GABA transporters (GATs) play a critical role in the translemmal transport of GABA in neurons and glial cells. Two major brain GATs, GAT-1 and GAT-3, are found in astrocytes in the adult brain. Astroglia demonstrate morphological and molecular changes in response to brain injury and deafferentation. The present study was designed to determine whether the expression of GATs changes after nerve deafferentation using the rat superior colliculus (SC) as a model. The immunoreactivity for GAT-1 and GAT-3, as well as GABA and glutamic acid decarboxylase (GAD)-65 and GAD-67, was studied in the SC of control rats and rats with unilateral optic nerve transections. Immunolabeling for both GAT-1 and GAT-3 was increased in the neuropil of the denervated SC as compared to that for the SC of control rats or for the unaffected SC of experimental rats. In contrast, immunoreactivity for GABA, GAD-65 and GAD-67 was not altered. The change in the immunolabeling of GAT-1 and GAT-3 was detectable at 1 day postlesion and became more evident between 3-30 days postlesion. At the electron microscopic level, immunoreactivity for both GAT-1 and GAT-3 in the unaffected SC was localized to astrocytic processes, whereas GAT-1 immunolabeling was also present in synaptic terminals. In the deafferented SC, immunolabeling for both GATs was elevated in the somata and processes of hypertrophied astrocytes as compared to that in the unaffected SC, whereas GAT-1 labeling in neuronal profiles was largely unchanged. A substantial increase of GAT-1 and GAT-3 in astrocytes following optic nerve transection suggests that these cells play a role in modulating GABA's action in the deafferented SC.

The cerebellar cortex contains several classes of GABAergic neurons. Previous studies have shown that most GABAergic neurons in this region possess the capacity for gamma-aminobutyric acid (GABA) uptake. The present study determined the postnatal expression of two GABA transporters, GAT-1 and GAT-3, in the cerebellar cortex and deep nuclei of the rat by using immunocytochemistry. Immunoreactivity for GAT-1 and GAT-3 appears at postnatal day 7 (P7), emerges centroperipherally across the cerebellum during the following 2 weeks and reaches an adult-like pattern by P30. The mature patterns are fully established by P45, which for GAT-1 is characterized by immunolabeled profiles localized exclusively to neuropil, mostly in the molecular layer and the pinceaux deep to the Purkinje cell bodies, and for GAT-3 as immunoreactivity distributed in the neuropil of mainly the granular layer. Before the adult patterns are completed, GAT-1 immunoreactivity is present in the somata of Purkinje, Golgi, basket and stellate cells between P7 and P21, while GAT-3 immunoreactivity is distinct in astrocytic somata which are organized in regularly spaced clusters. During this period, there is also a banding pattern in the sagittal plane of GAT-1 immunoreactivity in developing Purkinje cells. The postnatal development of GAT-1 and GAT-3 in the rat cerebellar cortex shares a similar spatiotemporal pattern with other GABAergic parameters, including the GABA synthesizing enzyme, GABA content and uptake. Specifically, the transient expression of GAT-1 in the somata and dendrites of cerebellar GABAergic neurons appears to correlate with the supra-adult levels of whole-tissue GABA uptake capability during development. Further, GAT-1 expression in immature Purkinje cells may play a unique role in regulating GABA's function during development, since mature Purkinje cells do not express GAT-1 or take up GABA.

In the adult cerebral cortex, GABA transporters (GATs) are expressed by both neurons and astrocytes. GAT-1 immunoreactivity is found in axon terminals of GABAergic neurons and astrocytes, while GAT-3 immunolabeling occurs only in the latter. The present study was designed to determine whether the expression of GAT-1 and GAT-3 in the adult rat cerebrum changes after needle lesion and colchicine infusion. Following a needle puncture or a saline injection, immunolabeling for GAT-1 and GAT-3 was slightly increased in an area around the needle track. Not only was the neuropil labeling for both GATs increased, but also a few neuronal somata were found to be immunoreactive for GAT-1. Colchicine injections induced a striking increase in immunolabeling for both GATs in the neuropil in an area adjacent to the needle path and surrounding it. A homologous region of the contralateral hemisphere also showed a moderate increase of immunoreactivity in the neuropil for both GATs. Furthermore, this contralateral site showed many neuronal somata immunolabeled for GAT-1. These changes were mainly detected during the first 5 days following intracortical lesions. These results indicate that (1) the upregulation of GAT-1 and GAT-3 in cortical interneurons and astrocytes is caused by both mechanical and chemical factors associated with the injections; (2) increased GAT-1 and GAT-3 expression contralateral to the site of colchicine injection is mediated by transcellular signaling across the corpus callosum; and (3) the lesion-induced GAT expression may play a protective role by helping to balance excitatory and inhibitory neuronal activities.

The present study shows the distribution of calcitonin gene-related peptide (CGRP)-immunolabeled neuronal somata and fibers in the accessory optic system of adult rats. CGRP-immunoreactive cell bodies were small to medium-sized and mostly fusiform or oval-shaped. Both immunolabeled somata and fibers were found in the dorsal and lateral terminal nuclei as well as in the interstitial nucleus of the superior fasciculus (posterior fibers); whereas only immunoreactive fibers were found in the ventral division of the medial terminal nucleus, particularly its rostral portion. These results indicate that CGRP-containing neurons are present in all nuclear components of the accessory optic system and suggest that this neuropeptide may play a neuromodulative role in eye movements.

The immunoreactivity for a gamma-aminobutyric acid (GABA) membrane transporter, GAT-1, was examined in the neocortex and hippocampal formation of developing rats from the day of birth (postnatal day 0, P0) to the adult stage. The immunolabeling was mainly localized to the neuropil, but was also in a select population of cell bodies during a limited time period. Layers I and VIb of neocortex exhibited relatively high reactivity at birth, but diminished their staining with development. In contrast, GAT-1 immunoreactivity in the neuropil in the cortical plate and its derivatives was light at birth, but increased rapidly during the first 2-3 postnatal weeks in an inside-out order. An adult pattern with immunoreactive puncta more densely distributed in layers II to IV than the deeper layers was completed by P30-45. The neuropil reactivity in the hippocampal formation at P0 was greater than that in the neocortex, densely localized in a supragranular band, and less densely in the hilus of the dentate gyrus and the strata radiatum and oriens of the hippocampus. This pattern was basically maintained at later stages except that the immunoreactivity in the supragranular band diminished, whereas that in the subgranular zone was enhanced. A population of cell bodies morphologically characteristic of cortical and hippocampal interneurons was substantially immunolabeled for GAT-1 by P5 and remained until P30. At the electron microscopic level, GAT-1 immunoreactivity was localized mainly to axon terminals and astrocytes between P5 and P45, but was also found in neuronal somata and their dendrites between P5 and P30. Our data show a differential postnatal development of GAT-1 immunoreactivity in the rat cerebral cortex, including a transient presence of immunoreactivity in the somata of a subpopulation of cerebral interneurons and a developmental downregulation of GAT-1 expression in the earliest generated cortical elements (layers 1 and VIb). The findings in the present study suggest that GAT-1 expression in the neocortex and hippocampus may relate to the functional maturation of the GABAergic system.

The stereotyped morphology of dentate granule cells in rodents consists of apical dendrites arborizing in the molecular layer and an axon arising from the opposite pole of the soma. Recently, we showed that epilepsy induces the formation of basal dendrites on granule cells and that these dendrites extend into the hilus of the dentate gyrus. The present Golgi study of granule cells from adult rats shows two atypical features for granule cells in control rats. One is the occurrence of recurrent basal dendrites (RBDs) that are defined as basal dendrites arising at or near the hilar pole of the soma and then curving back to the molecular layer. The frequency of granule cells with RBDs was 3.8% in control rats. The second is apical axons of granule cells that were observed to originate from either the apical pole of the soma or an apical dendrite. The incidence of these "apical" axons was about 1%. These morphological findings in the present study suggest that rat granule cells are more heterogeneous than previously indicated. Furthermore, their frequency was not increased in epileptic rats.

Purpose

The gamma-aminobutyric acid (GABA) degradation blocker gamma-vinyl-GABA (VGB) is used clinically to treat seizures in both adult and immature individuals. The mechanism by which VGB controls developmental seizures is not fully understood. Specifically, whether the anticonvulsant properties of VGB arise only from its elevation of brain GABA levels and the resulting activation of GABA receptors, or also from associated mechanisms, remains unresolved. Corticotropin-releasing hormone (CRH), a neuropeptide present in many brain regions involved in developmental seizures, is a known convulsant in the immature brain and has been implicated in some developmental seizures. In certain brain regions, it has been suggested that CRH synthesis and release may be regulated by GABA. Therefore we tested the hypothesis that VGB decreases CRH gene expression in the immature rat brain, consistent with the notion that VGB may decrease seizures also by reducing the levels of the convulsant molecule, CRH.

Methods

VGB was administered to immature, 9-day-old rats in clinically relevant doses, whereas littermate controls received vehicle.

Results

In situ hybridization histochemistry demonstrated a downregulation of CRH mRNA levels in the hypothalamic paraventricular nucleus but not in other limbic regions of VGB-treated pups compared with controls. In addition, VGB-treated pups had increased CRH peptide levels in the anterior hypothalamus, as shown by radioimmunoassay.

Conclusions

These findings are consistent with a reduction of both CRH gene expression and secretion in the hypothalamus, but do not support an indirect anticonvulsant mechanism of VGB via downregulation of CRH levels in limbic structures. However, the data support a region-specific regulation of CRH gene expression by GABA.

Corticotropin-releasing hormone (CRH) excites hippocampal neurons and induces death of selected CA3 pyramidal cells in immature rats. These actions of CRH require activation of specific receptors that are abundant in CA3 during early postnatal development. Given the dramatic effects of CRH on hippocampal neurons and the absence of CRH-containing afferents to this region, we hypothesized that a significant population of CRHergic neurons exists in developing rat hippocampus. This study defined and characterized hippocampal CRH-containing cells by using immunocytochemistry, ultrastructural examination, and colocalization with gamma-aminobutyric acid (GABA)-synthesizing enzyme and calcium-binding proteins. Numerous, large CRH-immunoreactive (ir) neurons were demonstrated in CA3 strata pyramidale and oriens, fewer were observed in the corresponding layers of CA1, and smaller CRH-ir cells were found in stratum lacunosum-moleculare of Ammon's horn. In the dentate gyrus, CRH-ir somata resided in the granule cell layer and hilus. Ultrastructurally, CRH-ir neurons had aspiny dendrites and were postsynaptic to both asymmetric and symmetric synapses. CRH-ir axon terminals formed axosomatic and axodendritic symmetric synapses with pyramidal and granule cells. Other CRH-ir terminals synapsed on axon initial segments of principal neurons. Most CRH-ir neurons were coimmunolabeled for glutamate decarboxylase (GAD)-65 and GAD-67 and the majority also contained parvalbumin, but none were labeled for calbindin. These results confirm the identity of hippocampal CRH-ir cells as GABAergic interneurons. Further, a subpopulation of neurons immunoreactive for both CRH and parvalbumin and located within and adjacent to the principal cell layers consists of basket and chandelier cells. Thus, axon terminals of CRH-ir interneurons are strategically positioned to influence the excitability of the principal hippocampal neurons via release of both CRH and GABA.

Febrile seizures are the most common seizure type in young children. Whether they induce death of hippocampal and amygdala neurons and consequent limbic (temporal lobe) epilepsy has remained controversial, with conflicting data from prospective and retrospective studies. Using an appropriate-age rat model of febrile seizures, we investigated the acute and chronic effects of hyperthermic seizures on neuronal integrity and survival in the hippocampus and amygdala via molecular and neuroanatomical methods. Hyperthermic seizures-but not hyperthermia alone-resulted in numerous argyrophilic neurons in discrete regions of the limbic system; within 24 hr of seizures, a significant proportion of neurons in the central nucleus of the amygdala and in the hippocampal CA3 and CA1 pyramidal cell layer were affected. These physicochemical alterations of hippocampal and amygdala neurons persisted for at least 2 weeks but were not accompanied by significant DNA fragmentation, as determined by in situ end labeling. By 4 weeks after the seizures, no significant neuronal dropout in these regions was evident. In conclusion, in the immature rat model, hyperthermic seizures lead to profound, yet primarily transient alterations in neuronal structure.

Corticotropin releasing hormone (CRH) has been localized to interneurons of the mammalian cerebral cortex, but these neurons have not been fully characterized. The present study determined the extent of co-localization of CRH with glutamate decarboxylase (GAD) and calcium-binding proteins in the infant rat neocortex using immunocytochemistry. CRH-immunoreactive (ir) neurons were classified into two major groups. The first group was larger and consisted of densely CRH-immunostained small bipolar cells, predominantly localized to layers II and III. The second group of CRH-ir cells was lightly labeled and included multipolar neurons mainly found in deep cortical layers. Co-localization studies indicated that the vast majority of CRH-ir neurons, including both bipolar and multipolar types, was co-immunolabeled for GAD-65 and GAD-67. Most multipolar, but only some bipolar, CRH-ir neurons also contained parvalbumin, while CRH-ir neurons rarely contained calbindin or calretinin. These results indicate that virtually all CRH-ir neurons in the rat cerebral cortex are GABAergic. Furthermore, since parvalbumin is expressed by cortical basket and chandelier cells, the co-localization of CRH and parvalbumin suggests that some cortical CRH-ir neurons may belong to these two cell types.