Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism.

No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.

Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates.

Injection of adeno-associated virus (AAV) into the cerebrospinal fluid (CSF) offers a means to achieve widespread transgene delivery to the central nervous system, where the doses can be readily translated from small to large animals. In contrast to studies with other serotypes (AAV2, AAV4 and AAV5) in rodents, we report that a naturally occurring capsid (AAV9) and rationally engineered capsid (AAV2.5) are able to achieve broad transduction throughout the brain and spinal cord parenchyma following a single injection into the CSF (via cisterna magna or lumbar cistern) in non-human primates (NHP). Using either vector at a dose of ∼2 × 10(12) vector genome (vg) per 3-6 kg animal, approximately 2% of the entire brain and spinal cord was transduced, covering all regions of the central nervous system (CNS). AAV9 in particular displayed efficient transduction of spinal cord motor neurons. The peripheral organ biodistribution was highly reduced compared with intravascular delivery, and the presence of circulating anti-AAV-neutralizing antibodies up to a 1:128 titer had no inhibitory effect on CNS gene transfer. Intra-CSF delivery effectively translates from rodents to NHPs, which provides encouragement for the use of this approach in humans to treat motor neuron and lysosomal storage diseases.

The influence of epileptic neuropathology and prior peripheral immunity on CNS transduction by rAAV2 and rAAV5.

Adeno-associated virus (AAV) provides a promising platform for clinical treatment of neurological disorders owing to its established efficacy and lack of apparent pathogenicity. To use viral vectors in treating neurological disease, however, transduction must occur under neuropathological conditions. Previous studies in rodents have shown that AAV5 more efficiently transduces cells in the hippocampus and piriform cortex than AAV2. Using the kainic acid (KA) model of temporal lobe epilepsy and AAV2 and 5 carrying a hybrid chicken ß-actin promoter driving green fluorescent protein (GFP), we found that limbic seizure activity caused substantial neuropathology and resulted in a significant reduction in subsequent AAV5 transduction. Nonetheless, this reduced transduction still was greater than AAV2 transduction in control rats. Although KA seizures compromise blood-brain barrier function, potentially increasing exposure of target tissue to circulating neutralizing antibodies, we observed no interaction between KA seizure-induced damage and immunization status on AAV transduction. Finally, while we confirmed the near total neuronal-specific transgene expression for both serotypes in control rats, AAV5-GFP expression was increasingly localized to astrocytes in seizure-damaged areas. Thus, the pathological milieu of the injured brain can reduce transduction efficacy and alter viral tropism- both relevant concerns when considering viral vector gene therapy for neurological disorders.

Delivering multiple gene products in the brain from a single adeno-associated virus vector.

For certain gene therapy applications, the simultaneous delivery of multiple genes would allow for novel therapies. In the case of adeno-associated virus (AAV) vectors, the limited packaging capacity greatly restricts current methods of carrying multiple transgene cassettes. To address this issue, a recombinant AAV (rAAV) vector was designed such that a furin proteolytic cleavage site (RKRRKR) was placed between the coding sequences of two genes (green fluorescent protein (GFP) and galanin), to allow cleavage of the chimeric protein into two fragments. In addition, these constructs contained the fibronectin secretory signal sequence that causes the gene products to be constitutively secreted from transduced cells. In vitro studies show that after transfection of HEK293 cells, the appropriate cleavage and constitutive secretion occurred regardless of the order of the genes in the transgene cassette. In vivo, infusion of rAAV vectors into the piriform cortex resulted in both GFP expression and significant galanin attenuation of kainic acid-induced seizure activity. Thus, the present results establish the utility of a proteolytic approach for the expression and secretion of multiple gene products from a single AAV vector transgene cassette.

Adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 suppresses seizure activity in vivo.

Neuropeptide Y (NPY) is a 36-amino-acid peptide that attenuates seizure activity following direct infusion or adeno-associated virus (AAV)-mediated expression in the central nervous system. However, NPY activates all NPY receptor subtypes, potentially causing unwanted side effects. NPY13-36 is a C-terminal peptide fragment of NPY that primarily activates the NPY Y2 receptor, thought to mediate the antiseizure activity. Therefore, we investigated if recombinant adeno-associated virus-mediated expression and constitutive secretion of NPY or NPY13-36 could alter limbic seizure sensitivity. Rats received bilateral piriform cortex infusions of AAV vectors that express and constitutively secrete full-length NPY (AAV-FIB-NPY) or NPY13-36 (AAV-FIB-NPY13-36). Control rats received no infusion, as we have previously shown that vectors expressing and secreting reporter genes like GFP (AAV-FIB-EGFP), as well as vectors expressing peptides that lack secretion sequences (AAV-GAL) have no effect on seizures. One week later, all animals received kainic acid (10 mg kg(-1), intraperitoneally), and the latencies to wet dog shakes and limbic seizure behaviors were determined. Although both control and vector-treated rats developed wet dog shake behaviors with similar latencies, the latencies to class III and class IV limbic seizures were significantly prolonged in both NPY- and NPY13-36-treated groups. Thus, AAV-mediated expression and constitutive secretion of NPY and NPY13-36 is effective in attenuating limbic seizures, and provides a platform for delivering therapeutic peptide fragments with increased receptor selectivity.

Generation of a stable cell line producing high-titer self-inactivating lentiviral vectors.

To facilitate the generation of SIN lentivirus vector-producer cell lines, we have developed a novel conditional SIN (cSIN) lentivirus vector, which retains its SIN properties in normal target cells yet can be produced at high titers from tetracycline-regulated packaging cell lines. The design of the cSIN vector is based on replacing the vector U3 transcription regulatory elements with the Tet-responsive element, which allows vector production exclusively in cells expressing the synthetic Tet-regulated transactivator (tTA). In contrast minimal vector production ( approximately 200 IU/ml) is obtained in target cells that do not express the tTA, even in the presence of all HIV-1 proteins. Following transduction of the Tet-regulated SODk1 lentivirus vector-packaging cell line with the cSIN vector, high titers of cSIN recombinant vector (>10(6) IU/ml) could be generated, which efficiently transduced terminally differentiated neurons in normal rat brain.

Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element.

Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604-1611, 1998) demonstrated the use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the "off" state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other cis-acting elements (AAV terminal repeats [TR]) were contributing to this activity. In the present study, we identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using deletion analysis and specific PCR-derived TR reporter gene templates, we mapped this activity to a 37-nucleotide stretch in the A/D elements of the TR. Although the mRNA derived from the TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green fluorescent protein expression both in vitro and in vivo in brain by using recombinant virus carrying only the TR element. Since the AAV terminal repeat is a necessary component of all recombinant AAV vectors, this TR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel TR split gene vectors currently being considered for genes too large to be packaged.

Interactive role for neurosteroids in ethanol enhancement of gamma-aminobutyric acid-gated currents from dissociated substantia nigra reticulata neurons.

Although previous in vivo electrophysiological studies demonstrated a consistent ethanol enhancement of gamma-aminobutyric acid (GABA) responsiveness from substantia nigra reticulata (SNR) neurons, ethanol applied in vitro to dissociated neurons from the SNR had an inconsistent effect on GABA function. One source for the disparity between these contrasting in vivo and in vitro results could be an endogenous factor (acting on an auxiliary site on GABA(A) receptors) that was not available to the isolated SNR neurons. Because neurosteroids are present in vivo and act on an auxiliary site, it was hypothesized that the presence of a neurosteroid was important for a consistent effect of ethanol on GABA responsiveness from neurons studied in vitro. Alone, the neurosteroid analog alphaxalone produced a significant, concentration-related enhancement of GABA responsiveness from isolated SNR neurons. In contrast to an inconsistent action of 100 mM ethanol on GABA responsiveness in the absence of alphaxalone, the presence of 30 and 100 nM alphaxalone resulted in the majority of isolated neurons responding to this ethanol level. At a concentration of alphaxalone as low as 30 nM, ethanol produced a robust concentration-related increase in GABA-gated currents from this cell type. The neurosteroid 3alpha, 5alpha-tetrahydrodeoxycorticosterone (100 nM) also permitted a reliable concentration-dependent ethanol enhancement of responses to GABA from SNR cells, indicative that the effects of alphaxalone were not unique. This consistent neurosteroid-induced ethanol enhancement of GABA responsiveness from dissociated SNR neurons supports the view that neurosteroids may play a key role in the action of ethanol on postsynaptic GABA(A) receptor function.

Selective and rapid uptake of adeno-associated virus type 2 in brain.

Recombinant adeno-associated virus (AAV) vectors effectively transfer and express foreign genes in the brain. The transferred genes, however, are selectively expressed in neurons, and the cause of this specificity is not understood. To address this question, wild-type AAV-2 capsids were covalently labeled with the fluorophore, Cy3, and infused into the inferior colliculus or the hippocampus. Using antibodies to identify neurons (NeuN), astrocytes (GFAP), or oligodendrocytes (OX-42), clear neuron-specific uptake of the virus was observed as early as 6 min after the start of the infusion. By 30 min postinfusion, AAV particles were present in the nucleus of neurons, yet in both the inferior colliculus and hippocampus, a subset of neurons did not take up the virus particles. No AAV particles were found in astrocytes 1.5 min or 24 hr after virus infusion. Interestingly, 1 hr postinfusion, no AAV particles were found in microglia, yet by 24 hr postinfusion, a punctate pattern of AAV particles was found in microglia. To test whether virus uptake correlated with vector-transduced cells, an rAAV-CMV-GFP virus was infused. By 3 days postinfusion, GFP was localized to neuronal populations with no expression in astrocytes or microglia, similar to that of fluorescent virus uptake. These findings demonstrate that in brain, AAV particles rapidly bind and enter primarily neurons with a pattern similar to that of in vivo vector transduction. In addition, these studies indicate that viral binding and uptake, independent of promoter tropism, can explain the specificity of AAV brain transduction. Thus, this first description of AAV kinetic disposition in vivo should facilitate targeted application of this vector for human brain gene therapy.

Inducible long-term gene expression in brain with adeno-associated virus gene transfer.

Recombinant adeno-associated virus (rAAV) vectors hold promise for treating a number of neurological disorders due to the ability to deliver long-term gene expression without toxicity or immune response. Critical to these endeavors will be controlled expression of the therapeutic gene in target cells. We have constructed and tested a dual cassette rAAV vector carrying a reporter gene under the control of the tetracycline-responsive system and the tetracycline transactivator. Transduction in vitro resulted in stable expression from the vector that can be suppressed 20-fold by tetracycline treatment. In vivo experiments, carried out to 6 weeks, demonstrated that vector-transduced expression is sustained until doxycycline administration upon which reporter gene expression is reduced. Moreover, the suppression of vector-driven expression can be reversed by removal of the drug. These studies demonstrate long-term regulated gene expression from rAAV vectors. This system will provide a valuable approach for controlling vector gene expression both in vitro and in vivo.

Inferior collicular seizure generalization produces site-selective cortical induction of cyclooxygenase 2 (COX-2).

Given the potential role of mitogen-inducible cyclooxygenase (COX-2) in CNS damage, patterns of COX-2 induction were determined both before and after seizure generalization from the inferior collicular cortex into the forebrain. With midbrain seizures, no change was found in COX-2-like immunoreactivity, even at the site of seizure genesis. However, upon forebrain seizure generalization, dramatic, ipsilateral increases in COX-2-like immunoreactivity were found in layers II and II of perirhinal, entorhinal and temporal cortex, just dorsal to the perirhinal fissure, coursing from the level of the medial geniculate to the level of the inferior colliculus. No changes in COX-2-like immunoreactivity were found in contralateral cortical regions, retrosplenial cortex, dentate gyrus, subiculum, tenia tectum or inferior colliculus. Thus, initial seizure generalization into the forebrain induces COX-2 expression in a highly specific area of the cerebral cortex.

Adeno-associated virus (AAV) vector antisense gene transfer in vivo decreases GABA(A) alpha1 containing receptors and increases inferior collicular seizure sensitivity.

In the inferior colliculus, adeno-associated virus (AAV) vectors are capable of gene transfer and stable, long-term expression, but it remained to be shown if this in vivo gene transfer could alter focal seizure sensitivity in the inferior colliculus. Because GABA receptors directly modulate inferior collicular seizures, AAV vectors were constructed with a cytomegalovirus (CMV) promoter and a truncated, human GABA(A) alpha1 cDNA in both the sense and antisense orientations. Seven days after collicular microinjection of the sense vectors (1 microl; 3 x 10(9) particles/microl), neurons exhibited GABA(A) alpha-like immunoreactivity in amounts far exceeding endogenous concentrations. Unilateral or bilateral sense vector infusion had no effect on inferior collicular seizure parameters or on [3H]zolpidem binding. In contrast, bilateral infusion of the antisense AAV-GABA(A) alpha1 vector (1 microl; 3 x 10(8) particles/microl) caused a 137% increase in the seizure duration. Moreover, unilateral antisense vector infusion produced a localized, 48% decrease in [3H]zolpidem binding. Thus, in the inferior colliculus, antisense AAV-CMV vectors can reduce a specific receptor subunit protein and change receptor function that directly influences in vivo seizure sensitivity.

Gene transfer by adeno-associated virus vectors into the central nervous system.

Adeno-associated virus (AAV) vectors are derived from a nonpathogenic and defective human parvovirus. Although currently unable to display the integration specificity featured by its wild-type parent, the recombinant AAV (rAAV) system has continued to attract enormous interest primarily due to its unique features such as safety, high titers, broad host range, transduction of quiescent cells, and vector integration. Recently, rAAV-mediated in vivo gene transfers have demonstrated efficient long-term transduction (from 3 months to more than 1.5 years) and lack of cytotoxicity and cellular immune responses in the target tissues, especially in the CNS. Alternative approaches using rAAV plasmid DNA in nonviral gene delivery systems also generated promising results. Propelled by various efforts to improve the system, rAAV vectors will provide numerous opportunities to explore the potential therapeutic applications in humans.

Action of zolpidem on responses to GABA in relation to mRNAs for GABA(A) receptor alpha subunits within single cells: evidence for multiple functional GABA(A) isoreceptors on individual neurons.

The relationship between zolpidem sensitivity and GABA(A) receptor alpha subunits was studied in individual dissociated neurons from rat brain. Using whole-cell recording, similar EC50 values were demonstrated for the effect of gamma-aminobutyric acid (GABA) on gated-chloride currents from substantia nigra reticulata (SNR) and lateral septal neurons. Subsequently, many neurons from both the SNR or lateral septum were found to exhibit enhanced GABA-gated chloride currents across concentrations of zolpidem ranging from 10 to 300 nM. Some neurons exhibited a greater than 20% increase in responsiveness to GABA at 30 nM of zolpidem without further increase at higher concentrations of zolpidem. Conversely, zolpidem enhancement of GABA from another group of neurons was not observed at 30 nM zolpidem, but between 100 and 300 nM the response to GABA increased greater than 20%. Finally, a third group of neurons reached both of these criteria for zolpidem enhancement of GABA. This latter spectrum of responses to GABA after varying concentrations of zolpidem was consistent with the presence of either two GABA(A) receptors or a single receptor with differing affinities for zolpidem on an individual neuron. Following determination of the sensitivity of neurons from SNR or lateral septum to zolpidem, cytoplasm was extracted from some individual cells to allow identification of cellular mRNAs for the alpha1, alpha2 and alpha3 GABA(A) receptor subunits with RT-PCR. Those neurons that responded to the 30 nM zolpidem concentration invariably expressed the alpha1-GABA(A) receptor subunit. This result is consistent with the GABA(A) alpha1-receptor subunit being an integral part of a functional high-affinity zolpidem type 1-BZD receptor complex on neurons in brain. Those neurons which showed enhancement of GABA from 100 to 300 nM zolpidem contained mRNAs for the alpha2 and/or the alpha3 receptor subunits, a finding consistent with these alpha subunits forming type 2-BZD receptors. Some individual dissociated SNR neurons were sensitive to both low and high concentrations of zolpidem and contained mRNAs for all three alpha-receptor subunits. These latter individual neurons are proposed to have at least two functional GABA(A) receptor subtypes. Thus, the present investigation emphasizes the importance of characterizing the relationship between endogenous GABA(A) receptor function and the presence of specific structural components forming GABA(A) receptor subtypes on neurons.

Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector.

Safe, long-term gene expression is a primary criteria for effective gene therapy in the brain, so studies were initiated to evaluate adeno-associated virus (AAV) vector transfer of a reporter gene into specific sites of the rat brain. In the 4 day old rat, site infusions of AAV-CMV-lacZ (1 microliter; 5 x 10(4) particles) produced neuronal beta-galactosidase gene expression 3 weeks later in the hippocampus and inferior colliculus, but not in the cerebral cortex. Seven days after infusion of AAV-CMV-lacZ viral vectors (1 microliter) in the adult rat, beta-galactosidase gene expression was found in the olfactory tubercle, caudate, hippocampus, piriform cortex and inferior colliculus. primarily in multipolar neurons close to the infusion site. Three months after vector microinfusion, similar levels of gene expression remained in the olfactory tubercle and the inferior colliculus, with some reduction found in the caudate, but substantial reductions in beta-galactosidase gene expression occurred in the hippocampus and piriform cortex. In no case were obvious signs of toxicity noted. Therefore, AAV vectors can transfer foreign genes into the adult and neonatal CNS, but the pattern and longevity of gene expression depends upon the area of brain being studied.

Metabolic and functional mapping of the neural network subserving inferior collicular seizure generalization.

The sensory-motor portion of the inferior collicular cortex is capable of seizure genesis that is characterized initially by coincident wild running behaviors and localized electrographic afterdischarge. With repeated stimulations, this seizure activity spreads into the forebrain, producing generalized tonic-clonic or myoclonic seizure activity. In order to characterize the neural network subserving this caudal-rostral seizure generalization, three mapping techniques were used: 2-deoxyglucose (2-DG) utilization, c-fos expression and local anesthetic microinjection. Kindled seizure generalization from the inferior collicular cortex produced a global increase in 2-DG accumulation, while relative 2-DG increases were found in the inferior collicular cortex, dorsal lateral lemniscus, dorsal central gray, peripeduncular nucleus, medial geniculate nucleus, substantia nigra, entopeduncular nucleus, ventroposterior and centromedian thalamus and tenia tectum, as well as the perirhinal, somatosensory and frontal cortices. Kindled seizure generalization also increased c-fos-like immunoreactivity (FLI) in the inferior collicular cortex, cuneiform nucleus, dorsal lateral nucleus of the lateral lemniscus, peripeduncular nucleus, caudal central gray, dentate gyrus of the hippocampus, rhinal fissure area of the perirhinal cortex and the frontal cortex. Microinjections of procaine into the amygdala, perirhinal cortex, entopeduncular nucleus, substantia nigra, peripeduncular nucleus, dorsal central gray, and pontine reticular nucleus all prevented generalized seizure behaviors, but had no effect on the wild running seizures. Conversely, procaine microinjection into the area of the cuneiform nucleus/pedunculopontine tegmental nucleus prevented the wild running seizure but did not block the generalized seizure activity. Neither wild running, nor generalized seizures were altered following procaine microinjections into the anterior thalamus, sub-thalamus, lateral hypothalamus, hippocampus or deep superior colliculus. Thus, specific forebrain sites form a widespread neural network that mediates the generalization of seizure activity from the inferior collicular cortex into the forebrain.

Effect of zolpidem on gamma-aminobutyric acid (GABA)-induced inhibition predicts the interaction of ethanol with GABA on individual neurons in several rat brain regions.

Previous investigations have suggested a relationship between zolpidem binding within specific brain regions and the ability of ethanol or zolpidem to enhance gamma-aminobutyric acid (GABA)-induced inhibition. The purpose of the present study was to extend our electrophysiological analysis to additional brain sites with high levels of zolpidem binding. In the brain regions chosen, red nucleus and globus pallidus, GABA-induced inhibition was shown to be enhanced by either ethanol or zolpidem on some, but not all, neurons. These findings led to the hypothesis that the effect of zolpidem on GABA-induced inhibition would predict the action of ethanol on responses to GABA for that neuron. When zolpidem and ethanol were applied individually to the same neurons in the red nucleus and globus pallidus, those neurons sensitive to zolpidem enhancement of GABA also were sensitive to ethanol. Conversely, if zolpidem did not enhance responses to GABA, ethanol did not enhance responses to GABA at these brain sites. A similar relationship between the abilities of zolpidem and ethanol to enhance GABA-induced inhibition was obtained in 90% of the neurons studied in the medial septum/diagonal band and ventral pallidum. These studies provide further support for the contention that the zolpidem-sensitive GABAA-benzodiazepine isoreceptor also responds to ethanol. Finally, the expression of GABAA subunit mRNAs was analyzed by polymerase chain reaction from micropunches of several brain regions that contain zolpidem binding sites and exhibit sensitivity to ethanol. Polymerase chain reaction analysis proved more sensitive than in situ hybridization in the detection of receptor subunit mRNAs. Several subunits (alpha 1, alpha 2, alpha 3, beta 2, beta 3 and gamma 2) were common to all brain regions in which ethanol and zolpidem enhanced GABA responses. GABAA receptor alpha 4/5, alpha 6, beta 1, gamma 1, gamma 3 and delta subunits were not consistently expressed in association with the presence of zolpidem binding. These data are consistent with the view that one native GABAA receptor to which zolpidem binds, and on which ethanol acts, contains the GABAA receptor subunits alpha 1, beta 2 and gamma 2; however, the present investigation did not preclude the possibility that other subunit combinations can contribute to ethanol and zolpidem enhancement of responses to GABA.

Distribution of [3H]zolpidem binding sites in relation to messenger RNA encoding the alpha 1, beta 2 and gamma 2 subunits of GABAA receptors in rat brain.

Localization of the messenger RNAs that encode the alpha 1, beta 2 and gamma 2 subunits of GABAA showed a distinct topographic pattern in rat brain which corresponded with [3H]zolpidem binding in most brain regions. The close topographic correspondence between the specific receptor subunits examined and the distribution of [3H]zolpidem binding sites provides support for the hypothesis that this benzodiazepine type 1 selective ligand binds to a GABAA receptor that consists of alpha 1, beta 2 and gamma 2 subunits in the rat brain. Brain regions with relatively high densities of alpha 1, beta 2 and gamma 2 subunits of GABAA and [3H]zolpidem binding included olfactory bulb, medial septum, ventral pallidum, diagonal band, inferior colliculus, substantia nigra pars reticulata and specific layers of the cortex. Two areas with low [3H]zolpidem binding and a virtual absence of these GABAA receptor subunit messenger RNAs were the lateral septum and the striatum. In contrast to the discrete pattern observed for alpha 1 and beta 2 subunit messenger RNAs, the gamma 2 subunit messenger RNA was distributed more diffusely in brain. Only the hippocampus, layer 2 of the piriform cortex and the cerebellum showed a strong concentration of the gamma 2 subunit messenger RNA. It was determined with a polymerase chain reaction assay that both long and short variants of the gamma 2 subunit messenger RNAs were present within several of the brain sites selected for examination. Sites with high densities of [3H]zolpidem binding sites had a greater relative abundance of the gamma 2 long splice variant, compared to the gamma 2 short variant. There were some regions that expressed high levels of alpha 1, beta 2 and gamma 2S subunit messenger RNAs but low [3H]zolpidem binding, suggesting that gamma 2 splice variant expression may modulate high-affinity [3H]zolpidem binding. To determine relationships between in vitro [3H]zolpidem binding and functional sensitivity in vivo, interactions between zolpidem and GABA were assessed in brain regions that contained high and low densities of [3H]zolpidem binding sites. In the medial septum, a brain region with a high concentration of [3H]zolpidem binding sites, iontophoretic application of zolpidem enhanced the inhibitory effect of GABA responses on 70% of the neurons examined. In the lateral septum, which contains very low densities of [3H]zolpidem binding sites, neurons were not sensitive to zolpidem enhancement of GABA-induced inhibition. These electrophysiological results demonstrate a correspondence between the regional distribution of [3H]zolpidem binding in vitro and functional sensitivity to the drug in vivo.

A potential contribution to ethanol withdrawal kindling: reduced GABA function in the inferior collicular cortex.

Because multiple withdrawals from chronic ethanol treatment facilitate the rate of kindling from the inferior collicular cortex, the following studies sought to identify potential sources of this long-term change in seizure sensitivity. When rats received 6 or 10 withdrawals from a 5-day ethanol liquid diet, a significant decrease was found in the threshold frequency for seizure genesis, 6-7 days post-withdrawal. The magnitude of this change was related to the number of withdrawals (6 withdrawals, -2.0 +/- 0.3 Hz; 10 withdrawals, -3.2 +/- 1.2 Hz). Thus, multiple ethanol withdrawals increased seizure sensitivity within the inferior collicular cortex. On the following day in the same animals, changes in inhibitory or excitatory function were evaluated within the inferior collicular cortex. We found a withdrawal-related increase in the effectiveness of bicuculline to reduce the seizure threshold current within the inferior collicular cortex. Seizure sensitivity to collicular N-methyl-D-aspartic acid (NMDA) microinjection was decreased after 6 ethanol withdrawals, and increased after 10 withdrawals, but control liquid diet animals exhibited similar responses to collicular NMDA microinjection. Therefore, multiple withdrawals from ethanol alters the seizure sensitivity within the inferior collicular cortex. One possible contribution to this change is a local decrease in GABA inhibitory function.

Molecular basis for regionally specific action of ethanol on gamma-aminobutyric acidA receptors: generalization to other ligand-gated ion channels.

The present investigation provides evidence that there is neuroanatomical specificity for ethanol enhancement of gamma-aminobutyric acid (GABA)-induced inhibition in mammalian brain and that the expression of a specific GABAA isoreceptor is associated with this regional action of ethanol. Ethanol enhanced responses to iontophoretically applied GABA in the medial septum, inferior colliculus, substantia nigra reticulata, ventral pallidum and the diagonal band of Broca. In contrast to these results, responses to GABA applied to cells in the lateral septum, ventral tegmental area and the hippocampus were not affected by ethanol. In those brain regions where ethanol enhanced responses to GABA, a high concentration of zolpidem binding was found, whereas zolpidem binding was much lower or absent in brain regions where ethanol did not enhance GABA. These observations support the hypothesis that ethanol enhances GABA within specific regions of brain by affecting a GABAA receptor with specific structural components. From data obtained with in situ hybridization, there was a strong relationship between the regional distribution of zolpidem binding and the expression of specific mRNAs for the alpha-1, beta-2 and gamma-2 GABAA receptor subunits at sites where ethanol enhanced responses to GABA. The mRNA for the long and short variants of the gamma-2 subunit were found in brain regions both sensitive and insensitive to the action of ethanol on GABA-induced inhibition. These data were not able to address whether the gamma-2 long variant in combination with the alpha-1 and beta-2 subunits is essential for ethanol enhancement of responses to GABA. However, the observation that the long version of the gamma-2 subunit is present in brain areas where ethanol did not affect GABA function suggests that the presence of the long variant of the gamma-2 subunit alone is not sufficient for ethanol's action to enhance responses to GABA. Rather it is concluded that the appropriate combination of GABAA receptor subunits is critical for this action of ethanol. Because the GABAA receptor belongs to a superfamily of ligand-gated ion channels, the action of ethanol was examined on responses to agonists acting on receptors linked to other ion channels. As noted for GABA, local application of ethanol altered responses to NMDA, nicotine and glycine when applied to some, but not all, neurons.(ABSTRACT TRUNCATED AT 400 WORDS)