Levels Of AMPA Receptor Subunits Research Articles

Learning differentially increases the synaptic levels of AMPA receptor subunits (GluA1‐GluA4) in the rodent hippocampus: a single‐synapse approach

AbstractBackgroundDecades of experimental work support the idea that synapses are the anatomical substrate for experience‐dependent plasticity in the brain. Changes in the synaptic strength underlie learning and memory via the accumulation of glutamate AMPA receptors (AMPAR) at the surface of excitatory synapses. AMPAR are tetrameric receptors constituted by multiple combination of four subunits: GluA1 GluA2, GluA3, and GluA4. However, the relative contribution of each AMPAR subunit on learning and memory is relatively unexplored.MethodHere, we introduce Fluorescence Analysis of Single‐Synapse Potentiation induced by Learning (FASS‐PiL), a flow cytometry‐based method to quantify surface levels of all four GluA‐AMPAR subunits, in parallel, in isolated synaptosomes after a learning episode in rodents. We evaluated surface levels of all four AMPAR subunits in synaptosomes of mice trained in a learning protocol commonly used for studying episodic memory in the hippocampus (e.g., Object Location Memory (OLM) and Object Recognition Memory (ORM) tasks). Briefly, after exploring novel objects in a novel environment for 10 min, mice were returned to their home cage for 60 min. After hippocampus dissection, synaptosome isolation, and immunostaining, samples were analyzed via flow cytometry.ResultWe first demonstrated that FASS‐PiL is a simple and sensitive method to track all four GluAs at the synaptosome surface (each GluA subunit was paired with the presynaptic marker Neurexin‐1beta to focus on synaptosome particles containing both pre‐ and postsynaptic compartments). Notably, by profiling hundreds of events, our data showed that the OLM training increases the number of hippocampal synaptosomes expressing high levels of GluA1 and GluA2, but not GluA3 and GluA4 (vs control animals).ConclusionOur results indicate that plasticity‐related mechanisms underlying learning are AMPA‐subunit‐specific in hippocampal synapses. Our approach could provide the basis for protocols to study behavioral‐relevant mechanisms of plasticity directly at the synapse, with single‐event resolution.

Melamine impairs working memory and reduces prefrontal activity associated with inhibition of AMPA receptor GluR2/3 subunit expression

Recent studies have reported that melamine can accumulate in several regions of the brain including the medial prefrontal cortex (mPFC). Although melamine accumulation in the hippocampus has been verified to induce cognitive impairments, whether it can cause mPFC-dependent working memory deficits is still unknown. After chronic treatment with melamine (150 (Mel(150)) or 300 (Mel(300)) mg/kg), rats were tested during both delay nonmatching-to-sample spatial and odor discrimination tasks. Levels of AMPA receptor subunits in the mPFC were detected using western blotting. To further explore the mechanism at the cellular level, prefrontal activity was recorded during the odor discrimination. The working memory of Mel(150) rats was found to be significantly impaired in a 3-minute delay odor discrimination task (control: n = 6, Mel(150): n = 6; P < 0.05). Compared with the control group (n = 6), rats in the 300 mg/kg Mel(300)-treated group (n = 8) displayed working memory deficits in 60-second delay Y-maze task (P < 0.05), 1-minute and 3-minute delay odor discrimination tasks (both P < 0.05). The levels of AMPA receptor mGluR2/3 subunit were significantly decreased in rats of the Mel(150) (n = 7) and Mel(300) (n = 7) groups (both P < 0.05). Exposure to 150 (n = 7) or 300 mg/kg (n = 7) melamine resulted in significant inhibition of the regular-spiking neuron activity during the delay period of the memory test (both P < 0.05). Intraperitoneal (n = 7) and intra-mPFC (n = 6) infusions of GluR2/3 agonists, effectively enhanced the neural correlate (both P < 0.05) while rescuing cognitive deficits in Mel(300)-treated rats (both P < 0.05). Collectively, these findings suggested that melamine could induce prefrontal dysfunction and cause cognitive impairments.

Quantitative Immunoblotting Analyses Reveal that the Abundance of Actin, Tubulin, Synaptophysin and EEA1 Proteins is Altered in the Brains of Aged Mice

Optimal synaptic activity is essential for cognitive function, including memory and learning. Evidence indicates that cognitive decline in elderly individuals is associated with altered synaptic function. However, the impact of aging on the expression of neurotransmitter receptors and accessory proteins in brain synapses remains unclear. To fill this knowledge gap, we investigated the effect of aging on the mouse brain by utilizing a subcellular brain tissue fractionation procedure to measure protein abundance using quantitative Western Blotting. Comparing 7-month- (control) and 22-month- (aged) old mouse tissue, no significant differences were identified in the levels of AMPA receptor subunits between the experimental groups. The abundance of GluN2B NMDA receptor subunits decreased in aged mice, whereas the levels of GluN2A did not change. The analysis of cytoskeletal proteins showed an altered level of actin and tubulin in aged mice while PSD-95 protein did not change. Vesicle protein analysis revealed that synaptophysin abundance is decreased in older brains whereas EEA1 was significantly increased. Thus, our results suggest that physiological aging profoundly impacts the abundance of molecules associated with neurotransmitter release and vesicle cycling, proteins implicated in cognitive function.

The glutamatergic system in the preoptic area is involved in the retention of maternal behavior in maternally experienced female rats.

Maternally experienced female rats show high maternal behavior performance for a long time after acquisition of maternal experience, although the mechanisms responsible for the retention of maternal behavior are not well understood. The medial preoptic area (MPOA) plays an important role in the onset and maintenance of maternal behavior in female rats. We aimed to determine whether maternal experience affects the glutamatergic system in the MPOA for the retention of maternal behavior in female rats. First, to determine the effects of maternal experience in the postpartum period on dendritic spines, which are the postsynaptic component of excitatory glutamatergic neurotransmission, we examined the number of dendritic spines on MPOA neurons of primiparous mothers that had experienced mothering until weaning (sufficiently experienced mothers) and of primiparous mothers that were separated from their pups on the day of parturition (insufficiently experienced mothers). The number of mushroom spines, but not other types of spine, was significantly greater in the sufficiently experienced mothers compared with that in the insufficiently experienced mothers. Next, to determine the effects of maternal experience in the postpartum period on the expression of ionotropic glutamate receptors, we measured the mRNA levels of AMPA receptor subunits (GluA1-A4) and NMDA receptor subunits (GluN1, GluN2A-2D) in the MPOA of primiparous female rats that were kept with pups until brain sampling. As a result, we found that the mRNA levels of GluA3 and GluN2B were significantly higher in primiparous females on the day of weaning compared with those in primiparous females on the day of parturition. Additionally, we examined the effects of CNQX, an AMPA receptor antagonist, and MK-801, an NMDA receptor antagonist, injected into the MPOA on maternal behavior in maternally experienced primiparous female rats. Maternal behavioral activity was significantly reduced when CNQX or MK-801 was injected into the MPOA. These findings indicate that long-term maternal experience in the postpartum period up-regulates glutamatergic neurotransmission by increasing the number of mushroom spines and glutamate receptor expression, which may be involved in the retention of maternal behavior in maternally experienced female rats.

Disruption of the NMDA receptor GluN2A subunit abolishes inflammation-induced depression

Recent reports have demonstrated that lipopolysaccharide (LPS)-induced depressive-like behaviour is mediated via NMDA receptor. In this study, we further investigated the role of GluN2 A subunit of NMDA receptor in synaptic processes in the prefrontal cortex (PFC) and hippocampus of GluN2 A knockout (KO) mice in LPS-induced depressive-like behavior. Our data suggest that LPS-treated mice, lacking GluN2 A subunit, did not exhibit depressive-like behaviour. This was accompanied by unaltered levels of IL-6 and significant changes in neuroplasticity markers and glutamate receptor subunits composition in PFC and hippocampus. In particular, an immune challenge in GluN2 A KO mice resulted in unchanged PSA-NCAM levels and proBDNF increase in both brain structures as well as in increase in BDNF levels in hippocampus. Furthermore, the absence of GluN2 A resulted in increased levels of all NCAM isoforms in PFC upon LPS which was followed with a decrease in GluN1 and GluN2B subunits. The levels of AMPA receptor subunits (GluA1, GluA3, and GluA4) in the hippocampus of GluN2 A mice were unaltered upon the treatment and abundantly present in the PFC of KO mice.These results indicate that the GluN2 A subunit is critical in neuroinflammation-related depression, that its absence abolishes LPS-induced depressive phenotype, sustains PSA-NCAM levels, increases proBDNF signalling in the PFC and hippocampus and potentiates synaptic stabilization through NCAM in the PFC upon an immune challenge.

Alterations in mRNA expression of glutamate receptor subunits and excitatory amino acid transporters following pilocarpine-induced seizures in rats

Temporal lobe epilepsy is the most prevalent form of complex partial seizure, and it is frequently triggered by an initial brain-damaging insult. The prevention of epileptogenesis after a primary event could be a key innovative approach to epilepsy treatment. Therefore, it is critical to understand the pathogenic mechanisms of this process in detail. Multiple mechanisms are involved in epileptogenesis, including alterations in the expression of synaptic receptors and transporters. The present study aimed to investigate the mRNA expression of excitatory amino acid transporters 1–3 (EAATs) and the subunits of the NMDA (GluN1, GluN2a, and GluN2b) and AMPA (GluA1 and GluA2) glutamate receptors following status epilepticus in a rat lithium-pilocarpine model. The analysis of the mRNA was performed via qRT-PCR one week after pilocarpine injections (the period of epileptogenesis) into the ventral and dorsal hippocampus and the entorhinal, temporal, and medial prefrontal cortexes – brain areas that are differentially involved in the pathogenesis of TLE.We found that increased EAAT2 mRNA levels in the medial prefrontal cortex and dorsal hippocampus may represent compensatory neuroprotective changes. Alterations in the gene expression levels of AMPA receptor subunits were found in the ventral hippocampus and temporal cortex. The reduced expression of the GluN2a subunit was observed in the temporal and entorhinal cortical areas and the ventral hippocampus, which may result in the predominance of GluN2b-containing NMDA receptors in these areas. The receptors with this altered subunit composition may be involved in pathophysiological mechanisms related to epileptogenesis. These data provide a better understanding of the pathogenesis of epilepsy.

Altered glutamate response and calcium dynamics in iPSC-derived striatal neurons from XDP patients

X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder endemic to Panay Island (Philippines). Patients present with generalizing dystonia and parkinsonism. Genetic changes surrounding the TAF1 (TATA-box binding protein associated factor 1) gene have been associated with XDP inducing a degeneration of striatal spiny projection neurons. There is little knowledge about the pathophysiology of this disorder.Our objective was to generate and analyze an in-vitro model of XDP based on striatal neurons differentiated from induced pluripotent stem cells (iPSC).We generated iPSC from patient and healthy control fibroblasts (3 affected, 3 controls), followed by directed differentiation of the cultures towards striatal neurons. Cells underwent characterization of immunophenotype as well as neuronal function, glutamate receptor properties and calcium dynamics by whole-cell patch-clamp recordings and calcium imaging. Furthermore, we evaluated expression levels of AMPA receptor subunits and voltage-gated calcium channels by quantitative real-time PCR.We observed no differences in basic electrophysiological properties. Application of the AMPA antagonist NBQX led to a more pronounced reduction of postsynaptic currents in XDP neurons. There was a higher expression of AMPA receptor subunits in patient-derived neurons. Basal calcium levels were lower in neurons derived from XDP patients and cells with spontaneous calcium transients were more frequent. Our data suggest altered glutamate response and calcium dynamics in striatal XDP neurons.

Persistent pain alters AMPA receptor subunit levels in the nucleus accumbens.

BackgroundA variety of pain conditions have been found to be associated with depressed mood in clinical studies. Depression-like behaviors have also been described in animal models of persistent or chronic pain. In rodent chronic neuropathic pain models, elevated levels of GluA1 subunits of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the nucleus accumbens (NAc) have been found to inhibit depressive symptoms. However, the effect of reversible post-surgical pain or inflammatory pain on affective behaviors such as depression has not been well characterized in animal models. Neither is it known what time frame is required to elicit AMPA receptor subunit changes in the NAc in various pain conditions.ResultsIn this study, we compared behavioral and biochemical changes in three pain models: the paw incision (PI) model for post-incisional pain, the Complete Freund’s Adjuvant (CFA) model for persistent but reversible inflammatory pain, and the spared nerve injury (SNI) model for chronic postoperative neuropathic pain. In all three models, rats developed depressive symptoms that were concurrent with the presentation of sensory allodynia. GluA1 levels at the synapses of the NAc, however, differed in these three models. The level of GluA1 subunits of AMPA-type receptors at NAc synapses was not altered in the PI model. GluA1 levels were elevated in the CFA model after a period (7 d) of persistent pain, leading to the formation of GluA2-lacking AMPA receptors. As pain symptoms began to resolve, however, GluA1 levels returned to baseline. Meanwhile, in the SNI model, in which pain persisted beyond 14 days, GluA1 levels began to rise after pain became persistent and remained elevated. In addition, we found that blocking GluA2-lacking AMPA receptors in the NAc further decreased the depressive symptoms only in persistent pain models.ConclusionOur study shows that while both short-term and persistent pain can trigger depression-like behaviors, GluA1 upregulation in the NAc likely represents a unique adaptive response to minimize depressive symptoms in persistent pain states.

Depalmitoylation preferentially downregulates AMPA induced Ca2+ signaling and neurotoxicity in motor neurons

Excessive activation of AMPA receptor has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). However, it is not clear why motor neurons are preferentially sensitive to AMPA receptor mediated excessive [Ca2+]i rise and excitotoxicity. In the present study we examined whether palmitoylation regulates Ca2+ permeability of AMPA receptor and excitotoxicity in cultured spinal cord neurons. We adapted chronic 2-bromopalmitate (2-BrP) treatment to achieve depalmitoylation and examined its effect on the cytotoxicity in spinal cord neurons exposed to AMPA. The change in AMPA induced signaling and cytotoxicity in motor neurons and other spinal neurons under identical conditions of exposure to AMPA was studied. 2-BrP treatment inhibited AMPA induced rise in [Ca2+]i and cytotoxicity in both types of neurons but the degree of inhibition was significantly higher in motor neurons as compared to other spinal neurons. The AMPA induced [Na+]i rise was moderately affected in both type of neurons on depalmitoylation. Depalmitoylation reduced the expression levels of AMPA receptor subunits (GluR1 and GluR2) and also PSD-95 but stargazin levels remained unaffected. Our results demonstrate that 2-BrP attenuates AMPA receptor activated Ca2+ signaling and cytotoxicity preferentially in motor neurons and suggest that AMPA receptor modulation by depalmitoylation could play a significant role in preventing motor neuron degeneration.

Elevated glucose concentration changes the content and cellular localization of AMPA receptors in the retina but not in the hippocampus

Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

Neuronal nitric oxide synthase inhibition attenuates the development of L-DOPA-induced dyskinesia in hemi-Parkinsonian rats

Long-term treatment with the dopamine precursor levodopa (l-DOPA) frequently induces dyskinesia in Parkinson's disease patients, which is a major complication of this therapy. Previous studies using animal models show that repeated administration of l-DOPA results in alterations of some signaling molecules, including ΔFosB, phospho-DARPP32 and phosoho-GluA1 (also referred to as GluR1 or GluR-A) AMPA receptor subunits. Moreover, an in vivo microdialysis study showed that l-DOPA increases nitric oxide (NO) production in the striatum. However, it is not known whether NO is involved in the development of dyskinesia. The present study examined the effects of NOS inhibitors on the development of l-DOPA-induced dyskinesia in the rats. Dyskinesia symptoms were triggered by daily administration of l-DOPA for 3–4weeks in unilateral 6-hydroxydopamine lesioned rats. Repeated treatments, 30min prior l-DOPA administration, of the nonselective NOS inhibitor, NG-nitro-l-arginine methyl ester, and the nNOS inhibitor 7-nitroindazole, but not the inducible NOS inhibitor aminoguanidine, attenuated the development of l-DOPA-induced dyskinesia. In agreement with the behavioral analysis, 7-nitroindazole reduced the l-DOPA-induced increases in ΔFosB, phospho-DARPP32 and phospho-GluA1 AMPA receptor subunit levels in the striatum of 6-hydroxydopamine-lesioned rats. Furthermore, aminoguanidine did not affect ΔFosB or phospho-GluA1 AMPA receptor subunit levels. These findings suggest that nNOS-derived NO is involved in the development of l-DOPA-induced dyskinesia through a post-synaptic mechanism.

Cochlear nucleus neurons redistribute synaptic AMPA and glycine receptors in response to monaural conductive hearing loss

Neurons restore their function in response to external or internal perturbations and maintain neuronal or network stability through a homeostatic scaling mechanism. Homeostatic responses at synapses along the auditory system would be important for adaptation to normal and abnormal fluctuations in the sensory environment. We investigated at the electron microscopic level and after postembedding immunogold labeling whether projection neurons in the cochlear nucleus responded to modifications of auditory nerve activity. After unilaterally reducing the level of auditory inputs by ∼20 dB by monaural earplugging, auditory nerve synapses on bushy cells somata and basal dendrites of fusiform cells of the ventral and dorsal cochlear nucleus, respectively, upregulated GluR3 AMPA receptor subunit, while inhibitory synapses decreased the expression of GlyRα1 subunit. These changes in expression levels were fully reversible once the earplug was removed, indicating that activity affects the trafficking of receptors at synapses. Excitatory synapses on apical dendrites of fusiform cells (parallel fibers) with different synaptic AMPA receptor subunit composition, were not affected by sound attenuation, as the expression levels of AMPA receptor subunits were the same as in normal hearing littermates. GlyRα1 subunit expression at inhibitory synapses on apical dendrites of fusiform cells was also found unaffected. Furthermore, fusiform and bushy cells of the contralateral side to the earplugging upregulated the GluR3 subunit at auditory nerve synapses. These results show that cochlear nucleus neurons innervated by the auditory nerve, are able to respond to small changes in sound levels by redistributing specific AMPA and glycine receptor subunits.

Repeated 4-aminopyridine induced seizures diminish the efficacy of glutamatergic transmission in the neocortex

Systemic administration of the potassium channel blocker 4-aminopyridine (4-AP) elicits acute convulsions. Synchronized tonic–clonic activity develops during the first hour after the treatment. However, subsequent chronic spontaneous seizures do not appear which suggests changes in neuronal excitability. The aim of our present work was to evaluate alterations in the glutamatergic transmission in the somatosensory cortex of rats following daily, brief convulsions elicited by 4-AP treatment. Changes in general neuronal excitability and pharmacological sensitivity of glutamate receptors were tested in ex vivo electrophysiological experiments on brain slices. In parallel studies quantitative changes in subunit composition of glutamate receptors were determined with immunohistoblot technique, together with the analysis of kainate induced Co 2+ uptake. The results of our coordinated electrophysiological, receptor-pharmacological and histoblot studies demonstrated that repeated, daily, short convulsions resulted in a significant decrease of the general excitability of the somatosensory cortex together with changes in ionotropic glutamate receptor subunits. The relative inhibitory effect of the AMPA receptor antagonist, however, did not change. The NMDA receptor antagonist exerted somewhat stronger effect in the slices from convulsing animals. 4-AP pretreatment resulted in the attenuation of kainate induced Co 2+ uptake, which suggests either reduction in non-NMDA receptors numbers or reduction in their Ca 2+ permeability. Repeated seizures decreased GluR1–4 AMPA receptor subunit levels in all cortical layers with a relaitve increase in GluR1 subunits. While the principle NR1 NMDA receptor subunit showed no significant change, the staining density of NR2A subunit increased. These changes in ionotropic glutamate receptors are consistent with reduced excitability at glutamatergic synapses following repeated 4-AP induced seizures.

The Effects of Aging and Caloric Restriction upon Rat CA3 Hippocampus: A Three‐Dimensional Ultrastructural Study

We have previously shown an age‐related decline in glutamate receptor subunit levels in hippocampal CA3 that is altered by caloric restriction (CR). Specifically, CR beginning at 4 months of age results in a decline in NMDA and AMPA receptor subunits by 10 months of age to levels that remain stable across life‐span. By 28 months of age, subunits in ad libitum (AL) fed rats have declined to levels significantly lower than observed in CR animals. Moreover, cognitive performance in 28 month‐old CR rats is significantly better than in age‐matched AL animals. Because NMDA and AMPA receptor subunit levels as well as neural function have been correlated with area and complexity of hippocampal excitatory synapses, we used serial 3‐D reconstruction to assess size and morphology of postsynaptic densities (PSDs) in CA3 of young‐AL, aged‐AL and aged‐CR male F344×BN rats. Preliminary analysis reveals no difference in mean PSD surface area among groups. We also are quantifying the effects of age and CR on the overall synapse population in CA3 as well as on those with functionally‐relevant morphologies such as perforated synapses and multiple spine boutons.

Methamphetamine induces alterations on hippocampal NMDA and AMPA receptor subunit levels and impairs spatial working memory

Methamphetamine (METH) is a powerful psychostimulant that increases glutamate (Glu) levels in the mammalian brain and it is currently known that hippocampi are particularly susceptible to METH. Moreover, it is well established that the overactivation of N-methyl- d-aspartate (NMDA) and AMPA ionotropic Glu receptors causes excitotoxicity. In the present study, we investigated the effect of acute (30 mg/kg) versus escalating dose (ED) administration of METH on NMDA receptor 1, NMDA receptor 2 and glutamate receptor 2 (GluR2) subunit expression in the hippocampus and on memory. Adult Sprague–Dawley rats were injected s.c. during six consecutive days with saline (control and acute groups) or with a growing dose of METH (10, 15, 15, 20, 20, 25 mg/kg/day; ED group). On the 7th day, both METH groups were injected with a ‘bolus’ of 30 mg/kg METH whereas controls received saline. Western blot analysis showed an increase of GluR2 and NR2A expression levels and no alterations on NR1 subunit in the acute group. On the other hand, in the ED group, GluR2 and NR2A expression levels were unaltered and there was a decrease on NR1 levels. Moreover, we did not observe neurodegeneration with both administration paradigms, as assessed by Fluoro-Jade C staining, but we did observe a strong astrogliosis in the acute administration group by using both immunohistochemistry and Western blot analysis. The impact of METH on working memory was evaluated using the Y maze test and revealed significant mnemonic deficit in the rats acutely treated with the drug. Overall, our results suggest a protection mechanism under conditions of METH administration by decreasing permeability and/or functionality of NMDA and AMPA receptors, which has implications on memory. So, the participation of the glutamatergic system should be considered as an important pharmacological target to design new strategies to prevent or diminish the harmful effect of drug consumption.

Caloric restriction eliminates the aging-related decline in NMDA and AMPA receptor subunits in the rat hippocampus and induces homeostasis

Caloric restriction (CR) extends life span and ameliorates the aging-related decline in hippocampal-dependent cognitive function. In the present study, we compared subunit levels of NMDA and AMPA types of the glutamate receptor and quantified total synapses and multiple spine bouton (MSB) synapses in hippocampal CA1 from young (10 months), middle-aged (18 months), and old (29 months) Fischer 344×Brown Norway rats that were ad libitum (AL) fed or caloric restricted (CR) from 4 months of age. Each of these parameters has been reported to be a potential contributor to hippocampal function. Western blot analysis revealed that NMDA and AMPA receptor subunits in AL animals decrease between young and middle age to levels that are present at old age. Interestingly, young CR animals have significantly lower levels of glutamate receptor subunits than young AL animals and those lower levels are maintained across life span. In contrast, stereological quantification indicated that total synapses and MSB synapses are stable across life span in both AL and CR rats. These results indicate significant aging-related losses of hippocampal glutamate receptor subunits in AL rats that are consistent with altered synaptic function. CR eliminates that aging-related decline by inducing stable NMDA and AMPA receptor subunit levels.

Sequential changes in BDNF mRNA expression and synaptic levels of AMPA receptor subunits in rat hippocampus after chronic antidepressant treatment

Increased expression of brain-derived neurotrophic factor (BDNF) appears to be involved in the mechanism of action of antidepressant drugs. It has also been proposed that potentiation of the AMPA receptor (AMPAR) function may be useful in the treatment of depression. Here we looked for the time course of the effect of different doses of two antidepressants, desipramine (DMI) and paroxetine (PAR), which differentially affect monoamine reuptake, on BDNF mRNA expression in hippocampal subfields (CA1, CA3 and dentate gyrus) and levels of AMPAR subunits in total and membrane-enriched extracts from rat hippocampus. Acute antidepressant treatment changed neither BDNF mRNA expression nor AMPAR subunit levels. In chronic treatments, rats were treated daily with the antidepressants for 7–21 days. PAR produced a time- and dose-dependent increase of BDNF expression in the three hippocampal subfields examined. On the contrary, the effect of DMI on BDNF mRNA was neither dose- nor time-dependent. In rats receiving the same chronic antidepressant treatments, PAR produced a dose-dependent increase of GluR1 and GluR2/3 levels in the membrane fraction after a 3-week treatment, and not at earlier times. DMI increased the membrane levels of AMPAR subunits after a 3-week treatment with the lower dose tested. However, a higher dose, 15 mg/kg, did not produce any change in AMPAR subunits and reduced membrane levels of α-tubulin and PSD-95, possibly indicating a disorganization of membrane scaffolding proteins. The results suggest that paroxetine, but not desipramine, enhances synaptic plasticity in the hippocampus by increasing BDNF mRNA expression, which determines a later AMPAR subunit trafficking to synaptic membranes.

Enhanced expression of AMPA receptor protein at perforated axospinous synapses.

We performed a quantitative electron microscopic analysis of the middle third of the molecular layer in the dentate gyrus of rat, using material processed with postembedding gold labeling for the glutamate receptor subunits GluR1, GluR2/3, or NMDAR1. Perforated axospinous synapses were at least twice as likely as non-perforated ones to express detectable levels of AMPA receptor subunits, whereas no significant differences in NMDA receptor expression were observed. These data imply that perforated synapses may be especially potent, and are consistent with the hypothesis that insertion of AMPA receptor protein into the postsynaptic membrane of previously silent synapses contributes to long-term potentiation.

AMPA Receptor Development in Rat Telencephalon: [3H]AMPA Binding and Western Blot Studies

Telencephalic membranes from rats of different embryonic (E16, E19) and postnatal (P2, P7, P14, adult) ages were assessed for alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H] AMPA) binding and for immunoreactivity levels of AMPA receptor subunits (GluR1, GluR2/3, and GluR4). In addition, the synaptic markers synaptophysin and NCAM140 (a neural cell adhesion molecule isoform) were examined by immunoblot. The density of [3H]AMPA binding sites increased steadily with advancing age. This increase was due mainly to the development of the large low-affinity component (KD = 400 nM) that dominates the [3H]AMPA binding profile of adult rat brain membranes. As resolved by two-site regression analysis, the high-affinity component (KD = 15 nM) of the [3H]AMPA binding increased by approximately twofold from E16 to adult, whereas the low-affinity component increased by 25-fold. Staining for GluR1 and GluR2/3 increased steadily with increasing age at all time points examined; synaptophysin and NCAM140 exhibited similar ontogenic immunostaining profiles. GluR4 immunoreactivity was first evident at P14 and increased by adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)