Evaluation of the Cepheid Xpert Flu Assay for rapid identification and differentiation of influenza A, influenza A 2009 H1N1, and influenza B viruses.

The Xpert Flu Assay cartridge is a next-generation nucleic acid amplification system that provides multiplexed PCR detection of the influenza A, influenza A 2009 H1N1, and influenza B viruses in approximately 70 min with minimal hands-on time. Six laboratories participated in a clinical trial comparing the results of the new Cepheid Xpert Flu Assay to those of culture or real-time PCR with archived and prospectively collected nasal aspirate-wash (NA-W) specimens and nasopharyngeal (NP) swabs from children and adults. Discrepant results were resolved by DNA sequence analysis. After discrepant-result analysis, the sensitivities of the Xpert Flu Assay for prospective NA-W specimens containing the influenza A, influenza A 2009 H1N1, and influenza B viruses compared to those of culture were 90.0%, 100%, and 100%, respectively, while the sensitivities of the assay for prospective NP swabs compared to those of culture were 100%, 100%, and 100%, respectively. The sensitivities of the Xpert Flu Assay for archived NA-W specimens compared to those of Gen-Probe ProFlu+ PCR for the influenza A, influenza A 2009 H1N1, and influenza B viruses were 99.4%, 98.4%, and 100%, respectively, while the sensitivities of the Xpert Flu Assay for archived NP swabs compared to those of ProFlu+ were 98.1%, 100%, and 93.8%, respectively. The sensitivities of the Xpert Flu Assay with archived NP specimens compared to those of culture for the three targets were 97.5%, 100%, and 93.8%, respectively. We conclude that the Cepheid Xpert Flu Assay is an accurate and rapid method that is suitable for on-demand testing for influenza viral infection.

Phenotypic heterogeneity and genetic modification of P102L inherited prion disease in an international series.

The largest kindred with inherited prion disease P102L, historically Gerstmann-Sträussler-Scheinker syndrome, originates from central England, with émigrés now resident in various parts of the English-speaking world. We have collected data from 84 patients in the large UK kindred and numerous small unrelated pedigrees to investigate phenotypic heterogeneity and modifying factors. This collection represents by far the largest series of P102L patients so far reported. Microsatellite and genealogical analyses of eight separate European kindreds support multiple distinct mutational events at a cytosine-phosphate diester-guanidine dinucleotide mutation hot spot. All of the smaller P102L kindreds were linked to polymorphic human prion protein gene codon 129M and were not connected by genealogy or microsatellite haplotype background to the large kindred or each other. While many present with classical Gerstmann-Sträussler-Scheinker syndrome, a slowly progressive cerebellar ataxia with later onset cognitive impairment, there is remarkable heterogeneity. A subset of patients present with prominent cognitive and psychiatric features and some have met diagnostic criteria for sporadic Creutzfeldt-Jakob disease. We show that polymorphic human prion protein gene codon 129 modifies age at onset: the earliest eight clinical onsets were all MM homozygotes and overall age at onset was 7 years earlier for MM compared with MV heterozygotes (P = 0.02). Unexpectedly, apolipoprotein E4 carriers have a delayed age of onset by 10 years (P = 0.02). We found a preponderance of female patients compared with males (54 females versus 30 males, P = 0.01), which probably relates to ascertainment bias. However, these modifiers had no impact on a semi-quantitative pathological phenotype in 10 autopsied patients. These data allow an appreciation of the range of clinical phenotype, modern imaging and molecular investigation and should inform genetic counselling of at-risk individuals, with the identification of two genetic modifiers.

Association of a null allele of SPRN with variant Creutzfeldt-Jakob disease.

BACKGROUND: No susceptibility genes have been identified in human prion disase, apart from the prion protein gene (PRNP). The gene SPRN, encodes Shadoo (Sho, shadow of prion protein) which has protein homology and possible functional links with the prion protein. METHODS: A genetic screen was carried out of the open reading frame of SPRN by direct sequencing in 522 patients with prion disease, including 107 with variant Creutzfeldt-Jakob disease (vCJD), and 861 healthy controls. RESULTS: A common coding variant of SPRN, two further single nucleotide polymorphisms (SNPs) and three rare insertion or deletion variants were found. A single base-pair insertion at codon 46, predicted to cause a frameshift and potentially a novel protein, was found in two patients with vCJD but not in controls (p = 0.01). Two linked SNPs, one in intron 1 and the other a missense variant at codon 7, were associated with risk of sporadic CJD (p = 0.009). CONCLUSION: These data justify the functional genetic characterisation of SPRN and support the involvement of Shadoo in prion pathobiology.

Anxiety responses, plasma corticosterone and central monoamine variations elicited by stressors in reactive and nonreactive mice and their reciprocal F1 hybrids.

Stressor-provoked anxiety, plasma corticosterone, and variations of brain monoamine turnover are influenced by genetic factors, but may also be moderated by early life experiences. To evaluate the contribution of maternal influences, behavioral and neurochemical stress responses were assessed in strains of mice that were either stressor-reactive or -resilient (BALB/cByJ and C57BL/6ByJ, respectively) as well as in their reciprocal F(1) hybrids. BALB/cByJ mice demonstrated poorer maternal behaviors than did C57BL/6ByJ dams, irrespective of the pups being raised (inbred or F(1) hybrids). The BALB/cByJ mice appeared more anxious than C57BL/6ByJ mice, exhibiting greater reluctance to step-down from a platform and a greater startle response. Although the F(1) behavior generally resembled that of the C57BL/6ByJ parent strain, in the step-down test the influence of maternal factors were initially evident among the F(1) mice (particularly males) with a BALB/cByJ dam. However, over trials the C57BL/6ByJ-like behavior came to predominate. BALB/cByJ mice also exhibited greater plasma corticosterone elevations, 5-HT utilization in the central amygdala (CeA), and greater NE turnover in the paraventricular nucleus of the hypothalamus (PVN). Interestingly, among the F(1)'s corticosterone and 5-HIAA in the CeA resembled that of the BALB/cByJ parent strain, whereas MHPG accumulation in the PVN was more like that of C57BL/6ByJ mice. It seems that, to some extent, maternal factors influenced anxiety responses in the hybrids, but did not influence the corticosterone or the monoamine variations. The inheritance profiles suggest that anxiety was unrelated to either the corticosterone or monoamine changes.

The neurosteroid THDOC differentially affects spatial behavior and anesthesia in Slow and Fast kindling rat strains.

Rats selectively bred for "Fast" or "Slow" kindling epileptogenesis express different GABA(A) receptor subunits that may account for differences in their miniature inhibitory postsynaptic currents (mIPSCs). The neurosteroid tetrahydrodeoxycorticosterone (THDOC), an endogenous modulator of GABA-mediated inhibition with anesthetic properties and effects on mnemonic processes, preferentially enhances the mIPSCs recorded from the interneurons of Fast rats. Here we show that the anesthetic effect of 20 mg/kg THDOC was reduced in Fast compared to Slow rats. Further, as the strains have previously been shown to differ in their spatial learning abilities, we subsequently examined the effect of a lower dose (5 mg/kg) of THDOC on their performance in the Morris water maze using a matching-to-place paradigm. THDOC injection deteriorated the usually superior mnemonic capabilities of the Slow rats, i.e., concept learning as well as working and reference memory, while marginally improving these behaviors in Fast rats. These outcomes may reflect the divergent expression of GABAA receptors or disinhibition on interneurons versus principal cells that have been observed between the two strains. Possible mechanisms are discussed.

Regeneration of dopaminergic function in 6-hydroxydopamine-lesioned rats by neuroimmunophilin ligand treatment.

Nonimmunosuppressant immunophilin ligands have been found previously to stimulate neurite growth in culture and to promote regeneration of peripheral and central nerve fibers in vivo. To further characterize the effectiveness of these ligands, we have investigated the effect of the immunophilin ligand GPI-1046 in 6-hydroxydopamine (6-OHDA)-lesioned rats. In unlesioned rats, tetanic stimulation of the white matter induced long-term potentiation (LTP) of corticostriatal synaptic transmission as indicated by a 40-100% increase in the field potential amplitudes recorded in striatal brain slices. Unilateral microinjection of 6-OHDA into the substantia nigra resulted in a loss of corticostriatal LTP and in significant abnormality of motor behavior as assessed by amphetamine-induced ipsilateral rotations. Daily treatment of 6-OHDA-lesioned rats with GPI-1046 (10 mg/kg, s.c.) for 1 week reduced amphetamine-induced rotations by 75% and greatly restored the striatal tyrosine hydroxylase immunostaining. In addition, GPI-1046 almost completely restored corticostriatal LTP in 6-OHDA-lesioned animals. LTP in normal animals and that restored by GPI-1046 in lesioned animals were both blocked by the NMDA receptor antagonist APV, suggesting mediation by NMDA receptors. Both LTPs were sensitive to dopamine (DA) receptor antagonists. The nonselective DA receptor antagonist chlorpromazine and the selective D1-D5 receptor antagonist SCH23390 reduced the LTP by 90%. These results demonstrate that the immunophilin ligand GPI-1046 can reverse the abnormalities in the substantia nigra-striatal dopaminergic system that are caused by 6-OHDA, thus providing a potential therapeutic agent for Parkinson's disease.

Differential expression of alpha1, alpha2, alpha3, and alpha5 GABAA receptor subunits in seizure-prone and seizure-resistant rat models of temporal lobe epilepsy.

Temporal lobe epilepsy remains one of the most widespread seizure disorders in man, the etiology of which is controversial. Using new rat models of temporal lobe epilepsy that are either prone or resistant to develop complex partial seizures, we provide evidence that this seizure susceptibility may arise from arrested development of the GABAA receptor system. In seizure-prone (Fast kindling) and seizure-resistant (Slow kindling) rat models, both the mRNA and protein levels of the major alpha subunit expressed in adult brain (alpha1), as well as those highly expressed during development (alpha2, alpha3, and alpha5), were differentially expressed in both models compared with normal controls. We found that alpha1 subunit mRNA expression in the Fast kindling strain was approximately half the abundance of control rats, whereas in the Slow kindling strain, it was approximately 70% greater than that of controls. However, Fast rats overexpressed the alpha2, alpha3, and alpha5 ("embryonic") subunits, having a density 50-70% greater than controls depending on brain area, whereas the converse was true of Slow rats. Using subunit-specific antibodies to alpha1 and alpha5 subunits, quantitative immunoblots and immunocytochemistry revealed a concordance with the mRNA levels. alpha1 protein expression was approximately 50% less than controls in the Fast strain, whereas it was 200% greater in the Slow strain. In contrast, alpha5 subunit protein expression was greater in the Fast strain than either the control or Slow strain. These data suggest that a major predispositional factor in the development of temporal lobe epilepsy could be a failure to complete the normal switch from the GABAA receptor alpha subunits highly expressed during development (alpha2, alpha3, and alpha5) to those highly expressed in adulthood (alpha1).

The pathogenesis of clinical depression: stressor- and cytokine-induced alterations of neuroplasticity.

Stressful events promote neurochemical changes that may be involved in the provocation of depressive disorder. In addition to neuroendocrine substrates (e.g. corticotropin releasing hormone, and corticoids) and central neurotransmitters (serotonin and GABA), alterations of neuronal plasticity or even neuronal survival may play a role in depression. Indeed, depression and chronic stressor exposure typically reduce levels of growth factors, including brain-derived neurotrophic factor and anti-apoptotic factors (e.g. bcl-2), as well as impair processes of neuronal branching and neurogenesis. Although such effects may result from elevated corticoids, they may also stem from activation of the inflammatory immune system, particularly the immune signaling cytokines. In fact, several proinflammatory cytokines, such as interleukin-1, tumor necrosis factor-alpha and interferon-gamma, influence neuronal functioning through processes involving apoptosis, excitotoxicity, oxidative stress and metabolic derangement. Support for the involvement of cytokines in depression comes from studies showing their elevation in severe depressive illness and following stressor exposure, and that cytokine immunotherapy (e.g. interferon-alpha) elicited depressive symptoms that were amenable to antidepressant treatment. It is suggested that stressors and cytokines share a common ability to impair neuronal plasticity and at the same time altering neurotransmission, ultimately contributing to depression. Thus, depressive illness may be considered a disorder of neuroplasticity as well as one of neurochemical imbalances, and cytokines may act as mediators of both aspects of this illness.

Common polymorphism in a highly variable region upstream of the human lactase gene affects DNA-protein interactions.

In most mammals lactase activity declines after weaning when lactose is no longer part of the diet, but in many humans lactase activity persists into adult life. The difference responsible for this phenotypic polymorphism has been shown to be cis-acting to the lactase gene. The causal sequence difference has not been found so far, but a number of polymorphic sites have been found within and near to the lactase gene. We have shown previously that in Europeans there are two polymorphic sites in a small region between 974 bp and 852 bp upstream from the start of transcription, which are detectable by denaturing gradient gel electrophoresis (DGGE). In this study, analysis of individuals from five other population groups by the same DGGE method reveals four new alleles resulting from three additional nucleotide changes within this very small region. Analysis of sequence in four primate species and comparison with the published pig sequence shows that the overall sequence of this highly variable human region is conserved in pigs as well as primates, and that it lies within a 1kb region which has been shown to control lactase downregulation in pigs. Electrophoretic mobility shift assay (EMSA) studies were carried out to determine whether common variation affected protein-DNA binding and several binding activities were found using this technique. A novel two base-pair deletion that is common in most populations tested, but is not present in Europeans, caused no change in binding activity. However, a previously published C to T transition at -958bp dramatically reduced binding activity, although the functional significance of this is not clear.

Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells.

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR1(1XX); NR1(0XX), and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.

Ontogeny of GABAA receptor subunit mRNAs in rat spinal cord and dorsal root ganglia.

Relatively little is known about the development of GABAA receptor subunits and their gene expression in mammalian spinal cord. The expression of mRNAs encoding 13 GABAA receptor subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) in embryonic, postnatal, and adult rat spinal cord and dorsal root ganglia (DRG) cells were studied by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Both techniques revealed the presence of all subunit mRNAs originally found in the rat brain, except for alpha 6, which was not detectable, and delta, which was weakly detected only by RT-PCR. Two anatomically distinctive sets of subunit mRNAs were found by in situ hybridization within the ventricular zone (VZ) and mantle zone (MZ). The trio of alpha 4, beta 1, and gamma 1 subunit mRNAs emerged exclusively in neuroepithelial cells at embryonic day 13 (E13) and remained detectable in the VZ until E17. In the MZ, beta 3 subunit mRNA was first detected at E12, while alpha 2, alpha 3, alpha 5, beta 2, gamma 2, and gamma 3 transcripts appeared at E13. Expressions of the subunit mRNAs in the MZ rapidly increased and expanded in a ventrodorsal sequence from motoneurons to dorsal horn neurons before reaching a peak in the late embryonic/early postnatal period. The mRNA expressions declined during postnatal development, by region-selective depletion, with alpha 4, alpha 5, beta 1, beta 2, gamma 1, and gamma 3 subunit mRNAs becoming barely detectable. In contrast, alpha 2, alpha 3, beta 3, and gamma 2 transcripts persisted into adulthood with distinct anatomical distributions. RT-PCR analysis revealed unique developmental patterns in the intensities of PCR products, most of which were in good agreement with developmental changes in the densities of hybridized mRNA signals. However, RT-PCR amplified minute amounts of mRNAs for alpha 1, alpha 4, alpha 5, beta 1, beta 2, gamma 1, gamma 3, and delta subunits in adults, which were not found in film autoradiograms, but could be detected in a few grain-positive cells in emulsion-dipped sections. DRG cells expressed alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2 subunit mRNAs during embryogenesis but only alpha 2, beta 3, and gamma 2 subunit mRNAs were reliably detected in the adult.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterisation and distribution of vasoactive intestinal polypeptide binding sites in human brain.

The binding of 125I-vasoactive intestinal polypeptide (VIP) to human brain membranes has been studied. The binding was saturable with a dissociation constant (KD) of 4.4 +/- 2.0 nM and maximum binding value (Bmax) of 8.6 +/- 6.2 fmoles/mg tissue (mean +/- SD, n = 4), and was displaced by unlabelled VIP with an IC50 of 4 nM; secretin was much less potent with an IC50 of approximately 8 microM. The regional distribution of 125I-VIP binding in human brain revealed highest binding values to be in frontal and temporal cortices with intermediate values in amygdala, caudate and cerebellum, with lowest values in hippocampus, substantia nigra and hypothalamus.

Neurotransmitter receptors and monoamine metabolites in the brains of patients with Alzheimer-type dementia and depression, and suicides.

In patients with Alzheimer-type dementia, in addition to the well-known losses of cholinergic neurones, there is evidence of degeneration of the noradrenergic and serotonergic innervation of the cerebral cortex. While noradrenergic and cholinergic receptors are preserved there is a loss of serotonin S1 and S2 receptors, particularly in the temporal lobe. The loss of serotonin S2 receptors may occur at an early stage of the disease and, in temporal and frontal cortex, is correlated with the loss of somatostatin immunoreactivity. In patients dying in hospital with depression, and in individuals committing suicide, there are no consistent changes in monoamine metabolites. Noradrenergic, serotonergic, and other neurotransmitter receptors were found to be unchanged, although there was a moderate decrease in imipramine binding in a small group (n = 6) of subjects with a history of depression, who had committed suicide.

Different voltage-dependent potassium conductances regulate action potential repolarization and excitability in frog myelinated axon.

Intracellular microelectrode recordings were used to examine the effects of the potassium channel blockers: 4-aminopyridine, a selective blocker of fast potassium conductances gKf1 and gKf2,13 and tetraethylammonium, a blocker of gKf1, gKf2 and the slow conductance gKs,13 on the repetitive activity of large myelinated axons of frog. The blockers were applied intracellularly by diffusional leak of the agents from the recording microelectrode containing either 4-aminopyridine or a mixture of 4-aminopyridine and tetraethylammonium. A decrease in outward rectification, a measure of the block of the potassium conductances, was evident within 5 min of axon impalement. Within 30 min 80% of maximal blockade was observed during prolonged recording sessions (> 1 h). Parallel with the resistance increase, the action potential duration increased (up to 5 ms). This was attributed to the block of gKf2. The excitability regularly increased, manifested as a train of action potentials (a decrease in accommodation) for a maximum of 200 ms (54 +/- 8 vs 111 +/- 22, 4-aminopyridine vs 4-aminopyridine-tetraethylammonium, respectively, n = 8 and 6, P < 0.006). The presence of 4-aminopyridine-tetraethylammonium in the microelectrodes decreased the spike frequency adaptation (the instantaneous action potential frequency per spike interval number) observed in fibres treated with 4-aminopyridine alone (32 +/- 9 vs 7 +/- 1 Hz; 4-aminopyridine vs 4-aminopyridine-tetraethylammonium, n = 8 and 6, P < 0.04). This effect was attributed to block of gKs by the tetraethylammonium.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a sodium-dependent potassium conductance in frog myelinated axon.

After blockade of the voltage-dependent potassium conductances by intracellular application of 4-aminopyridine and tetraethylammonium in frog myelinated axons, a set of brief (0.1 ms) intracellular depolarizing pulses or a long (200 ms) depolarizing pulse evoked a train of action potentials. Under both experimental conditions a hyperpolarizing afterpotential appeared (duration 367 ms +/- 34, mean +/- S.E., n = 15). The purpose of this study was to investigate the properties of this hyperpolarizing afterpotential. It was found that the hyperpolarizing afterpotential increases in amplitude with: (1) the number of sodium-dependent action potentials; (2) action potential broadening (following potassium channels blockade); and (3) the level of depolarization during a current step. Application of tetrodotoxin prevented the activation of the hyperpolarizing afterpotential by any of the above stimuli. The hyperpolarizing afterpotential was unaffected by: (1) 8-acetyl-strophanthidin, an agent that poisons the electrogenic pumping in the axon; (2) blocking calcium influx with extracellular 10 mM magnesium or 2 mM manganese; and (3) buffering of the intracellular calcium, using EGTA in the recording microelectrode. Extracellular application of tetraethylammonium, but not 4-aminopyridine, reduced the hyperpolarizing afterpotential. The hyperpolarizing afterpotential reversed at >> -92 mV. Increasing the external potassium concentration from 2 to 10 mM shifted the reversal potential +14.5 mV, indicating that the hyperpolarizing afterpotential is a potassium mediated conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroimmunophilins: a novel drug therapy for the reversal of neurodegenerative disease?

Neuroimmunophilin ligands (NILs) are drugs derived from the immunosuppressant FK506 (tacrolimus) that have been shown to have variable efficacy in reversing neuronal degeneration and preventing cell death. In a wide range of animal models mimicking Parkinson's disease, dementia and even surgical nerve damage they induce re-sprouting, are neurotrophic or prevent nerve damage. The neurotrophic mechanism of action of these compounds is not known and may be dependent on the type of damage and genetic variability at the species or cellular level. Some evidence suggests that NILs may act through a family of proteins called FK506 binding proteins, some of which may regulate steroid hormone receptors. Other evidence suggests that NILs may protect neurons by upregulating the antioxidant glutathione and stimulating nerve regrowth by inducing the production of neurotrophic factors. Initial clinical trials have had mixed success. In one, patients with moderately severe Parkinson's disease showed no overall improvement in fine motor skills following 6 months of treatment by the neuroimmunophilin GPI 1485. But these patients did exhibit decreased loss of dopaminergic nerve terminals with a low dose of GPI 1485 and in fact some increase in dopaminergic terminals within 6 months of the higher dose of GPI 1485 drug treatment. As a result, a second phase II clinical trial using a patient population with less severe degeneration has been initiated concurrent with an investigation of GPI 1485 and other neuroprotective therapies funded by the National Institute of Neurological Disorders and Stroke. Another clinical trial ongoing at this time is exploring the use of a neuroimmunophilin ligand to prevent nerve degeneration and erectile dysfunction resulting from prostatectomy. In summary, neuroimmunophilins show promise to reverse some forms of neurodegeneration but exact factors that predict outcome have not been identified.

GABA(A) receptor subunit messenger RNA expression in the enteric nervous system of the rat: implications for functional diversity of enteric GABA(A) receptors.

GABAergic neurons occur in the myenteric plexus and submucosa and their innervations of the gut, where GABA stimulates motor neurons, and non-neural cells via "central type" GABA(A) receptors. These receptors occur on half of the neurons in the rat intestine. The GABA(A) receptor is a ligand-gated chloride channel constructed from different subunit families (alpha, beta, gamma, delta, epsilon). In rat these exist as subtypes, alpha1-6, beta1-3, gamma1-3 and delta, defining the clinically relevant pharmacological features of GABA(A) receptors. However, the identity, distribution, and abundance of enteric GABA(A) receptor subunits are unknown. To identify and map the regional expression of GABA(A) receptor subunit messenger RNAs in the enteric nervous system, we assayed enteric RNA from the ileum of Sprague-Dawley rats by reverse transcription-polymerase chain reaction for alpha1-6, beta 1-3, gamma1-3, and delta subunit messenger RNAs. Subunit messenger RNA localization, was probed by in situ hybridization. Reverse transcription-polymerase chain reaction analysis of RNA from myenteric and submucosal nerve layers revealed the expression alpha1, alpha3, beta2, beta3, gamma1 and gamma3 subunit messenger RNAs. Little alpha4 and alpha6 and no alpha2, beta1, gamma2 or delta subunit messenger RNA were detected. In situ hybridization revealed that transcripts for alpha1, alpha3, alpha5 and beta2 subunits occur in both myenteric and submucous ganglia. However, beta3 messenger RNA was found only in myenteric plexus. The gamma1 subunit messenger RNA was also restricted to the cells in the myenteric plexus while gamma3 was found in cells of both nerve layers. In this study of the subunit messenger RNA expression profile of GABA(A) receptors within the enteric nerve layers we show an abundant, diverse and widespread distribution that is unique in comparison to the CNS. The distinctive and heterogeneous distribution of enteric GABA(A) subunits may be important in the integration of neural control of gut function.

Co-existent expression of GABAA receptor beta 2, beta 3 and gamma 2 subunit messenger RNAs during embryogenesis and early postnatal development of the rat central nervous system.

The expression of beta 1, beta 2, beta 3, gamma 2 and delta subunit messenger RNAs of the GABAA receptor was followed by in situ hybridization histochemistry using radiolabeled oligodeoxynucleotide probes in sections of embryonic (E12-21) and early postnatal (P1-5) rat. beta 2, beta 3 and gamma 2 subunit messenger RNAs were first detectable at E15 in the spinal cord (ventral > dorsal) and lower central nervous system regions (e.g. pons, medulla and thalamus). beta 3 subunit messenger RNA was abundantly expressed in olfactory bulb neurons at E15. At E17, the expression pattern of these subunit messenger RNAs continued in the lower central nervous system. In the upper central nervous system, beta 2, beta 3, and gamma 2 subunit messenger RNAs were first detectable in the outer layer of the hippocampal and entire cortical neuroepithelium. The expression for both beta 3 and gamma 2 subunit messenger RNAs increased significantly over that observed at E15, whereas beta 2 subunit messenger RNA increased to a lesser extent and was more discretely expressed in inferior colliculus, cerebellar neuroepithelium and spinal cord (ventral = dorsal). By E19, messenger RNAs for beta 2, beta 3 and gamma 2 subunits a widespread and abundant co-existent distribution throughout the central nervous system. Exceptions to this co-expression were the absence of beta 2 messenger RNA in the dentate gyrus and beta 3 messenger RNA in entorhinal cortex, areas in which they are present in adult. There was also a differential distribution of subunit messenger RNAs in developing olfactory bulb at E19-20: the glomerular cells preferentially expressed beta 3 and gamma 2 subunit messenger RNAs; the mitral cells preferentially expressed beta 2 subunit messenger RNA; inner granule cells expressed moderate levels of beta 2, beta 3 and gamma 2 subunit messenger RNAs. Expression of beta 2, beta 3 and gamma 2 messenger RNAs was also anatomically co-existent at P5. In addition, significant expression of beta 1 and delta subunit messenger RNAs was apparent in hippocampus and entorhinal cortex. The identity of the gamma 2 expressed between E15 and E21 was shown to be mostly the short isoform of gamma 2 subunit messenger RNA. Expression of both forms was evident beginning around P3-5. These results indicate that during the late embryonic and early postnatal period of development, beta 2, beta 3 and gamma 2 subunit messenger RNAs are abundantly expressed and co-localized to most central nervous system regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Search for linkage to schizophrenia on the X and Y chromosomes.

Markers for X chromosome loci were used in linkage studies of a large group of small families (n = 126) with at least two schizophrenic members in one sibship. Based on the hypothesis that a gene for schizophrenia could be X-Y linked, with homologous loci on both X and Y, our analyses included all families regardless of the pattern of familial inheritance. Lod scores were computed with both standard X-linked and a novel X-Y model, and sib-pair analyses were performed for all markers examining the sharing of maternal alleles. Small positive lod scores were obtained for loci pericentromeric, from Xp11.4 to Xq12. Lod scores were also computed separately in families selected for evidence of maternal inheritance and absence of male to male transmission of psychosis. The lod score for linkage to the locus DXS7 reached a maximum of 1.83 at 0.08% recombination, assuming dominant inheritance on the X chromosome in these families (n = 34). Further investigation of the X-Y homologous gene hypothesis focussing on this region is warranted.

Male siblings with schizophrenia share alleles at the androgen receptor above chance expectation.

In families that included two or more siblings with schizophrenia or schizo-affective disorder male-male pairs were found to share alleles at the androgen receptor (AR) gene (in Xq11.2-q12) above chance expectation (p < 0.003); female-female and mixed sex pairs showed no such tendency. The findings are compatible with X-Y linkage or with an X-linked contribution to liability in males.