Expression Of Different Subunits Research Articles

Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster.

Ecdysone, an essential insect steroid hormone, promotes larval metamorphosis by coordinating growth and maturation. In Drosophila melanogaster, prothoracicotropic hormone (PTTH)-releasing neurons are considered to be the primary promoting factor in ecdysone biosynthesis. Recently, studies have reported that the regulatory mechanisms of PTTH release in Drosophila larvae are controlled by different neuropeptides, including allatostatin A and corazonin. However, it remains unclear whether neurotransmitters provide input to PTTH neurons and control the metamorphosis in Drosophila larvae. Here, we report that the neurotransmitters acetylcholine (ACh) affect larval development by modulating the activity of PTTH neurons. By downregulating the expression of different subunits of nicotinic ACh receptors in PTTH neurons, pupal volume was significantly increased, whereas pupariation timing was relatively unchanged. We also identified that PTTH neurons were excited by ACh application ex vivo in a dose-dependent manner via ionotropic nicotinic ACh receptors. Moreover, in our Ca2+ imaging experiments, relatively low doses of OA caused increased Ca2+ levels in PTTH neurons, whereas higher doses led to decreased Ca2+ levels. We also demonstrated that a low dose of OA was conveyed through OA β-type receptors. Additionally, our electrophysiological experiments revealed that PTTH neurons produced spontaneous activity in vivo, which provides the possibility of the bidirectional regulation, coming from neurons upstream of PTTH cells in Drosophila larvae. In summary, our findings indicate that several different neurotransmitters are involved in the regulation of larval metamorphosis by altering the activity of PTTH neurons in Drosophila.

Utility of Immunohistochemistry and Immunofluorescence in Determining the Pathogenic Variants of Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder of phagocytes due to defects in any of the five subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. An initial diagnosis of CGD is made by flow cytometry-based dihydrorhodamine assay or nitro blue tetrazolium test, which is further confirmed by molecular assays. Expression of five subunits of NADPH oxidase components by either flow cytometric or western blot analysis provides clues toward the potential gene targets which are subsequently confirmed by various genetic assays. Immunohistochemistry (IHC) and immunofluorescence (IF) have never been earlier used to determine the expression of different subunits of NADPH oxidase system. We evaluated the utility of IHC and IF in determining the underlying pathogenic variants of CGD. Twelve genetically confirmed cases of CGD, comprising of biopsy specimens (n = 6), tissue blocks from autopsy cases (n = 3), and cellblocks of cell pellet prepared from peripheral blood (n = 4) were included. IHC for p67phox and p47phox subunits and IF for cytochrome b558 were performed. All 4 cases with pathogenic variation of NCF2 gene showed loss of expression for p67phox subunit. Two cases with pathogenic variation of NCF1 gene showed loss of expression for p47phox subunit. Five cases, except a single case with CYBB gene pathogenic variation, showed loss of expression for cytochrome b558 on IF. Thus, loss of expression consistently matched with the underlying genetic defects assessed by sequencing. Our results confirm our hypothesis that IHC and IF are two rapid, economical, pathologist-friendly techniques providing pertinent information regarding the underlying pathogenic variants and such immuno-analysis can be easily performed on the tissue.

Deciphering the effect of Endoplasmic Reticulum (ER) stress on near‐mitochondrial localized translation

Local synthesis of proteins near their active site is a critical cellular phenomenon. Recent studies have demonstrated that a subpopulation of cytoplasmic ribosomes is localized to organelles such as the endoplasmic reticulum and mitochondria. Due to the presence of different membranous compartments within eukaryotic cells, proper mRNA localization or targeted translation is required to focus the expensive process of translation to where it is needed most. During stress while global translation has been downregulated translation of stress responsive proteins as well other essential proteins remains active. During ER stress, substantial amounts of remodeling happen near ER localized translation populations. Due to its physical and functional relation with mitochondria it is also essential to regulate the translation process of mitochondrial destined proteins during the ER stress condition.In the current study, a digitonin based extraction method has been successfully established to separate the free cytoplasmic ribosome and mitochondria bound ribosome from HEK293 cells and to perform ribosome profiling in order to capture the actively translating polysome populations of free and mitochondria bound translating ribosome populations. This method can successfully separate the two ribosomal populations and it is enriched with known near‐mitochondria localized ribosome. Previous studies identified a population of mRNAs which are translating near mitochondrial surface in yeast and most of them has been found to be conserved in higher organisms. Interestingly, a set of known near mitochondria localized RNA has been found to involve in active translation in our global riboseq data collected from Hela upon ER stress. It indicates a correlation between the near mitochondrial localized translation and ER stress regulation. Specifically, higher expression of different sub units of oxidative phosphorylation complexes has been found to be enriched upon ER stress. This motivates further detailed study to understand organeller cross talk at translation level during stress. Understanding the near mitochondria localized populations in presence and absence of ER stress in higher organisms would be helpful to understand the complex ER‐mitochondrial cross talk events.Support or Funding InformationNIHR01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

Changes in synaptic plasticity and expression of glutamate receptor subunits in the CA1 and CA3 areas of the hippocampus after transient global ischemia

Excess glutamate release from the presynaptic membrane has been thought to be the major cause of ischemic neuronal death. Although both CA1 and CA3 pyramidal neurons receive presynaptic glutamate input, transient cerebral ischemia induces CA1 neurons to die while CA3 neurons remain relatively intact. This suggests that changes in the properties of pyramidal cells may be the main cause related to ischemic neuronal death. Our previous studies have shown that the densities of dendritic spines and asymmetric synapses in the CA1 area are increased at 12h and 24h after ischemia. In the present study, we investigated changes in synaptic structures in the CA3 area and compared the expression of glutamate receptors in the CA1 and CA3 hippocampal regions of rats after ischemia. Our results demonstrated that the NR2B/NR2A ratio became larger after ischemia although the expression of both the NR2B subunit (activation of apoptotic pathway) and NR2A subunit (activation of survival pathway) decreased in the CA1 area from 6h to 48h after reperfusion. Furthermore, expression of the GluR2 subunit (calcium impermeable) of the AMPA receptor class significantly decreased while the GluR1 subunit (calcium permeable) remained unchanged at the same examined reperfusion times, which subsequently caused an increase in the GluR1/GluR2 ratio. Despite these notable differences in subunit expression, there were no obvious changes in the density of synapses or expression of NMDAR and AMPAR subunits in the CA3 area after ischemia. These results suggest that delayed CA1 neuronal death may be related to the dramatic fluctuation in the synaptic structure and relative upregulation of NR2B and GluR1 subunits induced by transient global ischemia.

Altered expression of ATP-sensitive K+ channels in Hirschsprung's disease

PurposeHirschsprung's disease-associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung's disease (HSCR). Altered intestinal epithelial barrier function has been suggested to play a role in the causation of HAEC. In rodent experimental models of colitis, a decreased expression of K(ATP) channels (Subunits: Kir6.1/6.2 and SUR1/2) is reported. We designed this study to determine if K(ATP) channels exist within the human colon and to investigate the expression of different subunits in Hirschsprung's disease. MethodsWe investigated Kir6.1, Kir6.2, SUR1, and SUR2 expression in ganglionic and aganglionic bowel of HD patients (n=5) and controls (n=5). Western blotting and confocal immunofluorescence were performed. Main resultsWestern blot analysis revealed that Kir6.1, Kir6.2, SUR1, and SUR2 are strongly expressed in the normal human colon. Kir6.1, Kir6.2, SUR1, and SUR2 expression was significantly decreased in the aganglionic bowel compared to ganglionic bowel and controls. Kir6.1 and SUR1 expression were also significantly decreased in the ganglionic bowel of HSCR patients compared to controls. ConclusionWe demonstrate for the first time the existence of K(ATP) channels in the human colon. The decreased K(ATP) channel expression in HSCR specimens suggests that an altered K(ATP) expression may interfere with intestinal epithelium barrier function and predispose to HAEC.

Паттерн нейрональной экспрессии транскрипционных факторов NF-kb при предъявлении различных режимов гипобарической гипоксии

Transcription factor NF-kappaB plays a pivotal role in mechanisms of brain neuron survival and degeneration under injurious stimuli, first of all different types of hypoxia. In the present work, using quantitative immunohystochemistry, we provide analysis of expression of different subunits of NF-kappaB (p65 and c-Rel) in the rat neocortex in response to severe injurious hypobaric hypoxia (HH) or after a single or multiple sessions of mild protective HH. Severe hypoxia (SH), resulting in loss of brain neurons, has no effect on the level of expression of p65 but suppresses expression of c-Rel. Multiple (but not single one) trials of preconditioning using mild HH which reduce neuronal damage promote p65 expression and prevent suppression of c-Rel level after SH. Triple session of mild HH itself when applied as a preconditioning stimulus upregulate expression of both subunits, while single administration or sixfold trials has no effect on the level of immunoreactivity of both subunits. The revealed peculiarities of the expression of p65 and c-Rcl implies that these subunits of NF-kappaB appear to contribute to the mechanisms of brain tolerance to SH.

Expression of Different Subunits of the Calcium-Regulated BK Channel in Rat Brain and Its Putative Cytoprotective Properties

BKCa is ubiquitously expressed in the plasma membrane of both excitable and non-excitable cells. Although it was extensively studied mainly in plasma membrane, its counterpart exists also in mitochondria (mitoBKCa). It was identified in mitochondria of several cell types including astrocytes and neurons.Potassium channels are notorious for their role in cytoprotection but the mechanisms of these properties remain to be elucidated. However, it is suspected that attenuation of ROS generation and mild uncoupling in mitochondria occurring upon activation of the mitoBKCa may play some role in this phenomenon.In the current study we have evaluated the expression pattern of different subunits of the BKCa channel within certain areas of rat brain. We could show that there is a correlation between the mtDNA copy number and the expression level of the BKCa β4 auxiliary subunit. The expression of the β1, β2 and β3 subunits in different brain areas does not seem to correlate with the mtDNA copy number.This strongly suggest that β4 auxiliary subunit might be a constituent of mitoBKCa in rat brain. This finding supports the recent report about the expression of this subunit in the astrocytoma U-87 MG cell line1.The presence of the mitoBKCa in brain can explain why NS-1619 (a BKCa opener) inhibits ROS generation rate and causes mild dissipation of the membrane potential in succinate energized rat brain mitochondria.1. Bednarczyk, P. et al. Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain. PloS one8, e68125 (2013).This study is supported by the International PhD Program in Neurobiology coordinated by The Foundation for Polish Science and financed from the European Funds.

The K + channel GIRK2 is both necessary and sufficient for peripheral opioid‐mediated analgesia

The use of opioid agonists acting outside the central nervous system (CNS) is a promising therapeutic strategy for pain control that avoids deleterious central side effects such as apnea and addiction. In human clinical trials and rat models of inflammatory pain, peripherally restricted opioids have repeatedly shown powerful analgesic effects; in some mouse models however, their actions remain unclear. Here, we investigated opioid receptor coupling to K+ channels as a mechanism to explain such discrepancies. We found that GIRK channels, major effectors for opioid signalling in the CNS, are absent from mouse peripheral sensory neurons but present in human and rat. In vivo transgenic expression of GIRK channels in mouse nociceptors established peripheral opioid signalling and local analgesia. We further identified a regulatory element in the rat GIRK2 gene that accounts for differential expression in rodents. Thus, GIRK channels are indispensable for peripheral opioid analgesia, and their absence in mice has profound consequences for GPCR signalling in peripheral sensory neurons.GIRK channels are indispensable for peripheral opioid analgesia. The absence of GIRK channels from mouse dorsal root ganglion neurons questions the predictive validity of mice as a model organism for investigating peripheral GPCRmediated analgesia.

Glutamate modulates the firing rate in oculomotor nucleus motoneurons as a function of the recruitment threshold current

Studies in alert preparations have demonstrated that ocular motoneurons exhibit a phasic–tonic firing rate related to eye velocity and position, respectively. The slopes of these relationships are higher in motoneurons with higher recruitment threshold and have been proposed to depend upon synaptic input. To investigate this hypothesis, motoneurons of the rat oculomotor nucleus were recorded in a brain slice preparation in control conditions and during glutamate (5 μm) application to the bath. Glutamate did not affect membrane potential or input resistance, but produced a decrease in rheobase and depolarization voltage as a function of the current needed for generating a maintained repetitive discharge (recruitment threshold current). In addition, glutamate compressed the range of recruitment threshold current (0.1–0.4 nA) as compared to the control (0.15–0.7 nA). Glutamate exposed motoneurons showed an increase in the tonic frequency gain and the peak frequency. Such increments depended on the recruitment threshold current and the last recruited motoneurons almost doubled the tonic frequency gain (35.2 vs. 57.9 spikes s(−1) nA(−1)) and the peak frequency (52.4 vs. 102.6 spikes s(−1)). Finally, glutamate increased the spike frequency adaptation due to a significant increase in the phasic firing component as compared to the tonic one. In conclusion, glutamate modulates tonic and phasic discharge properties as a function of the recruitment threshold current and, presumably, motoneuron size. These findings contribute to understand the link between cellular functions and motoneuron discharge during oculomotor behaviour.

An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse.

Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the 'levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER.

Дослідження експресії різних субодиниць еукаріотного фактора елонгації трансляції eEF1 у гліальних пухлинах головного мозку людини

Дослідження експресії різних субодиниць еукаріотного фактора елонгації трансляції eEF1 у гліальних пухлинах головного мозку людини

Chloroplast translation regulation

Chloroplast gene expression is primarily controlled during the translation of plastid mRNAs. Translation is regulated in response to a variety of biotic and abiotic factors, and requires a coordinate expression with the nuclear genome. The translational apparatus of chloroplasts is related to that of bacteria, but has adopted novel mechanisms in order to execute the specific roles that this organelle performs within a eukaryotic cell. Accordingly, plastid ribosomes contain a number of chloroplast-unique proteins and domains that may function in translational regulation. Chloroplast translation regulation involves cis-acting RNA elements (located in the mRNA 5' UTR) as well as a set of corresponding trans-acting protein factors. While regulation of chloroplast translation is primarily controlled at the initiation steps through these RNA-protein interactions, elongation steps are also targets for modulating chloroplast gene expression. Translation of chloroplast mRNAs is regulated in response to light, and the molecular mechanisms underlying this response involve changes in the redox state of key elements related to the photosynthetic electron chain, fluctuations of the ADP/ATP ratio and the generation of a proton gradient. Photosynthetic complexes also experience assembly-related autoinhibition of translation to coordinate the expression of different subunits of the same complex. Finally, the localization of all these molecular events among the different chloroplast subcompartments appear to be a crucial component of the regulatory mechanisms of chloroplast gene expression.

Brain Region Specific Alterations in the Protein and mRNA Levels of Protein Kinase A Subunits in the Post-Mortem Brain of Teenage Suicide Victims

Protein kinase A (PKA), a critical component of the adenylyl cyclase signaling system, phosphorylates crucial proteins and has been implicated in the pathophysiology of depression and suicide. The objective of the study was to examine if changes in PKA activity or in the protein and messenger RNA (mRNA) expression of any of its subunits are related to the pathophysiology of teenage suicide. We determined PKA activity and the protein and mRNA expression of different subunits of PKA in cytosol and membrane fractions obtained from the prefrontal cortex, (PFC) hippocampus, and nucleus accumbens (NA) of post-mortem brain from 17 teenage suicide victims and 17 nonpsychiatric control subjects. PKA activity was significantly decreased in the PFC but not the hippocampus of teenage suicide victims as compared with controls. However, the protein and mRNA expression of only two PKA subunits, that is, PKA RIalpha and PKA RIbeta, but not any other subunits were significantly decreased in both membrane and cytosol fractions of the PFC and protein expression of RIalpha and RIbeta in the NA of teenage suicide victims as compared to controls. A decrease in protein and mRNA expression of two specific PKA subunits may be associated with the pathogenesis of teenage suicide, and this decrease may be brain region specific, which may be related to the specific behavioral functions associated with these brain areas. Whether these changes in PKA subunits are related to suicidal behavior or are a result of suicide or are specific to suicide is not clear at this point.

Developmental changes in inhibin alpha and inhibin/activin betaA and betaB mRNA levels in the gonads during post-hatch prepubertal development of male and female chickens.

Dimeric inhibins and activins are barely detectable in the plasma during prepubertal development of male and female chickens. This may be misconstrued to indicate that the proteins are not produced in the gonads and have no functional significance during this period. Very few studies have actually determined the mRNA expression profile of the inhibin and activin subunits in the gonads prior to puberty in order to establish their secretion at the local level and postulate potential roles for the inhibin and activins at this developmental stage. In this study, the expression of the mRNA for the alpha-, betaA-, and betaB-subunits was determined in the ovary and testis of chickens during prepubertal development. Gene expression was determined at 3, 5, 6, 8, 10, 12, 16, and 18 weeks of age by RT-PCR. Messenger RNA level was quantified by competitive RT-PCR at 3, 6, 12, and 18 weeks of age in order to detect any changes with development, suggest potential relationship to the profile of dimeric inhibins and activins reported previously and to suggest potential paracrine and endocrine roles for them. The results show that all the inhibin/activin subunit mRNAs are expressed in the testis of the chicken throughout the period of prepubertal development up to 18 weeks of age. However, in the ovary, only the betaA- and betaB-subunits were detected at all ages whereas the alpha-subunit mRNA could only be detected just before puberty. Quantification of the mRNA levels showed variation of each subunit with age. These temporal changes suggest relationship with paracrine functional role in the ovary or the testis. Quantitative changes in expression levels also suggests that there may be some relationship between mRNA levels and the type and amount of dimeric inhibins and activins produced at any developmental stage. There are major differences between the male and female gonads in the timing of the expression of different subunits. In conclusion, the expression of the mRNA subunits in the testis and ovary suggests that inhibins and activins are being produced but may be principally involved in autocrine/paracrine function within the gonads.

Alterations of markers related to synaptic function in aging rat brain, in normal conditions or under conditions of long-term dietary manipulation

Neurochemical alterations of markers related to synaptic function are potential candidates for age-related impairment of brain function and cognition. The process of aging, including brain aging, can be counteracted to some degree by maintaning animals in long-term conditions of caloric restriction, or supplementing their diet with antioxidant substances. We report here that the age-related decline of the cholinergic and GABAergic systems, that takes place in some CNS regions of aged rats, is not affected by maintaining them under conditions of dietary restriction and, therefore, of reduced calorie intake, from the 12th to the 30th month of age. We also notice the same lack of effect by adding, during the same period, the aging rat diet with the potential antioxidant substance, N-acetylcysteine (NAC). The same dietary manipulations are also unable to counteract the derangement of the first step of the main biosynthetic pathway for polyamines, putative neuromodulators in the CNS, that occurs in the aged spinal cord. Some age-related alterations in the expression of different subunits of the NMDA-type glutamate receptors in some CNS regions of aged rats were instead, at least in some cases, counteracted by long-term dietary manipulation.

Developmentally regulated expression of CaMKII and iGluRs in the rat retina

Calcium/calmodulin-dependent protein kinase II (CaMKII) and the ionotropic glutamate receptors (iGluRs) have been shown to be pivotal in the maturation of synapses during development of the central nervous system. The purpose of the current study was to assay the expression profiles of these molecules during the development of the rat retina. The mRNA levels of CaMKII were determined by the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. The protein levels of CaMKII were assayed in slot blots. The CaMKII enzyme activity was also measured. In addition, the protein levels of iGluRs in a retinal membrane-enriched fraction were evaluated in Western blots. The results show that the levels of CaMKII (mRNA, protein, and activity) and distinct subunits of iGluR proteins increased during the first 2 weeks after birth. The highest level of CaMKII was reached during the second postnatal week, coincident with the peak of synaptogenesis in the inner plexiform layer of the rat retina. The expressions of NMDAR-NR1 and -NR2A were relatively low in the first postnatal week but rose quickly thereafter. However, NMDAR-NR2B was relatively high at postnatal day 5 (P5) and increased steadily during the postnatal period. Thus, the subunit compositional profile of the retinal NMDARs was altered during retinal maturation. The developmental pattern of AMPAR-GluR1 was similar to that of NMDAR-NR2B, with high expression at P5, and modest increases thereafter. The patterns of CaMKII and NR1/NR2A were better correlated than were CaMKII and NR2B, or CaMKII and GluR1. The temporal differences in subunit expression of these synaptically relevant molecules suggest that they play distinct roles during the development of the retina.

Gamma-Aminobutyric acid(A) receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development.

Profound alterations in the function of GABA occur over the course of postnatal development. Changes in GABA(A) receptor expression are thought to contribute to these differences in GABAergic function, but how subunit changes correlate with receptor function in individual developing neurons has not been defined precisely. In the current study, we correlate expression of 14 different GABA(A) receptor subunit mRNAs with changes in the pharmacological properties of the receptor in individual hippocampal dentate granule cells over the course of postnatal development in rat. We demonstrate significant developmental differences in GABA(A) receptor subunit mRNA expression, including greater than two-fold lower expression of alpha1-, alpha4- and gamma2-subunit mRNAs and 10-fold higher expression of alpha5-mRNA in immature compared with adult neurons. These differences correlate both with regional changes in subunit protein level and with alterations in GABA(A) receptor function in immature dentate granule cells, including two-fold higher blockade by zinc and three-fold lower augmentation by type-I benzodiazepine site modulators. Further, we find an inverse correlation between changes in GABA(A) receptor zinc sensitivity and abundance of vesicular zinc in dentate gyrus during postnatal development. These findings suggest that developmental differences in subunit expression contribute to alterations in GABA(A) receptor function during postnatal development.

Declines in mRNA Expression of Different Subunits May Account for Differential Effects of Aging on Agonist and Antagonist Binding to the NMDA Receptor

The purpose of the present study was to determine whether some of the age-related changes that occur in binding to the NMDA receptor complex can be accounted for by changes in subunit expression during the aging process. In situ hybridization for the NMDA subunits zeta1, epsilon1, and epsilon2, and receptor autoradiography, using the agonist glutamate and the competitive antagonist [(+/-)-2-carboxypiperazin-4-yl] propyl-1-phosphonic acid (CPP), were performed on sections from C57Bl/6 mice representing three different age groups (3, 10, and 30 months of age). There was a significant overall decrease between 3 and 30 month olds in the density of mRNA for the zeta1 subunit in the cortex and hippocampus, but only a few individual brain regions exhibited significant declines. The mRNA for the epsilon2 subunit was significantly decreased in a majority of cortical regions and in the dentate granule cells. Emulsion analysis indicated that the change in the density of epsilon2 subunit mRNA in the inner frontal cortex was primarily attributable to a decrease in the amount of messages per cell. Age-related changes in mRNA density of the epsilon2 subunit correlated with changes in NMDA-displaceable [(3)H]glutamate binding, and mRNA density changes in the zeta1 subunit showed a significant relationship with changes in [(3)H]CPP binding in the 30-month-old mice. These results suggest that changes during aging in the expression of different subunits of the NMDA receptor may account for the differential effects of aging on agonist versus antagonist binding to the NMDA binding site.

Developmental switch in the expression of GABA A receptor subunits α 1 and α 2 in the hypothalamus and limbic system of the rat

The GABA A receptor is a pentameric ligand gated ion channel complex assembled from a family of at least 17 different subunits encoded by distinct genes. Two subunits, α 1 and α 2, exhibit age dependent expression throughout several areas of the brain. In general, the density of immunoreactive product for α 1 is greatest in the adult brain, while α 2 is highest in younger tissue. Since the developmental switch in α 1 and α 2 coincides with the end of the sensitive period for steroid-mediated sexual differentiation of the brain, we hypothesized that GABA A receptor subunit expression may be involved in this process. We have examined the age-dependent expression of α 1 and α 2 in discrete regions of the hypothalamus and limbic system of males and females. While we did not detect any dramatic sex differences in α 1 or α 2 immunoreactive density, each region exhibited a unique developmental profile. In the ventromedial nucleus of neonatal animals immunoreactivity is highest for α 1, while in the adult the signal for α 2 is greater; the opposite of that observed in the ventrolateral thalamus. There is no age dependent change for α 1 in the preoptic area, while α 2 shows a small, but significant increase. Immunoreactive densities for both subunits increase in the arcuate nucleus and the hippocampus, but decrease in the lateral amygdala. We conclude that these regional differences in subunit expression across development determine individual characteristics of brain areas and may play a role in establishing unique physiological responses to GABA.