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.

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)

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)

Transient expression of GABAA receptor subunit mRNAs in the cellular processes of cultured cortical neurons and glia.

In this study, we have studied by in situ hybridisation histochemistry the expression and intracellular distribution of the GABAA receptor subunit mRNAs in cultured neurons obtained from postnatal day 1-3 rats in order to determine how neurotransmitter receptor expression may be regulated during development of the nervous system. In postnatal cortical cells, we found that GABAA receptor subunit mRNAs coding for alpha2, alpha5, beta2, beta3 and gamma2 subunits were transiently expressed in the cellular processes and growth cones after 1-3 days in culture. These observations indicate that GABAA receptor subunit mRNAs are transported (or trafficked) into the cellular processes of early postnatal cortical cells. These selective localisations were rarely observed after 5 days in culture and only in cells which had not made cell-to-cell contact. The localisation of subunit mRNAs in the processes was more effectively maintained up to 5 days or even longer if cell-to-cell contact was avoided by culturing the cells at low density or by inhibiting neurite sprouting pharmacologically with the GABA receptor channel antagonist TBPS. Finally, immunocytochemistry revealed the expression of GABAA receptors in the growth cones of pyramidal neurons in culture. Thus, the expression of mRNA correlates to the expression of protein. These results suggest that the selective trafficking of GABAA receptor subunit mRNAs during synaptogenesis may be regulated by synapse formation and/or glial-neural communication.

Digital analysis of light microscope immunofluorescence: high-resolution co-localization of synaptic proteins in cultured neurons.

A protocol is presented for determining the subcellular distribution of fluorescently labeled proteins in neurons using deconvolved images gathered with a wide-field microscope. The protocol includes optimal settings for the numerical algorithm used to deconvolve the images and an objective method for thresholding the deconvolved images to retain only high-intensity, specific labeling. The effectiveness of the protocol is demonstrated using a fluorescent antibody stain directed towards the alpha1 subunit of the GABA(A) receptor in cultured neurons. We also show, using an antibody against the presynaptic vesicular protein synaptophysin, that the technique can detect presumptive regions of synaptic contact between neurons. Double-labeling with the anti-alpha1 and anti-synaptophysin antibodies in a cultured neuron reveals regions of both synaptic and non-synaptic alpha1 labeling. Thus, numerical postprocessing of wide-field images can be used to efficiently locate receptor proteins in neurons in relation to functionally important structures. This confocal-like functionality is attained without the excessive bleaching and phototoxicity associated with the intense laser excitation light used in confocal techniques.

Mesencephalic trigeminal neuron responses to gamma-aminobutyric acid.

Mesencephalic trigeminal neurons are primary sensory neurons which have cell somata located within the brain stem. In spite of the presence of synaptic terminals on and around the cell somata, applications of a variety of neurotransmitter substances in earlier studies have failed to demonstrate responses. Using intracellular recording in a brain slice preparation, we have observed prominent depolarizations and decreases in input resistance in response to applications of gamma-aminobutyric acid (GABA) in most recorded mesencephalic trigeminal neurons. Those cells failing to respond were located deeply within the slice, and the low responsiveness was shown to be related to uptake of GABA in the slice. The responses were direct, since they remained during perfusion with a low calcium, high magnesium solution that blocks synaptic transmission. The responses were mimicked by the GABA(A) receptor agonist isoguvacine, and blocked by GABA(A) receptor antagonists. The GABA(B) receptor agonist baclofen evoked no changes in membrane potential or input resistance in neurons exhibiting depolarizations with GABA application. Tests of neuronal excitability during GABA applications indicated that the excitatory effects of the depolarization prevail over the depressant effects of the increase in membrane conductance. In situ hybridization histochemistry indicated that the GABA(A) receptors in Me5 cells are comprised of alpha2, beta2 and gamma2 subunits.

Alteration in NMDA receptor subunit mRNA expression in vulnerable and resistant regions of in vitro ischemic rat hippocampal slices.

Brain insults, including cerebral ischemia, can alter glutamate receptor subunit expression in vulnerable neurons. Understanding these post-ischemic changes in glutamate receptors could enhance our ability to identify specific, novel neuroprotective compounds. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to quantify the altered expression of the N-methyl-D-aspartate (NMDA) NR2A, NR2B and NR2C subunits relative to one another in rat hippocampal slices in resistant and vulnerable regions following in vitro oxygen-glucose deprivation. Ninety minutes after re-oxygenation and return to 10 mM glucose, there was a significant increase in the expression of NR2C relative to NR2B and NR2A in the slice as a whole, as well as in the selectively vulnerable CA1 region and the resistant CA3 and dentate gyrus regions.

Brain derived neurotrophic factor induction of N-methyl-D-aspartate receptor subunit NR2A expression in cultured rat cortical neurons.

N-methyl-D-aspartate (NMDA) receptor subunit expression changes during development and following injury in several brain regions. These changes may be mediated by neurotrophic factors, such as brain derived neurotrophic factor (BDNF). Exposure of cultured cortical neurons to BDNF (100 ng/ml) for 24 h produced a significant decrease in the NMDA-induced whole-cell currents sensitive to the NR2B subunit selective NMDA receptor antagonist, CP-101,606, suggesting a relative decrease in NR2B subunit expression. There was a significant increase in NR2A by Western blot analysis. Consistent with the electrophysiology and Western blot analysis, reverse transcriptase-polymerase chain reaction (RT-PCR) amplification revealed that BDNF caused a significant increase in relative NR2A subunit expression, a significant decrease in relative NR2B subunit expression and no change in relative NR2C subunit expression. These results suggest that BDNF enhances NMDA receptor maturation, warranting further study of the mechanism of BDNF effects on NMDA receptor subunit expression and the role these effects play in development and neuronal injury.

Plasticity of the GABA(A) receptor subunit cassette in response to stressors in reactive versus resilient mice.

GABA(A) functioning has been implicated in anxiety and depressive disorders. In this regard, we suggested that in addition to analyzing GABA(A) and the subunits that comprise the GABA(A) receptor, it might be profitable to assess the coordinated expression of subunits that comprise the GABA(A) receptor cassette. We demonstrate that certain subunits within stress-sensitive brain regions were higher in stressor reactive BALB/cByJ than in hardy C57BL/6ByJ mice, and that a chronic, intermittent, variable stressor (6 days/week over 7 weeks) differentially influenced subunit expression in these strains. Further, mRNA expression of GABA(A) subunits were highly coordinated (inter-correlated), and markedly altered by stressors, once again varying with brain region. At the central amygdala of BALB/cByJ mice the ordinarily high subunit inter-relations were reduced in acutely stressed mice, and this outcome was exacerbated with a chronic stressor. In C57BL/6ByJ mice subunit inter-relations were lower than in BALB/cByJ mice; the acute stressor increased subunit organization, which returned to control levels with following a chronic stressor. The profile of amygdala subunit inter-relations was recapitulated in a step-down behavioral test; anxiety was increased by acute and chronic stressors in BALB/cByJ mice, but in the C57BL/6ByJ strain the elevated anxiety associated with an acute stressor was not apparent after chronic stressor treatment. The anxiety could be dissociated from apparent anhedonia (reflected by free sucrose consumption) where the preference for sucrose was reduced by an acute stressor, but this outcome was more pronounced following a chronic stressor, especially in BALB/cByJ mice. These findings support the view that analyses involving subunit organization, rather than just differences in absolute levels, may be expedient in assessing GABA(A) functioning in stressor-related psychological disturbances.

Developmental change in GABAA receptor desensitization kinetics and its role in synapse function in rat cortical neurons.

We examined the maturation of GABAA receptor synapses in cortical pyramidal neurons cultured from embryonic rats. The decay kinetics of GABAA receptor-mediated miniature postsynaptic currents (mPSCs) were compared with those of responses evoked by GABA in excised membrane patches. Fast perfusion of 1 or 10 mM GABA on membrane patches evoked currents with different desensitizing time courses in young and old neurons. For neurons older than 4 days in vitro (DIV), GABAA currents had a fast component of desensitization (median approximately 3 ms) seldom seen in patches from younger neurons. In contrast, mPSCs exhibited a substantial fast component of decay at 2-4 DIV that became more prominent with further development although the median value of its time constant remained unchanged. The selective alpha3 subunit positive modulator SB-205384 had no effect on mPSCs at any time in vitro but potentiated extrasynaptic activity. This suggests that synapse maturation does not proceed by a gradual exchange of early embryonic GABAA receptor subforms for adult forms. At all ages, the kinetic properties of mPSCs were heterogeneous. This heterogeneity extended to the level of mPSCs from single neurons and may be a normal aspect of synaptic functioning. These results suggest that inhibitory synapses in developing neurons are capable of selectively capturing GABAA receptors having fast desensitization kinetics. This functional preference probably reflects the developmental turning point from an inwardly looking trophic capacity of embryonic GABAA receptors to a role concerned with information processing.

Recombinant GABAA receptor desensitization: the role of the gamma 2 subunit and its physiological significance.

1. The purpose of these investigations was to examine the role that the gamma 2 subunit plays in human GABAA receptor desensitization. Two different recombinant GABAA receptors (alpha 1 beta 3 and alpha 1 beta 3 gamma 2) were compared by measuring the relaxation of whole-cell currents during the application of GABA, isoguvacine or taurine. 2. At concentrations which trigger a maximum response (100-500 microM GABA) the current relaxation usually fitted the sum of two exponentials. For alpha 1 beta 3 subunit receptors these values were tau 1 = 145 +/- 12 ms and tau 2 = 6.3 +/- 2.1 s (means +/- S.E.M.). Receptors consisting of alpha 1 beta 3 gamma 2 subunits desensitized faster: tau 1 = 41.6 +/- 8.3 ms and tau 2 = 2.4 +/- 0.6 s. 3. The Hill slope, determined for each receptor subunit combination, was the same and greater than 1.0, implying two binding steps in the activation of both receptor subunit combinations. 4. For alpha 1 beta 3 subunit receptors the fast desensitization rates were unaltered by reducing the GABA concentration from the EC100 (100 microM) to the approximate EC50 values (10-20 microM), whereas for alpha 1 beta 3 gamma 2 subunit receptors a significant slowing was observed. The fast desensitization disappeared at agonist concentrations below the EC50 for both subunit combinations. In contrast, the slow desensitization appeared at agonist concentrations near the EC20. This rate was dependent on agonist concentration reaching a maximum near the EC60 value of GABA. 5. The fast desensitization rates were unaltered by changing the holding potential of the cell during agonist application. However, for alpha 1 beta 3 gamma 2 subunit receptors the slow desensitization rate increased by approximately 15- to 20-fold over the range of voltages of -60 to +40 mV. This indicates that the gamma 2 subunit makes GABAA receptor desensitization voltage dependent. 6. Recovery from desensitization was also biphasic. The first recovery phase was faster for alpha 1 beta 3 gamma 2 than for alpha 1 beta 3 subunit receptors (0.13 vs. 0.03 s-1, respectively). The second phase of recovery for the two receptors were the same (approximately 0.003 s-1). 7. There was only a poor correlation between agonist potency and the degree or time course of desensitization. Isoguvacine (EC50 approximately to 10 microM) induced biphasic relaxation for both alpha 1 beta 3 and alpha 1 beta 3 gamma 2 subunit receptors (tau 1 = 288.6 +/- 43.3 and 167 +/- 15 ms, and tau 2 = 8.0 +/- 1.9 and 4.4 +/- 0.4 S, respectively, for each subunit combination). Taurine (EC50 approximately 7 mM) usually induced monophasic relaxation for both subunit combinations (tau 2 = 7.1 +/- 1.6 and 23.0 +/- 6.6 s, respectively). 8. A computer model was developed to examine the effect of the gamma 2 subunit on the time course of a synaptic potential. It was found that the gamma 2 subunit theoretically prolongs the time course of a synaptic potential by inducing desensitization more rapidly. The subsequent relaxation of the desensitized receptors through the open state increases Popen (the probability that the GABAA receptor is in an open conducting state) altering the time course of the modelled potential. alpha 1 beta 3 subunit receptors do not desensitize sufficiently rapidly to induce this desensitized state and, therefore, are shorter in time course. These data imply that the physiological role of the gamma 2 subunit is to increase synaptic efficacy by prolonging Popen.