N-methyl-D-aspartate Receptor-mediated Responses Research Articles

Specific factors in blood from young but not old mice directly promote synapse formation and NMDA-receptor recruitment

Aging drives a progressive decline in cognition and decreases synapse numbers and synaptic function in the brain, thereby increasing the risk for neurodegenerative disease. Pioneering studies showed that introduction of blood from young mice into aged mice reversed age-associated cognitive impairments and increased synaptic connectivity in brain, suggesting that young blood contains specific factors that remediate age-associated decreases in brain function. However, whether such factors in blood from young animals act directly on neurons to enhance synaptic connectivity, or whether they act by an indirect mechanism remains unknown. Moreover, which factors in young blood mediate cognitive improvements in old mice is incompletely understood. Here, we show that serum extracted from the blood of young but not old mice, when applied to neurons transdifferentiated from human embryonic stem cells, directly increased dendritic arborization, augmented synapse numbers, doubled dendritic spine-like structures, and elevated synaptic N-methyl-d-aspartate (NMDA) receptors, thereby increasing synaptic connectivity. Mass spectrometry revealed that thrombospondin-4 (THBS4) and SPARC-like protein 1 (SPARCL1) were enriched in serum from young mice. Strikingly, recombinant THBS4 and SPARCL1 both increased dendritic arborization and doubled synapse numbers in cultured neurons. In addition, SPARCL1 but not THBS4 tripled NMDA receptor-mediated synaptic responses. Thus, at least two proteins enriched in young blood, THBS4 and SPARCL1, directly act on neurons as synaptogenic factors. These proteins may represent rejuvenation factors that enhance synaptic connectivity by increasing dendritic arborization, synapse formation, and synaptic transmission.

Whole-Cell Patch-Clamp Electrophysiology to Study Ionotropic Glutamatergic Receptors and Their Roles in Addiction.

Development of the whole-cell patch-clamp electrophysiology technique has allowed for enhanced visualization and experimentation of ionic currents in neurons of mammalian tissue with high spatial and temporal resolution. Electrophysiology has become an exceptional tool for identifying single cellular mechanisms underlying behavior. Specifically, the role of glutamatergic signaling through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors underlying behavior has been extensively studied. Here we will discuss commonly used protocols and techniques for performing whole-cell patch-clamp recordings and exploring AMPA and NMDA receptor-mediated glutamatergic responses and alterations in the context of substance abuse.

Nonsteroidal anti-inflammatory drug, indomethacin improves spatial memory and NMDA receptor function in aged animals.

A redox-mediated decrease in N-methyl-D-aspartate (NMDA) receptor function contributes to psychiatric diseases and impaired cognition during aging. Inflammation provides a potential source of reactive oxygen species for inducing NMDA receptor hypofunction. The present study tested the hypothesis that the nonsteroidal anti-inflammatory drug indomethacin, which improves spatial episodic memory in aging rats, would enhance NMDA receptor function through a shift in the redox state. Male F344 young and aged rats were prescreened using a 1-day version of the water maze task. Animals were then treated with the indomethacin or vehicle, delivered in a frozen milk treat (orally, twice per day, 18days), and retested on the water maze. Indomethacin treatment enhanced water maze performance. Hippocampal slices were prepared for examination of CA3-CA1 synaptic responses, long-term potentiation, and NMDA receptor-mediated synaptic responses. No effect of treatment was observed for the total synaptic response. Long-term potentiation magnitude and NMDA receptor input-output curves were enhanced for aged indomethacin-treated animals. To examine redox regulation of NMDA receptors, a second group of aged animals was treated with indomethacin or vehicle, and the effect of the reducing agent, dithiothreitol ([DTT], 0.5mM) on NMDA receptor-mediated synaptic responses was evaluated. As expected, DTT increased the NMDA receptor response and the effect of DTT was reduced by indomethacin treatment. The results indicate that indomethacin acted to diminish the age-related and redox-mediated NMDA receptor hypofunction and suggest that inflammation contributes to cognitive impairment through an increase in redox stress.

The effect of ketamine on NMDA receptor-mediated LTP depends on ketamine effects on non-NMDA-mediated synaptic transmission in CA1 area of rat hippocampal slices

It has been reported that ketamine as an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist has also non-NMDA receptor antagonist properties. We recently found that ketamine (20 ?M) affected differently induction of NMDA receptor-mediated long-term potentiation (LTP) when administered 30 min prior to tetanic Primed-Bursts (PBs) stimulation. On the other hand, ketamine also influenced non-NMDA-mediated synaptic transmission in different manner. In the present study, in order to find the cause of different effects of ketamine on NMDA-mediated responses, we examined the probable relationship between the effects of ketamine on non-NMDA-mediated baseline responses and NMDA-mediated responses. Population spikes amplitude (PSA) was measured before and after tetanic stimulation in ketamine (1-100 µM) treated slices. We found that ketamine failed to inhibit NMDA receptor-mediated LTP, when baseline PSA enhanced in presence of ketamine. On the other hand, when ketamine not changed or decreased beseline PSA, caused LTP inhibition or even LTD induction. These findings indicated that ketamine effects on NMDA receptor-mediated responses depends on, ketamine effects on non-NMDA receptor-mediated synaptic transmission.

Phosphorylation-State-Dependent Regulation of NMDA Receptor Short-Term Plasticity Modifies Hippocampal Dendritic Ca2+ Transients

N-methyl-D-aspartate (NMDA) receptor-mediated currents are enhanced by phosphorylation. We have investigated effects of phosphorylation-dependent short-term plasticity of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) on the induction of long-term depression (LTD). We confirmed in whole cell clamped CA1 pyramidal neurons that LTD is induced by pairing stimulus protocols. However, after serine-threonine phosphorylation was modified by postsynaptic introduction of a protein phosphatase-1 (PP1) inhibitor, the same pairing protocol evoked long-term potentiation (LTP). We determined effects of modification of phosphatase activity on evoked NMDA EPSCs during LTD induction protocols. During LTD induction, using a protocol pairing depolarization to -40 mV and 0.5 Hz stimulation, NMDA receptor-mediated EPSCs undergo a short-term enhancement at the start of the protocol. In neurons in which PP1 activity was inhibited, this short-term enhancement was markedly amplified. We then investigated the effect of this enhancement on Ca(2+) entry during the start of the LTD induction protocol. Enhancement of NMDA receptor-mediated responses was accompanied by an amplification of induction protocol-evoked Ca(2+) transients. Furthermore, this amplification required synaptic activation during the protocol, consistent with an enhancement of Ca(2+) entry mediated by NMDA receptor activation. The sign of NMDA receptor-mediated long-term plasticity, whether potentiation or depression depends on the amplitude of the synaptic Ca(2+) transient during induction. We conclude that short-term phosphorylation-dependent plasticity of the NMDA receptor-mediated EPSCs contributes significantly to the effect of phosphatase inhibition on the subsequent induction of LTD or LTP.

Adjunctive α2-adrenoceptor blockade enhances the antipsychotic-like effect of risperidone and facilitates cortical dopaminergic and glutamatergic, NMDA receptor-mediated transmission

Compared to both first- and second-generation antipsychotic drugs (APDs), clozapine shows superior efficacy in treatment-resistant schizophrenia. In contrast to most APDs clozapine possesses high affinity for alpha2-adrenoceptors, and clinical and preclinical studies provide evidence that the alpha2-adrenoceptor antagonist idazoxan enhances the antipsychotic efficacy of typical D2 receptor antagonists as well as olanzapine. Risperidone has lower affinity for alpha2-adrenoceptors than clozapine but higher than most other APDs. Here we examined, in rats, the effects of adding idazoxan to risperidone on antipsychotic effect using the conditioned avoidance response (CAR) test, extrapyramidal side-effect (EPS) liability using the catalepsy test, brain dopamine efflux using in-vivo microdialysis in freely moving animals, cortical N-methyl-D-aspartate (NMDA) receptor-mediated transmission using intracellular electrophysiological recording in vitro, and ex-vivo autoradiography to assess the in-vivo alpha2A- and alpha2C-adrenoceptor occupancies by risperidone. The dose of risperidone needed for antipsychotic effect in the CAR test was approximately 0.4 mg/kg, which produced 11% and 17% in-vivo receptor occupancy at alpha2A- and alpha2C-adrenoceptors, respectively. Addition of idazoxan (1.5 mg/kg) to a low dose of risperidone (0.25 mg/kg) enhanced the suppression of CAR, but did not enhance catalepsy. Both cortical dopamine release and NMDA receptor-mediated responses were enhanced. These data propose that the therapeutic effect of risperidone in schizophrenia can be enhanced and its EPS liability reduced by adjunctive treatment with an alpha2-adrenoceptor antagonist, and generally support the notion that the potent alpha2-adrenoceptor antagonistic action of clozapine may be highly important for its unique efficacy in schizophrenia.

Potentiation of spinal NMDA-mediated nociception by cocaine- and amphetamine-regulated transcript peptide via PKA and PKC signaling pathways in rats

Our previous study showed that cocaine- and amphetamine-regulated transcript peptide fragment 55–102 (CARTp) specifically potentiated spinal N-methyl- d-aspartate (NMDA)-mediated nociceptive transmission in vivo and in vitro. The cellular mechanisms underlying CARTp potentiation of NMDA receptor function remains unclear. The present study was carried out to test the hypothesis that CARTp changes the phosphorylated state of NMDA receptors by activating intracellular signals and subsequently increasing the function of NMDA receptors. We found that the potentiating effect of CARTp on spinal NMDA-induced hyperalgesia in rats was reduced by intrathecal pretreatment with KT5720 (a selective PKA inhibitor) or GF109206X (a selective PKC inhibitor), but was increased by pretreatment with calyculin A (a protein phosphatase inhibitor). In the in vitro electrophysiological study, CARTp potentiation of NMDA-induced depolarizations was blocked by superfusion of PKA or PKC inhibitor applied 10 min before the application of CARTp. The levels of phosphoserine 897 on the NR1 subunit (pNR1-ser897) and phosphoserine 896 on the NR1 subunit (pNR1-ser896) in the dorsal horn of spinal lumbar segments significantly increased following CARTp superfusion in vitro or intrathecal injection in vivo. The increases in pNR1-ser897 and pNR1-ser896 in the in vivo and in vitro studies were inhibited by pretreatment with KT5720 and GF109206X, respectively. The results provide the first evidence that CARTp increases the phosphorylation of NMDA receptor NR1 subunit via activation of PKA and PKC signals, which may play a crucial role in CARTp regulation of spinal NMDA receptor-mediated nociceptive responses.

Glycine Site of NMDA Receptor Serves as a Spatiotemporal Detector of Synaptic Activity Patterns

Calcium influx associated with the opening of N-methyl-D-aspartate (NMDA) receptor channels is the major signal triggering synaptic and developmental plasticity. Controlling the NMDA receptor function is therefore critical for many functions of the brain. We explored the mechanisms of synaptic activation of the NMDAR glycine site by endogenous coagonist using whole cell voltage-clamp recordings from hippocampal neurons in mixed cultures, containing both neurons and glial cells, and, under more physiological conditions, in hippocampal slices. Here we show that the glycine site of the NMDA receptor at hippocampal synapses, both in culture and acute brain slices, is not saturated by the ambient coagonist concentration and is modulated through activity-dependent coagonist release. Augmentation of the NMDA receptor-mediated synaptic responses by local glutamate-induced coagonist release is spatially restricted and determined by spatiotemporal summation of synaptic events at neighboring synaptic inputs on a single dendritic branch. Therefore different spatiotemporal patterns of presynaptic activity could be translated into different levels of the NMDAR activation in specific afferent projections. These results suggest that the NMDA receptor glycine site may serve as a detector of the spatiotemporal characteristics of presynaptic activity patterns.

Control of GnRH expression in the olfactory lobe of Octopus vulgaris

In the cephalopod mollusk Octopus vulgaris, the gonadotropic hormone released by the optic gland controls sexual maturity. Several lobes of the central nervous system control the activity of this gland. In one of these lobes, the olfactory lobe, a gonadotropin releasing hormone (GnRH) neuronal system has been described. We assume that several inputs converge on the olfactory lobes in order to activate GnRH neurons and that a glutamatergic system mediates the integration of stimuli on these neuropeptidergic neurons. The presence of N-methyl- d-aspartate (NMDA) receptor immunoreactivity in the neuropil of olfactory lobes and in the fibers of the optic gland nerve, along with the GnRH nerve endings strongly supports this hypothesis. A distinctive role in the control of GnRH secretion has also been attributed, in vertebrates, to nitric oxide (NO). The lobes and nerves involved in the nervous control of reproduction in Octopus contain nitric oxide synthase (NOS). Using a set of experiments aimed at manipulate a putative l-glutamate/NMDA/NO signal transduction pathway, we have demonstrated, by quantitative real-time PCR, that NMDA enhances the expression of GnRH mRNA in a dose–response manner. The reverting effect of a selective antagonist of NMDA receptors (NMDARs), 2-amino-5-phosphopentanoic acid ( D-APV), confirms that such an enhancing action is a NMDA receptor-mediated response. Nitric oxide and calcium also play a positive role on GnRH mRNA expression. The results suggest that in Octopus l-glutamate could be a key molecule in the nervous control of sexual maturation.

Interleukin-2 inhibits NMDA receptor-mediated currents directly and may differentially affect subtypes

Using whole-cell patch-clamp recordings, this study investigated the effects of interleukin-2 (IL-2) on N-methyl- d-aspartate (NMDA) receptor-mediated currents ( I NMDA) in rat cultured hippocampal neurons and human embryonic kidney (HEK) 293 cells expressing recombinant NMDA receptors. We found that IL-2 (0.01–1 ng/ml) immediately and significantly decreased peak I NMDA in cultured neurons. Interestingly, the peak I NMDA induced in HEK 293 cells was also inhibited by IL-2. We also found that IL-2 differentially decreased the peak amplitudes of NR2A- and NR2B-containing NMDA receptor-mediated currents ( I NR2A and I NR2B) by 54 ± 5% and 30 ± 4%, respectively. These results provide new evidence that IL-2 induces rapid inhibition of peak currents of NMDA receptor-mediated responses with possible NR1/NR2A and NR1/NR2B subtype-differentiation, and suggest that the inhibition is mediated by direct interaction between IL-2 and NMDA receptors.

Subpopulation of Dorsal Horn Neurons Displays Enhanced N -methyl-d-aspartate Receptor Function after Chronic Morphine Exposure

Morphine tolerance may be attributed to enhancement of glutamatergic neurotransmission, in particular to increased function of the N-methyl-D-aspartate (NMDA) receptor. The cellular mechanisms responsible for these changes remain poorly defined. The authors identified and characterized a specific subpopulation of dorsal horn neurons, displaying NMDA receptor plasticity in response to chronic morphine administration. The authors undertook current clamped and voltage clamped recordings of NMDA receptor-mediated responses from cultured rat dorsal horn neurons that were untreated or treated for 7 days with 1 or 100 microm morphine. Smaller (capacitance < or = 22 pF), tonic firing neurons showed a significantly enhanced NMDA receptor-mediated peak current after prolonged morphine treatment, whereas larger and phasic firing neurons showed no enhancement. With high-concentration but not low-concentration morphine treatment, Mg2+ blockade of NMDA receptors at resting membrane potentials was reduced. Furthermore, the chronic opioid-induced increase in NMDA current was attenuated by pretreatment with either a mu-opioid receptor inhibitor (naloxone) or an NMDA receptor inhibitor (2-amino-5-phosphonovalerate) (low-concentration > high-concentration morphine). In an electrophysiologically defined subpopulation of dorsal horn neurons, enhanced NMDA receptor function after chronic morphine exposure was shown to be mechanistically dependent on morphine concentration and sensitive to both NMDA and mu-opioid receptor antagonism. Therefore, these changes observed in this population of sensory spinal neurons can be used to study the development and prevention of opioid tolerance described in multiple laboratory and clinical reports.

Neuron-derived D-Serine Release Provides a Novel Means to Activate N-Methyl-D-aspartate Receptors

D-serine is a coagonist of N-methyl-D-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of D-serine is carried out by serine racemase, which converts L- to D-serine. D-serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of D-serine. We now report significant amounts of serine racemase and D-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and D-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and D-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived D-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous D-serine by addition of the recombinant enzyme D-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal D-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked D-serine release. Release of D-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. D-serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of D-serine. Glutamate-induced neuronal D-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.

Dynamin-dependent NMDAR endocytosis during LTD and its dependence on synaptic state

BackgroundThe N-methyl-D-aspartate (NMDA)-type glutamate receptor expressed at excitatory glutamatergic synapses is required for learning and memory and is critical for normal brain function. At a cellular level, this receptor plays a pivotal role in triggering and controlling synaptic plasticity. While it has been long recognized that this receptor plays a regulatory role, it was considered by many to be itself immune to synaptic activity-induced plasticity. More recently, we and others have shown that NMDA receptor-mediated synaptic responses can be subject to activity-dependent depression.ResultsHere we show that depression of synaptic transmission mediated by NMDA receptors displays a state-dependence in its plasticity; NMDA receptors are resistant to activity-induced changes at silent and recently-silent synapses. Once synapses transition to the active state however, NMDA receptors become fully 'plastic'. This state-dependence is identical to that shown by the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor. Furthermore, the down-regulation of NMDAR-mediated responses during synaptic depression is prevented by disruption of dynamin-dependent endocytosis.ConclusionNMDA receptor-mediated synaptic responses are plastic in a state-dependent manner. Depending on the plasticity state in which a synapse currently resides, NMDA receptors will either be available or unavailable for down-regulation. The mechanism underlying the down-regulation of NMDA receptor-mediated synaptic responses is endocytosis of the NMDA receptor. Other potential mechanisms, such as receptor diffusion along the plane of the membrane, or changes in the activity of the channel are not supported. The mechanisms of AMPA receptor and NMDA receptor endocytosis appear to be tightly coupled, as both are either available or unavailable for endocytosis in the same synaptic states. Endocytosis of NMDA receptors would serve as a potent mechanism for metaplasticity. Such state-dependent regulation of NMDAR endocytosis will provide fundamental control over downstream NMDA receptor-dependent plasticity of neuronal circuitry.

An Opiate Cocktail that Reduces Morphine Tolerance and Dependence

Morphine is an exceptionally effective analgesic whose utility is compromised by the development of tolerance and dependence to the drug. Morphine analgesia and dependence are mediated by its activity at the mu opioid peptide (MOP) receptor [1]. The MOP receptor is activated not only by morphine, but also by other opiate drugs such as methadone and endogenous opioids such as endorphins. Morphine, however, is a unique opioid agonist ligand because it fails to induce endocytic trafficking of the MOP receptor [2], whereas the endogenous ligands and methadone do facilitate endocytosis [3]. Using the unique pharmacology of the MOP receptor and its proposed existence as an oligomeric structure [4], we designed a pharmacological cocktail that facilitates endocytosis of the MOP receptor in response to morphine. This cocktail consists of morphine and a small dose of methadone. Importantly, this cocktail, while retaining full analgesic potency, does not promote morphine dependence. We further demonstrate that dependence is reduced, at least in part, because endocytosis of the MOP receptor in response to morphine prevents the upregulation of N-methyl-D-aspartate (NMDA) receptors.

Estradiol increases delayed, N-methyl- d-aspartate receptor-mediated excitation in the hippocampal CA1 region

Hippocampal functions, e.g. synaptic plasticity and hippocampal-dependent behavior, are influenced by the circulating levels of ovarian steroids in adult, female rats. The mechanisms underlying this estradiol-dependent modulation, however, are poorly understood. One possibility is that estradiol alters N-methyl- d-aspartate (NMDA)-receptor functioning in the hippocampus. Here, using the in vitro hippocampal slice preparation, we evaluate estradiol-dependent changes in the NMDA receptor- and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated components of excitatory postsynaptic potentials (EPSPs) evoked in CA1 by Schaffer collateral test stimulation. Using established experimental conditions [J Neurosci 17 (1997) 1848], we replicate the observation that estradiol pretreatment of ovariectomized rats increases a pharmacologically isolated NMDA receptor-mediated EPSP evoked by Schaffer collateral stimulation. However, using different conditions that optimize study of this evoked response, the estradiol-dependent increase in the monosynaptic NMDA receptor-mediated EPSP is eliminated. Low-intensity test stimulation of the Schaffer collaterals in this optimized medium reveals a novel, late NMDA receptor-mediated EPSP in CA1 from estradiol-pretreated rats. The mechanism(s) underlying this estradiol-dependent increase in a late, NMDA receptor-mediated EPSP is not known, but enhanced CA1–CA1 excitatory circuitry and glutamate spillover could contribute to this response. We conclude that estradiol pretreatment enhances NMDA receptor function in the female hippocampus by increasing not the monosynaptic, but rather a late NMDA receptor-mediated response. Variations in the magnitude of this late response may well contribute to ovarian steroid-dependent modulation of hippocampal synaptic plasticity.

Striatal neurons but not nigral dopaminergic neurons in neonatal primary cell culture express endogenous functional N-methyl- d-aspartate receptors

Developmental expression of N-methyl- d-aspartate (NMDA) receptor subunits were determined and compared in striatal and nigral neurons in neonatal primary cell cultures. In striatal neurons, NR1, NR2A and NR2B mRNAs and immunoreactivity, and NR2D mRNA were found and the maximal levels of NR1 mRNA and immunoreactivity expression were found at 6 day-in-vitro (DIV). NMDA receptors found at this stage in striatal neurons are likely to contain NR1 plus NR2A, NR2B and NR2D subunits. In nigral neurons, NR1 and NR2B mRNAs and immunoreactivity, and NR2D mRNA were found and the maximal level of NR1 immunoreactivity expression was found at 10 DIV. Unlike striatal neurons, NMDA receptors found in nigral neurons are likely to contain NR1 plus NR2B and NR2D subunits only. NMDA-induced toxicity assays showed that striatal neurons were most susceptible to cell death at around 10 DIV but nigral neurons were not susceptible to NMDA-induced cell death at all stages. In addition, patch clamp analysis revealed that functional NMDA receptors could only be found in striatal neurons but not in nigral dopaminergic neurons in vitro. The present results indicate that striatal and nigral neurons are programmed to express distinct NMDA receptor subunits during their endogenous development in cell cultures. Despite dopaminergic neurons in culture display NMDA receptor subunits, functional NMDA receptors are not assembled. The present findings have demonstrated that dopaminergic neurons in vitro may behave very differently to their counterparts in vivo in terms of NMDA receptor-mediated responses. Our results also have implications in transplantations using dopaminergic neurons in vitro in treatments of Parkinson's disease.

Potentiation of NMDA receptor-mediated synaptic responses by microglia

To study the influence of microglia on glutamatergic synaptic transmission in the acute phase of neuronal injury, we first examined the effects of primary cultured microglia transferred onto the organotypic cortical slice cultures. In these microglia-transferred cortical slice cultures, stimulation of the subcortical white matter induced fast excitatory postsynaptic potentials followed by N-methyl- d-aspartate (NMDA) receptor-mediated plateau-like potentials that were never observed in control slice cultures. A similar potentiation of NMDA receptor-mediated postsynaptic responses was also observed by an application of a microglial-conditioned medium (MCM, 10% v/v) in acute cortical slices. These effects of MCM disappeared after boiling or incubation with proteinase K. After fractionation of MCM by anion-exchange chromatography, the enhancing activity of each fraction was quantitated electrophysiologically. When each fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the fraction 24 which showed the most potent enhancing activity on NMDA receptor-mediated responses contained a relatively strong protein band with a molecular mass of approximately 70 kDa. MCM also enhanced both glutamate- and NMDA-induced inward currents recorded from acutely isolated cortical neurons. It was also noted that glutamate and NMDA induced transient large inward currents during an application of MCM, which were never observed in the control condition. These observations strongly suggest that NMDA receptor-mediated responses can be potentiated by both heat- and protease-labile (presumably 70-kDa proteins) molecules released from microglia.

Genetics of Childhood Disorders: LIV. Learning and Memory, Part 7: Maintenance of Long-Term Potentiation

Before moving on to the processes that contribute to the persistence of long-term potentiation (LTP), we need to address an apparently straightforward issue: which side of the synapse expresses LTP? We have seen that the main events of LTP induction occur in postsynaptic signaling pathways. Does this mean that the locus of LTP expression is also postsynaptic? At the CA3–CA1 synapse, the preponderance of electrophysiological evidence favors the postsynaptic side of the synapse, but this has been a contentious area of research and some data support the hypothesis of increased transmitter release in LTP. The problem of the site of LTP expression has been approached using several different strategies. One method is to determine the rate of glutamate release. The efflux of glutamate from a hippocampal slice can be directly measured, and early work showed an elevation during LTP. However, synaptic release represents only a fraction of total glutamate efflux, and metabolic changes could be largely responsible for the observed increases. A more sophisticated experimental approach exploits the active uptake of synaptic glutamate by glial cells, resulting in graded glial transporter currents that can be recorded. These currents are not increased during LTP, suggesting that the locus of the potentiation is postsynaptic. The hypothesis that LTP expression is restricted to the postsynaptic cell has been challenged by experiments in which vesicle recycling at the CA3–CA1 synapse was monitored using the presynaptic loading of a fluorescent dye. An increase in vesicle recycling would indicate that glutamate-containing vesicles were being turned over at a higher rate, presumably due to increased synaptic release. Such an effect occurred following LTP-inducing stimulation, supporting a presynaptic contribution to the expression of LTP. A second approach to identifying the locus of LTP exploits the voltage-dependent block of N-methyl-D-aspartate (NMDA) receptors described in the previous column, a property that is not shared by (AMPA)-type receptors. This difference can be used to separate the synaptic response into AMPA and NMDA components because the contribution of the NMDA-mediated current will depend upon the postsynaptic membrane voltage. If an increase in glutamate release is responsible for LTP, then the AMPA and NMDA receptor-mediated responses should increase equally. But most studies show that only the AMPA component is potentiated during LTP, consistent with a postsynaptic change that is restricted to AMPA receptors.

Modulation of nociceptive transmission by pituitary adenylate cyclase activating polypeptide in the spinal cord of the mouse.

Superficial layers of the dorsal horn receive a dense plexus of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide (PACAP). In vivo experiments were conducted in the mice to evaluate the effects of PACAP-38, herein referred to as PACAP, PACAP receptor antagonist PACAP(6-38) and PACAP-antiserum on nociceptive behaviors induced by radiant heat, intrathecally administered N-methyl-D-aspartate (NMDA) or intraplantarly administered formalin. PACAP (0.05-0.5 microg) dose-dependently decreased the paw-withdrawal latencies induced by thermal stimulation and enhanced the aversive licking and biting behaviors induced by intrathecally injected NMDA. Pretreatment with the PACAP receptor antagonist PACAP(6-38) (0.5-2 microg) or PACAP-antiserum (1:500-2,000 dilution) dose-dependently attenuated the second phase, but not the first phase, of nociceptive responses to formalin. Next, the effects of PACAP on NMDA- and kainate-induced currents evoked in single dorsal horn neurons were studied. Whole-cell patch recordings were made from superficial dorsal horn neurons of spinal cord slices from 14- to 20-day-old mice. PACAP at the concentrations of 100 and 200 nM, which caused no significant change of holding currents, increased NMDA-but not kainate-induced currents in superficial dorsal horn neurons. Our results suggest that exogenously applied PACAP sensitizes the dorsal horn neurons to formalin stimulation, and facilitates NMDA receptor-mediated nociceptive response. As a corollary, PACAP, which may be released from primary afferent fibers potentiates nociceptive transmission to the dorsal horn by interacting primarily with NMDA receptors.

Gain control of N-methyl-D-aspartate receptor activity by receptor-like protein tyrosine phosphatase alpha.

Src kinase regulation of N-methyl-D-aspartate (NMDA) subtype glutamate receptors in the central nervous system (CNS) has been found to play an important role in processes related to learning and memory, ethanol sensitivity and epilepsy. However, little is known regarding the mechanisms underlying the regulation of Src family kinase activity in the control of NMDA receptors. Here we report that the distal phosphatase domain (D2) of protein tyrosine phosphatase alpha (PTPalpha) binds to the PDZ2 domain of post-synaptic density 95 (PSD95). Thus, Src kinase, its activator (PTPalpha) and substrate (NMDA receptors) are linked by the same scaffold protein, PSD95. Removal of PTPalpha does not affect the association of Src with NMDA receptors, but turns off the constitutive regulation of NMDA receptors by the kinase. Further more, we found that application of the PTPalpha catalytic domains (D1 + D2) into neurones enhances NMDA receptor-mediated synaptic responses. Conversely, the blockade of endogenous PTPalpha inhibits NMDA receptor activity and the induction of long-term potentiation in hippocampal neurones. Thus, PTPalpha is a novel up-regulator of synaptic strength in the CNS.