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.

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.

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.

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.

High resolution autoradiographic determination of the topographic distribution of radioactivity in the hippocampal formation after injection of [1-14C]glucose or 2-deoxy[14C]glucose.

Using high resolution autoradiography, the accumulation of radioactivity after intravenous injection of [1-14C]glucose was measured in the corpus callosum, hippocampus, dorsal hippocampal commissure, somatosensory cortex, inferior colliculus and pontine periaqueductal grey. Autoradiograms were prepared by thaw-mounting 4 micron frozen sections on nuclear emulsion-coated slides, and were evaluated quantitatively with a computer-assisted video system for automated counting of silver grains. In all brain regions examined, silver grain densities were greater in rats killed 30 min after injection of [1-14C]glucose compared to rats killed 10 min after injection. After intravenous injection of [1-14C]glucose or 2-deoxy[14C]glucose, the relative uptake and retention of radioactivity in different hippocampal subregions was compared. Striking differences were found in the hippocampus between 2-deoxy[14C]glucose and [1-14C]glucose autoradiograms. After injection of 2-deoxy[14C]glucose, there were large variations in the uptake and retention of radioactivity among different pyramidal cell fields. The CA 3 pyramidal cell field retained considerably more radioactivity than other pyramidal cell fields after injection of 2-deoxy[14C]glucose, while after injection of [1-14C]glucose, the retention of radioactivity was similar in all pyramidal cell fields. After [1-14C]glucose injection, the dentate gyrus contained relatively high levels of radioactivity and more 14C accumulated in the granular layer, compared to the molecular layer. In contrast, after 2-deoxy[14C]glucose injection, there was uniformly less radioactivity throughout the dentate gyrus when compared to rats injected with [1-14C]glucose and there was no preferential accumulation of 2-deoxy[14C]glucose in the granular layer compared to the molecular layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroanatomical characterization of inferior collicular seizure genesis: 2-deoxyglucose and stimulation mapping.

Previous work has demonstrated that the cortical sub-division of the inferior colliculus is capable of seizure genesis, so the present studies delineated the neuroanatomical extent of this seizure genesis using two mapping techniques, [14C]2-deoxyglucose (2-DG) uptake and electrical stimulation. When 4 inferior collicular seizures were elicited over a 5 min period, the stimulated side of the brain showed highly selective increases in the [14C]2-DG uptake in comparison to the unstimulated side. Although the substantial change in [14C]2-DG uptake occurred over the entire inferior collicular cortex, electrical stimulation mapping delineated a specific area capable of seizure genesis within the inferior collicular cortex. The electrical stimulation also identified a number of regions that would support electrically dependent seizure behaviors: the cuneiform nucleus, the ventrolateral inferior colliculus, portions of the dorsal central gray, and the peripeduncular nucleus. In concert, marked unilateral increases in [14C]2-DG uptake were found on the stimulated side in the peripeduncular/substantia nigra lateralis area, the medial geniculate and a specific region of the dorsal central gray. These studies verify the asymmetric nature of inferior collicular seizure genesis, identify areas of seizure modulation, and delineate a region in the inferior collicular cortex that modulates sensory-motor integration.

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.

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.

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.

Brainstem localization of a thyrotropin-releasing hormone-induced change in respiratory function.

When rats received microinjections of 100 ng thyrotropin-releasing hormone (TRH) into the medial portions of the nucleus tractus solitarius and 12th nucleus or raphe obscurus, at the level of the obex, a significant decrease in the inspiratory time was found. Examination of TRH immunocytochemistry revealed a high density of TRH-positive nerve terminals in these regions, especially the more caudal aspects. If serotonin was depleted by neonatal 5,7-dihydroxytryptamine treatment, the respiratory response of the adults to TRH appeared potentiated. Even though the neonatal 5,7-dihydroxytryptamine reduces the occurrence of TRH-positive cell bodies, TRH-positive fibers were not appreciably altered. These results are discussed with regard to a possible role of endogenous TRH in the brainstem on rhythmic respiratory activity.

Effects of apamin and nicotinic acetylcholine receptor antagonists on inferior collicular seizures.

These studies compared the actions of apamin and nicotinic acetylcholine antagonists on seizure genesis within the inferior collicular cortex. In vitro alpha-bungarotoxin, d-tubocurarine and gallamine all competitively displaced [125I]apamin binding to brain sections through the inferior colliculus, while intracollicular microinjection of intermediate doses of apamin (21 pmol), alpha-bungarotoxin (0.3 nmol), d-tubocurarine (0.22 nmol) or gallamine (1.7 nmol) all significantly reduced the seizure initiation threshold current. However, higher doses of apamin did not cause spontaneous seizure activity, while higher doses of the nicotinic acetylcholine antagonists caused spontaneous seizures. Carbamylcholine also produced spontaneous seizures, but did not alter the seizure threshold current. N-Methyl-atropine caused a dose-related elevation of the seizure threshold current, yet microinjection of N-methyl-atropine (10 nmol) into the inferior collicular cortex reversed the effects of alpha-bungarotoxin on seizure threshold, partially opposed the effects of gallamine and d-tubocurarine, and had no effect on the ability of apamin to reduce the seizure threshold current. Thus, both the apamin-sensitive potassium channel and a variety of distinct cholinergic mechanisms contribute in vivo to seizure genesis within the inferior collicular cortex, but not through the same mechanisms.

A centrally elicited respiratory stimulant effect by bombesin in the rat.

The effects of the tetradecapeptide bombesin on respiratory regulation in the rat were studied using a whole body plethysmographic model. Application of the peptide was made intracerebroventricularly (i.c.v.) as well as via microinjections into specific brainstem areas. I.c.v. injection (0.1-5 micrograms) resulted in a dose-dependent increase in tidal volume while respiratory frequency was decreased only at higher doses. The respiratory duty cycle remained unchanged while the respiratory drive was significantly increased. The respiratory effects were blunted by bilateral section of the tenth cranial nerve. Studies employing the occluded breath technique indicated a change in the threshold to afferent vagal signals while the time-setting for inspiration remained unchanged. Similar ventilation changes were elicited when the peptide was injected into the area of the nucleus ambiguous but not in several other areas of the brainstem. Such bombesin sensitive areas are consistent with a recent immunohistochemical study describing a dense pattern of immunoreactive somata in this area of the brainstem. The ventilatory stimulant effect seems to depend on an intact afferent vagal innervation.

The role of the inferior collicular cortex in the neonatal rat: sensorimotor modulation.

A tail pinch in rats up to 10 days of age produces a spectrum of motor behaviors characterized by forelimb paddling, hindlimb treading and occasional curling and rolling of the torso, a behavioral pattern similar to the seizure behaviors electrically-elicited from the inferior collicular cortex of 5- or 10-day-old rats. In 5-day-old rats, these tail pinch-induced paddling and treading behaviors coincided with afterdischarge-like EEG activity recorded from the seizure-sensitive site in the inferior collicular cortex. In contrast, no change in the EEG activity occurred in an adjacent seizure-insensitive site during these tail pinch-induced behaviors. Similar electrographic-behavioral synchrony was found in 10-day-old rats, but by 16 days of age, as in the adult rat, a tail pinch stimulus did not induce post-stimulus behavioral changes or afterdischarge-like EEG activity. Since auditory function does not develop until 12-14 days of age in the rat, we propose that the inferior collicular cortex modulates sensorimotor integration in the neonatal rat, prior to assumption of this function by the cerebral cortex.

Seizure interactions between the inferior collicular cortex and the deep prepiriform cortex.

In rats, electrographic seizure activity was recorded from both the inferior collicular cortex and the deep prepiriform after i.p. administration of 15 mumol/kg bicuculline, a dose which produced a variety of seizure behaviors. electrographic spikes recorded from the deep prepiriform cortex coincided with myoclonic jerks, while both brain areas exhibited an increase in afterdischarge frequency at the onset of forelimb tonic extension. Conversely, afterdischarge activity was recorded in both brain regions, while no seizure behaviors were apparent, providing examples of dissociation between electrographic and behavioral seizure activity. However, when functional interactions were assessed, the tonic-clonic behaviors produced by kindling stimulation of the inferior collicular cortex were prevented by microinjections of procaine into the deep prepiriform cortex. Thus, a forebrain site, that has been associated with limbic seizure activity, also can modulate seizure generalization from the inferior collicular cortex into the forebrain.

Unilateral kindling of the inferior collicular cortex does not transfer to the contralateral seizure sensitive site or alter [3H]flunitrazepam and [35S]TBPS binding.

Acute electrical stimulation of a specific area in the inferior collicular cortex produced bilateral collicular afterdischarge and symmetrical wild running seizures. However, generalized seizures induced by kindling the inferior collicular cortex did not alter the kindling rate in the contralateral side. Furthermore, after both sides of the inferior collicular cortex have been kindled unilateral electrolytic lesions did not alter the seizure initiation or generalization elicited from the contralateral side. Since GABAergic function has been implicated in inferior collicular seizures, potential seizure-induced changes were measured for the chloride channel ([35S]TBPS) and the benzodiazepine receptor ([3H]flunitrazepam). Prior kindling did not alter [35S]TBPS or [3H]flunitrazepam binding in the central nucleus or cortex of the inferior colliculus, the medial geniculate, or the deep prepiriform cortex. Thus, the permanent neural change that subserves seizure generalization from the inferior collicular cortex is unilateral, but this change is not reflected by altered binding characteristics of the GABAA receptor complex.

Changes in cerebrospinal fluid homovanillic acid in children with Ondine's curse.

The cerebrospinal fluid (CSF) concentrations of three acid monoamine metabolites, two purines, and a group of amino acids were determined in two children with chronic central alveolar hypoventilation (Ondine's curse). The levels of all assayed neuroactive substances, metabolites, and amino acids, with one exception, were normal compared to an age-matched group of neurologically healthy children. The levels of the dopamine metabolite homovanillic acid in the children with Ondine's curse were approximately 2.4 times higher than expected for age range. The present findings may indicate a link between central nervous system dopamine activity and chronic central alveolar hypoventilation. Among other possible explanations, the changes seen might represent a primary alteration in dopamine activity or may reflect a change in dopamine turnover resulting from the chronic hypoventilation.