Extra-axial Endoscopic Third Ventriculostomy: A Novel Treatment for Managing Hydrocephalus Due to Vertebrobasilar Dolichoectasia.

BACKGROUND: Vertebrobasilar dolichoectasia, a rare vascular anomaly, rarely presents with hydrocephalus. The traditional treatment for hydrocephalus is a ventriculoperitoneal shunt. Conventional endoscopic third ventriculostomy can avoid shunt-related complications but is considered risky due to the presence of the dolichoectatic vessel. A subfrontal extra-axial fenestration of the lamina terminalis can circumvent this anatomic constraint and establish cerebrospinal fluid communication between the third ventricle and subarachnoid space. METHODS: We performed an extra-axial endoscopic third ventriculostomy to manage hydrocephalus due to vertebrobasilar dolichoectasia in a 26-year-old male. The clinical description, surgical technique, outcome, and rationale are described. RESULTS: The patient had symptomatic improvement in his headaches and vision. There was also improvement in the postoperative ventricular indices: Evans index-19% reduction, frontal occipital horn ratio-14.1% reduction, and third ventricle index-39.5% reduction. A cine-phase magnetic resonance image showed cerebrospinal fluid flow void through the lamina terminalis fenestration, suggesting patency. CONCLUSIONS: Extra-axial endoscopic third ventriculostomy may be a suitable treatment alternative to circumvent anatomic constraints produced by vertebrobasilar dolichoectasia in performing conventional endoscopic third ventriculostomy.

Leishmania donovani secretory protein nucleoside diphosphate kinase b localizes in its nucleus and prevents ATP mediated cytolysis of macrophages.

Leishmania donovani pathogenicity is closely linked to its ability to live and replicate in the hostile environment of macrophages. All protozoan parasites, including Leishmania, are unable to synthesize purines de novo, and nucleoside diphosphate kinases (NDKs) are enzymes required to preserve the intracellular nucleoside phosphate equilibrium. For some pathogens, secretion of ATP-utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor mediated, ATP-induced death of infected macrophages. Here, Leishmanaia donovani nucleoside diphosphate kinase (LdNDKb) was cloned, expressed and purified by Ni2+-NTA affinity chromatography to elucidate its biological significance. The presence of secreted form of LdNDKb in the medium was confirmed by Western blot analysis. Interestingly, cellular localization by confocal microscopy showed that this protein was localized in the nucleus, inner leaflet of membrane and on the flagella of this parasite which indicates its multiple role in the life cycle of Leishmania donovani. Its possibility to bind with DNA was confirmed by gel retardation assay and electrophoretic mobility shift assay (EMSA) which show the binding with linear and supercoiled is not sequence specific. Further, treatment of J774 macrophages with recombinant LdNdKb and periodate oxidized ATP - a P2X7 receptor antagonist, inhibited ATP-induced cytolysis in vitro, as determined by lactate dehydrogenise release from J774 macrophages. Thus, LdNDKb prevents ATP-mediated host-cell plasma membrane permeabilization by hydrolyzing extracellular ATP, thereby, preserving the integrity of the host cells for the benefit of the parasite. This study indicates that LdNDKb could be explored for its potentiality as a drug/vaccine target against visceral leishmaniasis.

In vitro antidiabetic, antioxidant activities and GC-MS analysis of Rhynchostylis Retusa and Euphorbia Neriifolia leaf extracts.

This study aimed to assess the antidiabetic, and antioxidant potential of Rhynchostylis retusa and Euphorbia neriifolia, well known for traditional ethnomedicinal uses in North-east India. Leaf extracts prepared in water, methanol and petroleum ether were evaluated for in vitro antidiabetic and antioxidant assay using α-amylase inhibition, glucose diffusion method and DPPH radical scavenging activity. The α-amylase inhibition with E. neriifolia methanolic extract at 400 µg/ml (66.67%) and R. retusa aqueous extract at 300 µg/ml (58.15%) were stronger than in equivalent concentrations of acarbose, i.e., 62.17, and 51.52%, respectively. Aqueous extract R. retusa showed a maximum 67.65% inhibition of glucose diffusion at 180 min in comparison to control without leaf extract. The DPPH radical scavenging activity of E. neriifolia extract in methanol was significantly better than equivalent aqueous or ether extract. However, the solvent choice had little impact on antioxidant activity in R. retusa. GC-MS analysis revealed the presence of a large number of phytochemicals in methanol fraction of E. neriifolia aqueous extracts in comparison to R. retusa. Though the in vitro α-amylase inhibition or glucose diffusion retardation implied potential medicinal use of endangered orchid R. retusa and E. neriifolia, further investigation may be warranted for identification of relevant bio-active compounds and in vivo validation of their pharmacological properties.

Intra-arterial onyx embolisation of sphenobasilar sinus fistula using pressure cooker technique: case report and review of the literature.

Dural arterio-venous fistulas of the middle cranial fossa may occur within the dura of lesser or greater sphenoid wings. Lesser sphenoid wing fistulas rarely recruit cortical venous drainage and mostly drain in the cavernous sinus. On the other hand, greater sphenoid wing dural fistulas, also known as paracavernous fistulas or sphenobasilar and sphenopetrosal sinus fistulas, are much more notorious as they almost always connect with the superficial middle cerebral vein resulting in secondary cortical venous reflux and varix formation. Curative transarterial or transvenous endovascular embolisation of fistulous connection is the primary therapeutic strategy, particularly using onyx via the transarterial approach. In the present case we describe a 62-year-old man who presented with significant subarachnoid haemorrhage, intraparenchymal and intra-ventricular bleed. Digital subtraction angiography showed a middle cranial fossa dural arteriovenous fistula in the region of the sphenobasilar sinus with cortical venous reflux and varix formation. The patient underwent successful transarterial endovascular embolisation with complete elimination of the fistula using onyx 34, onyx 18, squid 12 and a Scepter XC balloon using the pressure cooker technique. We also report the development of facial nerve palsy due to inadvertent reflux of onyx in the petrosal branch of the middle meningeal artery.

Mechanistic Paradigms of Natural Plant Metabolites as Remedial Candidates for Systemic Lupus Erythromatosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder involving a dysregulated immune response which ultimately leads to multiple organ failure. Several immunological and cellular checkpoints are available as drug targets. However, the available chemosynthetic drugs such as non-steroidal anti-inflammatory drugs and corticosteroids provide limited therapy with extreme toxicities. Moreover, the disease heterogeneity in SLE is very difficult to manage by a single drug component. Hence, it is imperative to utilize the holistic capabilities of natural plant products as immunomodulators and intracellular signaling regulators, thereby providing an auxiliary option of treatment. Additionally, the herbal drugs also serve as symptomatic relief providers, thereby serving as a prophylactic remedy in case of cerebrovascular, hepatic, nephropathological, hematological, cardiopulmonary, mucocutaneous and musculoskeletal manifestations of SLE. The present review attempts to showcase the current state of knowledge regarding the utility of plant-derived phyto-metabolites with their probable mechanistic roles in treating SLE, by means of targeting the signaling cascade, proinflammatory cytokine production and B-T cell co-stimulation. It is hoped that further preclinical and clinical studies will be embarked upon in order to understand the underlying therapeutic and mechanistic aspects of these medicinal herbs.

Seroepidemiology of Spotted Fever Rickettsiosis in Uttar Pradesh: A Prospective Study.

INTRODUCTION: Spotted Fever Rickettsiosis (SFR), an acute febrile illness caused by Rickettsia rickettsii, R. conorii and R. akari which is associated with considerable morbidity and mortality. SFR is one of the most covert emerging infections of the present time which is prevalent in various parts of India as shown by the increase in the number of clinically diagnosed patients in various states except Uttar Pradesh. AIM: To diagnose SFR in clinically suspected patients using serological tests and recognition of common epidemiologic situations and clinical manifestations of SFR in the state of Uttar Pradesh. MATERIALS AND METHODS: Patients of all age groups presented with a diagnosis of Pyrexia of Unknown Origin (PUO) from May 2013 to February 2015 were evaluated. Testing was done using a nonspecific Weil felix test followed by more specific Enzyme Linked Immunosorbent Assay (ELISA) and a gold standard Immunofluorescence Assay (IFA) test for specific IgM antibodies against Rickettsia conorii. The data was statistically analysed on Graph Pad Prism (5.0) software by using Chi-square test. RESULTS: Of the 432 patient samples tested by non specific Weil felix test, 200 (46.29 %) samples showed titre 1:80 or more and were taken as positive. Similarly out of the 432 blood samples tested by both ELISA and IFA based test against Rickettsiaconorii IgM antibody, only 115 (26.62%) samples were found to be positive and these samples were also positive by Weil felix. The common symptoms noted were fever, hepatomegaly, thrombocytopenia, lymphadenopathy and rashes, nausea followed by icterus, cyanosis, headache, oedema and abdominal pain. Eschar was found in only four (3.4%) patients. We also found that 31 patients with SFR also had associated co-infections like typhoid, malaria, dengue and hepatitis. CONCLUSION: Our findings demonstrated that Weil Felix test can fill in as an underlying yet not sole strategy to perceive and analyse rickettsial ailments, as it needs specificity. So, it may be used to assess the burden in the area and later on other tests like ELISA or IFA can be added, as these are more specific diagnostic tests. Further, our results also showed that if a patient tests positive for the more common endemic infections, we must test for rickettsiosis so that appropriate treatment could be administered.

Withania somnifera chemotype NMITLI 101R significantly increases the efficacy of antileishmanial drugs by generating strong IFN-γ and IL-12 mediated immune responses in Leishmania donovani infected hamsters.

BACKGROUND: Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most important medicinal plant in the traditional Indian medical systems. Pharmacological studies have established that root extracts of W. somnifera contain several bioactive constituents called withanolides. The plant has long been used for its several beneficial properties and recently as an immunomodulator. HYPOTHESIS/PURPOSE: A combination therapy including a potential and safe immunostimulant with lower doses of effective drug, which can reduce the parasitic burden and simultaneously can produce an enhancement of adaptive immunity, has proven to be significantly a more effective approach than immunotherapy or drug therapy alone. STUDY DESIGN: Evaluation of the immunostimulatory effect of W. somnifera chemotype NMITLI 101R when used in combination with ED50 doses of antileishmanial drugs in Leishmania donovani infected hamsters. METHODS: Infected animals were administered with chemotype 101R(30mg/kg × 15 days) either alone or in combination with ED50 doses of miltefosine (10mg/kg × 5 days), paromomycin (30mg/kg × 5 days) or amphotericin B (0.5mg/kg × 5 days). The treated animals were euthanized on days 30 and 60 post-treatment (p.t.) and checked for parasite clearance, delayed type hypersensitivity (DTH) response, cytokine and inducible nitric oxide synthase levels by real-time PCR, nitric oxide (NO) production, reactive oxygen species (ROS) generation, lymphoproliferative and antibody responses. RESULTS: The group of animals that received 101R and ED50 dose of miltefosine showed optimum inhibition of parasite multiplication (∼98%) by day 60 p.t. followed by the group that received 101R plus paromomycin (∼94%) and 101R plus amphotericin B (∼93%). The efficacy was well supported by the increased inducible NO synthase mRNA transcript, strong IFN-γand IL-12 mediated Th1 immune responses and significantly suppressed levels of Th2 cytokines (IL-4, IL-10 and TGF-ß). Additionally, same therapy also induced significant increase in the level of NO production, ROS generation, Leishmania specific IgG2 antibody along with profound DTH and strong T-cell responses as compared with all the other treated groups. CONCLUSION: Our results suggest that combination of chemotype 101R with ED50 doses of antileishmanial drugs may provide a promising alternative for the cure of visceral leishmaniasis with significant restoration of the host immune response.

Electrochemical Characterization of a Novel Exoelectrogenic Bacterium Strain SCS5, Isolated from a Mediator-Less Microbial Fuel Cell and Phylogenetically Related to Aeromonas jandaei.

A facultative anaerobic bacterium, designated as strain SCS5, was isolated from the anodic biofilm of a mediator-less microbial fuel cell using acetate as the electron donor and α-FeOOH as the electron acceptor. The isolate was Gram-negative, motile, and shaped as short rods (0.9-1.3 µm in length and 0.4-0.5 µm in width). A phylogenetic analysis of the 16S rRNA, gyrB, and rpoD genes suggested that strain SCS5 belonged to the Aeromonas genus in the Aeromonadaceae family and exhibited the highest 16S rRNA gene sequence similarity (99.45%) with Aeromonas jandaei ATCC 49568. However, phenotypic, cellular fatty acid profile, and DNA G+C content analyses revealed that there were some distinctions between strain SCS5 and the type strain A. jandaei ATCC 49568. The optimum growth temperature, pH, and NaCl (%) for strain SCS5 were 35°C, 7.0, and 0.5% respectively. The DNA G+C content of strain SCS5 was 59.18%. The isolate SCS5 was capable of reducing insoluble iron oxide (α-FeOOH) and transferring electrons to extracellular material (the carbon electrode). The electrochemical activity of strain SCS5 was corroborated by cyclic voltammetry and a Raman spectroscopic analysis. The cyclic voltammogram of strain SCS5 revealed two pairs of oxidation-reduction peaks under anaerobic and aerobic conditions. In contrast, no redox pair was observed for A. jandaei ATCC 49568. Thus, isolated strain SCS5 is a novel exoelectrogenic bacterium phylogenetically related to A. jandaei, but shows distinct electrochemical activity from its close relative A. jandaei ATCC 49568.

Comparative Analysis of Cellular Immune Responses in Treated Leishmania Patients and Hamsters against Recombinant Th1 Stimulatory Proteins of Leishmania donovani.

Our prior studies demonstrated that cellular response of T helper 1 (Th1) type was generated by a soluble antigenic fraction (ranging from 89.9 to 97.1 kDa) of Leishmania donovani promastigote, in treated Leishmania patients as well as hamsters and showed significant prophylactic potential against experimental visceral leishmaniasis (VL). Eighteen Th1 stimulatory proteins were identified through proteomic analysis of this subfraction, out of which 15 were developed as recombinant proteins. In the present work, we have evaluated these 15 recombinant proteins simultaneously for their comparative cellular responses in treated Leishmania patients and hamsters. Six proteins viz. elongation factor-2, enolase, aldolase, triose phosphate isomerase, protein disulfide isomerase, and p45 emerged as most immunogenic as they produced a significant lymphoproliferative response, nitric oxide generation and Th1 cytokine response in PBMCs and lymphocytes of treated Leishmania patients and hamsters respectively. The results suggested that these proteins may be exploited for developing a successful poly-protein and/or poly-epitope vaccine against VL.

Efficacy of Leishmania donovani trypanothione reductase, identified as a potent Th1 stimulatory protein, for its immunogenicity and prophylactic potential against experimental visceral leishmaniasis.

In visceral leishmaniasis (VL), Th1-type of immune responses play an important role which correlates with recovery from and resistance to disease resulting in lifelong immunity. Based on this rationale, the soluble leishmanial antigens that elicit cellular responses in peripheral blood mononuclear cells (PBMCs) from cured Leishmania patients were characterized through immunoproteomic approach which led to the identification of trypanothione reductase (TPR) (a cytosolic enzyme explored as a drug target), as one of the potent Th1 stimulatory protein. In this study, the immunogenicity of recombinant Leishmania donovani TPR (rLdTPR) was assessed in PBMCs of cured Leishmania-infected patients/hamsters and further evaluated its prophylactic efficacy against L. donovani challenges in hamsters. Substantial proliferative responses to rLdTPR, as compared to soluble L. donovani antigen, were observed in Leishmania-infected cured patients as well as in hamsters. Moreover, rLdTPR reasonably stimulated PBMCs of cured Leishmania patients to produce IFNγ, IL-12, and TNF-α but not IL-4 or IL-10. On the other hand, the protein downregulated LPS-induced IL-10 as well as soluble L. donovani antigen-induced IL-4 production in PBMCs of Leishmania patients. In case of cured hamsters, rLdTPR generates mixed Th1 and Th2 immune response. Vaccination with rLdTPR along with Bacillus Calmette-Guerin (BCG) was able to provide considerably good prophylactic efficacy (~60%) against L. donovani challenge in hamsters. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFNγ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-ß. Since rLdTPR protein is an important target, further attempts towards determination of immunodominant regions for designing fusion peptides may be taken up to optimize its prophylactic efficacy.

Leishmania donovani triose phosphate isomerase: a potential vaccine target against visceral leishmaniasis.

Visceral leishmaniasis (VL) is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI), a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9-97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI) was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT), nitric oxide (NO) production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%). The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.

A novel recombinant Leishmania donovani p45, a partial coding region of methionine aminopeptidase, generates protective immunity by inducing a Th1 stimulatory response against experimental visceral leishmaniasis.

The development of a vaccine against visceral leishmaniasis (VL) conferring long-lasting immunity remains a challenge. Identification and proteomic characterization of parasite proteins led to the detection of p45, a member of the methionine aminopeptidase family. To our knowledge the present study is the first known report that describes the molecular and immunological characterization of p45. Recombinant Leishmania donovani p45 (rLdp45) induced cellular responses in cured hamsters and generated Th1-type cytokines from peripheral blood mononuclear cells of cured/endemic VL patients. Immunization with rLdp45 exerted considerable prophylactic efficacy (∼85%) supported by an increase in mRNA expression of iNOS, IFN-γ, TNF-α and IL-12 and decrease in TGF-ß and IL-4, indicating its potential as a vaccine candidate against VL.

Evaluation of Leishmania donovani protein disulfide isomerase as a potential immunogenic protein/vaccine candidate against visceral Leishmaniasis.

In Leishmania species, Protein disulfide isomerase (PDI)--a redox chaperone, is reported to be involved in its virulence and survival. This protein has also been identified, through proteomics, as a Th1 stimulatory protein in the soluble lysate of a clinical isolate of Leishmania donovani (LdPDI). In the present study, the molecular characterization of LdPDI was carried out and the immunogenicity of recombinant LdPDI (rLdPDI) was assessed by lymphocyte proliferation assay (LTT), nitric oxide (NO) production, estimation of Th1 cytokines (IFN-γ and IL-12) as well as IL-10 in PBMCs of cured/endemic/infected Leishmania patients and cured L. donovani infected hamsters. A significantly higher proliferative response against rLdPDI as well as elevated levels of IFN-γ and IL-12 were observed. The level of IL-10 was found to be highly down regulated in response to rLdPDI. A significant increase in the level of NO production in stimulated hamster macrophages as well as IgG2 antibody and a low level of IgG1 in cured patient's serum was observed. Higher level of IgG2 antibody indicated its Th1 stimulatory potential. The efficacy of pcDNA-LdPDI construct was further evaluated for its prophylactic potential. Vaccination with this construct conferred remarkably good prophylactic efficacy (∼90%) and generated a robust cellular immune response with significant increases in the levels of iNOS transcript as well as TNF-α, IFN-γ and IL-12 cytokines. This was further supported by the high level of IgG2 antibody in vaccinated animals. The in vitro as well as in vivo results thus indicate that LdPDI may be exploited as a potential vaccine candidate against visceral Leishmaniasis (VL).

Subunit Compensation and Plasticity of Synaptic GABA(A) Receptors Induced by Ethanol in α4 Subunit Knockout Mice.

There is considerable evidence that ethanol (EtOH) potentiates γ-aminobutyric acid type A receptor (GABA(A)R) action, but only GABA(A)Rs containing δ subunits appear sensitive to low millimolar EtOH. The α4 and δ subunits co-assemble into GABA(A)Rs which are relatively highly expressed at extrasynaptic locations in the dentate gyrus where they mediate tonic inhibition. We previously demonstrated reversible- and time-dependent changes in GABA(A)R function and subunit composition in rats after single-dose EtOH intoxication. We concluded that early tolerance to EtOH occurs by over-activation and subsequent internalization of EtOH-sensitive extrasynaptic α4ßδ-GABA(A)Rs. Based on this hypothesis, any highly EtOH-sensitive GABA(A)Rs should be subject to internalization following exposure to suitably high EtOH doses. To test this, we studied the GABA(A)Rs in mice with a global deletion of the α4 subunit (KO). The dentate granule cells of these mice exhibited greatly reduced tonic currents and greatly reduced potentiation by acutely applied EtOH, whereas synaptic currents showed heightened sensitivity to low EtOH concentrations. The hippocampus of naive KO mice showed reduced δ subunit protein levels, but increased α2, and γ2 levels compared to wild-type (WT) controls, suggesting at least partial compensation by these subunits in synaptic, highly EtOH-sensitive GABA(A)Rs of KO mice. In WT mice, cross-linking and Western blot analysis at 1 h after an EtOH challenge (3.5 g/kg, i.p.) revealed increased intracellular fraction of the α1, α4, and δ, but not α2, α5, or γ2 subunits. By contrast, we observed significant internalization of α1, α2, δ, and γ2 subunits after a similar EtOH challenge in KO mice. Synaptic currents from naïve KO mice were more sensitive to potentiation by zolpidem (0.3 µM, requiring α1/α2, inactive at α4/5 GABA(A)Rs) than those from naïve WT mice. At 1 h after EtOH, synaptic currents of WT mice were unchanged, whereas those of KO mice were significantly less sensitive to zolpidem, suggesting decreases in functional α1/2ßγ GABA(A)Rs. These data further support our hypothesis that EtOH intoxication induces GABA(A)R plasticity via internalization of highly EtOH-sensitive GABA(A)Rs.

Spatiotemporal specificity of GABAA receptor-mediated regulation of adult hippocampal neurogenesis.

GABAergic transmission regulates adult neurogenesis by exerting negative feedback on cell proliferation and enabling dendrite formation and outgrowth. Further, GABAergic synapses target differentiating dentate gyrus granule cells prior to formation of glutamatergic connections. GABA(A) receptors (GABA(A) Rs) mediating tonic (extrasynaptic) and phasic (synaptic) transmission are molecularly and functionally distinct, but their specific role in regulating adult neurogenesis is unknown. Using global and single-cell targeted gene deletion of subunits contributing to the assembly of GABA(A) Rs mediating tonic (α4, δ) or phasic (α2) GABAergic transmission, we demonstrate here in the dentate gyrus of adult mice that GABA(A) Rs containing α4, but not δ, subunits mediate GABAergic effects on cell proliferation, initial migration and early dendritic development. In contrast, α2-GABA(A) Rs cell-autonomously signal to control positioning of newborn neurons and regulate late maturation of their dendritic tree. In particular, we observed pruning of distal dendrites in immature granule cells lacking the α2 subunit. This alteration could be prevented by pharmacological inhibition of thrombospondin signaling with chronic gabapentin treatment, shown previously to reduce glutamatergic synaptogenesis. These observations point to homeostatic regulation of inhibitory and excitatory inputs onto newborn granule cells under the control of α2-GABA(A) Rs. Taken together, the availability of distinct GABA(A) R subtypes provides a molecular mechanism endowing spatiotemporal specificity to GABAergic control of neuronal maturation in adult brain.

α4-Containing GABA(A) Receptors are Required for Antagonism of Ethanol-Induced Motor Incoordination and Hypnosis by the Imidazobenzodiazepine Ro15-4513.

Alcohol (ethanol) is widely consumed for its desirable effects but unfortunately has strong addiction potential. Some imidazobenzodiazepines such as Ro15-4513 are able to antagonize many ethanol-induced behaviors. Controversial biochemical and pharmacological evidence suggest that the effects of these ethanol antagonists and ethanol are mediated specifically via overlapping binding sites on α4/δ-containing GABA(A)-Rs. To investigate the requirement of α4-containing GABA(A)-Rs in the mechanism of action of Ro15-4513 on behavior, wildtype (WT) and α4 knockout (KO) mice were compared for antagonism of ethanol-induced motor incoordination and hypnosis. Motor effects of ethanol were tested in two different fixed speed rotarod assays. In the first experiment, mice were injected with 2.0 g/kg ethanol followed 5 min later by 10 mg/kg Ro15-4513 (or vehicle) and tested on a rotarod at 8 rpm. In the second experiment, mice received a single injection of 1.5 g/kg ethanol ± 3 mg/kg Ro15-4513 and were tested on a rotarod at 12 rpm. In both experiments, the robust Ro15-4513 antagonism of ethanol-induced motor ataxia that was observed in WT mice was absent in KO mice. A loss of righting reflex (LORR) assay was used to test Ro15-4513 (20 mg/kg) antagonism of ethanol (3.5 g/kg)-induced hypnosis. An effect of sex was observed on the LORR assay, so males and females were analyzed separately. In male mice, Ro15-4513 markedly reduced ethanol-induced LORR in WT controls, but α4 KO mice were insensitive to this effect of Ro15-4513. In contrast, female KO mice did not differ from WT controls in the antagonistic effects of Ro15-4513 on ethanol-induced LORR. We conclude that Ro15-4513 requires α4-containing receptors for antagonism of ethanol-induced LORR (in males) and motor ataxia.

Prototypic GABA(A) receptor agonist muscimol acts preferentially through forebrain high-affinity binding sites.

Muscimol has been regarded as a universal agonist for all gamma-aminobutyric acid type A receptor (GABA(A)-R) subtypes. However, brain regional distribution of muscimol's high-affinity binding sites greatly differs from those of other binding sites of the GABA(A)-R. To test whether behavioral effects of muscimol correlated with the density of high-affinity [(3)H]muscimol binding, we examined several GABA(A)-R subunit gene-modified mouse lines: alpha1, alpha4, or delta-knockouts (KO), alpha4+delta-double KO, and Thy1.2 promoter-driven alpha6 transgenic mice (Thy1alpha6). We determined the high-affinity [(3)H]muscimol binding in brain sections by quantitative autoradiography and sedative/ataxic effects induced in vivo by muscimol using a constant speed rotarod. alpha4-KO mice had reduced [(3)H]muscimol binding in the caudate-putamen, thalamus, and hippocampus, and were less sensitive to the behavioral impairment by muscimol. Similarly, delta-KO mice also had reduced binding to forebrain regions and a lower behavioral sensitivity to muscimol than their wild-type controls. In contrast, alpha1-KO mice had unaltered behavioral sensitivity to muscimol and unaltered [(3)H]muscimol binding, even though previous studies have demonstrated dramatically reduced binding to various other GABA(A)-R sites in these mice. Finally, Thy1alpha6 mice exhibited increased behavioral sensitivity to muscimol, and to another direct GABA-site agonist gaboxadol, and increased [(3)H]muscimol binding in the cerebral cortex and hippocampus. Thus, the differences in sedative and motor-impairing actions of muscimol in various mouse models correlated with the level of forebrain high-affinity [(3)H]muscimol binding. These data suggest that a small special population of GABA(A)-Rs, most likely extrasynaptic non-alpha1-containing receptors, strongly contributes to the in vivo pharmacological effects of muscimol.

Gamma-aminobutyric acid type A receptor alpha 4 subunit knockout mice are resistant to the amnestic effect of isoflurane.

BACKGROUND: General anesthesia produces multiple end points including immobility, hypnosis, sedation, and amnesia. Tonic inhibition via gamma-aminobutyric acid type A receptors (GABA(A)-Rs) may play a role in mediating behavioral end points that are suppressed by low concentrations of anesthetics (e.g., hypnosis and amnesia). GABA(A)-Rs containing the alpha4 subunit are highly concentrated in the hippocampus and thalamus, and when combined with delta subunits they mediate tonic inhibition, which is sensitive to low concentrations of isoflurane. METHODS: In this study, we used a GABA(A) alpha4 receptor knockout mouse line to evaluate the contribution of alpha4-containing GABA(A)-Rs to the effects of immobility, hypnosis, and amnesia produced by isoflurane. Knockout mice and their wild-type counterparts were assessed on 3 behavioral tests: conditional fear (to assess amnesia), loss of righting reflex (to assess hypnosis), and the minimum alveolar concentration of inhaled anesthetic necessary to produce immobility in response to noxious stimulation in 50% of subjects (to assess immobility). RESULTS: Genetic inactivation of the alpha4 subunit reduced the amnestic effect of isoflurane, minimally affected loss of righting reflex, and had no effect on immobility. CONCLUSIONS: These results lend support to the hypothesis that different sites of action mediate different anesthetic end points and suggest that alpha4-containing GABA(A)-Rs are important mediators of the amnestic effect of isoflurane on hippocampal-dependent declarative memory.

Investigation of ethanol-induced impairment of spatial memory in gamma2 heterozygous knockout mice.

GABA(A) receptors, the major inhibitory receptors in the mammalian central nervous system, are affected by a number of drug compounds, including ethanol. The pharmacological effects of certain drugs have been shown to be dependent upon specific GABA(A) receptor subunits. Because benzodiazepines and ethanol have similar effect signatures, it has been hypothesized that these drugs share the gamma2-containing GABA(A) receptors as a mechanism of action. To probe the involvement of the gamma2 subunit in ethanol's actions, spatial memory for the Morris water maze task was tested in gamma2 heterozygous knockout mice and wild type littermate controls following ethanol administration at the following doses: 0.0, 1.25, 1.75, and 2.25 g/kg. While baseline learning and memory were unaffected by reduction of gamma2 containing GABA(A) receptors, ethanol dose-dependently impaired spatial memory equally in gamma2 heterozygous knockouts and wild type littermate controls.