Receptor Subunit Protein Research Articles

Altered excitability and distribution of NMDA receptor subunit proteins in cortical layers of rat pups following multiple perinatal seizures

During a critical period of postnatal development the epileptogenic focus is thought to be of cortical origin. We used immunohistochemistry and Western blotting to elucidate potential mechanisms underlying an increased state of susceptibility to seizures in immature animals. Distribution patterns of N-methyl- d-aspartic acid (NMDA) (NR1 and NR2A/B) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) (GluR1 and GluR2) subunits were analyzed in retrosplenial, parietal and temporal cortices during the first two postnatal weeks following three episodes of status-epilepticus. Rat pups were injected three times with kainic acid (3× KA) on P6, P9, and P13 and subsequently sacrificed 48 h after the third seizure. Cortical electroencephalography (EEG) showed increased number of spikes and bursts of longer duration after 3× KA. Immunodensity measurements after 3× KA revealed a robust increase in NR2A/B labeling specific to cortical layer V throughout the retrosplenial, parietal, and temporal cortices, with no changes noted in piriform cortex. NR1 layer V immunoreactivity was also simultaneously increased in serial sections but to a lesser degree; heightened immunodensities were specific to retrosplenial and temporal cortices. The NR1:NR2 ratio was decreased in cortical layer V of the temporal and retrosplenial cortices but not in parietal cortex despite elevated immunoreactivity. Steady levels of GluR1 and GluR2 subunits were noted in all cortical areas studied in the same animals. Thus, recurrent perinatal seizures led to selective and layer-specific increases in NMDA receptor proteins. These changes may be responsible for lowering the seizure threshold in deeper cortical areas and eventually contribute to the cortical epileptogenic focus.

Exercise Training Prevents Arterial Baroreflex Dysfunction in Rats Treated With Central Angiotensin II

Angiotensin II (Ang II)-induced arterial baroreflex dysfunction is associated with superoxide generation in the brain. Exercise training (EX) improves baroreflex function and decreases oxidative stress in cardiovascular diseases linked to elevated central Ang II. The aim of this study was to determine whether previous EX prevents baroreflex impairment caused by central administration of exogenous Ang II via an Ang II-superoxide mechanism. Four groups of rats were used: non-EX artificial cerebrospinal fluid infused, non-EX Ang II infused, EX artificial cerebrospinal fluid infused, and EX Ang II infused. Rats were treadmill trained for 3 to 4 weeks and subjected to intracerebroventricular infusion of Ang II over the last 3 days of EX. Twenty-four hours after the end of EX, the arterial baroreflex was assessed in anesthetized rats. Compared with non-EX artificial cerebrospinal fluid-infused rats, Ang II significantly decreased baroreflex sensitivity (maximum gain: 3.0+/-0.2% of maximum per millimeter of mercury versus 1.6+/-0.1% of maximum per millimeter of mercury; P<0.01), which was abolished by acute intracerebroventricular infusion of the Ang II type 1 receptor antagonist losartan and the reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin. EX prevented the decrease in baroreflex sensitivity and downregulated Ang II type 1 receptor and NADPH oxidase subunit protein expression in the paraventricular nucleus of Ang II-infused rats. Finally, EX decreased superoxide production in the paraventricular nucleus of Ang II-infused rats. These results indicate that EX improves arterial baroreflex function in conditions of high brain Ang II, which is mediated by the central Ang II type 1 receptor and associated with a reduction in central oxidative stress.

Amyloid β-peptide preconditioning reduces glutamate-induced neurotoxicity by promoting endocytosis of NMDA receptor

Amyloid β-peptide (Aβ) and glutamate are generally believed to be closely related to the pathogenesis of Alzheimer’s disease and cerebrovascular disease, respectively. Recent advances suggest that risk factors linked to cerebrovascular disease significantly increase the risk of developing Alzheimer’s disease. In this study, we examined the effects of pretreatment of cultured hippocampal neurons with Aβ(1–42) (0.3, 0.5, and 1.0 μM) for 3 h (Aβ preconditioning) on glutamate-induced neurotoxicity. Aβ preconditioning significantly reduced both glutamate-induced neurotoxicity and the glutamate-induced increase in intracellular Ca 2+ concentration ([Ca 2+] i). Aβ preconditioning significantly reduced cell surface expression of N-methyl- d-aspartate (NMDA) glutamate receptor subunit protein NR1, although it exerted no significant effect on the total expression of NR1. These results suggest that Aβ preconditioning reduced glutamate-induced neurotoxicity by promoting endocytosis of NMDA receptor, followed by inhibition of the increase in [Ca 2+] i. Our results support the notion of an association between Alzheimer’s disease and cerebrovascular disease, and suggest a new mechanism for neuroprotection by promoting endocytosis of NMDA receptor.

Three Brief Epileptic Seizures Reduce Inhibitory Synaptic Currents, GABAA Currents, and GABAA‐Receptor Subunits

Cellular mechanisms activated during seizures may exacerbate epilepsy. gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in brain, and we hypothesized that brief epileptic seizures may reduce GABA function. We used audiogenic seizures (AGSs) in genetically epilepsy-prone rats (GEPRs) to investigate effects of seizures on GABA-mediated inhibition in the presence of epilepsy. GEPRs are uniformly susceptible to AGSs beginning at 21 postnatal days. AGSs are brief convulsions lasting approximately 20 s, and they begin in inferior colliculus (IC). We evoked three seizures in GEPRs and compared the results with those in seizure-naive GEPRs and nonepileptic Sprague-Dawley (SD) rats, the GEPR parent strain. Whole-cell recording in IC slices showed that GABA-mediated monosynaptic inhibitory postsynaptic currents (IPSCs) were reduced 55% by three brief epileptic seizures. Whole-cell recording in IC neuronal cultures showed that currents elicited by GABA were reduced 67% by three seizures. Western blotting for the alpha1 and alpha4 subunits of the GABA(A) receptor showed no statistically significant effects. In contrast, three brief epileptic seizures reduced gamma2 subunit levels by 80%. The effects of the very first seizures, in animals known to be epileptic, in an area of brain known to be critical to the seizure network, were studied. The results indicate that even brief epileptic seizures can markedly reduce IPSCs and GABA currents and alter GABA(A)-receptor subunit protein levels. The cause of the reductions in IPSCs and GABA currents is likely to be altered receptor subunit composition, with reduced gamma2 levels causing reduced GABA(A)-receptor sensitivity to GABA. Seizure-induced reductions in GABA-mediated inhibition could exacerbate epilepsy.

P2X2R purinergic receptor subunit mRNA and protein are expressed by all hypothalamic hypocretin/orexin neurons

Neurophysiologic data suggest that orexin neurons are directly excited by ATP through purinergic receptors (P2XR). Anatomical studies, though reporting P2XR in the hypothalamus, did not describe it in the perifornical hypothalamic area, where orexinergic neurons are located. Here we report the presence of the P2X(2)R subunit in the rat perifornical hypothalamus and demonstrate that hypothalamic orexin neurons express the P2X(2)R. Double immunohistochemistry showed that virtually all orexin-immunoreactive neurons are also P2X(2)R immunoreactive, whereas 80% of P2X(2)R-immunoreactive neurons are also orexin positive. Triple-labeling experiments, combining fluorescence in situ hybridization for P2X(2)R mRNA and P2X(2)R/orexin double immunofluorescence, confirmed these findings. In addition, in situ hybridization demonstrated that P2X(2)R mRNA is localized in cellular processes of orexinergic neurons. The present data support neurophysiologic findings on ATP modulation of orexinergic function and provide direct evidence that the entire population of orexin neurons expresses a P2XR subtype, namely, P2X(2)R. Thus, purinergic transmission might intervene in modulating key functions known to be controlled by the orexinergic system, such as feeding behavior and arousal.

Region Specific Regulation of NR1 in Rhesus Monkeys Following Chronic Antipsychotic Drug Administration

Altered NMDA receptor subunit protein levels have been reported in various regions of the schizophrenic brain; however, chronic antipsychotic administration in schizophrenic subjects may confound interpretation. The effects of chronic antipsychotic drug administration (haloperidol and clozapine) on protein levels of NR1, NR2A and NR2B proteins were evaluated in the nucleus accumbens (NAc), putamen (PUT), dorsolateral prefrontal cortex (DLPFC), superior temporal gyrus (STG), and entorhinal cortex (EC) of rhesus monkeys using Western blot analysis. Haloperidol administration significantly decreased NR1 expression in the DLPFC. In contrast, NR2B expression was not affected by antipsychotic administration in any brain region examined. NR2A was not reliably detected in any of the brain regions. Results indicate that the NR1 subunit in the DLPFC may be a substrate for antipsychotic action and that glutamatergic hypofunction in the DLPFC commonly associated with cognitive dysfunction in schizophrenia may be associated with haloperidol administration.

Expression of AMPA Receptor Subunit Proteins in Purified Retinal Ganglion Cells

To determine whether alpha-amino-3-hydroxy-5-methylisoxazole-4-propioate (AMPA) receptor (AMPAR) subunit proteins are expressed in cultured retinal ganglion cells (RGCs). RGCs were purified from dissociated rat retinal cells (postnatal days 6-8), using a modified two-step panning method and cultured in serum-free medium containing neurotrophic factors and forskolin. Immunohistochemistry was performed on cultured RGCs on days 1, 3, and 7 in vitro (1 DIV, 3 DIV, and 7 DIV) using specific antibodies against AMPAR subunits GluR1 to 4 and microtubule-associated protein (MAP) 2, which is a neuronal marker. Glutamate-induced Ca(2+) influx was measured with fura-2 acetoxymethyl ester fluorescence. GluR1 to 4 proteins were expressed in the cell body of RGCs on 1 DIV. RGCs showed strong GluR1 to 4 immunoreactivity in both cell bodies and processes on 3 DIV and 7 DIV, with the gradual spreading of expression and the growth of processes. At all time points examined, GluR2 immunoreactivity was equal to that of the other subunits. Accumulation of intracellular Ca(2+) levels in RGCs induced by glutamate occurred equally on both 3 DIV and 7 DIV. All AMPAR subunits are almost equally expressed in cultured RGCs.

Status Epilepticus: Danse Macabre in a Ballet of Subunits

Status Epilepticus Increases the Intracellular Accumulation of GABAA Receptors Goodkin HP, Yeh JL, Kapur J J Neurosci 2005;25:5511–5520 Status epilepticus is a neurological emergency that results in mortality and neurological morbidity. It has been postulated that the reduction of inhibitory transmission during status epilepticus results from a rapid modification of GABAA receptors. However, the mechanism(s) that contributes to this modification has not been elucidated. We report, using an in vitro model of status epilepticus combined with electrophysiological and cellular imaging techniques, that prolonged epileptiform bursting results in a reduction of GABA-mediated synaptic inhibition. Furthermore, we found that constitutive internalization of GABAA receptors is rapid and accelerated by the increased neuronal activity associated with seizures. Inhibition of neuronal activity reduced the rate of internalization. These findings suggest that the rate of GABAA receptor internalization is regulated by neuronal activity and its acceleration contributes to the reduction of inhibitory transmission observed during prolonged seizures. Trafficking of GABAA Receptors, Loss of Inhibition, and a Mechanism for Pharmacoresistance in Status Epilepticus Naylor DE, Liu H, Wasterlain CG J Neurosci 2005;25:7724–7733 During status epilepticus (SE), GABAergic mechanisms fail and seizures become self-sustaining and pharmacoresistant. During lithiumpilocarpine-induced SE, our studies of postsynaptic GABAA receptors in dentate gyrus granule cells show a reduction in the amplitude of miniature IPSCs (mIPSCs). Anatomical studies show a reduction in the colocalization of the β2/ β3 and γ2 subunits of GABAA receptors with the presynaptic marker synaptophysin and an increase in the proportion of those subunits in the interior of dentate granule cells and other hippocampal neurons with SE. Unlike synaptic mIPSCs, the amplitude of extrasynaptic GABAA tonic currents is augmented during SE. Mathematical modeling suggests that the change of mIPSCs with SE reflects a decrease in the number of functional postsynaptic GABAA receptors. It also suggests that increases in extracellular [GABA] during SE can account for the tonic current changes and can affect postsynaptic receptor kinetics with a loss of paired-pulse inhibition. GABA exposure mimics the effects of SE on mIPSC and tonic GABAA current amplitudes in granule cells, consistent with the model predictions. These results provide a potential mechanism for the inhibitory loss that characterizes initiation of SE and for the pharmacoresistance to benzodiazepines, as a reduction of available functional GABAA postsynaptic receptors. Novel therapies for SE might be directed toward prevention or reversal of these losses. Kainic Acid-Induced Status Epilepticus Alters GABA Receptor Subunit mRNA and Protein Expression in the Developing Rat Hippocampus Lauren HB, Lopez-Picon FR, Korpi ER, Holopainen IE J Neurochem 2005;94:1384–1394 Kainic acid-induced status epilepticus leads to structural and functional changes in inhibitory GABAA receptors in the adult rat hippocampus, but whether similar changes occur in the developing rat is not known. We have used in situ hybridization to study status epilepticus-induced changes in the GABAA α1– α5, β1– β3, γ1, and γ2 subunit mRNA expression in the hippocampus of 9-day-old rats during 1 week after the treatment. Immunocytochemistry was applied to detect the α1, α2, and β3 subunit proteins in the control and treated rats. In the saline-injected control rats, the α1 and α4 subunit mRNA expression significantly increased between the postnatal days 9 to 16, whereas those of α2, β3, and γ2 subunits decreased. The normal developmental changes in the expression of α1, α2, β3, and γ2 subunit mRNAs were altered after the treatment. The immunostainings with antibodies to α1, α2, and β3 subunits confirmed the in situ hybridization findings. No neuronal death was detected in any hippocampal subregion in the treated rats. Our results show that status epilepticus disturbs the normal developmental expression pattern of GABAA receptor subunit in the rat hippocampus during the sensitive postnatal period of brain development. These perturbations could result in altered functional and pharmacological properties of GABAA receptors.

Evidence for a functional role of GABAC receptors in the rat mature hippocampus

Both gamma-aminobutyric acid (GABA)(C) receptor subunit mRNA and protein are expressed in the stratum pyramidale in the CA1 area of the adult rat hippocampus, but so far no conclusive evidence about functional hippocampal GABA(C) receptors has been presented. Here, the contribution of GABA(C) receptors to stimulus-evoked postsynaptic potentials was studied in the hippocampal CA1 area with extracellular and intracellular recordings at the age range of 21-47 postnatal days. Activation of GABA(C) receptors with the specific agonist cis-4-aminocrotonic acid (CACA) suppressed postsynaptic excitability and increased the membrane conductance. The GABA(C) receptor antagonist 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic acid (TPMPA), but not the GABA(A) receptor antagonist bicuculline, inhibited the effects of CACA. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation of local inhibitory interneurons in the CA1 area in the presence of ionotropic glutamate receptor and GABA(B) receptor blockers were prolonged by TPMPA, indicating that GABA(C) receptors are activated under these conditions. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABA(C) receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABA(C) receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA.

Cocaine‐induced alterations in nucleus accumbens ionotropic glutamate receptor subunits in human and non‐human primates

Chronic cocaine and withdrawal induce significant alterations in nucleus accumbens (NAc) glutamatergic function in humans and rodent models of cocaine addiction. Dysregulation of glutamatergic function of the prefrontal cortical-NAc pathway has been proposed as a critical substrate for unmanageable drug seeking. Previously, we demonstrated significant up-regulation of NMDA, (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptor subunit mRNAs and protein levels in the ventral tegmental area (VTA), but not the substantia nigra, of cocaine overdose victims (COD). The present study was undertaken to examine the extent of altered ionotropic glutamate receptor (iGluR) subunit expression in the NAc and the putamen in cocaine overdose victims. Results revealed statistically significant increases in the NAc, but not in the putamen, of NMDA receptor subunit (NR)1 and glutamate receptor subunit (GluR)2/3 wit trends in GluR1 and GluR5 in COD. These results extend our previous finding and indicate pathway-specific alterations in iGluRs in COD. In order to determine that changes were related to cocaine intake and not to other factors in the COD victims, we examined the effects of cocaine intravenous self-administration in rhesus monkeys for 18 months (unit dose of 0.1 mg/kg/injection and daily drug intake of 0.5 mg/kg/session). Total drug intake for the group of four monkeys was 37.9 +/- 4.6 mg/kg. Statistically significant elevations were observed for NR1, GluR1, GluR2/3 and GluR5 (p < 0.05) and a trend towards increased NR1 phosphorylated at serine 896 (p = 0.07) in the NAc but not putamen of monkeys self-administering cocaine compared with controls. These results extend previous results by demonstrating an up-regulation of NR1, GluR2/3 and GluR5 in the NAc and suggest these alterations are pathway specific. Furthermore, these changes may mediate persistent drug intake and craving in the human cocaine abuser.

Differential regulation of ionotropic glutamate receptor subunits following cocaine self-administration

Previous examination of binge cocaine self-administration and 2 week withdrawal from cocaine self-administration on ionotropic glutamate receptor subunit (iGluRs) protein levels revealed significant alterations in iGluR protein levels that differed between the mesocorticolimbic and nigrostriatal pathways. The present study was undertaken to extend the examination of cocaine-induced alterations in iGluR protein expression by assessing the effects of acute withdrawal (15–16 h) from limited access cocaine self-administration (8 h/day, 15 days). Western blotting was used to compare levels of iGluR protein expression (NR1–3B, GluR1–7, KA2) in the mesolimbic (ventral tegmental area, VTA; nucleus accumbens, NAc; and prefrontal cortex, PFC) and nigrostriatal pathways (substantia nigra, SN and dorsal caudate–putamen, CPu). Within the mesolimbic pathway, reductions were observed in NR1 and GluR5 immunoreactivity in the VTA although no significant alterations were observed in any iGluR subunits in the NAc. In the PFC, NR1 was significantly upregulated while GluR2/3, GluR4, GluR5, GluR6/7, and KA2 were decreased. Within the nigrostriatal pathway, NR1, NR2A, NR2B, GluR1, GluR6/7 and KA2 were increased in the dorsal CPu, whereas no significant changes were observed in the SN. The results demonstrate region- and pathway-specific alterations in iGluR subunit expression following limited cocaine self-administration and suggest the importance for the activation of pathways that are substrates of the reinforcing and motoric effects of cocaine.

Epithelial Localization of Green Fluorescent Protein‐Positive Cells in Epididymis of the GAD67‐GFP Knock‐in Mouse

gamma-Aminobutyric acid (GABA), which is a major inhibitory neurotransmitter in the brain, is also found in many peripheral nonneuronal tissues, including male reproductive organs. However, the distribution of GABAergic cells in various organs is not known. The GAD67-GFP knock-in mouse is a useful model for studying the distribution and morphology of GABAergic neurons in the brain. We examined the male reproductive organs of GAD67-GFP knock-in mice by fluorescence microscopy and found cells with strong green fluorescent protein (GFP) signal exclusively in the epithelium of the initial segment and proximal caput of the epididymis. The characteristic cell morphology suggested that these were narrow cells. These GFP-positive narrow cells also expressed GAD67 and GABA. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that the predominant glutamic acid decarboxylase (GAD) isoform expressed in the epididymis is GAD67. RT-PCR analysis also revealed that mRNAs encoding the GABA(A) and GABA(B) receptor subunits necessary for the assembly of functional receptors are expressed in the epididymis. GABA(A) receptor subunit mRNAs detected in the proximal epididymis included alpha2, beta1, gamma1, and gamma3, and both the R1 and R2 subunit mRNAs of GABA(B) receptors were detected. Immunohistochemical analysis of GABA(A) receptor subunit proteins revealed that alpha2, beta1, and gamma subunits expressed in spermatozoa, whereas we did not detect these GABA(A) receptor subunits in epithelial cells. GABA(B) receptors were produced by narrow cells and spermatozoa of GAD67-GFP knock-in and wild-type Jcl:ICR mice. Our data suggest that the GABA system might have important functional roles in narrow cells and on spermatozoa in the lumen.

Cellular Localization of GABA and GABAB Receptor Subunit Proteins During Spermiogenesis in Rat Testis

The GABAergic system, a major inhibitory regulator in the central nervous system, may also play important roles in peripheral nonneuronal tissues and cells. Recent studies showed that GABAB receptor is expressed in testis and sperm. To understand the role of the GABAergic system in spermiogenesis, we examined cellular localization of GABA and GABAB receptor subunits in rat spermatids by immunocytochemistry. Immunoreactivity for GABA was detected around acrosomal granules of spermatids during the Golgi and cap phases. GABAB1 immunoreactivity was observed in the acrosomal vesicle of spermatids in Golgi phase, and during cap phase, this reactivity expanded to the entire region of the acrosome covering the nuclear membrane. The level of reactivity decreased gradually with maturation of spermatids. In contrast, GABAB2 immunoreactivity was not observed in spermatids during Golgi phase but was detected in the equatorial region during cap phase. Both GABA immunoreactivity and GABAB2 immunoreactivity were transferred to the residual cytoplasm during the release of spermatozoa. Electron microscopic immunocytochemistry revealed that, during cap phase, GABA and GABAB1 were distributed within the whole acrosomal vesicle but not in the acrosomal granule. GABAB2 immunoreactivity was observed in the narrow space between the inner acrosomal and nuclear membrane and was limited to the equatorial region of the spermatid head. These results indicate that the GABAergic system might be involved in regulation of spermiogenesis.

A model of bidirectional synaptic plasticity: From signaling network to channel conductance

In many regions of the brain, including the mammalian cortex, the strength of synaptic transmission can be bidirectionally regulated by cortical activity (synaptic plasticity). One line of evidence indicates that long-term synaptic potentiation (LTP) and long-term synaptic depression (LTD), correlate with the phosphorylation/dephosphorylation of sites on the alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit protein GluR1. Bidirectional synaptic plasticity can be induced by different frequencies of presynaptic stimulation, but there is considerable evidence indicating that the key variable is calcium influx through postsynaptic N-methyl-d-aspartate (NMDA) receptors. Here, we present a biophysical model of bidirectional synaptic plasticity based on [Ca2+]-dependent phospho/dephosphorylation of the GluR1 subunit of the AMPA receptor. The primary assumption of the model, for which there is wide experimental support, is that the postsynaptic calcium concentration, and consequent activation of calcium-dependent protein kinases and phosphatases, is the trigger for phosphorylation/dephosphorylation at GluR1 and consequent induction of LTP/LTD. We explore several different mathematical approaches, all of them based on mass-action assumptions. First, we use a first order approach, in which transition rates are functions of an activator, in this case calcium. Second, we adopt the Michaelis-Menten approach with different assumptions about the signal transduction cascades, ranging from abstract to more detailed and biologically plausible models. Despite the different assumptions made in each model, in each case, LTD is induced by a moderate increase in postsynaptic calcium and LTP is induced by high Ca2+ concentration.

Kainic acid‐induced status epilepticus alters GABAA receptor subunit mRNA and protein expression in the developing rat hippocampus

Kainic acid-induced status epilepticus leads to structural and functional changes in inhibitory GABAA receptors in the adult rat hippocampus, but whether similar changes occur in the developing rat is not known. We have used in situ hybridization to study status epilepticus-induced changes in the GABAAalpha1-alpha5, beta1-beta3, gamma1 and gamma2 subunit mRNA expression in the hippocampus of 9-day-old rats during 1 week after the treatment. Immunocytochemistry was applied to detect the alpha1, alpha2 and beta3 subunit proteins in the control and treated rats. In the saline-injected control rats, the alpha1 and alpha4 subunit mRNA expression significantly increased between the postnatal days 9-16, whereas those of alpha2, beta3 and gamma2 subunits decreased. The normal developmental changes in the expression of alpha1, alpha2, beta3 and gamma2 subunit mRNAs were altered after the treatment. The immunostainings with antibodies to alpha1, alpha2 and beta3 subunits confirmed the in situ hybridization findings. No neuronal death was detected in any hippocampal subregion in the treated rats. Our results show that status epilepticus disturbs the normal developmental expression pattern of GABAA receptor subunit in the rat hippocampus during the sensitive postnatal period of brain development. These perturbations could result in altered functional and pharmacological properties of GABAA receptors.

Chronic ethanol ingestion modulates proanxiety factors expressed in rat central amygdala

Withdrawal anxiety following chronic ethanol exposure is often associated with relapse in recovering alcoholics. It is likely that brain regions regulating anxiety-like behaviors adapt during chronic ethanol exposure to ultimately regulate such behaviors. The central amygdala contains numerous neurotransmitter systems that have been implicated in the regulation of anxiety-like behavior, including corticotropin releasing factor (CRF) and NMDA-type glutamate receptors. Chronic ethanol exposure causes functional adaptations in both CRF and NMDA receptors that are likely to regulate anxiety-like behaviors expressed during withdrawal. However, the molecular mechanisms governing these adaptations remain unexplored. We therefore evaluated these neurotransmitter systems in Sprague–Dawley rats during chronic ingestion of an ethanol-containing liquid diet. Quantitative real-time reverse transcription–PCR demonstrated that preproCRF mRNA was significantly upregulated by chronic ethanol exposure, whereas mRNA expression of CRF binding protein did not change. There were also no significant changes observed in any of the NMDA subunit mRNAs, although there was a trend toward greater NR2A mRNA expression during chronic ethanol exposure. Using Western blotting analysis we measured NMDA receptor subunit protein expression. Chronic ethanol exposure did not affect protein levels of the NR1 and NR2B subunits. Like the mRNA measures, chronic ethanol exposure did influence NR2A protein levels but the effects were modest. Our results demonstrate that NMDA receptor subunit mRNA and protein expressions are not strongly influenced by exposure to chronic ethanol. This suggests that the functional NMDA receptor adaptations identified in previous studies [Roberto, M., Schweitzer, P., Madamba, S. G., Stouffer, D. G., Parsons, L. H., & Siggins, G. R. (2004). Acute and chronic ethanol exposure alter glutamatergic transmission in rat central amygdala: an in vitro and in vivo analysis. J Neurosci 24, 1594–1603] are likely to be mediated by post-translational events. In contrast, enhanced levels of CRF during/after chronic ethanol exposure are likely to be mediated by increased levels of prepro CRF mRNA. Together, our findings suggest that adaptations to chronic ethanol exposure by proanxiety factors expressed in the central nucleus appear to be mediated by distinct cellular and molecular mechanisms.

Retinotectal transmission in the optic tectum of rainbow trout

Retinotectal transmission has not yet been well characterized at the cellular level in the optic tectum. To address this issue, we used a teleost, the rainbow trout, and characterized periventricular neurons as postsynaptic cells expected to receive the retinotectal inputs to the optic tectum. The somata of periventricular neurons are localized in the upper zone of the stratum periventriculare (SPV), whereas the lower zone of the SPV comprises the cell body layer of radial glial cells. Ca2+ imaging identified functional ionotropic glutamate receptors in periventricular neurons. We also cloned cDNAs encoding the NR1 subunit of N-methyl-D-aspartic acid (NMDA) receptors and the GluR2 subunit of (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors, and detected their mRNAs in periventricular neurons by in situ hybridization. The presence of the receptor subunit proteins was also confirmed in the dendrites of periventricular neurons by immunoblotting and immunohistochemistry. On the other hand, radial glial cells in the lower zone of the SPV did not respond to glutamate applications, and mRNA and immunoreactivities of ionotropic glutamate receptors were not detected in glial cells. The present findings suggest that glutamatergic transmission at synapses between retinotectal afferents and periventricular neurons is mediated by the functional NMDA and AMPA receptors.

Organization and regulation of paraventricular nucleus glutamate signaling systems: N‐methyl‐D‐aspartate receptors

Stress activation of the hypothalamo-pituitary-adrenocortical (HPA) axis is mediated in part by glutamatergic neurotransmission. The precise nature of glutamate effects on stress-integrative hypothalamic paraventricular nucleus (PVN) neurons remains to be determined. Therefore, the current study was designed to delineate the organization of glutamate/NMDA receptor systems in the PVN and to assess regulation of PVN glutamate receptor subunit expression by chronic intermittent stress and glucocorticoids. Immunohistochemical studies verified that N-methyl-D-aspartate (NMDA) receptor subunit proteins NR1 and NR2A/2B are expressed in the medial parvocellular PVN, indicating the potential for NMDA receptor regulation of corticotropin-releasing hormone (CRH) release. Dual-label confocal analysis revealed that CRH neurons are apposed by vesicular glutamate transporter 2 (VGLUT2)-containing terminals, consistent with glutamatergic innervation from hypothalamus and/or brainstem. In situ hybridization analysis revealed a significant and selective stress-induced decrease (37%) in NR2B subunit mRNA expression in the CRH-containing region of the PVN. No changes were observed for NR1 or NR2A mRNAs. In contrast, none of the subunits investigated showed altered expression following adrenalectomy with or without low/high-dose corticosterone replacement. Thus, the observed stress regulation is likely mediated by neurogenic mechanisms in the PVN and upstream stress-transducing neurocircuitry. Because a loss of NR2B subunit inclusion in NR receptors would likely confer increased Ca(++) conductance and faster deactivation kinetics, the stress-induced decrease in NR2B mRNA is consistent with enhanced glutamate signaling in the PVN following chronic stress and, perhaps, increased basal HPA activity and more rapid and/or more robust HPA responses to stress.

Aging and cholinergic deafferentation alter GluR1 expression in rat frontal cortex

Previously, we demonstrated that plasticity of frontal cortex is altered in aging rats: lesions of the nucleus basalis magnocellularis (NBM) produce larger declines in dendritic morphology in frontal cortex of aged rats compared to young adults. Cholinergic afferents from the NBM modulate glutamatergic transmission in neocortex, and glutamate is known to be involved in dendritic plasticity. To begin to identify possible mechanisms underlying age-related differences in plasticity after NBM lesion, we assessed the effect of cholinergic deafferentation on expression of the AMPA receptor subunit GluR1 in frontal cortex of young adult and aging rats. Young adult, middle-aged, and aged rats received sham or 192 IgG-saporin lesions of the NBM, and an unbiased stereological technique was used to estimate the total number of intensely GluR1-immunopositive neurons in layer II–III of frontal cortex. While the number of GluR1-positive neurons was increased in both middle-aged and aged rats, lesions markedly increased the number of intensely GluR1-immunopositive neurons in frontal cortex of young adult rats only. This age-related difference in lesion-induced expression of AMPA receptor subunit protein could underlie the age-related differences in dendritic plasticity after NBM lesions.

Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat.

N-methyl-D-aspartate (NMDA) receptor activation, at the level of the spinal cord, has been shown to play an important role in the facilitation of nociception in several animal models. However, the use of NMDA antagonists as analgesics is limited by serious side effects due to nonselective effects among the NMDA receptor subtypes. Recent discoveries revealed that the transfection of small interfering RNAs (siRNAs) into animal cells resulted in the potent, long-lasting, post-transcriptional silencing of specific genes. Thus, we investigated the effect of intrathecal (i.t.) injection of siRNAs targeting NMDA-R2B receptor subunit protein (NR2B) receptors, a subunit of NMDA receptor, for the modulation of pain. The results indicate that the use of siRNA targeting the NR2B subunit not only decreased the expression of NR2B mRNA and its associated protein, as demonstrated by real-time PCR and Western blotting, but also abolished formalin-induced pain behaviors in rat model. The peak effect occurred on day 3 for mRNA and day 7 for its protein, following i.t. injection of 5 microg of siRNA-NR2B. These data prove the feasibility of i.t. siRNAs in the investigation of functional gene expression in the context of whole animal behavior for the management of chronic pain.