Modulation of extrasynaptic GABAA alpha 5 receptors in the ventral hippocampus normalizes physiological and behavioral deficits in a circuit specific manner.

Hippocampal hyperactivity is correlated with psychosis in schizophrenia patients and likely attributable to deficits in GABAergic signaling. Here we attempt to reverse this deficit by overexpression of the α5-GABAA receptor within the ventral hippocampus (vHipp). Indeed, this is sufficient to normalize vHipp activity and downstream alterations in dopamine neuron function in the MAM rodent model. This approach also attenuated behavioral deficits in cognitive flexibility. To understand the specific pathways that mediate these effects, we used chemogenetics to manipulate discrete projections from the vHipp to the nucleus accumbens (NAc) or prefrontal cortex (mPFC). We found that inhibition of the vHipp-NAc, but not the vHipp-mPFC pathway, normalized aberrant dopamine neuron activity. Conversely, inhibition of the vHipp-mPFC improved cognitive function. Taken together, these results demonstrate that restoring GABAergic signaling in the vHipp improves schizophrenia-like deficits and that distinct behavioral alterations are mediated by discrete projections from the vHipp to the NAc and mPFC.

Stem cell-derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model.

An increasing literature suggests that schizophrenia is associated with a reduction in hippocampal interneuron function. Thus, we posit that stem cell-derived interneuron transplants may be an effective therapeutic strategy to reduce hippocampal hyperactivity and attenuate behavioral deficits in schizophrenia. Here we used a dual-reporter embryonic stem cell line to generate enriched populations of parvalbumin (PV)- or somatostatin (SST)-positive interneurons, which were transplanted into the ventral hippocampus of the methylazoxymethanol rodent model of schizophrenia. These interneuron transplants integrate within the existing circuitry, reduce hippocampal hyperactivity and normalize aberrant dopamine neuron activity. Further, interneuron transplants alleviate behaviors that model negative and cognitive symptoms, including deficits in social interaction and cognitive inflexibility. Interestingly, PV- and SST-enriched transplants produced differential effects on behavior, with PV-enriched populations effectively normalizing all the behaviors examined. These data suggest that the stem cell-derived interneuron transplants may represent a novel therapeutic strategy for schizophrenia.

Activation of a ventral hippocampus-medial prefrontal cortex pathway is both necessary and sufficient for an antidepressant response to ketamine.

A single sub-anesthetic dose of ketamine exerts rapid and sustained antidepressant effects. Here, we examined the role of the ventral hippocampus (vHipp)-medial prefrontal cortex (mPFC) pathway in ketamine's antidepressant response. Inactivation of the vHipp with lidocaine prevented the sustained, but not acute, antidepressant-like effect of ketamine as measured by the forced swim test (FST). Moreover, optogenetic as well as pharmacogenetic specific activation of the vHipp-mPFC pathway using DREADDs (designer receptors exclusively activated by designer drugs) mimicked the antidepressant-like response to ketamine; importantly, this was pathway specific, in that activation of a vHipp to nucleus accumbens circuit did not do this. Furthermore, optogenetic inactivation of the vHipp/mPFC pathway at the time of FST completely reversed ketamine's antidepressant response. In addition, we found that a transient increase in TrkB receptor phosphorylation in the vHipp contributes to ketamine's sustained antidepressant response. These data demonstrate that activity in the vHipp-mPFC pathway is both necessary and sufficient for the antidepressant-like effect of ketamine.

Isolation of a species-specific DNA probe for Neisseria gonorrhoeae using a novel technique particularly suitable for use with closely related species displaying high levels of DNA homology.

The use of nucleic acid probes has become an increasingly common method for detecting pathogenic micro-organisms in clinical specimens. In the course of our efforts to isolate species-specific DNA probes for bacterial pathogens, we encountered a special problem with regard to Neisseria gonorrhoeae. As a consequence of the high degree of DNA homology that this organism displays with its nearest relative, Neisseria meningitidis, the isolation of such probes could not be readily achieved. We therefore developed a novel method of probe isolation which overcomes this problem. This methodology relies upon the application of a 'sandwich' hybridization assay to screen an M13 'shotgun' library derived from N. gonorrhoeae genomic DNA. For this, genomic DNA from N. gonorrhoeae and N. meningitidis was immobilized on nitrocellulose filters and probed with recombinant phage DNA from candidate clones. Those clones which had hybridized to target sequences were then detected using labelled vector sequences in a second hybridization step. By this means we obtained a numerical assessment of the degree of specificity of candidate clones for the target organism as compared to one or more related species. Using this technique we have isolated three DNA probes which are highly specific for N. gonorrhoeae and which display no cross-reactivity with N. meningitidis or other members of the Neisseriaceae. This paper presents the basis of the methodology and describes the isolation and characterization of three N. gonorrhoeae-like specific probes.

DNA probes for mycobacteria. I. Isolation of DNA probes for the identification of Mycobacterium tuberculosis complex and for mycobacteria other than tuberculosis (MOTT).

Traditional methods used in identifying mycobacteria such as acid-fast bacillus stains and culture are often time-consuming, insensitive and non-specific. The isolation of DNA probes, coupled to a non-radioactive, e.g. biotin-based detection system, have the potential to foster the development of clinical assays for Mycobacterium tuberculosis and mycobacteria other than tuberculosis (MOTT) that are rapid, sensitive and specific. To this end, we have isolated two different probes: one which is specific for the Mtb complex and one which recognizes all other potentially pathogenic mycobacteria. The use of these probes in combination should allow the detection and differentiation of M. tuberculosis from MOTT. To isolate the first probe, we prepared a library of M. tuberculosis DNA fragments in a lambda EMBL phage vector. Recombinant phage were screened by plaque-lift hybridization procedures using nick-translated mycobacterial genomic DNA to identify sequences specific to the Mtb complex. Inserts from candidate recombinant phage were purified, nick-translated and hybridized against a wide variety of filter-bound mycobacterial and non-mycobacterial DNAs. Two clones were identified which hybridized to the closely related M. tuberculosis, M. bovis and M. microti but not to other species of mycobacteria. The second probe was isolated by preparing a library of M. malmoense DNA fragments in lambda EMBL and screening by plaque-lift hybridization. One clone was identified which, in addition to recognizing members of the Mtb complex, also hybridized to M. intracellulare, M. malmoense, M. scrofulaceum, M. simiae, M. xenopi, M. avium, M. szulgai, M. kansasii and M. haemophilum. None of the three clones hybridized to DNA from non-mycobacterial species.

Evidence that both growing DNA chains at a replication fork are synthesized discontinuously.

Escherichia coli, Bacillus subtilis, and T7-infected E. coli have been labeled with short pulses of [3H] thymidine, and the labeled DNA has been examined by sedimentation in alkaline sucrose. In all three systems, the great majority of the DNA labeled by a short pulse is found in the form of small DNA chains of 10S, the so-called Okazaki pieces. The B. subtilis and T7 nascent DNA fragments hybridize with equal efficiency to the separated strands of B. subtilis and T7 DNA, respectively. The results suggest that both growing DNA chains at a given replication fork are synthesized discontinuously in the case of E. coli, B. subtilis, and T7. We have found that the method used to terminate the pulse affects the size distribution of the labeled DNA; some methods allow joining of nascent DNA fragments after termination of the pulse. Previous reports of discontinuous DNA synthesis on only one growing DNA chain and continuous synthesis on the other DNA chain are probably due to preferential joining of Okazaki pieces on the DNA chain growing in the overall 5' leads to 3' direction.