NMDA Currents Research Articles

Enhanced Glutamatergic Inputs to Hypothalamic Presympathetic Neurons in Spontaneously Hypertensive Rats

Glutamatergic inputs in the paraventricular nucleus (PVN) maintain the increased sympathetic activity in hypertension. In this study, we determined the synaptic mechanisms of increased glutamatergic inputs to presympathetic PVN neurons in spontaneously hypertensive rats (SHR). The spinally projecting PVN neurons were retrogradely labeled by injection of Fluospheres into the intermediolateral cell column of the spinal cord. The basal frequency of spontaneous and miniature glutamatergic excitatory postsynaptic currents (EPSCs) of labeled PVN neurons are significantly greater in SHR than that in WKY rats. The amplitude of EPSCs was not significantly different between WKY and SHR. Furthermore, the postsynaptic NMDA currents elicited by either electrical stimulation or puff application of NMDA are significantly higher in SHRs than in WKY rats. However, AMPA currents evoked by the electrical stimulation and puff application in were not different between WKY and SHRs. In addition, ionotropic glutamate receptors selective antagonists kynurenic acid, AP5, and CNQX significantly decreased the basal firing activity of labeled PVN neurons in SHR, but failed to alter the firing activity of the PVN neurons in WKY rats. Thus, the glutamatergic synaptic inputs to presympathetic PVN neurons are augmented and contribute to the elevated firing activity of the PVN neruons in SHR.

Presenilin-1 mutation impairs cholinergic modulation of synaptic plasticity and suppresses NMDA currents in hippocampus slices

Presenilin-1 (PS1) mutations cause many cases of early-onset inherited Alzheimer's disease, in part, by increasing the production of neurotoxic forms of amyloid β-peptide (Aβ). However, Aβ-independent effects of mutant PS1 on neuronal Ca 2+ homeostasis and sensitivity to excitatory neurotransmitters have been reported. Here we show that cholinergic modulation of hippocampal synaptic plasticity is impaired in PS1 mutant knockin (PS1KI) mice. Whereas activation of muscarinic receptors enhances LTP at CA1 synapses of normal mice, it impairs LTP in PS1KI mice. Similarly, mutant PS1 impairs the ability of the cholinesterase inhibitor phenserine to enhance LTP. The NMDA current is decreased in CA1 neurons of PS1KI mice and is restored by intracellular Ca 2+chelation. Similar alterations in acetylcholine and NMDA receptor-mediated components of synaptic plasticity are evident in 3xTgAD mice with PS1, amyloid precursor protein and tau mutations, suggesting that the adverse effects of mutant PS1 on synaptic plasticity can occur in the absence or presence of amyloid and tau pathologies.

Key role of tissue plasminogen activator in neurovascular coupling

The increase in blood flow evoked by synaptic activity is essential for normal brain function and underlies functional brain imaging signals. Nitric oxide, a vasodilator released by NMDA receptor activation, is critical for the flow increase, but the factors linking NMDA receptor activity to nitric oxide-dependent hyperemia are poorly understood. Here, we show that tissue plasminogen activator (tPA), a serine protease implicated in NMDA receptor signaling, is required for the flow increase evoked by somatosensory stimulation. tPA acts by facilitating neuronal nitric oxide release, but this effect does not involve enhancement of NMDA currents or the associated intracellular Ca(2+) rise. Rather, the evidence suggests that tPA controls NMDA-dependent nitric oxide synthesis by influencing the phosphorylation state of neuronal nitric oxide synthase. These findings unveil a previously unrecognized role of tPA in vital homeostatic mechanisms coupling NMDA receptor signaling with nitric oxide synthesis and local cerebral perfusion.

Neuroactive steroid pregnenolone sulphate inhibits long-term potentiation via activation of α2-adrenoreceptors at excitatory synapses in rat medial prefrontal cortex

Pregnenolone sulphate (PREGS) is one of the most important neuroactive steroids. Previous study showed that PREGS enhanced long-term potentiation (LTP) via activation of post-synaptic NMDA receptors at excitatory synapses in the hippocampus. The present paper studied the effect of PREGS on LTP at excitatory synapses in the pyramidal cells of layers V-VI of the medial prefrontal cortex (mPFC) using whole-cell patch-clamp in slices and made a comparison with that in the hippocampus. We also studied the mechanism of the effect of PREGS in the mPFC. We found that PREGS inhibited induction of LTP in the mPFC and had no influence on NMDA currents, which was different from its effect in the hippocampus. Moreover, the effect of PREGS on LTP in the mPFC was cancelled by alpha2-adrenoreceptor antagonist, alpha2A-adrenoreceptor antagonist, Gi protein inhibitor, adenylate cyclase inhibitor and protein kinase A inhibitor. These results suggest that PREGS inhibits LTP via activation of the alpha2-adrenoreceptor-Gi protein-adenylate cyclase-protein kinase A signalling pathway in the mPFC.

Adenosine A2A Receptors Are Essential for Long-Term Potentiation of NMDA-EPSCs at Hippocampal Mossy Fiber Synapses

The physiological conditions under which adenosine A2A receptors modulate synaptic transmission are presently unclear. We show that A2A receptors are localized postsynaptically at synapses between mossy fibers and CA3 pyramidal cells and are essential for a form of long-term potentiation (LTP) of NMDA-EPSCs induced by short bursts of mossy fiber stimulation. This LTP spares AMPA-EPSCs and is likely induced and expressed postsynaptically. It depends on a postsynaptic Ca2+ rise, on G protein activation, and on Src kinase. In addition to A2A receptors, LTP of NMDA-EPSCs requires the activation of NMDA and mGluR5 receptors as potential sources of Ca2+ increase. LTP of NMDA-EPSCs displays a lower threshold for induction as compared with the conventional presynaptic mossy fiber LTP; however, the two forms of LTP can combine with stronger induction protocols. Thus, postsynaptic A2A receptors may potentially affect information processing in CA3 neuronal networks and memory performance.

Dopamine and Serotonin Interactions in the Prefrontal Cortex: Insights on Antipsychotic Drugs and Their Mechanism of Action

Diminished activity within the prefrontal cortex (PFC) has been associated with many of the cognitive deficits that are observed in schizophrenia. It has been hypothesized that antipsychotic drugs (APDs) used to treat schizophrenia restore normal activity by antagonizing the dopamine (DA) D2 receptor, which is also known to modulate key ionic currents in the PFC. However, the hypothesis that an under-active cortical DA system is responsible for schizophrenic symptoms has been challenged by evidence that newer atypical APDs are weak antagonists at the D2 receptor but potent antagonists at the serotonin (5-HT) 2A receptor . This review examines how DA and 5-HT modulate cortical activity and how they may interact in ways that are relevant to schizophrenia. It is concluded that although D2 receptor antagonism remains a critical factor in restoring impaired cortical activity, effects on 5-HT receptors may act in a synergistic manner on NMDA and GABA currents to potentiate antipsychotic actions in the PFC.

NMDA receptor mobility: cultures versus acute brain slices or neonatal versus mature synapses?

The determining factors for NMDAR functions in synapse formation, synaptic plasticity, learning and memory include receptor assembly and trafficking, subunit-specific associations with other proteins as well as subcellular localization at synaptic or extrasynaptic sites. NR2B-rich NMDARs occupy both locations in immature principal neurons. During postnatal maturation, NR2A expression increases, NR2A-containing NMDARs enrich in synapses and can be activated by single synaptic stimulation. In contrast, NR2B-rich NMDARs are activated by glutamate spillover or glutamate released from astrocytes (Kohr, 2006). For a long time, the anchoring of NMDARs opposite to presynaptic glutamate release sites has been taken as evidence for a relatively stable organization of the postsy-naptic membrane. Within the postsynaptic density (PSD), signalling molecules, scaffolding and adaptor proteins have been identified as associating with NMDARs and forming large macromolecular signalling complexes. Nevertheless, synaptic NMDARs can be replaced by extrasynaptic ones in 6- to 8-day-old (DIV6–8) autapses or organotypic cultures (Barria & Malinow, 2002; Tovar & Westbrook, 2002). This mobility could involve lateral diffusion and hence be distinct from exocytic–endocytic vesicular NMDAR trafficking (Lau & Zukin, 2007). Lateral mobility of NMDARs between synaptic and extrasynaptic pools was demonstrated in hippocampal autapses by use of the NMDAR open channel blocker MK-801. Repeated synaptic stimulation in the presence of MK-801 progressively and irreversibly blocked NMDAR-mediated EPSCs (NMDA EPSCs). As a subsequent substantial recovery of NMDA EPSCs occurred within minutes, Tovar & Westbrook (2002) suggested that at least 65% of the synaptic NMDARs are mobile. More recently, surface mobility of NMDARs was visualized in hippocampal dissociated cultures (Groc et al. 2006). NR2B-containing NMDARs switched between extrasynaptic and synaptic localizations about 36 times per minute at DIV8 and DIV15. At DIV15, more NR2A-containing NMDARs were present in the synapses, showing a slower diffusion rate than NR2B-containing NMDARs. Together, these studies in culture generated the view that NMDARs switch as rapidly as AMPARs and challenged trafficking experiments in intact brain tissue. In this issue of The Journal of Physiology Harris and Petit investigated the mobility of NMDARs at CA3-to-CA1 synapses in acute hippocampal slices of 2- to 3-week-old rats (P14–22). Initially, Harris & Pettit determined the spatial distribution of NMDARs within CA1 neurons using glutamate uncaging techniques to activate synaptic and extrasynaptic NMDARs within a circumscribed dendritic region before applying synaptic stimulation in the presence of MK-801 to block the synaptic NMDARs. Subsequently, glutamate uncaging activated the unblocked extrasynaptic NMDAR pool, which was found to be about one third of the total NMDAR pool at both proximal and distal dendritic regions, comparable to results from cultured neurons after DIV7. In acute slices, the subunit composition of the synaptic and extrasynaptic NMDARs was uniform (unlike DIV5–7 cultures), as judged by the sensitivity of NMDA currents to ifenprodil (3 μm), which is > 100-fold more selective for NR1–NR2B than for NR1–NR2A. Somewhat surprising, NMDA currents evoked by uncaging glutamate over the soma did not show higher ifenprodil sensitivity than currents evoked in dendrites, although NMDA field EPSPs showed the expected developmental decrease in ifenprodil sensitivity between P5 and P39–92. Finally and remarkably, extrasynaptic NMDARs in acute slices appeared not to exchange with synaptic NMDARs, as no recovery of NMDA EPSCs was observed after MK-801 blockade and washout. This important issue was examined after partial or complete MK-801 blockade by short or prolonged MK-801 incubations (2 versus 14 min). Recovery from MK-801 was tested at 46 versus 23 min, respectively, following a 20 min stimulation-free period. Considering the slice preparation, the MK-801 washout following the complete blockade of NMDA EPSCs was rather short, although prolonged washout could have caused some MK-801 unblock, thus feigning NMDAR mobility. Also, the number of mobility experiments was low (n= 4 at each incubation time), calling for more experiments at hippocampal and other synapses. In spite of this caveat, the mobility of NMDARs in acute slices appears to be less than in cultured autaptic neurons. Tovar & Westbrook (2002) used young autapses (starting at DIV6), which are more apt for NMDAR mobility, given that (i) extrasynaptic NMDARs outnumber synaptic NMDARs by 3 : 1 at DIV5–7 and (ii) molecules involved in synaptogenesis and synaptic maturation can be assumed to be incompletely expressed at that age. This immature synaptic architecture also occurs in acute slices of young rats, allowing activity-dependent, rapid NMDAR switching at P2–9 but not at P16–21 (Bellone & Nicoll, 2007), consistent with stable NMDAR pools at P14–22 (Harris & Pettit, 2007). Thus, rapid switching of synaptic NMDARs could be restricted to neonatal synapses, which are suitable to study the still unresolved mechanism for NMDAR mobility. In addition, it will be important to elucidate whether lateral mobility of NMDARs occurs in mature synapses and which role it plays during plasticity and remodeling.

NMDA Receptor Contribution to the Climbing Fiber Response in the Adult Mouse Purkinje Cell

Among integrative neurons displaying long-term synaptic plasticity, adult Purkinje cells seemed to be an exception by lacking functional NMDA receptors (NMDA-Rs). Although numerous anatomical studies have shown both NR1 and NR2 NMDA-R subunits in adult Purkinje cells, patch-clamp studies failed to detect any NMDA currents. Using more recent pharmacological and immunodetection tools, we demonstrate here that Purkinje cells from adult mice respond to exogenous NMDA application and that postsynaptic NMDA-Rs carry part of the climbing fiber-mediated EPSC (CF-EPSC), with undetectable contribution from presynaptic or polysynaptic NMDA currents. We also detect NR2-A/B subunits in adult Purkinje cells by immunohistochemistry. The NMDA-mediated CF-EPSC is barely detectable before 3 weeks postnatal. From the end of the third week, the number of cells displaying the NMDA-mediated CF-EPSC rapidly increases. Soon, this EPSC becomes detectable in all the Purkinje cells but is still very small. Its amplitude continues to increase until 12 weeks after birth. In mature Purkinje cells, we show that the NMDA-Rs contribute to the depolarizing plateau of complex spikes and increase their number of spikelets. Together, these observations demonstrate that mature Purkinje cells express functional NMDA receptors that become detectable in CF-EPSCs at approximately 21 d after birth and control the complex spike waveform.

Modulation of NMDA receptors by prion proteins

Background: The precise physiological function of endogenous cellular prion protein (PrPC) remains unclear. It has been shown that PrP-null mice exhibit reduced LTP and greater susceptability to seizure mortality in several in vivo (e.g. kainic acid) models of epilepsy. In addition, PrP-null mice exhibit greater exctitotoxic cell death in response to kainic acid exposure. Methods: In our study we investigated the synaptic properties of WT and PrP-null mice. 
 Results: Recordings in the CA1 layer of adult hippocampal slices showed that PrP-null mice exhibit a reduced threshold to evoked responses and no difference in paired-pulse facilitation relative to WT animals. In addition, greater excitability was observed in PrP-null slices in response to zero-Mg2+ induced seizure-like events. Recordings from mature hippocampal cultures showed slightly altered AMPA and GABAA miniature synaptic currents. NMDA mEPSCs were observed to be increased in amplitude and significantly prolonged in decay time. NMDA-evokved currents also exhibited markedly prolonged deactivation kinetics. This effect on evoked NMDA currents was reproduced in WT neurons by PrP-RNAi transfection, and eliminated by PrPC transfection into PrP-null neurons. 
 Conclusions: These data suggest enhanced NMDA activity in PrP-null neurons. Consistent with this finding, in vitro and in vivo excitotoxicity assays demonstrated increased neuronal cell death in PrP-null cultures and animals upon transient exposure to NMDA. The prolonged deactivation kinetics were most consistent with functional activity/augmentation of NR2D NMDA receptor subunits, and PrP coimmunoprecipiated with NR2D NMDA receptor subunits. This enhanced NMDA receptor function was paralleleld by increased excitotoxicy in Prp-null mice. Our findings demonstrate a novel functional role for PrP as a modulator of synaptic NMDA currents and attributes a neuroprotective function to PrP.

Takusan: A Large Gene Family that Regulates Synaptic Activity

We have characterized a rodent-specific gene family designated alpha-takusan (meaning "many" in Japanese). We initially identified a member of the family whose expression is upregulated in mice lacking the NMDAR subunit NR3A. We then isolated cDNAs encoding 46 alpha-takusan variants from mouse brains. Most variants share an approximately 130 aa long sequence, which contains the previously identified domain of unknown function 622 (DUF622) and is predicted to form coiled-coil structures. Single-cell PCR analyses indicate that one neuron can express multiple alpha-takusan variants and particular variants may predominate in certain cell types. Forced expression in cultured hippocampal neurons of two variants, alpha1 or alpha2, which bind either directly or indirectly to PSD-95, leads to an increase in PSD-95 clustering, dendritic spine density, GluR1 surface expression, and AMPAR activity. Conversely, treating cultured neurons with RNAi targeting alpha-takusan variants resulted in the opposite phenotype. Hence, alpha-takusan represents a large gene family that regulates synaptic activity.

The symptoms of schizophrenia related to the stability of attractor networks

We propose a hypothesis to account for the symptoms of schizophrenia in a statistical dynamical systems framework. We propose that the symptoms of schizophrenia are related to alterations in the depth of the basins of attraction, and the statistical effects of spiking neurons, which together influence the stability of cortical attractor neural networks. The cognitive symptoms such as distractibility, working memory deficits and poor attention could be caused by instability of persistent attractor states in prefrontal cortical networks. The negative symptoms, which include a lack of affect and reduction of emotions, may be related to the concomitant decreased firing rates in cortical areas such as the orbitofrontal cortex and anterior cingulate cortex that occur when the depth of the basins of attraction are reduced. The positive symptoms including delusions, paranoia, and hallucinations could arise because the basins of attraction are shallow in temporal lobe semantic memory networks. This would lead thoughts to move too freely around the attractor energy landscape, loosely from thought to weakly associated thought. These hypotheses were investigated in a model of a cortical attractor network based on integrate-and-fire neurons and synaptic currents activated by AMPA, NMDA and GABA receptors [1]. It was found that a decrease in the NMDA receptor conductance leads to a decrease in the stability of working memory states, an increase in distractibility, and lower firing rates, and can potentially account for both the cognitive and negative symptoms. We observed a flat energy landscape associated with the positive symptoms of schizophrenia when, in addition to a reduction in the NMDA conductance, the GABA conductance was reduced. This caused the dynamical system not only to switch between the high firing rate attractor states, but also sometimes to jump from spontaneous activity into one of the attractor states. The findings are consistent with the neurophysiological effects of dopamine, which can influence NMDA and GABA currents. In this approach we thus start with a statistical dynamical systems framework, and use this to help understand how some of the different symptoms of schizophrenia might arise.

Presynaptic G-Protein-Coupled Receptors Regulate Synaptic Cleft Glutamate via Transient Vesicle Fusion

When synaptic vesicles fuse with the plasma membrane, they may completely collapse or fuse transiently. Transiently fusing vesicles remain structurally intact and therefore have been proposed to represent a form of rapid vesicle recycling. However, the impact of a transient synaptic vesicle fusion event on neurotransmitter release, and therefore on synaptic transmission, has yet to be determined. Recently, the molecular mechanism by which a serotonergic presynaptic G-protein-coupled receptor (GPCR) regulates synaptic vesicle fusion and inhibits synaptic transmission was identified. By making paired electrophysiological recordings in the presence and absence of low-affinity antagonists, we now demonstrate that activation of this presynaptic GPCR lowers the peak synaptic cleft glutamate concentration independently of the probability of vesicle fusion. Furthermore, this change in cleft glutamate concentration differentially inhibits synaptic NMDA and AMPA receptor-mediated currents. We conclude that a presynaptic GPCR regulates the profile of glutamate in the synaptic cleft through altering the mechanism of vesicle fusion leading to qualitative as well as quantitative changes in neural signaling.

Phosphatidylinositol-4,5-Bisphosphate Regulates NMDA Receptor Activity through α-Actinin

Phosphatidylinositol-4,5-bisphosphate (PIP2) has been shown to regulate many ion channels, transporters, and other signaling proteins, but it is not known whether it also regulates neurotransmitter-gated channels. The NMDA receptors (NMDARs) are gated by glutamate and serve as a critical control point in synaptic function. Here we demonstrate that PIP2 supports NMDAR activity. In Xenopus oocytes, overexpression of phospholipase Cgamma (PLCgamma) or preincubation with 10 microm wortmannin markedly reduced NMDA currents. Stimulation of the epidermal growth factor receptor (EGFR) promoted the formation of an immunocomplex between PLCgamma and NMDAR subunits. Stimulation of EGFR or the PLCbeta-coupled M1 acetylcholine receptor produced a robust transient inhibition of NMDA currents. Wortmannin application blocked the recovery of NMDA currents from the inhibition. Using mutagenesis, we identified the structural elements on NMDAR intracellular tails that transduce the receptor-mediated inhibition, which pinpoint to the binding site for the cytoskeletal protein alpha-actinin. Mutation of the PIP2-binding residues of alpha-actinin dramatically reduced NMDA currents and occluded the effect of EGF. Interestingly, EGF or wortmannin affected the interaction between NMDAR subunits and alpha-actinin, suggesting that this protein mediates the effect of PIP2 on NMDARs. In mature hippocampal neurons, expression of the mutant alpha-actinin reduced NMDA currents and accelerated inactivation. We propose a model in which alpha-actinin supports NMDAR activity via tethering their intracellular tails to plasma membrane PIP2. Thus, our results extend the influence of PIP2 to the NMDA ionotropic glutamate receptors and introduce a novel mechanism of "indirect" regulation of transmembrane protein activity by PIP2.

The Neuregulin-1 Receptor ErbB4 Controls Glutamatergic Synapse Maturation and Plasticity

Neuregulin-1 (NRG1) signaling participates in numerous neurodevelopmental processes. Through linkage analysis, nrg1 has been associated with schizophrenia, although its pathophysiological role is not understood. The prevailing models of schizophrenia invoke hypofunction of the glutamatergic synapse and defects in early development of hippocampal-cortical circuitry. Here, we show that the erbB4 receptor, as a postsynaptic target of NRG1, plays a key role in activity-dependent maturation and plasticity of excitatory synaptic structure and function. Synaptic activity leads to the activation and recruitment of erbB4 into the synapse. Overexpressed erbB4 selectively enhances AMPA synaptic currents and increases dendritic spine size. Preventing NRG1/erbB4 signaling destabilizes synaptic AMPA receptors and leads to loss of synaptic NMDA currents and spines. Our results indicate that normal activity-driven glutamatergic synapse development is impaired by genetic deficits in NRG1/erbB4 signaling leading to glutamatergic hypofunction. These findings link proposed effectors in schizophrenia: NRG1/erbB4 signaling perturbation, neurodevelopmental deficit, and glutamatergic hypofunction.

MGluR5 stimulates gliotransmission in the nucleus accumbens

Although metabotropic glutamate receptor 5 (mGluR5) is essential for cocaine self-administration and drug-seeking behavior, there is limited knowledge of the cellular actions of this receptor in the nucleus accumbens (NAc). Although mGluR5 has the potential to regulate neurons directly, recent studies have shown the importance of mGluR5 in regulating Ca(2+) signaling in astrocytes and, as a consequence, the Ca(2+)-dependent release of excitatory transmitters from these glia. In this study, we demonstrate that activation of mGluR5 induces Ca(2+) oscillations in NAc astrocytes with the correlated appearance of NMDA receptor-dependent slow inward currents detected in medium spiny neurons (MSNs). Photolysis of caged Ca(2+) loaded specifically into astrocytes evoked slow inward currents demonstrating that Ca(2+) elevations in astrocytes are responsible for these excitatory events. Pharmacological evaluation of these glial-evoked NMDA currents shows that they are mediated by NR2B-containing NMDA receptors, whereas synaptic NMDA receptors rely on NR2A-containing receptors. Stimulation of glutamatergic afferents activates mGluR5-dependent astrocytic Ca(2+) oscillations and gliotransmission that is sustained for minutes beyond the initial stimulus. Because gliotransmission is mediated by NMDA receptors, depolarized membrane potentials exhibited during up-states augment excitation provided by gliotransmission, which drives bursts of MSN action potentials. Because the predominant mGluR5-dependent action of glutamatergic afferents is to cause the sustained activation of astrocytes, which in turn excite MSNs through extrasynaptic NMDA receptors, our results raise the potential for gliotransmission being involved in prolonged mGluR5-dependent adaptation in the NAc.

Spinal Cord Transection Enhances Afferent-Evoked Inhibition in Lamina II Neurons And Abolishes BDNF-Induced Facilitation of Their Sensory Input

We previously reported that the pronociceptive neurotrophin brain-derived neurotrophic factor (BDNF) induces facilitation of C-fiber evoked EPSCs and NMDA currents in lamina II neurons of rats up to P40. Here, patch-clamp recording was used to study BDNF-induced modification of synaptic and NMDA-evoked responses in transverse spinal slices from lumbar (L2-L5) spinal cord of rats from P3 to P21 following complete spinal cord transection at P2. After transection injury at either T13/L1 or L6/S1, BDNF failed to facilitate synaptic AMPA-kainate currents or agonist-induced NMDA currents. The evoked synaptic currents were smaller in amplitude, often consisting of a biphasic (excitatory-inhibitory) response. The EPSC decayed more rapidly in neurons from transected cords than in those from uninjured cords. In transected cords most neurons responded to the GABA(A) receptor antagonist bicuculline with a significant increase in duration of the excitatory synaptic response. This could subsequently be blocked by D-APV, suggesting an NMDA-receptor mediated component. These findings suggest that following spinal cord transection, BDNF spinal actions are no longer predominantly pronociceptive. It is possible that a diminished availability of full-length TrkB and an enhanced expression of truncated TrkB receptors in the injured cord could play an important role in reducing the effect of BDNF following injury. These data are compared to those obtained after contusion, and it is concluded that the physiological changes after spinal injury differ according to nature of the injury.

Reduced presynaptic efficiency of excitatory synaptic transmission impairs LTP in the visual cortex of BDNF‐heterozygous mice

The neurotrophin brain-derived neurotrophic factor (BDNF) plays an important role in neuronal survival, axonal and dendritic growth and synapse formation. BDNF has also been reported to mediate visual cortex plasticity. Here we studied the cellular mechanisms of BDNF-mediated changes in synaptic plasticity, excitatory synaptic transmission and long-term potentiation (LTP) in the visual cortex of heterozygous BDNF-knockout mice (BDNF(+/-)). Patch-clamp recordings in slices showed an approximately 50% reduction in the frequency of miniature excitatory postsynaptic currents (mEPSCs) compared to wild-type animals, in the absence of changes in mEPSC amplitudes. A presynaptic impairment of excitatory synapses from BDNF(+/-) mice was further indicated by decreased paired-pulse ratio and faster synaptic fatigue upon prolonged repetitive stimulation at 40 Hz. In accordance, presynaptic theta-burst stimulation (TBS) failed to induce LTP at layer IV to layers II-III synapses during extracellular field-potential recordings in BDNF(+/-) animals. Changes in postsynaptic function could not be detected, as no changes were observed in either the amplitudes of evoked EPSCs, the ratios of AMPA : NMDA currents or the kinetics of evoked AMPA and NMDA EPSCs. In line with this observation, an LTP pairing paradigm that relies on direct postsynaptic depolarization under patch-clamp conditions could be induced successfully in BDNF(+/-) animals. These data suggest that a chronic reduction in the expression of BDNF to nearly 50% attenuates the efficiency of presynaptic glutamate release in response to repetitive stimulation, thereby impairing presynaptically evoked LTP in the visual cortex.

D2‐class dopamine receptor inhibition of NMDA currents in prefrontal cortical neurons is platelet‐derived growth factor receptor‐dependent

NMDA receptor function is modulated by both G-protein-coupled receptors and receptor tyrosine kinases. In acutely isolated rat hippocampal neurons, direct activation of the platelet-derived growth factor (PDGF) receptor or transactivation of the PDGF receptor by D4 dopamine receptors inhibits NMDA-evoked currents in a phospholipase C (PLC)-dependent manner. We have investigated further the ability of D2-class dopamine receptors to modulate NMDA-evoked currents in isolated rat prefrontal cortex (PFC). We have demonstrated that, similar to isolated hippocampal neurons, the application of PDGF-BB or quinpirole to isolated PFC neurons induces a slow-onset and long-lasting inhibition of NMDA-evoked currents. However, in contrast to hippocampal neurons, the inhibition of NMDA-evoked currents by quinpirole in PFC neurons is dependent upon D2/3, rather than D4, dopamine receptors. In PFC slices, application of both PDGF-BB and quinpirole induced a phosphorylation of the PDGF receptor at the PLCgamma binding and activation site, Tyr1021. The PDGF receptor kinase inhibitor, tyrphostin A9, and the D2/3 dopamine receptor antagonist, raclopride, inhibited quinpirole-induced Tyr1021 phosphorylation. These finding suggest that quinpirole treatment inhibits NMDAR signaling via PDGF receptor transactivation in both the hippocampus and the PFC, and that the effects of quinpirole in these regions are mediated by D4 and D2/3 dopamine receptors, respectively.

Critical roles of Src family tyrosine kinases in excitatory neuronal differentiation of cultured embryonic stem cells

Embryonic stem (ES) cells have been tested for potential cell transplantation therapy for CNS disorders. Understanding their differentiation mechanism and identifying factors involved in driving excitatory and inhibitory neuron lineages should enhance the efficacy and efficiency of the cell transplantation therapy. We tested the hypothesis that selective expression of Src family tyrosine kinases is required for phenotype-specific differentiation and functional maturation of ES cell derived neurons. Cultured mouse pluripotent ES cells were treated with retinoic acid (RA) to induce neural differentiation. After RA induction, neurons derived from ES cells showed significant neurite growth, increased expression of Src, Fyn and Lck and an extension of Src kinase expression from cell body to neurite processes. ES cell derived neuron-like cells expressed neurofilament, synaptophysin, glutamate receptors, NMDA and kainate currents, became vulnerable to excitotoxicity and formed functional excitatory synapses. These developmental events were blocked or attenuated when cells were grown in the presence of Src family kinase inhibitor PP2. However, there was no change in the expression of GABAergic-specific protein GAD67 during PP2 treatment. Our data suggest that Src tyrosine kinases are involved in the terminal differentiation of excitatory neuronal phenotype during ES cell neural differentiation after RA induction.

Effect of a non-hydrolyzable analog of diadenosine polyphosphates on NMDA-mediated currents in isolated pyramidal neurons of the rat hippocampus

Using a patch-clamp technique under voltage clamp conditions, we studied the effect of a non-hydrolyzable analog of diadenosine polyphosphates (AppCH2ppAs) on chemoactivated transmembrane currents through NMDA channels (NMDA currents) in isolated pyramidal neurons of the rat hippocampal CA3 zone. In 55.7% of the cases, AppCH2ppAs caused an increase in the peak amplitude of the currents induced by application of aspartate. In 39.5% of the cases, the agent exerted no effect, while in 4.8% these currents were suppressed. When studying the pharmacological effect of an increase in the amplitude of NMDA currents, we found that potentiation of these currents is mediated, first of all, by activation of P2 purinoceptors and is prevented by a blocker of tyrosine kinases, genistein. Receptor-channel NMDA complexes, due to their ability to be blocked by divalent cations, also contribute to the above effect of AppCH2ppA. Based on the data obtained, we conclude that AppCH2ppA influences NMDA receptors via activation of the P2 receptors and subsequent activation of tyrosine kinases; this leads to the modification of receptor-channel NMDA complexes and to the removal of their tonic blocking by zinc ions.