Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia‐inducible factor‐2α

Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood–brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side‐effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia‐induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia‐induced increase in EPO mRNA expression in a concentration‐dependent manner. TNFα inhibition of hypoxia‐induced EPO expression was mediated primarily by hypoxia‐inducible factor (HIF)‐2α rather than HIF‐1α. The effects of TNFα in reducing hypoxia‐induced upregulation of EPO mRNA expression probably involve destabilization of HIF‐2α, which is regulated by the nuclear factor (NF)‐κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response.     

Autoantigens in human neuroblastoma cells

Neuroblastoma cells are frequently used as targets in studies of autoimmune diseases of the nervous system. We examined the human neuroblastoma cell line, LAN-5, for the presence of autoantigens that react with naturally occurring autoantibodies in human sera. Antibodies to the HNK-1 and Gal(beta 1-3)GalNAc epitopes, which have been implicated in human autoimmune neuropathy and motor neuron disease, respectively, immunostained the surface of the neuroblastoma cells, and antibodies to the 200 kDa high molecular weight neurofilament protein (NFH) immunostained the cytoplasm and cell processes. The NHK-1 and Gal(beta 1-3)GalNAc epitopes were associated with several glycoprotein bands in Western blots of the neuroblastoma cells, and the HNK-1 epitope was also shared by a glycolipid which co-migrated with 3-sulfoglucuronyl paragloboside (SGPG) from peripheral nerve, indicating that SGPG is synthesized in neuronal cells. Northern blot analysis revealed a single RNA band of 4800 bp for NFH in normal brain but two RNA species of 4800 and 3800 bp in both neuroblastoma and adrenal cells, confirming their common origin. The neuroblastoma cells appear to contain antigens that bind to naturally occurring autoantibodies in human serum and might therefore be useful for detecting and investigating the effects of anti-neuronal antibodies. The antibody populations being investigated, however, should be distinguished from other autoantibodies which might be present in the patients' serum.     

Combined administration of secretin and oxytocin inhibits chronic colitis and associated activation of forebrain neurons

Background The pathogenesis of inflammatory bowel disease is unknown; however, the disorder is aggravated by psychological stress and is itself psychologically stressful. Chronic intestinal inflammation, moreover, has been reported to activate forebrain neurons. We tested the hypotheses that the chronically inflamed bowel signals to the brain through the vagi and that administration of a combination of secretin (S) and oxytocin (OT) inhibits this signaling. Methods Three daily enemas containing 2,4,6‐trinitrobenzene sulfonic acid (TNBS), which were given to rats produced chronic colitis and ongoing activation of Fos in brain neurons. Key Results Fos was induced in neurons in the paraventricular nucleus of the hypothalamus, basolateral amygdala, central amygdala, and piriform cortex. Subdiaphragmatic vagotomy failed to inhibit this activation of Fos, suggesting that colitis activates forebrain neurons independently of the vagi. When administered intravenously, but not when given intracerebroventricularly, in doses that were individually ineffective, combined S/OT prevented colitis‐associated activation of central neurons. Strikingly, S/OT decreased inflammatory infiltrates into the colon and colonic expression of tumor necrosis factor‐α and interferon‐γ. Conclusions & Inferences These observations suggest that chronic colonic inflammation is ameliorated by the systemic administration of S/OT, which probably explains the parallel ability of systemic S/OT to inhibit the colitis‐associated activation of forebrain neurons. It is possible that S and OT, which are endogenous to the colon, might normally combine to restrict the severity of colonic inflammatory responses and that advantage might be taken of this system to develop novel means of treating inflammation‐associated intestinal disorders.     

The socially-isolated mouse: a model to study the putative role of allopregnanolone and 5α-dihydroprogesterone in psychiatric disorders

Allopregnanolone (3α,5α-TH PROG) and 5α-dihydroprogesterone (5α-DH PROG), the two most important neuroactive steroids synthesized in the brain, potently modulate neuronal activity by allosterically regulating GABA action at GABA_A receptors or by changing specific GABA_A receptor subunit gene expression, respectively. We recently reported [Proc. Natl. Acad. Sci. USA 95 (1998) 3239] that in patients with severe depression there is a decrease in the CSF levels of 3α,5α-TH PROG, which is normalized by treatment with drugs (i.e. fluoxetine) that improve psychopathology. The mechanism by which fluoxetine and other selective serotonin reuptake inhibitors normalize 3α,5α-TH PROG CSF levels appears to involve a direct stimulation of 3α-hydroxysteroidoxidoreductase (3α-HSD), an enzyme that catalyses the reduction of 5α-DH PROG into 3α,5α-TH PROG. Here, we propose the use of socially-isolated mice that have a downregulation of 3α,5α-TH PROG and of 5α-DH PROG expression to establish a model to study the behavioral consequences of this deficiency. After 4–6 weeks of isolation, these mice exhibit increased anxiety and aggressive behavior and also a decreased response to the administration of GABA-mimetic drugs. In these mice, the decrease in 3α,5α-TH PROG is selectively normalized by the use of fluoxetine in doses that reduce behavioral abnormalities. In addition, the expression of 5α-reductase Type I mRNA and protein was lower in socially-isolated mice than that in group-housed mice whereas 3α-HSD mRNA expression remained unchanged. The results of these studies may enable us to design drugs that specifically affect neurosteroidogenic enzymatic activities and may provide an efficacious treatment for the psychopathologies associated with psychiatric disorders.     

Genistein attenuates cognitive deficits and neuroapoptosis in hippocampus induced by ketamine exposure in neonatal rats

Ketamine is a frequently used anesthetic in pediatric patients that can cause cognitive impairment. Genistein, a bioactive component of soy products, has been shown to suppress neuronal death through regulating the expression of apoptosis related genes. In this study, we hypothesized that genistein could alleviate ketamine‐induced cognitive impairment by ameliorating hippocampal neuronal loss and tested this hypothesis in rats. Neonatal rats were treated with ketamine and genistein. Hippocampal tissue was harvested for histological and biochemical analysis to determine neuronal apoptosis and proteins involved in the apoptotic pathways. Behavioral assays including contextual fear conditioning test and Morris water maze test were performed to assess cognitive functions, including learning and memory. We found that in fear conditioning test, genistein restored freezing time in ketamine treated rats in a dose dependent manner. Similarly, genistein attenuated impaired learning and memory in Morris water maze test in rats treated with ketamine. Additionally, ketamine‐induced neuronal apoptosis in rat hippocampus was attenuated by genistein treatment. Finally, we found that genistein partially restored proteins associated with apoptosis, including Bax, Bcl‐2, cleaved caspase 3, and phosphorylated GSK‐3ß and Akt. Genistein suppresses hippocampal neuronal loss and cognitive disruption induced by ketamine in rats.     

The effect of increased NaCl intake on rat brain endogenous μ‐opioid receptor signalling

Numerous studies demonstrate the significant role of central β‐endorphin and its receptor, the μ‐opioid receptor (MOR), in sodium intake regulation. The present study aimed to investigate the possible relationship between chronic high‐NaCl intake and brain endogenous MOR functioning. We examined whether short‐term (4 days) obligatory salt intake (2% NaCl solution) in rats induces changes in MOR mRNA expression, G‐protein activity and MOR binding capacity in brain regions involved in salt intake regulation. Plasma osmolality and electrolyte concentrations after sodium overload and the initial and final body weight of the animals were also examined. After 4 days of obligatory hypertonic sodium chloride intake, there was clearly no difference in MOR mRNA expression and G‐protein activity in the median preoptic nucleus (MnPO). In the brainstem, MOR binding capacity also remained unaltered, although the maximal efficacy of MOR G‐protein significantly increased. Finally, no significant alterations were observed in plasma osmolality and electrolyte concentrations. Interestingly, animals that received sodium gained significantly less weight than control animals. In conclusion, we found no significant alterations in the MnPO and brainstem in the number of available cell surface MORs or de novo syntheses of MOR after hypertonic sodium intake. The increased MOR G‐protein activity following acute sodium overconsumption may participate in the maintenance of normal blood pressure levels and/or in enhancing sodium taste aversion and sodium overload‐induced anorexia.     

Glycogen synthase kinase‐3β phosphorylates synphilin‐1 in vitro

α‐Synuclein is known to be a major component of Lewy bodies and glial cytoplasmic inclusions in the brains of patients with α‐synucleinopathies. Synphilin‐1, an α‐synuclein‐associated protein, is also present in these inclusions. However, little is known about the post‐translational modifications of synphilin‐1. In the present study, it is reported that synphilin‐1 is phosphorylated by glycogen synthase kinase‐3βin vitro. It is well known that protein phosphorylation is involved in various physiological phenomena, including signal transduction and protein degradation. Therefore, phosphorylation of synphilin‐1 may play an important role in the function of this protein in the brain.     

Characterization of hypothalamic noradrenaline receptors in the supraoptic nucleus and periventricular region of the paraventricular nucleus of mice in vitro

In an attempt to determine the basis for apparently conflicting reports of the effects of noradrenaline (NA) on the neurohypophyseal system and its effects on the parvocellular periventricular region of the paraventricular nucleus (PVN), recordings were made from the neurons in the supraoptic nucleus (SON) and the periventricular region in the mouse hypothalamic slice preparation. Of 47 SON neurons, 43 (91%) were excited and two (4%) were inhibited by NA. Seven SON neurons increased the firing rate with increase of NA concentration (10(-7)-10(-4) M). Both the alpha 1-agonists phenylephrine and methoxamine also increased the activity of all SON neurons tested whereas application of the alpha 2-agonist clonidine and the beta-agonist isoproterenol had weak and inconsistent effects. While the alpha 2-antagonist yohimbine had no consistent influence, the alpha 1-antagonist prazosin blocked or reversed the effects of NA. Another group of 37 neurons in the periventricular region of the PVN was also tested; 13 (35%) were excited and 22 (59%) inhibited by application of NA (10(-5) M). When tested with phenylephrine or methoxamine, 6 of the 7 neurons were excited and one inhibited but all the 4 neurons tested were excited by isoproterenol. Clonidine strongly depressed the activity of all 12 neurons tested. The NA-induced excitatory effects were suppressed or reversed by pre-application of prazosin and the beta-antagonist propranolol while the inhibitory ones were suppressed or reversed by yohimbine. Synaptic blockade did not affect the excitatory responses of SON cells to NA nor the inhibitory responses of periventricular neurons to NA or clonidine. We conclude that SON neurons receive adrenergic excitatory effects mainly through alpha 1-receptors. The periventricular neurons receive the excitatory effects through alpha 1- or beta-receptors and receive the inhibitory effects through alpha 2-receptors.     

Early NFkappaB activation is inhibited during focal cerebral ischemia in interleukin-1beta-converting enzyme deficient mice

Our previous study demonstrated that the inhibition of interleukin-1beta (IL-1beta) reduces ischemic brain injury; however, the molecular mechanism of the action of IL-1 in cerebral ischemia is unclear. We are investigating currently the role of NFkappaB during focal cerebral ischemia, using mutant mice deficient in the interleukin-1 converting enzyme gene (ICE KO) in a middle cerebral artery occlusion (MCAO) model. Adult male ICE KO and wild-type mice (n = 120) underwent up to 24 hr of permanent MCAO. Cytoplasmic phospho-NFkappaB/p65 expression in ischemic brain was examined using Western blot analysis and immunohistochemistry. NFkappaB DNA-binding activity was detected using electrophoretic mobility shift assay (EMSA). Furthermore, ICAM-1 expression was examined in both the ICE KO and wild-type mice (WT). Western blot analysis and immunostaining showed that the level of cytosolic phosphorylated NFkappaB/p65 increased after 2 and 4 hr of MCAO in WT mice; however, NFkappaB/p65 was significantly reduced after MCAO in the ICE KO mice (P < 0.05). EMSA showed that NFkappaB DNA-binding activity increased after MCAO in WT mice; but this effect was reduced in the ICE KO mice. The number of ICAM-1-positive vessels in the ischemic hemisphere was greatly attenuated in the ICE KO mice (P < 0.05), which paralleled the results of immunohistochemistry. Our results demonstrate that NFkappaB phosphorylation is reduced in ICE KO mice, suggesting that ICE or IL-1 are involved in early NFkappaB phosphorylation. Because cerebral ischemia induced infarction is significantly reduced in ICE KO mice, we conclude that early NFkappaB phosphorylation plays a disruptive role in the ischemic process.     

Female Sex Hormones Influence the Febrile Response Induced by Lipopolysaccharide,Cytokines and Prostaglandins but not by Interleukin‐1β in Rats

There are differences in the immune response, and particularly fever, between males and females. In the present study, we investigated how the febrile responses induced by lipopolysaccharide (LPS) and different endogenous pyrogens were affected by female gonadal hormones. The febrile response to i.p. injection of LPS (50 μg/kg) was 40% lower in female rats compared to male or ovariectomised (OVX) female rats. Accordingly, oestrogen replacement in OVX animals reduced LPS‐induced fever. Treatment with the prostaglandin synthesis inhibitor indomethacin (2 mg/kg, i.p. 30 min before) reduced the febrile response induced by LPS in both OVX (88%) and sham‐operated (71%) rats. In line with the enhanced fever in OVX rats, there was increased expression of cyclooxygenase‐2 (COX‐2) in the hypothalamus and elevated levels of prostaglandin E₂ (PGE₂). In addition, OVX rats were hyper‐responsive to PGE₂ injected i.c.v. By contrast to the enhanced fever in response to LPS and PGE₂, the febrile response induced by i.c.v. injection of interleukin (IL)‐1β was unaffected by ovariectomy, whereas the responses induced by tumour necrosis factor (TNF)‐α and macrophage inflammatory protein (MIP)‐1α were completely abrogated. These results suggest that the mediators involved in the febrile response in females are similar to males, although the reduction of female hormones may decrease the responsiveness of some mediators such as TNF‐α and MIP‐1α. Compensatory mechanisms may be activated in females after ovariectomy such as an augmented synthesis of COX‐2 and PGE₂.     

Insulin-like growth factor-binding protein 6 inhibits survival and differentiation of rat oligodendrocyte precursor cells

Insulin-like growth factor 1 (IGF-1) is a growth and survival factor for oligodendrocyte lineage cells and promotes myelination. We demonstrate that IGF-binding protein 6 (IGFBP-6) is expressed and localized to the Golgi complex in rat oligodendrocyte precursor (O2A) cells. IGFBP-6 mRNA showed a developmentally regulated expression pattern, displaying a transient decrease during early development, and enhanced levels upon cell maturation. IGFBP-6 mRNA expression could be reduced by addition of basic fibroblast growth factor and progesterone while estrogen increased IGFBP-6 mRNA. IGF-1, platelet-derived growth factor, and insulin had no effect. When added exogenously, IGFBP-6 reduced O2A cell survival in the absence of IGF-1 and inhibited IGF-1-stimulated survival in a partially IGF-1-dependent and partially IGF-1-independent fashion. In addition, IGFBP-6 reduced the IGF-stimulated expression of two myelin proteins, CNPase and MAG. Taken together, the data show that IGFBP-6 is a new negative effector of oligodendrocyte survival and differentiation.     

Intrahypothalamic 5,7-dihydroxytryptamine: Temporal analysis of effects on 5-hydroxytryptamine content in brain nuclei and on facilitated lordosis behavior

The long-term relationship between serotonin (5-HT) levels in discrete hypothalamic nuclei and female rat sexual behavior, the lordosis response, was examined following intrahypothalamic injection of 5,7-dihydroxytryptamine (5,7-DHT). One week following 5,7-DHT injection, 5-HT levels in the ventromedial hypothalamic nucleus, dorsomedial nucleus, anterior hypothalamic nucleus and the medial preoptic nucleus were approximately 90% depleted as compared to sham animals. Other hypothalamic and preoptic areas including the arcuate-median eminence, vertical nucleus of diagonal band and lateral septal nucleus showed smaller reductions in 5-HT, from 40 to 70% of sham values. At this time estrogen-dependent lordosis behavior in the lesioned group was facilitated. Behavioral facilitation was greatest at 4 weeks post lesion when depletion of 5-HT in the VMN was maximal. 5-HT levels increased at 57 days after 5,7-DHT treatment in most areas, and by 71 days post lesion, no significant differences in 5-HT levels were found between sham and 5,7-DHT-treated groups. Concomitant with the increases in 5-HT, facilitated lordosis behavior gradually decreased. Loss of behavioral facilitation appeared to be most closely related to increases in content of 5-HT in the ventromedial nucleus. These results further support the hypothesis that 5-HT endings in the hypothalamus exert tonic inhibitory regulation over hormone-dependent lordosis in the female rat. They also indicate that regenerating 5-HT fibers in the hypothalamus can reinstate a normal pattern of hormone-dependent behavioral function.     

Age-related expression of sigma1 receptors and antidepressant efficacy of a selective agonist in the senescence-accelerated (SAM) mouse

The sigma1 receptor is a unique intracellular receptor whose activation results in an efficient modulation of several neurotransmitter responses. Its role as a target for the rapid nongenomic effects of neuro(active)steroids and the age-related diminutions in steroid levels suggested that targeting the sigma1 receptor might allow alleviation of age-related neuronal dysfunctions. We examined here the expression and behavioral efficacy of sigma1 receptors in the senescence-accelerated (SAM) mouse model. The sigma1 receptor mRNA expression was measured by using comparative RT-PCR in the olfactory bulb, hippocampus, hypothalamus, cortex, or cerebellum of senescence-prone SAMP/8 and senescence-resistant SAMR/1 control animals. No difference was observed between substrains in 6-, 9-, and 12-month-old (m.o.) mice. The sigma1 protein expression was analyzed by using immunohistochemical techniques. Labeling was intense in the olfactory bulb, hippocampus, hypothalamus, and midbrain of both SAMR/1 and SAMP/8 mice, and the distribution appeared unchanged in 6-, 9-, and 12-m.o. animals. The receptor's in vivo availability was examined by using in vivo [3H](+)-SKF-10,047 binding. No age-related difference was observed in the olfactory bulb, hippocampus, hypothalamus, cortex, cerebellum, and brainstem of 6- or 12-m.o. SAMR/1 or SAMP/8 mice. The antidepressant efficacy of the selective agonist igmesine was examined in the forced-swimming test. The compound decreased significantly the immobility duration at 60 mg/kg in 6- and 12-m.o. SAMR/1 and in 6-m.o. SAMP/8 mice. In 12-m.o. SAMP/8 mice, the drug efficacy was facilitated; a significant effect was measured at 30 mg/kg. Decreased neurosteroid levels, particularly of progesterone, were seen in 12-m.o. SAMP/8 mice that might explain the enhanced efficacy of igmesine. Preserved sigma1 receptor expression and enhanced behavioral efficacy of sigma1 agonists were measured in SAM animals, confirming the therapeutic opportunities for selective ligands against age-related mood disorders.     

Differentiation-dependent progesterone synthesis and metabolism in NT2-N human neurons

Human embryonic teratocarcinoma-derived Ntera2/cl.D1 (NT2) cells recapitulate many features of embryonic neuronal progenitor cells. Upon retinoic acid (RA) treatment they terminally differentiate into post-mitotic neuron-like cells (NT2-N), akin to human fetal neurons, thus representing an in vitro model of human neuron terminal differentiation. Experimental evidence also indicate NT2-N cultures as a potential source for cell transplantation therapy. The neurosteroids progesterone and its metabolite 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) promote neurogenesis and show anti-neurodegenerative properties. This study's aim was to assess the neurosteroidogenic competence of NT2 cells during RA-induced neuronal differentiation. Radioimmunoassay measurements revealed progesterone only in NT2-N cultures (4 week RA). Accordingly, progesterone synthesis from ³H-pregnenolone was absent in NT2 cells and increased during RA exposure, being highest in NT2-N. [³H]-pregnenolone metabolism, yielding [³H]-progesterone and [³H]-5α-dihydroprogesterone ([³H]-5α-DHP), was time-dependent and inhibited by trilostane, a 3β-hydroxysteroid-dehydrogenase (3β-HSD) inhibitor. Conversely, ³H-progesterone metabolism, which yielded [³H]-5α-DHP > [³H]-3β,5α-THP > [³H]-3α,5α-THP, occurred at all time points examined, though showing a nadir in cultures treated with RA for 1 and 2 weeks. The differentiation-dependent increase of progesterone accumulation matched 3β-HSD type I mRNA expression and 3β-HSD immunoreactivity, that co-localized with Map2a/b- and GAD67 in NT2-N. Hence, in vitro differentiated human neurons, while retaining progesterone metabolic activity, also become competent in progesterone synthesis. These findings suggest an autocrine/paracrine role of neuronal progesterone, either on its own or through its 5α-reduced metabolites, in fetal brain development and allow speculation that NT2-N-produced neurosteroids may contribute to the encouraging results of NT2-N transplants in animal models of neurodegenerative diseases.     

Electrophysiological actions of norepinephrine in rat lateral hypothalamus. II. An in vitro study of the effects of iontophoretically applied norepinephrine on LH neuronal responses to γ-aminobutyric acid (GABA)

The preceding studies demonstrated that norepinephrine (NE) can consistently augment synaptically mediated (70%) and γ-aminobutyric acid (GABA)-induced (69%) inhibitory responses of lateral hypothalamic (LH) neurons in vivo. The present experiments further characterized the interactions of NE with LH neuronal responses to GABA in terms of α- and ß-receptor mechanisms and demonsrated the utility of the in vitro LH tissue slice preparation as a model for future extra- and intracellular studies of NE modulatory phenomena. Extracellular activity of LH cells was recorded from diencephalic slices (450 μm) incubated in artificial cerebrospinal fluid at 33 °C. Interactions between iontophoretically applied NE, isoproterenol (ISO) or phenylephrine (PE) and responses of LH neurons (n = 64) to GABA microiontophoresis were quantitated and characterized using computer-generated ratemeter and histogram records. This analysis revealed two distinct actions of NE on GABA-induced responses of LH neurons. In 8 of 32 cells tested (25%), locally applied NE markedly enhanced inhibitory responses to GABA iontophoresis in a manner identical to that observed in vivo. However, in 20 cells (62.5%), iontophoretic application of NE produced a clear antagonism of GABA responses. NE also exerted dual effects on the background firing rate of LH neurons, causing both inhibition and excitation. Overall, in those cells where NE administration increased spontaneous discharge, it either antagonized or had no effect on GABA-mediated inhibition. In contrast, spontaneous firing rate was never elevated above control levels in those cases where NE potentiated GABA responses. Additional experiments demonstrated that the GABA potentiating actions of the benzodiazepine, flurazepam, were preserved in LH tissue slice preparations. In addition, iontophoretic application of the ß-agonist, ISO, routinely suppressed the spontaneous activity of LH neurons and mimicked the facilitating action of NE on GABA. Likewise, microiontophoretic application of 8-bromo cyclic adenosine monophosphate (AMP) enhanced GABA-induced inhibition of LH firing rate in each of 11 cells tested. On the other hand, local administration of the alpha agonist, PE, routinely produced NE-like antagonism of GABA inhibition along with increases in spontaneous firing rate. Taken together these findings indicate that the commonly observed in vivo phenomena of NE augmentation of GABA and suppression of LH neuron spontaneous firing can be demonstrated in vitro, and most likely result from activation of beta adrenoceptors and subsequent elevation of cyclic AMP levels. As such these results suggest that the in vitro preparation will be a useful tool for further investigation of the transmembrane and intracellular events associated with noradrenergically mediated enhancement of GABA. However, in contrast to results obtained in vivo, NE antagonism of GABA inhibition and excitatory effects on spontaneous activity were more frequently observed in LH slices and appear to be mediated by alpha receptor activation. The reduced capacity of NE to augment GABA in vitro might be related to changes in the balance between α- and ß-mediated effects rather than deficits in GABA-facilitating mechanisms, since ISO, cyclic AMP and the benzodiazepine were all routinely capable of enhancing GABAergic responses.