. We observed autoradiographic signal over the cytoplasm of neurons of many regions of the central nervous system. Several types of controls indicated specificity of the labeling. Neurons containing preproenkephalin mRNA were found in the piriform cortex, ventral tenia tecta, several regions of the neocortex, nucleus accumbens, olfactory tubercle, caudate-putamen, lateral septum, bed nucleus of the stria terminalis, diagonal band of Broca, preoptic area, amygdala (especially central nucleus, with fewer labeled neurons in all other nuclei), hippocampal formation, anterior hypothalamic nucleus, perifornical region, lateral hypothalamus, paraventricular nucleus, dorsomedial and ventromedial hypothalamic nuclei, arcuate nucleus, dorsal and ventral premamillary nuclei, medial mamillary nucleus, lateral geniculate nucleus, zona incerta, periaqueductal gray, midbrain reticular formation, ventral tegmental area of Tsai, inferior colliculus, dorsal and ventral tegmental nuclei of Gudden, dorsal and ventral parabrachial nuclei, pontine and medullary reticular formation, several portions of the raphe nuclei, nucleus of the solitary tract, nucleus of the spinal trigeminal tract (especially substantia gelatinosa), ventral and dorsal cochlear nuclei, medial and spinal vestibular nuclei, cuneate and external cuneate nuclei, gracile nucleus, superior olive, nucleus of the trapezoid body, some deep cerebellar nuclei, Golgi neurons in the cerebellum, and most laminae of the spinal cord. In most of these brain regions, the present results indicate that many more neurons contain preproenkephalin mRNA than have been appreciated previously on the basis of immunocytochemistry.

Combination of immunocytochemistry and in situ hybridization in the same tissue section of rat pituitary.

A procedure is described for combining avidin-biotinylated horseradish peroxidase immunocytochemistry, for localizing peptides or proteins, with in situ hybridization for localizing mRNA autoradiographically in the same cryostat section of paraformaldehyde-fixed rat pituitary. Protection against enzymatic degradation of target mRNAs during the immunocytochemical step was necessary and was accomplished by including an RNase inhibitor, 0.04% diethylpyrocarbonate, in primary and secondary antisera. This combination of methods may be useful in other tissues, as well, for (a) determining the relation of protein content to the concentration of its encoding mRNA, (b) proving the synthetic capacity of a cell in which a protein has been localized, (c) determining immunological or nucleic acid probe specificity, or (d) as an alternative to double-labeling immunocytochemical methods.

Contribution of the ICE family to neurodegeneration.

The ICE family of cysteine proteases mediates necrotic or apoptotic events in the nervous system as well as in other tissues. This suggests that inhibitors may be of therapeutic value in acute and, perhaps, chronic neurodegenerative disease. In addition, some members of this family may respond to intercellular signals controlling proliferation or differentiation. This possibility should be kept in mind as therapeutics are pursued.

Inhibition of the lordosis reflex in rats by intrahypothalamic infusion of neural excitatory agents: evidence that the hypothalamus contains separate inhibitory and facilitatory elements.

In attempts to activate lordosis-facilitating neural mechanisms in the ventromedial hypothalamus (VMH), neural excitatory agents were infused into the medial hypothalamus, and the effects of the infusions on the lordosis reflex and on the electrical activity of VMH neurons were studied. Surprisingly, bilateral intrahypothalamic infusion of glutamate (10 mM, 1.0 microliter/side) into behaving, ovariectomized, estrogen-treated rats displaying moderate lordotic responsiveness did not facilitate lordosis, but instead, resulted in a rapid (within a few minutes) and transient (recovery in about 20 min) inhibition of lordosis. Further experiments showed that this lordosis-inhibiting effect of glutamate was dose-related, and was completely blocked by prior infusion of a local anesthetic, procaine. Infusion of KC1 (1.5 or 15%, 1.0 microliter/side) also induced a dose-related, rapid and transient inhibition of lordosis, that was essentially identical to that induced by glutamate. Kainic acid (0.25 micrograms/0.5 microliter/side) also caused a rapid inhibition of lordosis, but the effect was long-lasting (days). The inhibition of lordosis by these agents was dissociated in time course, presence, and/or severity from effects on non-lordotic behaviors. Electrophysiological studies showed that all three agents tested could excite multiunit activity of the VMH, and that the time courses of these excitations were closely comparable to those of the inhibition of lordosis induced by the respective agents. Altogether, these studies indicated that the excitation of certain medial hypothalamic neurons can inhibit the lordosis reflex. The implied lordosis-inhibiting neural mechanisms are separate from facilitatory mechanism(s), according to differences in latency, duration, and procaine-sensitivity of response.

Haloperidol increases proenkephalin mRNA levels in the caudate-putamen of the rat: a quantitative study at the cellular level using in situ hybridization.

Previous immunocytochemical studies have shown that the opioid peptides, Met-enkephalin and Leu-enkephalin, are present in medium-sized, spiny projection neurons of the caudate-putamen. It has also been demonstrated that chronic treatment of rats with the dopamine receptor blocker, haloperidol, results in an increase in the levels of enkephalin peptides and proenkephalin mRNA in this brain region. To determine whether this increase in proenkephalin mRNA content is exhibited by all enkephalinergic neurons of the caudate-putamen or by only a subpopulation, we have used in situ nucleic acid hybridization to examine the haloperidol-induced increase in proenkephalin mRNA levels at the cellular level. Results of in situ hybridization suggest that all enkephalinergic neurons in the caudate-putamen can respond to haloperidol treatment with an increase in steady state levels of proenkephalin mRNA, and that the mean induction is an approximate 3-fold increase in the message levels. This suggests that dopamine exerts a tonic inhibitory effect on the expression of the proenkephalin gene in all of the enkephalinergic neurons of the caudate-putamen. Dot blot analysis indicated a 2.4-fold increase in the tissue levels of this mRNA. The agreement between the in situ hybridization results and dot blot analysis supports in situ hybridization as a reliable method for quantitative studies of alterations in neuropeptide precursor mRNAs in the brain.

Estrogenic maintenance of lordotic responsiveness: requirement for hypothalamic action potentials.

Studies were conducted to determine the requirement for hypothalamic, sodium current-based action potentials in the performance of a stereotypic, estrogen-dependent reflex, the lordosis response. Intrahypothalamic infusion of local anesthetics (50% procaine or 0.5% bupivacaine) into conscious rats had no effect on lordotic responsiveness, and, in a separate group of urethane-anesthetized rats, depressed multiunit electrical activity temporarily. Intrahypothalamic infusion of tetrodotoxin into conscious rats, however, resulted in a dose-dependent, reversible decline in lordotic responsiveness. The first significant drop in lordotic responsiveness occurred 40 min after infusion; the minimum was reached 2-4 h after infusion. Recovery of lordotic responsiveness to preinfusion levels was complete by 12-24 h after infusion. Electrophysiological studies in a separate group of urethane-anesthetized rats revealed that intrahypothalamic tetrodotoxin infusion in most cases suppressed multiunit activity completely usually within 6 min, and this suppression lasted for at least several hours. These data indicate that large, prolonged decreases in electrical activity in the hypothalamus, where estrogenic action is necessary and sufficient to induce lordosis, result in a gradual, reversible decline in lordotic responsiveness. These data are consistent with a 'tonic' rather than a 'mount-by-mount' role of hypothalamic neurons in lordosis. Furthermore, since lordotic responsiveness declined only when hypothalamic electrical activity had been disrupted severely for at least 40 min, it is postulated that the neuroactive products released by lordosis-relevant, hypothalamic neurons may have a duration of action of at least several minutes.

Intrahypothalamic colchicine infusions disrupt lordotic responsiveness in estrogen-treated female rats.

Since some estrogenic effects on lordotic responsiveness are mediated through hypothalamic protein synthesis, we conducted experiments to determine if axoplasmic transport in the hypothalamus is necessary for the induction and maintenance of this reflex by estrogen. Colchicine infusion into the hypothalamus, but not into the dorsal thalamus, of ovariectomized rats 24 h prior to administration of subcutaneous estrogen implants delayed the induction of lordotic responsiveness, as measured by the manual (cutaneous-pressure) method, by 2 days, as compared with vehicle-infused rats. In other experiments, colchicine infusion into the hypothalamus, but not into the dorsal thalamus, of conscious, ovariectomized, estrogen-implanted rats displaying maximal lordotic responsiveness resulted in a bimodal decline in lordotic responsiveness. An initial decline occurred 20-40 min after infusion, and was associated with general behavioral agitation and hyperactivity. A subsequent decline began 4 h after infusion and lasted for several days. Vehicle infusion did not decrease lordotic responsiveness. Colchicine infusion did not alter multiunit electrical activity recorded near hypothalamically directed cannulae tips over a period of several hours. Results suggest that axoplasmic transport within and/or from the hypothalamus is necessary for the estrogenic induction and maintenance of the lordosis reflex in rats.

Failure of urethane anesthetic to block induction of pineal serotonin N-acetyltransferase activity in the rat.

Ability of urethane anesthetic to block induction of pineal serotonin N-acetyltransferase (SNAT; E.C.2.3.1.5) activity was measured in individual rat pineal glands in animals receiving urethane (25% w/v, IP, 1.2 g/kg) or saline, 6 hr prior to sacrifice. Using a radioenzymatic assay, SNAT determinations were made twice daily (at 1200 or 2400 hr) immediately after the sacrifice of each animal. The results show that urethane had no effect on the induction of SNAT activity: (1) implying that the neural activity of those structures involved in induction of SNAT activity (e.g., suprachiasmatic nucleus) is not substantially altered by this anesthetic and (2) suggesting that the central blockade of ovulation by urethane does not include alterations in suprachiasmatic nucleus activity.