Receptor Subunits In Hippocampus Research Articles

Neurodevelopmental effects of prenatal Bisphenol A exposure on the role of microRNA regulating NMDA receptor subunits in the male rat hippocampus

Maternal bisphenol A (BPA) exposure has been reported to cause learning and memory deficits in born offspring. However, little is known that this impairment is potentially caused by epigenetic modulation on the development of NMDA receptor subunits. This study investigates the effect of prenatal BPA exposure on the hippocampal miR-19a and miR-539, which are responsible for regulating NMDA receptor subunits as well as learning and memory functions. Pregnant Sprague Dawley rats were orally administered with 5 mg/kg/day of BPA from pregnancy day 1 (PD1) until gestation day 21 (GD21), while control mothers received no BPA. The mothers were observed daily until GD21 for either a cesarean section or spontaneous delivery. The male offspring were sacrificed when reaching GD21 (fetus), postnatal days 7, 14, 21 (PND7, 14, 21) and adolescent age 35 (AD35) where their hippocampi were dissected from the brain. The expression of targeted miR-19a, miR-539, GRIN2A, and GRIN2B were determined by qRT-PCR while the level of GluN2A and GluN2B were estimated by western blot. At AD35, the rats were assessed with neurobehavioral tests to evaluate their learning and memory function. The findings showed that prenatal BPA exposure at 5 mg/kg/day significantly reduces the expression of miR-19a, miR-539, GRIN2A, and GRIN2B genes in the male rat hippocampus at all ages. The level of GluN2A and GluN2B proteins is also significantly reduced when reaching adolescent age. Consequently, the rats showed spatial and fear memory impairments when reaching AD35. In conclusion, prenatal BPA exposure disrupts the role of miR-19a and miR-539 in regulating the NMDA receptor subunit in the hippocampus which may be one of the causes of memory and learning impairment in adolescent rats.

Prenatal bisphenol A exposure impairs the aversive and spatial memory reduces the level of NMDA receptor subunits in the hippocampus of male Sprague Dawley rats

Memory impairment in children is an ongoing issue worldwide related to a learning disability. This neurobiological condition has been suggested to associate with bisphenol A (BPA) exposure during pregnancy. BPA is an inorganic compound used to produce polycarbonate plastics and epoxy resins. We conduct this study to investigate the effects of prenatal BPA exposure on the level of the N-methyl-D-aspartate (NMDA) receptor subunits, synaptic markers of the hippocampus and neurobehavioral outcomes in rats. The pregnant rats were given a daily dose of 5 mg/kg and 50 mg/kg of BPA with 0.5% Tween 80 orally from gestation day 2 until 21 (GD21). The level of GluN2A, GluN2B, PSD-95 and synapsin I in the hippocampus and its neurobehaviour outcomes were quantified and evaluated in the male foetus and adolescent rat. Prenatal BPA exposure reduced GluN2A, GluN2B, synapsin I and PSD-95 (Postsynaptic Density-95) in the male foetus and adolescent rat hippocampus compared to the control group. The prenatal BPA exposed rats demonstrated anxiety-related behaviour and impairment in aversive and spatial memory. The findings suggested that the impairment in neurobehavioral performance may inhibit the signalling pathway in the NMDA receptor subunits in the male foetus rat hippocampus leading to learning and memory deficits when reaching adolescence.

Hyperammonemia alters membrane expression of GluA1 and GluA2 subunits of AMPA receptors in hippocampus by enhancing activation of the IL-1 receptor: underlying mechanisms

BackgroundHyperammonemic rats reproduce the cognitive alterations of patients with hepatic encephalopathy, including altered spatial memory, attributed to altered membrane expression of AMPA receptor subunits in hippocampus. Neuroinflammation mediates these cognitive alterations. We hypothesized that hyperammonemia-induced increase in IL-1β in hippocampus would be responsible for the altered GluA1 and GluA2 membrane expression. The aims of this work were to (1) assess if increased IL-1β levels and activation of its receptor are responsible for the changes in GluA1 and/or GluA2 membrane expression in hyperammonemia and (2) identify the mechanisms by which activation of IL-1 receptor leads to altered membrane expression of GluA1 and GluA2.MethodsWe analyzed in hippocampal slices from control and hyperammonemic rat membrane expression of AMPA receptors using the BS3 cross-linker and phosphorylation of the GluA1 and GluA2 subunits using phosphor-specific antibodies. The IL-1 receptor was blocked with IL-Ra, and the signal transduction pathways involved in modulation of membrane expression of GluA1 and GluA2 were analyzed using inhibitors of key steps.ResultsHyperammonemia reduces GluA1 and increases GluA2 membrane expression and reduces phosphorylation of GluA1 at Ser831 and of GluA2 at Ser880. Hyperammonemia increases IL-1β, enhancing activation of IL-1 receptor. This leads to activation of Src. The changes in membrane expression of GluA1 and GluA2 are reversed by blocking the IL-1 receptor with IL-1Ra or by inhibiting Src with PP2.After Src activation, the pathways for GluA2 and GluA1 diverge. Src increases phosphorylation of GluN2B at Tyr14721 and membrane expression of GluN2B in hyperammonemic rats, leading to activation of MAP kinase p38, which binds to and reduces phosphorylation at Thr560 and activity of PKCζ, resulting in reduced phosphorylation at Ser880 and enhanced membrane expression of GluA2.Increased Src activity in hyperammonemic rats also activates PKCδ which enhances phosphorylation of GluN2B at Ser1303, reducing membrane expression of CaMKII and phosphorylation at Ser831 and membrane expression of GluA1.ConclusionsThis work identifies two pathways by which neuroinflammation alters glutamatergic neurotransmission in hippocampus. The steps of the pathways identified could be targets to normalize neurotransmission in hyperammonemia and other pathologies associated with increased IL-1β by acting, for example, on p38 or PKCδ.Graphical abstractIL-1β alters membrane expression of GluA1 and GluA2 AMPA receptor subunits by two difrerent mechanisms in the hippocampus of hyperammonemic rats.

Endosulfan and Cypermethrin Pesticide Mixture Induces Synergistic or Antagonistic Effects on Developmental Exposed Rats Depending on the Analyzed Behavioral or Neurochemical End Points.

Exposure to pesticides has been associated with neurodevelopmental toxicity. Usually people are exposed to mixtures of pesticides. However, most studies analyze the effects of individual pesticides. Developmental exposure to mixtures of pesticides may result in additive effects or in antagonistic or synergistic effects. The aim of this work was to compare the effects of developmental exposure of rats to cypermethrin or endosulfan with the effects of its mixture on cognitive and motor function and on some underlying mechanisms. Exposure to individual pesticides or the mixture was from gestational day 7 to postnatal day 21. We analyzed the effects, in males and females, on spatial learning and memory, associative learning, anxiety, motor coordination, and spontaneous motor activity. We also analyzed neuroinflammation and NMDA receptor subunits in hippocampus and extracellular GABA in cerebellum. Exposure to the mixture, but not to individual pesticides, impaired spatial memory in males, associative learning in females, and increased motor activity in males and females. This indicates a synergistic effect of cypermethrin and endolsufan exposure on these end points. In contrast, motor coordination was impaired by individual exposure to endosulfan or cypermethrin, associated with increased extracellular GABA in cerebellum, but these effects were prevented in rats exposed to the mixture, indicating an antagonistic effect of cypermethrin and endolsufan exposure on these end points. The results show different interaction modes (synergism or antagonism) of the pesticides, depending on the end point analyzed and the sex of the rats.

Developmental Exposure to Pesticides Alters Motor Activity and Coordination in Rats: Sex Differences and Underlying Mechanisms.

It has been proposed that developmental exposure to pesticides contributes to increasing prevalence of neurodevelopmental disorders in children, such as attention deficit with hyperactivity (ADHD) and to alterations in coordination skills. However, the mechanisms involved in these alterations remain unclear. We analyzed the effects on spontaneous motor activity and motor coordination of developmental exposure to a representative pesticide of each one of the four main chemical families: organophosphates (chlorpyrifos), carbamates (carbaryl), organochlorines (endosulfan), and pyrethroids (cypermethrin). Pesticides were administered once a day orally, in a sweet jelly, from gestational day 7 to post natal day 21. Spontaneous motor activity was assessed by an actimeter and motor coordination using the rotarod, when rats were adults. The effects were analyzed separately in males and females. Extracellular GABA in cerebellum and NMDA receptor subunits in hippocampus were assessed as possible underlying mechanisms of motor alterations. Motor coordination was impaired by developmental exposure to endosulfan, cypermethrin, and chlorpyrifos in females but not in males. The effect of endosulfan and cypermethrin would be due to increased extracellular GABA in cerebellum, which remains unaltered in male rats. Chlorpyrifos increased motor activity in males and females. Cypermethrin decreased motor activity mainly in males. In male rats, but not in females, expression of the NR2B subunit of NMDA receptor in hippocampus correlated with motor activity. These results show sex-specific effects of different pesticides on motor activity and coordination, associated with neurotransmission alterations. These data contribute to better understand the relationship between developmental exposure to the main pesticide families and motor disorders in children.

Maternal restraint stress delays maturation of cation-chloride cotransporters and GABAA receptor subunits in the hippocampus of rat pups at puberty

The GABAergic synapse undergoes structural and functional maturation during early brain development. Maternal stress alters GABAergic synapses in the pup's brain that are associated with the pathophysiology of neuropsychiatric disorders in adults; however, the mechanism for this is still unclear. In this study, we examined the effects of maternal restraint stress on the development of Cation-Chloride Cotransporters (CCCs) and the GABAA receptor α1 and α5 subunits in the hippocampus of rat pups at different postnatal ages. Our results demonstrate that maternal restraint stress induces a transient but significant increase in the level of NKCC1 (Sodium–Potassium Chloride Cotransporter 1) only at P14, followed by a brief, yet significant increase in the level of KCC2 (Potassium-Chloride Cotransporter 2) at P21, which then decreases from P28 until P40. Thus, maternal stress alters NKCC1 and KCC2 ratio in the hippocampus of rat pups, especially during P14 to P28. Maternal restraint stress also caused biphasic changes in the level of GABAA receptor subunits in the pup's hippocampus. GABAA receptor α1 subunit gradually increased at P14 then decreased thereafter. On the contrary, GABAA receptor α5 subunit showed a transient decrease followed by a long-term increase from P21 until P40. Altogether, our study suggested that the maternal restraint stress might delay maturation of the GABAergic system by altering the expression of NKCC1, KCC2 and GABAA receptor α1 and α5 subunits in the hippocampus of rat pups. These changes demonstrate the dysregulation of inhibitory neurotransmission during early life, which may underlie the pathogenesis of psychiatric diseases at adolescence.

Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus

Hippocampus-dependent learning memory is sensitive to sleep deprivation (SD). Although the ionotropic glutamate receptors play a vital role in synaptic plasticity and learning and memory, however, whether the expression of these receptor subunits is modulated by sleep loss remains unclear. In the present study, western blotting was performed by probing with specific antibodies against the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1, GluA2, GluA3, and against the N-methyl-d-aspartate (NMDA) glutamate receptor subunits GluN1, GluN2A, GluN2B. In hippocampus, down regulation of surface GluA1 and GluN2A surface expression were observed in both SD groups. However, surface expression level of GluA2, GluA3, GluN1 and GluN2B was significantly up-regulated in 8h-SD rats when compared to the 4h-SD rats. In parallel with the complex changes in AMPA and NMDA receptor subunit expressions, we found the 8h-SD impaired rat spatial working memory in 30-s-delay T-maze task, whereas no impairment of spatial learning was observed in 4h-SD rats. These results indicate that sleep loss alters the relative expression levels of the AMPA and NMDA receptors, thus affects the synaptic strength and capacity for plasticity and partially contributes to spatial memory impairment.

Expression of AMPA receptor subunits in hippocampus after status convulsion

The expression of 2-amino-3-(5-methyl-3-oxo-1, 2-oxazol-4-yl) propanoic acid receptor (AMPAR) subunits in the hippocampus of naive immature and adult rats (IRs, ARs) was investigated after status convulsion (SC). Seizures were induced in IRs and ARs with intraperitoneal injections of lithium and pilocarpine. Rats were killed at four time points (3h, 1day, 3days, and 7days) after SC. The proportion of apoptotic cells was quantified by Annexin V-FITC apoptosis detection. The location and type of apoptotic cells were assessed by using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Immunoblotting techniques were used to demonstrate changes in AMPAR subunit expression. Severe seizures induced neuronal apoptosis in the hippocampus. The proportion of apoptotic cells in IRs was consistently lower than that in ARs after SC. The expressions of four AMPAR subunits in IRs were consistently lower than those in ARs before and after SC. SC for 1h inhibited the expression of glutamate receptors (GluR1-4) in the hippocampus of IRs and ARs and altered the subunit composition of AMPARs. GluR2 was the predominant AMPAR subunit in the hippocampus of normal ARs, while the GluR2/3 subunits were predominantly expressed 7days after SC. GluR3/4 subunits were mainly expressed in the hippocampus of normal IRs, which had the lowest levels of GluR2. Immature brain was more resistant to seizure-induced neural damage. The time course of reduction and recovery differed for each subunit and was dependent on developmental stage. The increased expression of GluR2 could confer early but transient protection in the immature brain after SC.

Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-d-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague–Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1h after footshock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1h. While 24h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182,780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway.

Alteration of Epileptogenesis Genes

Retrospective studies suggest that precipitating events such as prolonged seizures, stroke, or head trauma increase the risk of developing epilepsy later in life. The process of epilepsy development, known as epileptogenesis, is associated with changes in the expression of a myriad of genes. One of the major challenges for the epilepsy research community has been to determine which of these changes contributes to epileptogenesis, which may be compensatory, and which may be noncontributory. Establishing this for any given gene is essential if it is to be considered a therapeutic target for the prevention or treatment of epilepsy. Our laboratories have examined alterations in gene expression related to inhibitory neurotransmission that have been proposed as contributing factors in epileptogenesis. The GABA(A) receptor mediates most fast synaptic inhibition, and changes in GABA(A) receptor subunit expression and function have been reported in adult animals beginning immediately after prolonged seizures (status epilepticus [SE]) and continue as animals become chronically epileptic. Prevention of GABA(A) receptor subunit changes after SE using viral gene transfer inhibits development of epilepsy in an animal model, suggesting that these changes directly contribute to epileptogenesis. The mechanisms that regulate differential expression of GABA(A) receptor subunits in hippocampus after SE have recently been identified, and include the CREB-ICER, JAK-STAT, BDNF, and Egr3 signaling pathways. Targeting signaling pathways that alter the expression of genes involved in epileptogenesis may provide novel therapeutic approaches for preventing or inhibiting the development of epilepsy after a precipitating insult.

Differential alteration of NMDA receptor subunits in the gerbil dentate gyrus and subiculum following seizure

In the present study, a chronological and comparative analysis of the immunoreactivities of N-methyl- d-aspartate (NMDA) receptor subunits in hippocampus of both seizure resistant (SR) and seizure sensitive (SS) gerbils was made in order to clarify the temporal and spatial alterations of NMDA receptor subunit expressions in the hippocampus complex. The changes in NMDA receptor immunoreactivity in the hippocampi of SS gerbils were restricted to both the dentate gyrus and the subiculum. At 30 min postictal, a decline in NMDA receptor subunit 1 (NR1) immunoreactivity in the suprablade of dentate gyrus was observed. This is in contrast to the enhancement of its immunodensity in the infrablade. At 3 h postictal the NR1 immunoreactivity in the infrablade also declined significantly. At 12 h postictal, its immunoreactivity in the hilar neurons was reduced. The NMDA receptor subunit 2A/B (NR2A/B) immunoreactivity did not alter until 12 h following seizure-onset, when it was slightly decreased in the granule cells and hilar neurons. In the subiculum, NR1 immunoreactivity was significantly decreased, and was almost undetectable in this region until 12 h postictal; in contrast the NR2A/B immunoreactivity in this region increased significantly in this time point. These results suggest that the altering NMDA receptor expression in both the dentate gyrus and subiculum may affect tissue excitability and have an important role in regulating seizure activity in SS gerbils.

Effects of Kainic Acid on mRNA Expression of GABA, Receptor Subunits in Rat Hippocampus

Purpose: Kainic acid (KA)‐induced seizures in rats have been studied as an animal inodel for human temporal lobe epilepsy. After KA induced acute status epilepticus, neuropathological examination shows neurodegeneration in the CAI and CA3 sector 01 the Ammon's horn and or interneurons i n the hilus of the dcntatc gyrus. Thc GABA, receptor is a ligand‐gated chloride channel consisting of live membrane‐spanning proteins for which al least five different gene families have been identified in the mammalian brain. We investigated changes in inRNA expression of 13 GABA, receptor subunits i n the hippocampus of rats at viirious time intervals after acutc KA‐induced status epilepticus. We also studied scizurc‐related neuronal cell loss during and after acquisition of spontaneous recurrent scizures. Methods: Male Sprague‐Dawlcy rats (250–350 g) were injected with 10 mg/kg of KA in buffered salinc or the corresponding amount of saline without KA as a control. Rats were decapitatcd 6 hours, 12 hours, 24 hours, 7 days, and 30 days after KA injection. The brains were cut into coronal sections (20 pm) and mountcd on chrome‐alumgelatin‐coated slides. For in situ hybridization, synthetic oligonucleotides were labeled with I'SI‐a‐thio‐ATP. Thc oligonucleotide sequences were mostly derived from those used by Wisden and coworkers. The frozen sections rapidly were immersed in 2% paraCormaldehyde, PBS for 10 minutes, immersed in 0.25% acetic anhydride in 0. IM triethylamine hydrochloride for 10 minutes, dehydrated by an ethanol series, and de‐lipidized with chloroform. They were hybridized with about 2nM of labeled oligonucleotide probe in Soy1 hybridization buffer at 42°C for 16–24 hours. Alter thc incubation, the slides were briefly rinsed twice in IxSSC, SO% formamidc, then they were washed four times in SO% formamidc i n 2xSSC (42°C 15 minutes), rinsed in IxSSC, dipped briefly into watcr then into 70% ethanol, dried, and exposed to 13‐max X‐ray film for one to two weeks. Autoradiographical images were digitalixd using an Appligenc Imager and evaluated using the public domain NIH‐image program. For each time interval, sections ohtained from thrcc to six cxperimenlal animals and from 14 controls were examined. Statistics were pel‐formed using the two‐tailed Student's T‐test. Results: In the granule cell layer, initial (after 6 to 12 hours) decreases in a2. a3, 015, P I, p3, y2, and 6 mRNAs (by about 25%) to 50%) were accompanied by increases (by about 50%) in a I, a4, and p2 mcssagcs. At later intervals (alter 7 to 30 days), cxpression of α12, α4, β3, and β2 mRNAs recovered to control values, w i h a5 and 6 mKNA still being reduced (by 15% and 40% below control levcls, respectively). Concentrations of the transcripts encoding for OL I, α3, (3 I, and p2 became markedly enhanced (between 20% and 50% of controls). Within the pyramidal cell layers CAI and CA3, decrcases in (α2, α4, α15, β 1–3, and γ2 mRNAs were detected after 7 to 30 days, reflecting pronounced neurodegcneration in these areas. The a I transcript W Z I decreased in CA3 after 24 hours and increased to control levcls indicating compensatory up‐regulation of this message after 7 days. mRNAs encoding for α3, βI, and y3‐subunits were detected at rather low levels.ot6 was not present in the hippocampus. Conclusions: Our data suggest fast, transient changes in thc expression of mRNAs encoding for different subunits of the GABA, receptor in the granule cell layer of thc dentate gyrus in response to KA‐induced seizures. Changes in the expression of GABA, receptor subunits may reflect altered GABAergic transmission during development of the epileptic syndrome. Changes within the pyramidal cell layer are mostly determined by concomitant neurodegencrative processes.

Changes in protein tyrosine phosphorylation in the rat brain after cerebral ischemia in a model of ischemic tolerance.

A brief period of sublethal cerebral ischemia, followed by several days of recovery, renders the brain resistant to a subsequent lethal ischemic insult, a phenomenon termed ischemic preconditioning or tolerance. Ischemic tolerance was established in the rat two-vessel occlusion model of ischemia, induced by occlusion of both carotid arteries in combination with hypotension. Ischemic preconditioning (3 minutes) provided maximal neuroprotection when induced 2 days prior to a lethal ischemic insult of 9-minute duration. Neuroprotection persisted for at least 8 weeks. Since neurotransmission has been implicated in ischemic cell death, the effect of ischemic preconditioning on tyrosine phosphorylation of proteins and on the levels of glutamate receptor subunits in hippocampus and neocortex was studied. Regional levels of tyrosine phosphorylation of proteins in general and the N-methyl-D-aspartate receptor subunit NR2 in particular are markedly enhanced after ischemia in nonconditioned brains, in both the synaptosomal fraction and the whole-tissue homogenate of rat neocortex and hippocampus, but recover to control levels only in the preconditioned brain. Ischemic preconditioning selectively induces a decrease in the levels of the NR2A and NR2B subunits and a modest decrease in the levels of NR1 subunit proteins in the synaptosomal fraction of the neocortex but not hippocampus after the second lethal ischemia. It was concluded that ischemic preconditioning prevents a persistent change in cell signaling as evidenced by the tyrosine phosphorylation of proteins after the second lethal ischemic insult, which may abrogate the activation of detrimental cellular processes leading to cell death.

Acute effects of morphine on the expression of mRNAs for NMDA receptor subunits in the rat hippocampus, hypothalamus and spinal cord

The acute effects of subcutaneously injected morphine on transcripts of the NMDA receptor subunits NR1, NR2A and NR2B in certain areas of the central nervous system of male rats were examined by Northern blot analysis. The result clearly indicated that a single dose (10 mg/kg) of the opioid alters the expression of the mRNA for receptor subunits in the hippocampus and hypothalamus 4 h after drug injection. No change in the mRNA levels was observed 30 min following injection, and after 24 h most of the levels were restored to control values. The observation suggests that morphine affects this type of glutamate receptor already in the acute phase of its administration.

GABA A 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 α 2- and δ- and slight increases in α 1-, β 2- and β 3-immunoreactivities were observed at early intervals (12 to 24 h) after kainic acid injection. These changes were succeeded by marked increases in α 1-, α 2-, α 4-, α 5-, β 1-, β 3-, γ 2- and δ-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 α 1, α 2, α 5, β 1– β 3, γ 2 and α 4 (present only in CA1). Decreases in these subunits were followed by increased expression of α 2-, α 5-, β 3-, γ 2- and δ-subunits in the hippocampus proper notably in CA3 at later intervals (up to 30 days). α 1-, β 2-, γ 2- and δ-subunits were found in presumed GABA containing interneurons throughout the hippocampus. Their immunoreactivity was augmented after two to seven days. Some α 4-, γ 3- and δ-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.

GABA A 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 α 1-, α 2-, α 4-, β 3-, γ 2- and δ-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 α 1, α 2, α 5, β 3 and γ 2 that were located throughout the strata radiatum and oriens of CA1 to CA3. Immunocytochemical staining was there less prominent for α 4-, β 1-, β 2-, γ 3- and δ- subunits. In the hippocampus proper, the β 1 subunit was preferentially located in CA2. The α 4- and δ-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 α 1-, β 2-, γ 2- and/or δ-subunits. α 3 and γ 1 were present only in minute amounts and no α 6-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.

GABA A 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 Ammon's 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 ( α 2, α 3, α 5, β 1, β 3, γ 2 and δ messenger RNAs (by about 25 to 50%) were accompanied by increases (by about 50%) in α 1, α 4, and β 2 messages. At later intervals (after seven to 30 days), expression of α 2, α 4, β 3 and γ 2 messenger RNAs recovered to control values, with α 5 and δ messenger RNA still being reduced (by 15 and 40% below control levels, respectively). Concentrations of the transcripts encoding for α 1, α 3, β 1, β 2, became markedly enhanced (between 20 and 50% of controls). Within the pyramidal cell layers CA1 and CA3, decreases in α 2, α 4, α 5, β 1–3 and γ 2 messenger RNAs were detected after seven to 30 days, reflecting pronounced neurodegeneration in these areas. The α 1 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 α 3-, γ 1-, and γ 3-subunits were detected at rather low levels, α 6 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.

Effects of an anabolic-androgenic steroid on the regulation of the NMDA receptor NR1, NR2A and NR2B subunit mRNAs in brain regions of the male rat

The expression of the N-methyl- d-aspartate (NMDA) receptor subunits NR1, NR2A and NR2B mRNAs was examined in discrete areas of the male rat brain (including hippocampus, hypothalamus, nucleus accumbens and cortex) following 14 days daily intramuscular injections of high doses (5 and 15 mg/kg) of an anabolic-androgenic steroid (AAS) (nandrolone decanoate). The results indicated that the drug produced a significant decrease in the mRNA expression of the NR2A receptor subunit both in the hypothalamus and hippocampus. A decrease in the level of NR2B receptor mRNA was observed in hypothalamus at the lower dose of the AAS but in other areas examined, this receptor subunit mRNA was not affected. Except for a decreased expression in the nucleus accumbens at the higher dose of AAS the NR1 receptor subunit mRNA was not affected by the drug. The three subunit mRNAs in cortex were not significantly altered. The effects of the steroid on the mRNA expression for the NMDA receptor subunits in hippocampus and hypothalamus are suggested to be involved in the mechanism behind aggressive behaviour, a feature previously associated with AAS misuse. The downregulation of the mRNA for the NR1 receptor subunit in nucleus accumbens may relate to a mechanism involved in the recently suggested AAS-induced stimulation of the brain reward system.