Regulation Of Excitatory Synaptic Transmission Research Articles

Differential regulation of excitatory synaptic transmission in the hippocampus and anterior temporal lobe by cyclin dependent kinase 5 (Cdk5) in mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS)

Mesial temporal lobe epilepsy with hippocamapal sclerosis (MTLE-HS) is the most common form of drug resistant epilepsy (DRE). MTLE-HS is a distributed network disorder comprising of not only the hippocampus, but other anatomically related extrahippocampal regions. Excitatory synaptic transmission is differentially regulated in the hippocampal and extra-hippocampal regions of patients with MTLE-HS, but its mechanism not understood. Cyclin-dependent kinase 5 (Cdk5) is known to regulate synaptic transmission and plasticity through up-regulation of NMDA receptors by phosphorylating NR2Asubunits. The present study is designed to investigate whether Cdk5 differentially regulates the excitatory synaptic transmission in the hippocampus and anterior temporal lobe (ATL) samples obtained from patients of MTLE-HS. We have measured the Cdk5 kinase activity and the protein levels of Cdk5, p-Cdk5, p35/p25, NR2A, pNR2A in the hippocampal and ATL samples obtained from patients with MTLE-HS. We have also determined the effect of roscovitine, a Cdk5 antagonist, on spontaneous excitatory postsynaptic currents (EPSCs) recorded from the hippocampal and ATL using patch-clamp technique. We observed significant increase in the expression of Cdk5, p-Cdk5, p35/p25, NR2A, pNR2A in the ATL samples as compared to the hippocampal samples. Cdk5 activity was significantly higher in ATL samples as compared to the hippocampal samples. Magnitude of reduction in the frequency of EPSCs by roscovitine in the ATL samples was higher than that in the hippocampal samples. Our studies suggest that Cdk5 differentially regulates excitatory synaptic activity in the hippocampal and ATL region of patients with MTLE-HS.

Presynaptic NK1 Receptor Activation by Substance P Suppresses EPSCs via Nitric Oxide Synthesis in the Rat Insular Cortex

Substance P (SP) regulates inhibitory synaptic transmission mediated by GABAA receptors in the cerebral cortex; however, SP-mediated regulation of excitatory synaptic transmission remains poorly understood. We performed whole-cell patch-clamp recordings from pyramidal neurons to examine the effects of SP on excitatory postsynaptic currents (EPSCs) mediated via AMPA receptors in the insular cortex (IC), which is involved in nociceptive information processing. First, EPSCs evoked by minimal electrical stimulation (eEPSCs) including stepwise EPSCs and failure events, were examined. SP dose-dependently suppressed mean eEPSC amplitude, partially due to an increase in the failure rate of eEPSCs. The SP-induced suppression of eEPSCs was accompanied by an increase in the paired-pulse ratio and was inhibited by the preapplication of SR140333, an NK1 receptor antagonist. [Sar9,Met(O2)11]-substance P, an NK1 receptor-selective agonist, mimicked the effects of SP on eEPSCs and decreased the frequency of miniature EPSCs (mEPSCs) without changing the average mEPSC amplitude. Considering that most NK1 receptors in the cerebral cortex are expressed in nitric oxide synthase (NOS)-positive GABAergic neurons, the SP-induced suppressive effect on EPSCs may be mediated by nitric oxide (NO) in this subtype of GABAergic neurons. NO imaging using the fluorescent probe DAX-J2 Red supports this hypothesis: SP increased the fluorescence intensity of DAX-J2 Red in some GABAergic neurons. Furthermore, both L-NAME, an NOS inhibitor, and PTIO, an NO scavenger, diminished the SP-induced suppression of eEPSCs. These results suggest that the activation of presynaptic NK1 receptors contributes to SP-induced eEPSC suppression by activating the NO synthesis pathway in GABAergic neurons. (246 words)

Dorsal-Ventral Differences in Modulation of Synaptic Transmission in the Hippocampus.

Functional diversification along the longitudinal axis of the hippocampus is a rapidly growing concept. Modulation of synaptic transmission by neurotransmitter receptors may importantly contribute to specialization of local intrinsic network function along the hippocampus. In the present study, using transverse slices from the dorsal and the ventral hippocampus of adult rats and recordings of evoked field postsynaptic excitatory potentials (fEPSPs) from the CA1 stratum radiatum, we aimed to compare modulation of synaptic transmission between the dorsal and the ventral hippocampus. We found that transient heterosynaptic depression (tHSD, <2 s), a physiologically relevant phenomenon of regulation of excitatory synaptic transmission induced by paired stimulation of two independent inputs to stratum radiatum of CA1 field, has an increased magnitude and duration in the ventral hippocampus, presumably contributing to increased input segregation in this segment of the hippocampus. GABAB receptors, GABAA receptors, adenosine A1 receptors and L-type voltage-gated calcium channels appear to contribute differently to tHSD in the two hippocampal segments; GABABRs play a predominant role in the ventral hippocampus while both GABABRs and A1Rs play important roles in the dorsal hippocampus. Activation of GABAB receptors by an exogenous agonist, baclofen, robustly and reversibly modulated both the initial fast and the late slow components of excitatory synaptic transmission, expressed by the fEPSPslope and fEPSP decay time constant (fEPSPτ), respectively. Specifically, baclofen suppressed fEPSP slope more in the ventral than in the dorsal hippocampus and enhanced fEPSPτ more in the dorsal than in the ventral hippocampus. Also, baclofen enhanced paired-pulse facilitation in the two hippocampal segments similarly. Blockade of GABAB receptors did not affect basal paired-pulse facilitation in either hippocampal segment. We propose that the revealed dorsal-ventral differences in modulation of synaptic transmission may provide a means for specialization of information processing in the local neuronal circuits, thereby significantly contributing to diversifying neuronal network functioning along the dorsal-ventral axis of hippocampus.

The Familial dementia gene ITM2b/BRI2 facilitates glutamate transmission via both presynaptic and postsynaptic mechanisms

Mutations in the Integral membrane protein 2B (ITM2b/BRI2) gene, which codes for a protein called BRI2, cause familial British and Danish dementia (FBD and FDD). Loss of BRI2 function and/or accumulation of amyloidogenic mutant BRI2-derived peptides have been proposed to mediate FDD and FBD pathogenesis by impairing synaptic Long-term potentiation (LTP). However, the precise site and nature of the synaptic dysfunction remain unknown. Here we use a genetic approach to inactivate Itm2b in either presynaptic (CA3), postsynaptic (CA1) or both (CA3 + CA1) neurons of the hippocampal Schaeffer-collateral pathway in both female and male mice. We show that after CA3 + CA1 Itm2b inactivation, spontaneous glutamate release and AMPAR-mediated responses are decreased, while short-term synaptic facilitation is increased. Moreover, AMPAR-mediated responses are decreased after postsynaptic but not presynaptic deletion of Itm2b. In contrast, the probability of spontaneous glutamate release is decreased, while short-term synaptic facilitation is increased, primarily after presynaptic deletion of Itm2b. Collectively, these results indicate a dual physiological role of Itm2b in the regulation of excitatory synaptic transmission at both presynaptic termini and postsynaptic termini and suggest that presynaptic and postsynaptic dysfunctions may be a pathogenic event leading to dementia and neurodegeneration in FDD and FBD.

Age-dependent regulation of excitatory synaptic transmission at hippocampal temporoammonic-CA1 synapses by leptin.

The hippocampus is a key target for the hormone leptin and leptin regulation of excitatory synaptic transmission at Schaffer-collateral–CA1 synapses during aging are well documented. However, little is known about the age-dependent actions of leptin at the temporoammonic (TA) input to CA1 neurons. Here we show that leptin induces a novel form of N-methyl-D-aspartate receptor–dependent long-term depression (LTD) at adult (12–24 weeks old) TA-CA1 synapses. Leptin-induced LTD requires activation of canonical Janus tyrosine kinase 2- signal transducer and activator of transcription signaling and removal of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors from synapses. Moreover, leptin-induced LTD is occluded by activity-dependent LTD at TA-CA1 synapses. By contrast, leptin has no effect on excitatory synaptic transmission at aged (12–14 months old) TA-CA1 synapses, and low-frequency stimulation also fails to induce LTD at this age. These findings demonstrate clear age-related alterations in the leptin sensitivity of TA-CA1 synapses and provide valuable information on how the leptin system alters with age. As leptin has been linked to Alzheimer's disease, these findings have important implications for understanding of age-related disorders such as Alzheimer's disease.

Restoration of Glutamatergic Transmission by Dopamine D4 Receptors in Stressed Animals

The prefrontal cortex (PFC), a key brain region for cognitive and emotional processes, is highly regulated by dopaminergic inputs. The dopamine D4 receptor, which is enriched in PFC, has been implicated in mental disorders, such as attention deficit-hyperactivity disorder and schizophrenia. Recently we have found homeostatic regulation of AMPA receptor-mediated synaptic transmission in PFC pyramidal neurons by the D4 receptor, providing a potential mechanism for D4 in stabilizing cortical excitability. Because stress is tightly linked to adaptive and maladaptive changes associated with mental health and disorders, we examined the synaptic actions of D4 in stressed rats. We found that neural excitability was elevated by acute stress and dampened by repeated stress. D4 activation produced a potent reduction of excitatory transmission in acutely stressed animals and a marked increase of excitatory transmission in repeatedly stressed animals. These effects of D4 targeted GluA2-lacking AMPA receptors and relied on the bi-directional regulation of calcium/calmodulin kinase II activity. The restoration of PFC glutamatergic transmission in stress conditions may enable D4 receptors to serve as a synaptic stabilizer in normal and pathological conditions.

Homer1 gene products orchestrate Ca2+-permeable AMPA receptor distribution and LTP expression

We studied the role of Homer1 gene products on the presence of synaptic Ca2+-permeable AMPA receptors (AMPARs) and long-term potentiation (LTP) generation in hippocampal CA1 pyramidal neurons, using mice either lacking all Homer1 isoforms (Homer1 KO) or overexpressing the immediate early gene (IEG) product Homer1a (H1aTG). We found that Homer1 KO caused a significant redistribution of the AMPAR subunit GluA2 from the dendritic compartment to the soma. Furthermore, deletion of Homer1 enhanced the AMPAR-mediated component of glutamatergic currents at Schaffer collateral synapses as demonstrated by increased AMPA/NMDA current ratios. Meanwhile, LTP generation appeared to be unaffected. Conversely, sustained overexpression of Homer1a strongly reduced AMPA/NMDA current ratios and polyamine sensitivity of synaptic AMPAR, indicating that the proportion of synaptic GluA2-containing AMPAR increased relative to WT. LTP maintenance was abolished in H1aTG. Notably, overexpression of Homer1a in Homer1 KO or GluA2 KO mice did not affect LTP expression, suggesting activity-dependent interaction between Homer1a and long Homer1 isoforms with GluA2-containing AMPAR. Thus, Homer1a is essential for the activity-dependent regulation of excitatory synaptic transmission.

Exposure to Cocaine Alters Dynorphin-Mediated Regulation of Excitatory Synaptic Transmission in Nucleus Accumbens Neurons

Dysregulation of excitatory synaptic input to nucleus accumbens (NAc) medium spiny neurons (MSNs) underlies a key pathophysiology of drug addiction and addiction-associated emotional and motivational alterations. Dynorphin peptides, which exhibit higher affinity to κ type opioid receptors, are upregulated within the NAc upon exposure to cocaine administration, and the increased dynorphin-signaling in the NAc has been critically implicated in negative mood observed in cocaine- or stress-exposed animals. Despite such apparent behavioral significance of the NAc dynorphins, the understanding of how dynorphins regulate excitatory synaptic transmission in the NAc remains incomplete. We used electrophysiological recording in brain slices to examine the effects of dynorphins on excitatory synaptic transmission in the NAc. We focused on two key dynorphins, dynorphin A and B. Our current results show that dynorphin A and B differentially regulated excitatory postsynaptic currents (EPSCs) in NAc MSNs. Whereas perfusions of both dynorphin A and B to NAc slices decreased EPSCs in MSNs, the effect of dynorphin A but not dynorphin B was completely reversed by the κ receptor-selective antagonist nor-binaltorphimine. These results implicate κ receptor-independent mechanisms in dynorphin B-mediated synaptic effects in the NAc. Furthermore, repeated exposure to cocaine (15 mg/kg/day via intraperitoneal injection for 5 days, with 1, 2, or 14 days withdrawal) completely abolished dynorphin A-mediated modulation of EPSCs in NAc MSNs, whereas the effect of dynorphin B remained largely unchanged. Given the quantitatively higher abundance of dynorphin B in the NAc, our present results suggest that the dynorphin B-mediated, κ receptor-independent pathways predominate in the overall effect of dynorphins in cocaine-pretreated animals and potentially in cocaine-induced alterations in mood.

Modulation of NMDA and AMPA-mediated synaptic transmission by CB1 receptors in frontal cortical pyramidal cells

Although the endogenous cannabinoid system modulates a variety of physiological and pharmacological processes, the specific role of cannabinoid CB1 receptors in the modulation of glutamatergic neurotransmission and neural plasticity is not well understood. Using whole-cell patch clamp recording techniques, evoked or spontaneous excitatory postsynaptic currents (eEPSCs or sEPSCs) were recorded from visualized, layer II/III pyramidal cells in frontal cortical slices from rat brain. Bath application of the CB1 receptor agonist, WIN 55212-2 (WIN), reduced the amplitude of NMDA receptor-mediated EPSCs in a concentration-dependent manner. When co-applied with the specific CB1 antagonists, AM251 or AM281, WIN did not suppress NMDA receptor-mediated EPSCs. WIN also reduced the amplitude of evoked AMPA receptor-mediated EPSCs, an effect that was also reversed by AM251. Both the frequency and amplitude of spontaneous AMPA receptor-mediated EPSCs were significantly reduced by WIN. In contrast, WIN reduced the frequency, but not the amplitude of miniature EPSCs, suggesting that the suppression of glutamatergic activity by CB1 receptors in the frontal neocortex is mediated by a presynaptic mechanism. Taken together, these data indicate a critical role for endocannabinoid signaling in the regulation of excitatory synaptic transmission in frontal neocortex, and suggest a possible neuronal mechanism whereby THC regulates cortical function.

Endosomal sorting of AMPA receptors in hippocampal neurons

An important mechanism for the regulation of excitatory synaptic transmission in the hippocampus involves tight control of AMPAR [AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor] trafficking to alter the number or subtype of synaptic receptors. This is achieved via the multiple stages of the endosomal system. AMPARs constitutively cycle through early endosomes and recycling endosomes to maintain synaptic receptor numbers. However, on induction of synaptic plasticity, subtle alterations are made to this cycle by the action of specific AMPAR-interacting proteins and also via a number of additional proteins that regulate endosomal sorting more generally. During long-term depression, receptors are diverted to late endosomes and lysosomes rather than recycling back to the plasma membrane, hence reducing the number of receptors at the synapse. The increased number of synaptic AMPARs after induction of LTP (long-term potentiation) originates from the recycling compartment. In addition, transient changes in subunit composition may arise as a result of retention of AMPAR subtypes within the endosome during LTP. Aberrant trafficking after pathological insults such as oxygen/glucose deprivation or mechanical trauma also involves alterations in synaptic AMPAR subunit composition, leading to calcium influx that ultimately results in cell death.

Impaired muscarinic regulation of excitatory synaptic transmission in the APPswe/PS1dE9 mouse model of Alzheimer's disease

Cholinergic hypothesis and amyloid cascade hypothesis are mainly proposed for Alzheimer's disease; however, the relationship between these hypotheses is poorly understood. To address the question of whether amyloid β-peptide pathology affects cholinergic neurotransmission, we examined the effect of a cholinesterase inhibitor, physostigmine, on field excitatory postsynaptic potentials (EPSPs) evoked by single-pulse stimulation in the CA1 region of the hippocampus of various APPswe/PS1dE9 transgenic mice with different degrees of amyloid β-peptide pathology. Reduced field EPSPs by physostigmine in transgenic mice at 3 months of age, when the mice had negligible amyloid β-peptide levels and no amyloid β-peptide deposits, were indistinguishable from those in age-matched wild-type mice. In contrast, reduced field EPSPs by physostigmine in transgenic mice at 5 months of age, when the mice had low amyloid β-peptide levels and subtle amyloid β-peptide deposits, were significantly lower than those in age-matched wild-type mice. Next, we characterized acetylcholine receptors, which play important roles in cholinergic neurotransmission, because physostigmine resulted in increased acetylcholine levels in the synaptic cleft. Different reductions of field EPSPs by physostigmine between transgenic and wild-type mice at 5 months of age were not affected by a nicotinic receptor antagonist, mecamylamine; however, reduced field EPSPs by physostigmine in both transgenic and wild-type mice were restored to basal levels by a muscarinic receptor antagonist, atropine. These results indicate that cholinergic modulation of glutamatergic transmission is already impaired at the onset of the formation of amyloid β-peptide deposits, and muscarinic receptor dysfunction is one of the causes of this impairment.

NMDA receptor-mediated extracellular adenosine accumulation is blocked by phosphatase 1/2A inhibitors

We have previously demonstrated that NMDA receptor-mediated extracellular adenosine accumulation in neuronal cultures is receptor-mediated and requires calcium influx. Because protein kinase C (PKC) is a calcium-dependent enzyme, we hypothesized that activation of PKC might be involved in NMDA-mediated adenosine accumulation. PKC inhibitors, however, did not block NMDA-evoked adenosine accumulation, but rather, stimulated basal adenosine accumulation. These data suggested the possibility that NMDA receptor-mediated adenosine accumulation involves net dephosphorylation rather than phosphorylation of one or more substrates. Thus, inhibition of kinases would be expected to increase adenosine accumulation and inhibition of phosphatases would be expected to block adenosine accumulation. To test this hypothesis, we used the phosphatase 1/2A inhibitors calyculin A and okadaic acid. Both inhibitors significantly reduced NMDA-evoked adenosine accumulation. In contrast phosphatase 2B inhibitors did not block NMDA-evoked adenosine accumulation. These data suggest that NMDA-evoked adenosine accumulation is mediated by activation of phosphatase 1/2A. We have established previously that NMDA-mediated adenosine accumulation is associated with adenosine kinase inhibition. However, adenosine kinase is not a direct substrate for phosphatase 1/2A because inhibition of phosphatase 1/2A did not abolish NMDA-evoked adenosine kinase inhibition. Okadaic acid also had no effect on NO donor-evoked adenosine accumulation, which previously has been shown to be associated with adenosine kinase inhibition. Dephosphorylation of one or more proteins other than adenosine kinase as a consequence of NMDA receptor activation might play an important role in extracellular adenosine regulation, with important consequences for the regulation of excitatory synaptic transmission, plasticity, epileptogenesis, and excitotoxicity.

Regulation of excitatory synaptic transmission by glial glutamate transporters

Regulation of excitatory synaptic transmission by glial glutamate transporters

Altered long-term synaptic plasticity and kainate-induced Ca 2+ transients in the substantia gelatinosa neurons in GLU K6-deficient mice

Functional kainate receptors are expressed in the spinal cord substantia gelatinosa region, and their activation contributes to bi-directional regulation of excitatory synaptic transmission at primary afferent synapses with spinal cord substantia gelatinosa neurons. However, no study has reported a role(s) for kainate receptor subtypes in long-term synaptic plasticity phenomena in this region. Using gene-targeted mice lacking glutamate receptor 5 (GLU K5) or GLU K6 subunit, we here show that GLU K6 subunit, but not GLU K5 subunit, is involved in the induction of long-term potentiation of excitatory postsynaptic potentials, evoked by two different protocols: (1) high-frequency primary afferent stimulation (100 Hz, 3 s) and (2) low-frequency spike-timing stimulation (1 Hz, 200 pulses). In addition, GLU K6 subunit plays an important role in the expression of kainate-induced Ca 2+ transients in the substantia gelatinosa. On the other hand, genetic deletion of GLU K5 or GLU K6 subunit does not prevent the induction of long-term depression. These results indicate that unique expression of kainate receptors subunits is important in regulating spinal synaptic plasticity and thereby processing of sensory information, including pain.

Differential Regulation of AMPA Receptor Subunit Trafficking by Palmitoylation of Two Distinct Sites

Modification of AMPA receptor function is a major mechanism for the regulation of synaptic transmission and underlies several forms of synaptic plasticity. Post-translational palmitoylation is a reversible modification that regulates localization of many proteins. Here, we report that palmitoylation of the AMPA receptor regulates receptor trafficking. All AMPA receptor subunits are palmitoylated on two cysteine residues in their transmembrane domain (TMD) 2 and in their C-terminal region. Palmitoylation on TMD 2 is upregulated by the palmitoyl acyl transferase GODZ and leads to an accumulation of the receptor in the Golgi and a reduction of receptor surface expression. C-terminal palmitoylation decreases interaction of the AMPA receptor with the 4.1N protein and regulates AMPA- and NMDA-induced AMPA receptor internalization. Moreover, depalmitoylation of the receptor is regulated by activation of glutamate receptors. These data suggest that regulated palmitoylation of AMPA receptor subunits modulates receptor trafficking and may be important for synaptic plasticity.

Calcium/calmodulin-dependent protein kinase II and synaptic plasticity

A prominent role for calcium/calmodulin-dependent protein kinase II (CaMKII) in regulation of excitatory synaptic transmission was proposed two decades ago when it was identified as a major postsynaptic density protein. Since then, fascinating mechanisms optimized to fine-tune the magnitude and locations of CaMKII activity have been revealed. The importance of CaMKII activity and autophosphorylation to synaptic plasticity in vitro , and to a variety of learning and memory paradigms in vivo has been demonstrated. Recent progress brings us closer to understanding the regulation of dendritic CaMKII activity, localization, and expression, and its role in modulating synaptic transmission and cell morphology.

Modulation of excitatory synaptic transmission in the spinal substantia gelatinosa of mice deficient in the kainate receptor GluR5 and/or GluR6 subunit.

Functional kainate (KA) receptors (KARs) are expressed in the spinal cord substantia gelatinosa (SG) region, and their activation has a capacity to modulate excitatory synaptic transmission at primary afferent synapses with SG neurones. In the present study, we have used gene-targeted mice lacking KAR GluR5 and/or GluR6 subunits to determine the identity of the receptor subunits involved in the KA-induced modulation of excitatory transmission. Our findings reveal that KARs comprising GluR5 or GluR6 subunits can either suppress or facilitate glutamatergic excitatory transmission in the SG of acutely prepared adult mouse spinal cord slices. In the absence of synaptic inhibition mediated by GABA(A) and glycine receptors, a biphasic effect of kainate is characteristic with facilitation apparent at a low concentration (30 nM) and depression at a higher concentration (3 microM). In addition, GluR6-KARs, localizing pre- and postsynaptically, are critically involved in inhibiting transmission at both A delta and C fibre monosynaptic pathways, whereas presynaptic GluR5-KARs play a limited role in inhibiting the C fibre-activated pathway. The results obtained support the hypothesis that KARs are involved in bi-directional regulation of excitatory synaptic transmission in the spinal cord SG region, and that these actions may be of critical importance for nociception and the clinical treatment of pain.

Thymosin alpha-1 modulate excitatory synaptic transmission in cultured hippocampal neurons in rats

Thymosin alpha-1 (Tα1) not only possesses immunoregulatory properties in periphery but also is expressed in the central nervous system (CNS) and affects the function of the CNS. To further elucidate the role of Tα1 in the CNS, the whole-cell recording technique was used to observe the effect of Tα1 on the spontaneous excitatory synaptic transmission in cultured rat hippocampal neurons. The results showed that acute treatment with Tα1 significantly enhanced the frequency of AMPA-mediated spontaneous excitatory postsynaptic current (sEPSC) at the concentrations of 1 and 10 μg/ml and also enhanced the frequency of AMPA-mediated miniature excitatory postsynaptic current (mEPSC) at 10 μg/ml. However, the amplitude of both sEPSC and mEPSC were not changed by Tα1. Those results suggested that Tα1 involves in the regulation of excitatory synaptic transmission in hippocampal neurons, which contribute to its neurophysiological function in the CNS.

Control of glutamatergic neurotransmission in the rat spinal dorsal horn by the nucleoside transporter ENT1

Adenosine modulates nociceptive processing in the superficial dorsal horn of the spinal cord. In other tissues, membrane transporters influence profoundly the extracellular levels of adenosine. To investigate the putative role of nucleoside transporters in the regulation of excitatory synaptic transmission in the dorsal horn, we employed immunohistochemistry and whole-cell patch-clamp recording of substantia gelatinosa neurons in slices of rat spinal cord in vitro. The rat equilibrative nucleoside transporter (rENT1) was revealed by antibody staining to be abundant in neonatal and mature dorsal horn, especially within laminae I-III. This was confirmed by immunoblots of dorsal horn homogenate. Nitrobenzylthioinosine (NBMPR), a potent non-transportable inhibitor of rENT1, attenuated synaptically evoked EPSCs onto lamina II neurons in a concentration-dependent manner. Application of an adenosine A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine produced a parallel rightward shift in the NBMPR concentration-effect curve. The effects of NBMPR were partially reversed by adenosine deaminase, which facilitates the metabolic degradation of adenosine. The modulation by NBMPR of evoked EPSCs was mimicked by exogenous adenosine or the selective A1 receptor agonist, 2-chloro-N6-cyclopentyl adenosine. NBMPR reduced the frequency but not the amplitude of spontaneous miniature EPSCs and increased the paired-pulse ratio of evoked currents, an effect that is consistent with presynaptic modulation. These data provide the first direct evidence that nucleoside transporters are able to critically modulate glutamatergic synaptic transmission.

Control of glutamatergic neurotransmission in the rat spinal dorsal horn by the nucleoside transporter ENT1.

Adenosine modulates nociceptive processing in the superficial dorsal horn of the spinal cord. In other tissues, membrane transporters influence profoundly the extracellular levels of adenosine. To investigate the putative role of nucleoside transporters in the regulation of excitatory synaptic transmission in the dorsal horn, we employed immunohistochemistry and whole-cell patch-clamp recording of substantia gelatinosa neurons in slices of rat spinal cord in vitro. The rat equilibrative nucleoside transporter (rENT1) was revealed by antibody staining to be abundant in neonatal and mature dorsal horn, especially within laminae I-III. This was confirmed by immunoblots of dorsal horn homogenate. Nitrobenzylthioinosine (NBMPR), a potent non-transportable inhibitor of rENT1, attenuated synaptically evoked EPSCs onto lamina II neurons in a concentration-dependent manner. Application of an adenosine A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine produced a parallel rightward shift in the NBMPR concentration-effect curve. The effects of NBMPR were partially reversed by adenosine deaminase, which facilitates the metabolic degradation of adenosine. The modulation by NBMPR of evoked EPSCs was mimicked by exogenous adenosine or the selective A1 receptor agonist, 2-chloro-N6-cyclopentyl adenosine. NBMPR reduced the frequency but not the amplitude of spontaneous miniature EPSCs and increased the paired-pulse ratio of evoked currents, an effect that is consistent with presynaptic modulation. These data provide the first direct evidence that nucleoside transporters are able to critically modulate glutamatergic synaptic transmission.