Christoph Schwarzer

GABAA receptors : Immunocytochemical distribution of 13 subunits in the adult rat brain

Abstract GABAA receptors are ligand-operated chloride channels assembled from five subunits in a heteropentameric manner. Using immunocytochemistry, we investigated the distribution of GABAA receptor subunits deriving from 13 different genes (α1–α6, β1–β3, γ1–γ3 and δ) in the adult rat brain. Subunit α1-, β1-, β2-, β3- and γ2-immunoreactivities were found throughout the brain, although differences in their distribution were observed. Subunit α2-, α3-, α4-, α5-, α6-, γ1- and δ-immunoreactivities were more confined to certain brain areas. Thus, α2-subunit-immunoreactivity was preferentially located in forebrain areas and the cerebellum. Subunit α6-immunoreactivity was only present in granule cells of the cerebellum and the cochlear nucleus, and subunit γ1-immunoreactivity was preferentially located in the central and medial amygdaloid nuclei, in pallidal areas, the substantia nigra pars reticulata and the inferior olive. The α5-subunit-immunoreactivity was strongest in Ammon’s horn, the olfactory bulb and hypothalamus. In contrast, α4-subunit-immunoreactivity was detected in the thalamus, dentate gyrus, olfactory tubercle and basal ganglia. Subunit α3-immunoreactivity was observed in the glomerular and external plexiform layers of the olfactory bulb, in the inner layers of the cerebral cortex, the reticular thalamic nucleus, the zonal and superficial layers of the superior colliculus, the amygdala and cranial nerve nuclei. Only faint subunit γ3-immunoreactivity was detected in most areas; it was darkest in midbrain and pontine nuclei. Subunit δ-immunoreactivity was frequently co-distributed with α4 subunit-immunoreactivity, e.g. in the thalamus, striatum, outer layers of the cortex and dentate molecular layer. Striking examples of complementary distribution of certain subunit-immunoreactivities were observed. Thus, subunit α2-, α4-, β1-, β3- and δ-immunoreactivities were considerably more concentrated in the neostriatum than in the pallidum and entopeduncular nucleus. In contrast, labeling for the α1-, β2-, γ1- and γ2-subunits prevailed in the pallidum compared to the striatum. With the exception of the reticular thalamic nucleus, which was prominently stained for subunits α3, β1, β3 and γ2, most thalamic nuclei were rich in α1-, α4-, β2- and δ-immunoreactivities. Whereas the dorsal lateral geniculate nucleus was strongly immunoreactive for subunits α4, β2 and δ, the ventral lateral geniculate nucleus was predominantly labeled for subunits α2, α3, β1, β3 and γ2; subunit α1- and α5-immunoreactivities were about equally distributed in both areas. In most hypothalamic areas, immunoreactivities for subunits α1, α2, β1, β2 and β3 were observed. In the supraoptic nucleus, staining of conspicuous dendritic networks with subunit α1, α2, β2, and γ2 antibodies was contrasted by perykarya labeled for α5-, β1- and δ-immunoreactivities. Among all brain regions, the median emminence was most heavily labeled for subunit β2-immunoreactivity. In most pontine and cranial nerve nuclei and in the medulla, only subunit α1-, β2- and γ2-immunoreactivities were strong, whereas the inferior olive was significantly labeled only for subunits β1, γ1 and γ2. In this study, a highly heterogeneous distribution of 13 different GABAA receptor subunit-immunoreactivities was observed. This distribution and the apparently typical patterns of co-distribution of these GABAA receptor subunits support the assumption of multiple, differently assembled GABAA receptor subtypes and their heterogeneous distribution within the adult rat brain.

GABAA receptor subunits in the rat hippocampus I: Immunocytochemical distribution of 13 subunits

The GABA(A) receptor is a ligand-operated chloride channel. It has a pentameric structure. In mammalian brain different subunits are recruited from four gene subfamilies. Using immunocytochemistry, we investigated the distribution of the 13 GABA(A) receptor subunits in the hippocampus of the rat. GABA(A) receptor subunits were heterogeneously distributed within different hippocampal subfields. High concentrations of alpha1-, alpha2-, alpha4-, beta3-, gamma2- and delta-immunoreactivities were observed within the molecular layer of the dentate gyrus, representing the dendritic area of the granule cells. In the hippocampus proper, the predominant GABA(A) receptor subunits were alpha1, alpha2, alpha5, beta3 and gamma2 that were located throughout the strata radiatum and oriens of CA1 to CA3. Immunocytochemical staining was there less prominent for alpha4-, beta1-, beta2- gamma3- and delta- subunits. In the hippocampus proper, the beta1 subunit was preferentially located in CA2. The alpha4- and delta-subunits were somewhat more abundant in CA1 than in CA3. Numerous local circuit neurons in the hippocampus proper and the hilus of the dentate gyrus contained alpha1-, beta2-, gamma2- and/or delta-subunits. Alpha3 and gamma1 were present only in minute amounts and no alpha6-IR was detected in the hippocampal formation. The distribution of the GABA(A) receptor subunits indicates the existence of heterogenously constituted GABA(A) receptor complexes within various hippocampal subfields, which may exert different physiological or pharmacological properties upon stimulation by GABA or its agonists.

Neuropeptide Y inhibits potassium‐stimulated glutamate release through Y2 receptors in rat hippocampal slices in vitro

1 We investigated the effects of neuropeptide Y (NPY), peptide YY (PYY), NPY13–36, NPY18–36, [Leu31][Pro34]NPY and of pancreatic polypeptide Y (PPY) on calcium‐dependent, potassium‐stimulated glutamate release in superfused rat hippocampal slices. 2 NPY, PYY and the Y2 receptor agonist NPY13–36 equipotently inhibited the release of glutamate. The half‐maximal response was observed at about 10 nm in a dose‐dependent manner (3 to 100 nm). Maximal inhibition of 50 to 60% was obtained at 100 nm. At higher concentrations of the peptides (300 nm and 1 μm) this inhibition was partially or entirely reversed. Porcine NPY13–36 and NPY18–36, inhibited glutamate release by about 44% at 100 nm. 3 The specific Y1 receptor agonist, [Leu31][Pro34]NPY, caused an insignificant increase in glutamate release at 100 to 300 nm concentrations. PPY had no effect on potassium‐evoked glutamate release in hippocampal slices at concentrations of 30 nm to 1 μm. 4 The experiments support previous electrophysiological data. They suggest a potent inhibitory action of NPY through NPY‐Y2 receptors on the release of the excitatory amino acid glutamate in rat hippocampus. Especially under conditions of increased NPY synthesis, such as in epilepsy, this mechanism may be of pathophysiological relevance.

Hippocampal granule cells express glutamic acid decarboxylase-67 after limbic seizures in the rat.

Temporal lobe epilepsy is the most common form of epilepsy. Decreased GABA-ergic inhibition has been suggested as one cause of hyperexcitability. On the other hand, increased expression of glutamic acid decarboxylase, the rate-limiting enzyme of GABA synthesis, has been found in interneurons of the hippocampus in patients with temporal lobe epilepsy and in rats after kainic acid-induced limbic seizures, indicating increased GABA-ergic transmission. Here we report differential expression of two genes encoding different molecular forms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, after kainic acid-induced seizures in the rat. There is a rapid but transient elevation of GAD67 mRNA levels in granule cells 6-24 h after kainic acid injection, followed by enhanced GAD immunoreactivity in the terminal field of mossy fibers. In interneurons in the hilus of the dentate gyrus, a sustained and progressing increase in the expression of both GAD65 and GAD67 messenger RNA occurs. These observations indicate that consitutively glutamatergic mossy fibers may be capable of synthetizing and utilizing the inhibitory transmitter GABA in sustained limbic seizures. Enhanced expression of glutamic acid decarboxylases within interneurons and in granule cells/mossy fibers suggest augmented GABA-ergic neurotransmission supporting selfprotective, anticonvulsive mechanisms in limbic epilepsy.

Somatostatin, neuropeptide Y, neurokinin B and cholecystokinin immunoreactivity in two chronic models of temporal lobe epilepsy

Somatostatin-, neuropeptide Y-, neurokinin B- and cholecystokinin-containing neurons were investigated in the rat hippocampus in two chronic models of temporal lobe epilepsy, i.e. 30 days after rapid kindling or electrically induced status epilepticus (post-status epilepticus). After rapid kindling, somatostatin immunoreactivity was strongly increased in interneurons and in the outer and middle molecular layer of the dentate gyrus. In four of six post-status epilepticus rats (status epilepticus I rats), somatostatin immunoreactivity was slightly increased in the dorsal but decreased in the ventral dentate gyrus and molecular layer. Somatostatin immunoreactivity decreased in neurons of the dorsal hilus in the two other post-status epilepticus rats investigated, while a complete loss was found in the respective ventral extension (status epilepticus-II rats). These changes were associated with a different extent of neurodegeneration as assessed by Nissl staining. Similarly, neuropeptide Y immunoreactivity was enhanced in neurons of the hilus and in the middle and outer molecular layer of the dentate gyrus in the dorsal hippocampus of rapidly kindled and status epilepticus-I rats. Neuropeptide Y and neurokinin B immunoreactivity was enhanced in the mossy fibers of all post-status epilepticus rats, but not in the rapidly kindled rats. In status epilepticus-II rats, neuropeptide Y-and neurokinin B-positive fibers were also detected in the infrapyramidal region of the stratum oriens of CA3 and in the inner molecular layer of the dentate gyrus in the dorsal and ventral hippocampus respectively, labeling presumably sprouted mossy fibers. Increased staining of neuropeptide Y and neurokinin B was found in the alveus after rapid kindling. Cholecystokinin immunoreactivity was markedly increased in the cerebral cortex, Ammons horn and the molecular layer of the dentate gyrus in the ventral hippocampus of rapidly kindled and post-status epilepticus rats. The lasting changes in the immunoreactive pattern of various peptides in the hippocampus may reflect functional modifications in the corresponding peptide-containing neurons. These changes may be involved in chronic epileptogenesis, which evolves in response to limbic seizures.

GABAA receptor subunits in the rat hippocampus II: Altered distribution in kainic acid-induced temporal lobe epilepsy

Intraperitoneal injection of kainic acid in the rat represents a widely used animal model of human temporal lobe epilepsy. Injection of kainic acid induces acute limbic seizures which are accompanied by seizure-induced brain damage and late spontaneous recurrent seizures. There is considerable evidence for an altered transmission of GABA in human temporal lobe epilepsy and in the kainic acid model. We therefore investigated by immunocytochemistry the distribution of 13 GABA receptor subunits in the hippocampus of rats 12 h, 24 h, and two, seven and 30 days after injection of kainic acid. Within the molecular layer of the dentate gyrus, decreases in alpha2- and delta- and slight increases in alpha1, beta2- and beta3-immunoreactivities were observed at early intervals (12 to 24 h) after kainic acid injection. These changes were succeeded by marked increases in alpha1-, alpha2-, alpha4-, alpha5-, beta1-, beta3-, gamma2- and delta-immunoreactivities in the same area after seven to 30 days. Within the hippocampus proper, changes in expression of GABA(A) receptor subunits were demarcated by considerable neurodegeneration of CA1 and CA3 pyramidal neurons. All subunits present within dendritic areas of CA1 and CA3 were affected. These were alpha1, alpha2, alpha5, beta1-beta3, gamma2 and alpha4 (present only in CA1). Decreases in these subunits were followed by increased expression of alpha2-, alpha5-, beta3-, gamma2- and delta-subunits in the hippocampus proper notably in CA3 at later intervals (up to 30 days). Alpha1-, beta2-, gamma2- and delta-subunits were found in presumed GABA containing interneurons throughout the hippocampus. Their immunoreactivity was augmented after two to seven days. Some alpha4-, gamma3- and delta-immunoreactivity was also found in astrocytes 48 h after kainic acid injection. Our data indicate an impairment of GABA-mediated neurotransmission due to a lasting loss of GABA(A) receptor containing cells after kainic acid-induced seizures. The seizure-induced loss in GABA(A) receptors within the hippocampus may in part be compensated by increased expression of GABA(A) receptor subunits within the molecular layer of the dentate gyrus and in pyramidal cells.

Neuropeptides-immunoreactivity and their mRNA expression in kindling: functional implications for limbic epileptogenesis

Recent studies have demonstrated that neuropeptide expression in forebrain neurons is responsive to changes in physiological activity. This is particularly true in the hippocampus where the expression of various neuropeptides has been reported to change in distinct neuronal populations in response to seizure activity. The aim of this work is to review and integrated the information on the pathological changes and functional modifications in neuropeptide systems of the hippocampal formation in kindling and other models of limbic epilepsy. This will be done by presenting a study in which we investigated the changes in the expression of somatostatin, neuropeptide Y (NPY), neurokinin B (NKB) and cholecystokinin-octapeptide (CCK) in the rat hippocampal principal neurons during and after kindling of the hippocampus using immunocytochemistry and in situ hybridization analysis of mRNA. NPY-IR was transiently expressed in the granule cells/mossy fibres after the preconvulsive stage 2 and 2 days but not 1 week after three consecutive tonic-clonic seizures (stage 5). A more pronounced increase was observed in NKB-IR lasting 1 week after kindling acquisition. Only the NKB mRNA expression was enhanced in granule cells at these intervals. At stages 2 and 5, somatostatin- and NPY-IR and their mRNA levels were markedly increased in interneurons in the deep hilus and in the polymorphic cell layer and their presumed projections to the outer molecular layer of the dentate gyrus. NKB- and CCK-IR and their mRNAs were highly expressed in basket cells at both stages of kindling. Their IR was increased in the inner molecular layer of the dentate gyrus in the ventral hippocampus. Peptide-containing neurons in the hilus appeared well preserved in spite of a reduction of Nissl stained cells by 24 % in the stimulated and contralateral hippocampus at stage 5. In the hippocampus proper, somatostatin and NPY-IR were enhanced in the stratum lacunosum molecular while CCK-IR fibres and its mRNA were particularly expressed in the pyramidal cell layer. The number of Somatostatin-, NKB- and CCK-IR cells was increased in the subiculum. The intensity of these changes was similar 2 days after stages 2 or 5 of kindling. Less pronounced effects were observed 1 week after kindling completion. These results, in the frame of the literature data, suggest that lasting functional changes occur in distinct neuropeptide-containing neurons during limbic epileptogenesis. This may have profound effects on synaptic transmission and contribute to modulate hippocampal excitability.

Distribution of the major gamma-aminobutyric acid(A) receptor subunits in the basal ganglia and associated limbic brain areas of the adult rat.

Within the basal ganglia, γ‐aminobutyric acid (GABA) exerts a fundamental role as neurotransmitter of local circuit and projection neurons. Its fast hyperpolarizing action is mediated through GABAA receptors. These ligand‐gated chloride channels are assembled from five subunits, which derive from multiple genes. Using immunocytochemistry, we investigated the distribution of 12 major GABAA receptor subunits (α1–5, β1–3, γ1–3, and δ) in the basal ganglia and associated limbic brain areas of the rat. Immunoreactivity for an additional subunit (subunit α6) was not observed. The striatum, the nucleus accumbens, and the olfactory tubercle displayed strong, diffuse staining for the subunits α2, α4, β3, and δ presumably located on dendrites of the principal medium spiny neurons. Subunit α1‐, β2‐, and γ2‐immunoreactivities were apparently mostly restricted to interneurons of these areas. In contrast, the globus pallidus, the entopeduncular nucleus, the ventral pallidum, the subthalamic nucleus, and the substantia nigra pars reticulata revealed dense networks of presumable dendrites of resident projection neurons, which were darkly labeled for subunit α1‐, β2‐, and γ2‐immunoreactivities. The globus pallidus, ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata, all areas receiving innervations from the striatum, displayed strong subunit γ1‐immunoreactivity compared to other brain areas. In the substantia nigra pars compacta and in the ventral tegmental area, numerous presumptive dopaminergic neurons were labeled for subunits α3, γ3, and/or δ. This highly heterogeneous distribution of individual GABAA receptor subunits suggests the existence of differently assembled, and presumably also functionally different, GABAA receptors within individual nuclei of the basal ganglia and associated limbic brain areas. J. Comp. Neurol. 433:526–549, 2001.

GABAA receptor subunits in the rat hippocampus III: altered messenger RNA expression in kainic acid-induced epilepsy

Kainic acid-induced seizures in rats represent an established animal model for human temporal lobe epilepsy. The neuropathological sequelae include acute status epilepticus followed by neurodegeneration in the CA1 and CA3 sector of the Ammons horn and of interneurons in the hilus of the dentate gyrus. After about three weeks spontaneous recurrent seizures become manifest. We investigated changes in messenger RNA expression of 13 GABA(A) receptor subunits in the hippocampus of rats in the initial phase (6 h, 12 h and 24 h) after acute kainic acid-induced status epilepticus and seizure-related neuronal cell damage during and after acquisition of spontaneous recurrent seizures (seven and 30 days after kainic acid injection). In the granule cell layer, initial (after 6 to 12 h) decreases in (alpha2, alpha3, alpha5, beta1, beta3, gamma2 and delta messenger RNAs (by about 25 to 50%) were accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages. At later intervals (after seven to 30 days), expression of alpha2, alpha4, beta3 and gamma2 messenger RNAs recovered to control values, with alpha5 and delta messenger RNA still being reduced (by 15 and 40% below control levels, respectively). Concentrations of the transcripts encoding for alpha1, alpha3, beta1, beta2, became markedly enhanced (between 20 and 50% of controls). Within the pyramidal cell layers CA1 and CA3, decreases in alpha2, alpha4, alpha5, beta(1-3) and gamma2 messenger RNAs were detected after seven to 30 days, reflecting pronounced neurodegeneration in these areas. The alpha1 transcript was decreased in CA3 after 24 h and increased to control levels indicating compensatory up-regulation of this message after seven days. Messenger RNAs encoding for alpha3-, gamma1-, and gamma3-subunits were detected at rather low levels, alpha6 was not present in the hippocampus. Our data suggest a fast but transient change in the expression of messenger RNAs encoding for different subunits of the GABA(A) receptor in the granule cell layer of the dentate gyrus. This is followed by a lasting augmentation of messenger RNAs encoding different GABA(A) receptor subunits in the same cell layer indicating long-lasting GABAergic inhibition. Changes within the pyramidal cell layer are mostly determined by concomitant neurodegenerative processes.

Seizure susceptibility and epileptogenesis are decreased in transgenic rats overexpressing neuropeptide Y

Functional studies in epileptic tissue indicate that neuropeptide Y and some of its peptide analogs potently inhibit seizure activity. We investigated seizure susceptibility in transgenic rats overexpressing the rat neuropeptide Y gene under the control of its natural promoter. Seizures were induced in adult transgenic male rats and their wild-type littermates by i.c.v. injection of 0.3 microg kainic acid or by electrical kindling of the dorsal hippocampus. Transgenic rats showed a significant reduction in the number and duration of electroencephalographic seizures induced by kainate by 30% and 55% respectively (P<0.05 and 0.01). Transgenic rats were also less susceptible to epileptogenesis than wild-type littermates as demonstrated by a 65% increase in the number of electrical stimuli required to induce stage 5 seizures (P<0.01). This phenotype was associated with a strong and specific expression of neuropeptide Y mRNA in area CA1, a brain area involved in the seizure network. We conclude that endogenous neuropeptide Y overexpression in the rat hippocampus is associated with inhibition of seizures and epileptogenesis suggesting that this system may be a valuable target for developing novel antiepileptic treatments.