An adenovirus vector for gene transfer into neurons and glia in the brain.

The efficient introduction of genetic material into quiescent nerve cells is important in the study of brain function and for gene therapy of neurological disorders. A replication-deficient adenoviral vector that contained a reporter gene encoding beta-galactosidase infected rat nerve cells in vitro and in vivo. beta-Galactosidase was expressed in almost all sympathetic neurons and astrocytes in culture. After stereotactic inoculations into the rat hippocampus and the substantia nigra, beta-galactosidase activity was detected for 2 months. Infected cells were identified as microglial cells, astrocytes, or neurons with anatomical, morphological, and immunohistochemical criteria. No obvious cytopathic effect was observed.

Is the acetylcholine releasing protein mediatophore present in rat brain?

Mediatophore is a protein purified from the nerve terminal membranes of Torpedo electric organ. It confers to artificial membranes a calcium-dependent mechanism that translocates acetylcholine. When similar reconstitution experiments are applied to rat brain synaptosomal membranes they reveal the presence of mediatophore activity with properties close to those described for the Torpedo protein (extractability, sensitivity to calcium, and effect of the drug cetiedil). The activity was more abundant in synaptosomal membranes than in mitochondrial or myelinic membranes and in cholinergic areas as compared to cerebellum.

Recurrent seizures and hippocampal sclerosis following intrahippocampal kainate injection in adult mice: electroencephalography, histopathology and synaptic reorganization similar to mesial temporal lobe epilepsy.

Human mesial temporal lobe epilepsy is characterized by hippocampal seizures associated with pyramidal cell loss in the hippocampus and dispersion of dentate gyrus granule cells. A similar histological pattern was recently described in a model of extensive neuroplasticity in adult mice after injection of kainate into the dorsal hippocampus [Suzuki et al. (1995) Neuroscience 64, 665-674]. The aim of the present study was to determine whether (i) recurrent seizures develop in mice after intrahippocampal injection of kainate, and (ii) the electroencephalographic, histopathological and behavioural changes in such mice are similar to those in human mesial temporal lobe epilepsy. Adult mice receiving a unilateral injection of kainate (0.2 microg; 50 nl) or saline into the dorsal hippocampus displayed recurrent paroxysmal discharges on the electroencephalographic recordings associated with immobility, staring and, occasionally, clonic components. These seizures started immediately after kainate injection and recurrid for up to eight months. Epileptiform activities occurred most often during sleep but occasionally while awake. The pattern of seizures did not change over time nor did they secondarily generalize. Glucose metabolic changes assessed by [14C]2-deoxyglucose autoradiography were restricted to the ipsilateral hippocampus for 30 days, but had spread to the thalamus by 120 days after kainate. Ipsilateral cell loss was prominent in hippocampal pyramidal cells and hilar neurons. An unusual pattern of progressive enlargement of the dentate gyrus was observed with a marked radial dispersion of the granule cells associated with reactive astrocytes. Mossy fibre sprouting occurred both in the supragranular molecular layer and infrapyramidal stratum oriens layer of CA3. The expression of the embryonic form of the neural cell adhesion molecule coincided over time with granule cell dispersion. Our data describe the first histological, electrophysiological and behavioural evidence suggesting that discrete excitotoxic lesions of the hippocampus in mice can be used as an isomorphic model of mesial temporal lobe epilepsy.

Identification of two proteolipid antigens with different localization at torpedo neuromuscular synapses.

The cholinergic nerve terminals of Torpedo electric organ are a rich source of a hydrophobic protein, the mediatophore, which endows artificial membranes with a calcium dependent mechanism that translocates acetylcholine. An antiserum raised against purified mediatophore permitted the identification of two different proteolipids of 14 and 15 kDa molecular weight. Mediatophore activity paralleled the abundance of the 15 kDa proteolipid. Antibodies to this 15 kDa proteolipid were affinity purified and the distribution of this antigen was studied at neuromuscular junction, where it was only detected at nerve terminals. An antisynaptic vesicle monoclonal antibody was produced and used as a nerve terminal specific marker. Its binding pattern was very similar to that of anti 15 kDa proteolipid antibodies. Their distribution is markedly different from that of the 14 kDa proteolipid which is associated with Schwann cells and endothelium of blood capilliaries as shown using affinity purified antibodies to the 14 kDa proteolipid.

Fos expression in GHB-induced generalized absence epilepsy in the thalamus of the rat.

Using the model of gamma-hydroxybutyrate (GHB)-induced generalized absence epilepsy, the present work investigated the distribution of fos oncoprotein expression in the rat thalamus with fos antibody immunohistochemistry. Thirty minutes after absence-like seizures, some fos-immunoreactive cell nuclei were found in bilateral thalamic paraventricular nuclei (PV). After a further 30 min, a massive bilateral induction of fos was observed in the lateral habenular nucleus (LHb), the PV, the rhomboid thalamic nucleus, and the intralaminar nuclei of the thalamus. These results suggest that the LHb and the midline and intralaminar thalamic nuclei may very likely be involved in the pathophysiology of absence seizures.

The use of adenovirus vectors for intracerebral grafting of transfected nervous cells.

Grafting genetically-modified cells into the brain is a promising approach to address fundamental and clinical issues in neurobiology. Despite recent substantial progress, most of the methods used for introducing DNA sequences into donor cells result in weak efficacy or transient gene expression after transplantation. We tested whether the use of adenovirus as the vector for foreign genes avoided these problems. A replication-defective adenovirus vector carrying a reporter gene encoding for beta-galactosidase was used to transfect primary astrocytes. After grafting into various brain structures, transfected cells exhibited robust survival and expressed the transgene for at least five months. These results demonstrate the advantage of adenovirus-mediated gene transfer for prolonged transgene expression in grafted primary cells.

Audiogenic seizures evoked in DBA/2 mice induce c-fos oncogene expression into subcortical auditory nuclei.

Recently the nuclear proto-oncogene c-fos has been shown to be rapidly and transiently expressed following seizures in many types of epilepsies. Until now, immunohistochemical as well as in situ hybridization studies have reported that the dentate gyrus of the hippocampus and most of the cortical areas were invariably heavily labeled. In order to see whether this distribution was reproduced or not in a model of epilepsy which has been proved to not involve these structures, a study was performed on genetically epilepsy-prone DBA/2 mice. Here we show that following audiogenic seizures, c-fos oncoprotein is not expressed in cortical and limbic structures but rather mapped the subcortical auditory nuclei.

Decreased epileptic susceptibility correlates with neuropeptide Y overexpression in a model of tolerance to excitotoxicity.

Prior epileptic episodes have been shown to decrease markedly the neuronal damage induced by a second epileptic episode, similar to the tolerance following an episode of mild ischemia. Endogenous neuroprotective effects mediated by various mechanisms have been put forward. This study investigated whether neuroprotection against the excitotoxic damage induced by re-exposure to an epileptic challenge can reflect a change in epileptic susceptibility. Tolerance was elicited in rats by a preconditioning session using intrahippocampal kainic acid (KA) administration followed at 1, 7 and 15-day intervals by a subsequent intraventricular KA injection. The degree of pyramidal cell loss in the vulnerable CA3 subfield contralateral to the KA-injected hippocampus was extensively reduced in animals experiencing KA ventricular administration. This neuroprotection was highly significant 1 and 7 days after injection, but not 15 days after injection. In preconditioned animals, the after-discharge threshold was assessed as an index of epileptic susceptibility. It increased significantly from 1 to 15 days after intrahippocampal KA administration. Finally, an enhancement of neuropeptide Y expression in both non-principal cells and mossy fibers was detected, occurring at the same time as the decrease in epileptic susceptibility. These results provide further evidence of an 'epileptic tolerance' as shown by the substantial neuroprotective effect of a prior episode of epileptic activity upon subsequent epileptic insult and suggest that the prevention of excitotoxic damage after preconditioning results from an endogenous neuroprotective mechanism against hyperexcitability and seizures.

Amygdaloid lesion increases the toxicity of intrahippocampal kainic acid injection and reduces the late occurrence of spontaneous recurrent seizures in rats.

Spontaneous recurrent seizures (SRS) following intrahippocampal kainic acid (KA) injection have been described in a previous paper from our laboratory. The SRSs are clinically similar to the seizures induced by kindling the amygdala and we suggested that the amygdala plays a role in initiating the SRSs. Accordingly, the present paper examines the effect of amygdaloid lesions on intrahippocampal KA-treated rats. There were short- and long-term effects. (1) Short-term: the toxicity of KA was increased in lesioned animals. Status epilepticus followed by death of the animals was evoked with half of the dose required to cause the same effect in intact rats. Moreover, a gross haematuria was encountered 6-12 h after KA injection. This was not observed in non-lesioned rats even following the highest KA doses. (2) Long-term: amygdaloid lesions delayed the occurrence of the SRSs, reduced their incidence and modified their expression. In lesioned animals seizures began with a period of tonic immobility with no sign of the masticatory movements seen in intact animals. Histological examination of the KA-induced lesions did not show any major differences between lesioned and intact animals. It is suggested that the short-term effects are due to an unspecific effect on homeostatic mechanisms, whereas the long-term ones reflect a specific involvement of the amygdala in the late appearing seizures.

Choline acetyltransferase and acetylcholinesterase containing projections from the basal forebrain to the amygdaloid complex of the rat.

The origin of the cholinergic innervation to the amygdaloid complex was investigated with the use of acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) assay of microdissected nuclei. Visualization of AChE-positive neurones in the ventral forebrain was facilitated by pretreatment of rats with 1.5 mg/kg di-isopropyl phosphofluoridate (DFP). The AChE-positive neurones in the ventral forebrain are distributed in a continuous system from the septum through the lateral preoptic area to the entopeduncular nucleus caudally. Knife cuts or kainic acid injections (1.5 microgram/l microliter) placed in the lateral preoptic area resulted in substantial depletion of the AChE and ChAT content of the amygdala nuclei. Kainic acid injections (1.5 microgram/l microliter) in the diagonal band area or cuts through the stria terminalis dorsally did not significantly modify the AChE staining or ChAT content of the amygdala (although diagonal band injections partially depleted the hippocampus of ChAT). Knife cuts severing both the so-called ventral pathway and the stria terminalis did not produce significantly greater ChAT depletion in the amygdala than those produced by the knife cuts or kainic acid injections in the lateral preoptic area. Parasagittal knife cuts undercutting the lateral pyriform cortex also failed to modify the AChE or ChAT content of the amygdala, but they depleted the undercut cortex of both ChAT and AChE; AChE-positive material accumulated ventrally and medially to the knife cut. It is suggested that the major source of the cholinergic innervation of the amygdala is the magnocellular AChE-positive neurones in the lateral preoptic area and adjacent regions of the ventral forebrain.

Adenoviral vectors as functional retrograde neuronal tracers.

Adenoviruses have been recently recognized as a highly efficient system for gene delivery to various tissues. The ability of replication-defective recombinant adenovirus to transfer the lacZ reporter gene encoding beta-galactosidase to nerve cells in various brain structures has been demonstrated. Here, on the continuation of these studies, we present evidence that the adenovirus can be transported in a retrograde manner to nerve cell bodies from axonal terminals. This method may be of great value for infecting selected subsets of specific neurons for either anatomo-functional studies or even therapeutic purposes.

Histamine synthesizing afferents within the amygdaloid complex and bed nucleus of the stria terminalis of the rat.

The regional distribution of histidine decarboxylase (HD) activity has been studied in the amygdaloid complex and the bed nucleus of the stria terminalis (BST) of the rat. The central and medial nuclei of the amygdala had 2-fold higher HD activity levels than the remaining nuclei of the complex. HD activity was exceptionally high in the BST, particularly in its ventral part. A lesion of the stria terminalis had no effect on this distribution whereas a combined lesion of the stria terminalis and the so-called ventral pathway induced a decrease of approximately 60% in all the amygdaloid nuclei, but not in the BST. On the other hand, a lesion of the medial forebrain bundle (MFB) induced a similar decrease in both the amygdaloid nuclei and the BST. These results confirm that HD-containing fibres are present in the MFB. On the one hand these project massively to the BST and on the other penetrate in the amygdala ventromedially along the ansa peduncularis and preferentially innervate the more medially located nuclei.

Reversal of the anticonvulsant effects of diazepam on amygdaloid-kindled seizures by a specific benzodiazepine antagonist: RO 15-1788.

The benzodiazepine antagonist RO 15-1788 was tested in an animal model of experimental epilepsy, the kindling effect. In animals fully kindled from repeated electrical amygdaloid stimulation, RO 15-1788 reversed the anticonvulsant effects of diazepam (2 mg/kg) from doses of 0.4-0.5 mg/kg. Doses up to 20 mg/kg also antagonized the preventive action of diazepam. RO 15-1788 alone had neither proconvulsant effects on the amygdaloid afterdischarges nor benzodiazepine-like activity on the kindled seizures.

The partial benzodiazepine agonist properties of Ro 15-1788 in pentylenetetrazol-induced seizures in cats.

The effects of Ro 15-1788, a specific benzodiazepine antagonist, were studied on pentylenetetrazol-induced seizures in cats. Ro 15-1788 decreased the number of myoclonic jerks induced by a subconvulsive dose of pentylenetetrazol (12.5 mg/kg, i.m.). Ro 15-1788 suppressed generalized convulsive seizures induced by a minimal convulsive dose of pentylenetetrazol (25-35 mg/kg), but did not block the effects of higher doses (35-45 mg/kg). These results indicate that Ro 15-1788 is not a pure benzodiazepine antagonist, but has partial agonistic properties.

Long-lasting and sequential increase of c-fos oncoprotein expression in kainic acid-induced status epilepticus.

Increased but transient expression of the proto-oncogene c-fos has been recently reported in metrazol and kindling-induced seizures. Here we tested whether kainic acid-induced status epilepticus may result in a long-term increase of this oncogene. A specific pattern of immunoreactive c-fos material was observed with the development of the seizures. Intense labeling first appeared in the dentate gyrus of the hippocampus and the entorhinal cortex. Pyramidal cell layer CA3, CA4 and CA1 as well as other limbic structures were then positively stained during status epilepticus. In addition, the duration of c-fos expression was different according to the anatomical sites. In the dentate gyrus labeling did not exceed 4-5 h whereas the pyramidal cell layer CA1 exhibited increased c-fos expression for as long as 24 h. Here we propose that c-fos which has been related to growth and differentiation in previous studies, could be involved in processes inducing long-term plastic alterations in the limbic system.

Abortive amygdaloid kindled seizures following microinjection of gamma-vinyl-GABA in the vicinity of substantia nigra in rats.

The effects of microinjection of a GABA-elevating substance (gamma-vinyl-GABA) in the substantia nigra were assessed on kindled convulsive seizures induced by daily appropriate amygdaloid stimulation in the rat. Bilateral administration of 20 micrograms of gamma-vinyl-GABA strongly reduced the afterdischarge duration of the seizures without significantly modifying the motor convulsions. This effect was noted 24 h after injection and lasted for up to 48 h. Administration of gamma-vinyl-GABA in structures 1.5 mm distant from the substantia nigra had no effect on kindled seizures. It is suggested that the substantia nigra may intervene in a negative feedback system that tends to suppress the paroxysmal activity initiated from the amygdala.

Regional distribution of substance P within the amygdaloid complex and bed nucleus of the stria terminals.

The distribution of substance P (SP) within the amygdaloid complex and bed nucleus of the stria terminalis has been studied using a sensitive radioimmunoassay. Particularly high levels of SP were found in the medial nucleus of the amygdala. Large amounts of SP were also found in the various subdivisions of the bed nucleus. These results are in good agreement with immunohistochemical data.

Fos oncoprotein expression in the rat forebrain following muscimol-induced absence seizures.

Fos oncoprotein expression is a marker of neuronal activation following seizures. Here, using this method we examined the anatomical locations of muscimol-induced absence seizures in the rat forebrain. Six hours after a systemic injection of muscimol a massive Fos immunoreactivity appeared in the olfactory system, retrosplenial cortex and paraventricular thalamic nucleus, whereas other cortical areas contained low level of Fos expression. These results provide the first functional morphological evidence suggesting that these forebrain structures with Fos expression may play an important role in the pathophysiology of muscimol-induced absence seizures.

Regional distribution of met-enkephalin within the amygdaloid complex and bed nucleus of the stria terminals.

The distribution of met-enkephalin (Met-E) within the amygdaloid complex and bed nucleus of the stria terminalis has been studied using a sensitive and highly specific radioimmunoassay. Particularly high levels of Met-E were found in the central nucleus of the amygdala. Large amounts of Met-E were also found in the various subdivisions of the bed nucleus of the stria terminalis (BST). These results are in good agreement with immunohistochemical data.

Pattern of electroencephalographic activity during light induced seizures in genetic epileptic chicken and brain chimeras.

Genetic epilepsy was studied in Fayoumi epileptic (F.Epi) chickens and in neural chimeras obtained by selective substitution of embryonic brain vesicles of F.Epi donors in normal recipient chickens. Typical motor seizures accompanied by convulsions were evoked by intermittent light stimulation in F.Epi and in chimeras having embryonic substitution of the prosencephalon and the mesencephalon. The motor seizure was less severe in chimeras receiving only the prosencephalon. In the F.Epi, as well as in all the chimeras, the EEG during seizures was characterized by a desynchronized (or a flattening) pattern of activity. F.Epi and chimeras had a lower threshold to Metrazol induced seizures than control chickens. The experimental animals show that, in this model, large prosencephalic and mesencephalic areas are involved in the epileptic disease. The epileptic character of this genetic dysfunction is discussed.