C. C. G. Naus

Upregulation of gap junction connexin 32 with epileptiform activity in the isolated mouse hippocampus.

Gap junctions, which serve as intercellular channels providing direct cytoplasmic continuity and ionic current flow between adjacent cells, are constituted by connexin proteins. Using an in vitro model of bicuculline-induced epileptiform activity, we asked whether increased connexin levels occur during epileptiform activity in the intact whole hippocampus, freshly isolated from young (15-day-old) mouse brain. Exposure to bicuculline (10 microM), for 2-10 h, induced persistent changes in electrical activities that included enhanced spontaneous field activity (4 h), an epileptiform response to single electrical stimulation (6 h), and spontaneous epileptiform activity (6 h). These electrophysiological changes were not reversed by up to 60 min perfusion with normal artificial cerebrospinal fluid, but were greatly depressed by the gap junction uncoupler, carbenoxolone (120 microM, 10 min). Data from RNase protection assay and immunoblotting showed that among several detected gap junctions, only connexin 32 was affected. After 2-6 h exposure to bicuculline, the connexin 32 mRNA expression was upregulated to 2-3-fold control (P < 0.01), and its protein level was significantly elevated the following 6 h (P < 0.01), at which time electrophysiologically measured evidence of clearly epileptiform activity was apparent. In addition, the transcription factor, c-fos protein, but not the cAMP response element-binding protein, was also found to be increased at the early stage of bicuculline exposure (2 h) compared to control (P < 0.05).Thus, we have found that exposing the acutely isolated hippocampus to bicuculline, induced increased c-fos protein, followed by increased connexin 32 transcript and protein, and concurrently, persistent epileptiform activity that was depressed by carbenoxolone.

An anterograde HRP-WGA study of aberrant corticorubral projections following neonatal lesions of the rat sensorimotor cortex

SummaryAnterograde transport of horseradish peroxidase — wheat germ agglutinin (HRP-WGA) was used to examine the effect of unilateral neonatal ablation of the sensorimotor cortex on the remaining corticofugal projections to the midbrain in the rat. In unlesioned animals, the sensorimotor cortical efferents to the midbrain were entirely ipsilateral, terminal labeling being evident in the red nucleus, the midbrain reticular formation, the periaqueductal gray, the intermediate gray layer of the superior colliculus, the nucleus parafascicularis prerubralis and the perilemniscal area. Corticorubral fibers were seen to reach the midbrain through the thalamus or the cerebral peduncle. In the red nucleus, terminal labeling was essentially restricted to the parvocellular region. In neonatally lesioned adults, aberrant corticofugal fibers crossed the midline to terminate in the contralateral red nucleus, the midbrain reticular formation, the periaqueductal gray, the nucleus parafascicularis prerubralis and the intermediate gray layer of the superior colliculus. The aberrant projections maintained the topographic specificity of the normal ipsilateral projections. This was most evident in the corticorubral projection, where the aberrant contralateral fibers terminated in the parvocellular area of the red nucleus.

Topographic specificity of aberrant cerebellorubral projections following neonatal hemicerebellectomy in the rat.

Anterograde transport of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) was used to examine the topographic specificity of ascending cerebellar efferent projections in adult rats which were hemicerebellectomized at birth. The results were compared to similar cerebellar projections in unlesioned adults. HRP-WGA placement in the nucleus interpositus of control rats resulted in a dense projection of labeled fibers which decussated in the midbrain, caudal to the red nucleus. In the red nucleus, dense terminal labeling was confined to the magnocellular region, while retrogradely labeled rubrocerebellar neurons were present throughout both parvo- and magnocellular areas. Similar HRP-WGA placements in the nucleus lateralis gave rise to fewer labeled fibers which terminated in the parvocellular red nucleus. In addition to the cerebellorubral projection, other areas of terminal labeling included the mid-brain reticular formation, nucleus parafascicularis prerubralis, zona incerta, fields of Forel and ventral thalamus. In neonatally lesioned adults, aberrant cerebellorubral and cerebellothalamic projections were observed deflecting ipsilaterally at the decussation of the normal contralateral projection. Topographic specificity of the aberrant ipsilateral cerebellorubral projection mirrored that of the normal contralateral fibers. In addition, an ipsilateral projection from the cerebellum could be followed rostral to the red nucleus, to terminate in the ipsilateral ventral thalamus. Lesioned animals also demonstrated marked cell loss in the red nucleus contralateral to the hemicerebellectomy.

Characterization of CarG-binding protein A initially identified by differential display

While investigating differences in the pattern of gene expression in functionally distinct areas of the rat caudate-putamen employing differential display, we identified a gene that is highly enriched in tissue adjacent to the lateral ventricle. To characterize the gene, a complementary DNA containing the complete coding sequence was obtained and sequenced. In addition, radiolabelled DNA and riboprobes were generated to examine the expression levels and anatomical distribution of the identified gene in the brain. The sequencing data suggests that the identified gene is a member of the heterogeneous nuclear ribonucleoprotein family and likely represents the rat homolog of CArG-binding protein A initially isolated from mouse C2 myogenic cells. CArG-binding protein A is widely distributed and moderately expressed in the rat brain and present within both neurons and astrocytes. Since the CArG box motif forms the core of the serum response element and the serum response element is involved in immediate early gene regulation, the expression level of CArG-binding protein A was examined following treatment of PC12 cells with nerve growth factor and correlated with changes in c-fos and zif268 expression. The results show that CArG-binding protein A is up-regulated following nerve growth factor treatment and that the up-regulation of CArG-binding protein A can be correlated with the down-regulation of c-fos and zif268. The results of the current study leads us to suggest that CArG-binding protein A may be involved in brain development and the regulation of the serum response element.

Somatostatin and the patch/matrix compartments of the rat caudate-putamen.

In the caudate‐putamen of the rat, two subpopulations of medium aspiny neurons exist that contain somatostatin. The first subpopulation contains somatostatin 14, somatostatin 28, and somatostatin 28(1–12). The other subpopulation contains only somatostatin 28. To examine the relationship between somatostatin‐containing neurons and the patch/matrix compartments, a series of double‐labelling experiments using antisera directed against different somatostatin peptides and calbindin were used. Sections stained in this manner were examined with the aid of a confocal microscope. The results of these experiments indicate that somatostatin 28(1–12)‐containing neurons may play a role in matrix integration, with some input directed from the patch compartment. In addition, somatostatin 28‐containing neurons are more numerous in the patch compartment than somatostatin 28(1–12)‐containing neurons, suggesting a possible role for these neurons in patch integration.

Changes in CArG-binding protein A expression levels following injection(s) of the D1-dopamine agonist SKF-82958 in the intact and 6-hydroxydopamine-lesioned rat.

We recently characterized the rat brain homolog of mouse muscle CArG-binding protein A initially identified in C2 myogenic cells and showed an inverse temporal correlation between increased expression levels of this messenger RNA, c-fos and zif268 messenger RNA levels following the addition of nerve growth factor to PC12 cells. In addition, we found an inverse correlation between c-Fos protein and CArG-binding protein A messenger RNA levels in the lateral caudate-putamen of rats treated acutely and chronically with the D2 receptor antagonist fluphenazine (phenothiozine typical psychotic). To determine whether D1 receptor stimulation is also capable of inducing CArG-binding protein A up-regulation, drug naive or dopamine-depleted (i.e. 6-hydroxydopamine-lesioned) D1 hypersensitized rats (i.e. rats given repeated daily injections of SKF-82958 for 14days) were acutely injected with the D1 agonist SKF-82958 and examined using a combination of in situ hybridization for CArG binding protein A and immunocytochemistry for c-Fos. Both acutely treated animals and dopamine-depleted hypersensitized animals showed increases in CArG-binding protein A. Moderate increases were found in the medial caudate-putamen and nucleus accumbens core and shell regions following acute treatment whereas large increases in CArG-binding protein A expression levels were found in the medial and lateral caudate-putamen and the shell and core of the nucleus accumbens following hypersensitization. No change in CArG-binding protein A expression level was found in the dopamine-depleted, drug naive animals relative to controls. Regions of the basal ganglia where increases in CArG-binding protein A were detected following each treatment correlated perfectly with c-Fos protein induction. The results demonstrate that CArG-binding protein A responds to SKF-82958 and that the changes in CArG-binding protein A match perfectly with the pattern of c-Fos induction induced by the D1 agonist.

Effects of graft-derived dopaminergic innervation on the target neurons of patch and matrix compartments of the striatum.

Fetal dopaminergic neurons grafted into the dopamine-depleted striatum have previously been shown to normalize neurochemical and behavioural abnormalities. However, the extent of graft-induced recovery of striatal compartments, which differ in their ontogeny, neurochemical properties and function, is still not clear. The striosome and matrix compartments of the striatum provide a segregated projection to somatostatin-containing GABAergic neurons of the rostral part of the entopeduncular nucleus and somatostatin-negative GABAergic neurons of the caudal part of the entopeduncular nucleus, respectively. In the present study, preprosomatostatin and glutamate decarboxylase messenger RNA levels in the rostral and caudal parts of the entopeduncular nucleus were determined six and 18 months postgrafting in rats with complete recovery of rotational behaviour following apomorphine challenge, and in rats with unilateral 6-hydroxydopamine lesions or sham lesions and no grafts. Sections were processed for in situ hybridization using 35S-labelled cRNA probes for glutamate decarboxylase (67,000 mol. wt isoform; GAD67) and preprosomatostatin. Autoradiographs showed a marked increase in preprosomatostatin messenger RNA within the ipsilateral entopeduncular nucleus in 6-hydroxydopamine-lesioned rats, and a substantially lower increase six months postgrafting. At 18 months postgrafting, the preprosomatostatin messenger RNA levels were symmetrical within the entopeduncular nucleus. Unilateral depletion of striatal dopamine resulted in a moderate increase in GAD67 messenger RNA levels within the ipsilateral entopeduncular nucleus, along with a substantial decrease in GAD67 levels within the contralateral nucleus. By six months postgrafting, the GAD67 levels had decreased considerably within the ipsilateral entopeduncular nucleus, while the messenger RNA levels had returned to normal within the contralateral nucleus. Interestingly, at 18 months postgrafting, the GAD67 levels remained decreased within the ipsilateral entopeduncular nucleus and were significantly lower than the normal value. The results indicate that fetal nigral grafts placed within the dopamine-depleted striatum can restore the neurochemical alterations seen in striatal target areas such as the entopeduncular nucleus. This may form the neurochemical basis of graft-induced behavioural recovery, as the normalization of neurotransmitter messenger RNA levels in the entopeduncular nucleus reflects the restoration of overall activity in both direct and indirect striatal output pathways. The results also indicate that the graft-derived dopaminergic innervation restores the output of both striosome and matrix compartments of the striatum. The present results also showed a progressive recovery leading to over-compensation of neurotransmitter messenger RNA levels following grafting, perhaps indicating the importance of feedback regulation of grafted dopaminergic neurons by the host.

Quantitative analysis of rubral degeneration following neonatal deafferentation.

The effect of neonatal hemicerebellectomy on the cytoarchitecture of the red nucleus was investigated in the rat. Quantitative analysis revealed a 31% loss of neurons in the magnocellular red nucleus contralateral to the lesion. This cell loss was accompanied by a similar decrease in the cross-sectional area of the red nucleus. To provide insight into this degenerative process, the neonatal status of the normal cerebellorubral projection was determined using anterograde transport of HRP-WGA which was injected into the deep cerebellar nuclei at various times during the first 10 postnatal days. A definitive cerebellorubral projection was not detectable before postnatal day 8. The data indicate that the rubral afferent fibers from the cerebellum were removed before they reached their target. These results are significant in light of the aberrant projections that develop after neonatal lesions.