Increased expression of gamma-aminobutyric acid transporter-1 in the forebrain of infant rats with corticotropin-releasing hormone-induced seizures but not in those with hyperthermia-induced seizures

Abstract

High affinity, gamma-aminobutyric acid (GABA) plasma membrane transporters (GATs) influence the availability of GABA, the main inhibitory neurotransmitter in the brain. Recent studies suggest a crucial role for GATs in maintaining levels of synaptic GABA in normal as well as abnormal (i.e., epileptic) adult brain. However, the role of GATs during development and specifically changes in their expression in response to developmental seizures are unknown. The present study examined GAT-1-immunolabeling in infant rats with two types of developmental seizures, one induced by corticotropin-releasing hormone (CRH) lasting about 2 h and the other by hyperthermia (a model of febrile seizures) lasting only 20 min. The number of GAT-1-immunoreactive (ir) neurons was increased in several forebrain regions 24 h after induction of seizures by CRH as compared to the control group. Increased numbers of detectable GAT-1-ir cell bodies were found in the hippocampal formation including the dentate gyrus and CA1, and in the neocortex, piriform cortex and amygdala. In contrast, hyperthermia-induced seizures did not cause significant changes in the number of detectable GAT-1-ir somata. The increase in GAT-1-ir somata in the CRH model and not in the hyperthermia model may reflect the difference in the duration of seizures. The brain regions where this increase occurs correlate with the occurrence of argyrophyllic neurons in the CRH model.

Introduction

The majority of seizures occurring in the developing human are not spontaneous; i.e., they are not related to inherent abnormalities in the balance of neuronal excitation and inhibition. Instead, most developmental seizures are provoked by injurious or stressful stimuli (Baram and Hatalski, 1998). Thus, seizures during development may be induced by hypoxia (Jensen et al., 1998) or fever (see Baram et al., 1997b for review). Febrile seizures represent an important type of developmental seizure. They are extremely common in the human and have been modeled in the immature rat (Baram et al., 1997b, Toth et al., 1998, Chen et al., 1999, Dube et al., 2000). The mechanisms by which these stressful stimuli cause seizures are not clear. However, recently a role for the stress-activated excitatory neuropeptide, corticotropin-releasing hormone (CRH)(Vale et al., 1981), has been suggested in developmental seizures (Baram and Hatalski, 1998). Stress activates the expression of CRH in several brain regions that are involved in the circuitry for stressful stimuli (Hatalski et al., 1998). The limbic system, particularly the amygdala and hippocampus, is rich in neuronal populations that either synthesize CRH or possess CRH receptors (Swanson et al., 1983, De Souza et al., 1985, Avishai-Eliner et al., 1996). CRH functions as an excitatory neuromodulator (Aldenhoff et al., 1983, Ehlers et al., 1983, Hollrigel et al., 1998). In fact, picomolar amounts of CRH induce severe and prolonged seizures within minutes in young rats (Baram and Schultz, 1991, Baram et al., 1992, Baram and Schultz, 1995). Furthermore, repeated administration of CRH doses (150 × 10⁻¹² mole), which results in status epilepticus, leads to enhanced excitability of the limbic circuit (Baram and Hatalski, 1998) and to excitotoxic injury in select hippocampal and amygdala neurons in the infant rat (Baram and Ribak, 1995, Ribak and Baram, 1996). This information suggests that some stressors (i.e., hypoxia and hyperthermia) may provoke seizures early in life by increasing the levels of CRH in limbic structures. CRH-induced seizures are a model of developmental seizures because they are far more robust during the second posnatal week in rats than in adults (Baram et al., 1992, Baram and Hatalski, 1998). This may be partially due to high levels of the CRH receptor in limbic regions at this time (Avishai-Eliner et al., 1996).

Many previous neurophysiological studies have indicated that a failure of inhibition is a principal reason for neuronal hyperexcitability during epileptic seizures (Gean et al., 1989, Kamphuis et al., 1987, Kapur et al., 1989, Morimoto et al., 1987a, Morimoto et al., 1987b, Ribak, 1991). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. Extracellular GABA concentrations in the synaptic cleft are regulated by the activity of GABAergic neurons and by specific Na⁺ dependent transporter proteins on presynaptic terminals and glial cells involved in GABA uptake (Iversen and Kelly, 1975). Molecular studies have revealed at least four subtypes of GABA transporters: GAT-1, GAT-2, GAT-3 and BGT-1 (Borden et al., 1992, Guastella et al., 1990, Clark et al., 1992). These transporters have several possible functional roles, including the inactivation of GABA's action in synaptic transmission by removing GABA from the vicinity of its receptors (Iversen and Kelly, 1975, Schousboe, 1981, Isaacson et al., 1993).

GAT-1 has been demonstrated within neurons and astrocytes in various regions of the rat brain, including the olfactory bulb, neocortex, hippocampus, ventral pallidum and cerebellum (Durkin et al., 1995, Minelli et al., 1995, Ribak et al., 1996). The neuronal localization of GAT-1 in the adult hippocampus is exclusively within axon terminals forming symmetric synapses, including those from GABAergic basket and chandelier cells (Ribak et al., 1978, Ribak et al., 1996). In young rats at 10–30 postnatal days (PND) of age, GAT-1 is also localized to the somata and dendrites of GABAergic interneurons in the neocortex and hippocampus (Yan et al., 1997). The number of labeled somata varied with the age of the rat during this period. A role for GATs in adult seizures has been established (Akbar et al., 1998, During et al., 1995, Hirao et al., 1998, Mathern et al., 1999). Here we tested the hypothesis that seizures influence the normal distribution of GAT-1-immunolabeling in the immature brain. Thus, the present study addresses the question of whether an alteration in transporter distribution occurred after two types of developmental seizures: CRH-induced and hyperthermia-induced seizures. Immunocytochemical techniques were used to assess regional differences in the number of GAT-1 labeled neurons in rats with both types of seizure, and those data were compared to that of vehicle-treated controls.