Extracellular pH modulation of excitatory synaptic transmission in hippocampal CA3 neurons.

In this study, the effect of extracellular pH on glutamatergic synaptic transmission was examined in mechanically dissociated rat hippocampal CA3 pyramidal neurons using a whole-cell patch-clamp technique under voltage-clamp conditions. Native synaptic boutons were isolated without using any enzymes, using a so-called "synapse bouton preparation," and preserved for the electrical stimulation of single boutons. Both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were found to decrease and increase in response to modest acidic (~pH 6.5) and basic (~pH 8.5) solutions, respectively. These changes in sEPSC frequency were not affected by the addition of TTX but completely disappeared by successive addition of Cd2+. However, changes in sEPSC amplitude induced by acidic and basic extracellular solutions were not affected by the addition of neither TTX nor Cd2+. The glutamate-induced whole-cell currents were decreased and increased by acidic and basic solutions, respectively. Acidic pH also decreased the amplitude and increased the failure rate (Rf) and paired-pulse rate (PPR) of glutamatergic electrically evoked excitatory postsynaptic currents (eEPSCs), while a basic pH increased the amplitude and decreased both the Rf and PPR of eEPSCs. The kinetics of the currents were not affected by changes in pH. Acidic and basic solutions decreased and increased voltage-gated Ca2+ but not Na+ channel currents in the dentate gyrus granule cell bodies. Our results indicate that extracellular pH modulates excitatory transmission via both pre- and postsynaptic sites, with the presynaptic modulation correlated to changes in voltage-gated Ca2+ channel currents.NEW & NOTEWORTHY The effects of external pH changes on spontaneous, miniature, and evoked excitatory synaptic transmission in CA3 hippocampal synapses were examined using the isolated nerve bouton preparation, which allowed for the accurate regulation of extracellular pH at the synapses. Acidification generally reduced transmission, partly via effects on presynaptic Ca2+ channel currents, while alkalization generally enhanced transmission. Both pre- and postsynaptic sites contributed to these effects.

Growth and excitability at synapsin II deficient hippocampal neurons.

Synapsins are neuronal phosphoproteins that fine-tune synaptic transmission and suppress seizure activity. Synapsin II (SynII) deletion produces epileptic seizures and overexcitability in neuronal networks. Early studies in primary neuronal cultures have shown that SynII deletion results in a delay in synapse formation. More recent studies at hippocampal slices have revealed increased spontaneous activity in SynII knockout (SynII(-)) mice. To reconcile these observations, we systematically re-examined synaptic transmission, synapse formation, and neurite growth in primary hippocampal neuronal cultures. We find that spontaneous glutamatergic synaptic activity was suppressed in SynII(-) neurons during the initial developmental epoch (7 days in vitro, DIV) but was enhanced at later times (12 and18 DIV). The density of synapses, transmission between connected pairs of neurons, and the number of docked synaptic vesicles were not affected by SynII deletion. However, we found that neurite outgrowth in SynII(-) neurons was suppressed during the initial developmental epoch (7 DIV) but enhanced at subsequent developmental stages (12 and18 DIV). This finding can account for the observed effect of SynII deletion on synaptic activity. To test whether the observed phenotype resulted directly from the deletion of SynII we expressed SynII in SynII(-) cultures using an adeno-associated virus (AAV). Expression of SynII at 2 DIV rescued the SynII deletion-dependent alterations in both synaptic activity and neuronal growth. To test whether the increased neurite outgrowth in SynII(-) observed at DIV 12 and18 represents an overcompensation for the initial developmental delay or results directly from SynII deletion we performed "late expression" experiments, transfecting SynII(-) cultures with AAV at 7 DIV. The late SynII expression suppressed neurite outgrowth at 12 and 18 DIV to the levels observed in control neurons, suggesting that these phenotypes directly depend on SynII. These results reveal a novel developmentally regulated role for SynII function in the control of neurite growth.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling.

Delayed dendritic development in newly generated dentate granule cells by cell-autonomous expression of the amyloid precursor protein.

Neuronal connectivity and synaptic remodeling are fundamental substrates for higher brain functions. Understanding their dynamics in the mammalian allocortex emerges as a critical step to tackle the cellular basis of cognitive decline that occurs during normal aging and in neurodegenerative disorders. In this work we have designed a novel approach to assess alterations in the dynamics of functional and structural connectivity elicited by chronic cell-autonomous overexpression of the human amyloid precursor protein (hAPP). We have taken advantage of the fact that the hippocampus continuously generates new dentate granule cells (GCs) to probe morphofunctional development of GCs expressing different variants of hAPP in a healthy background. hAPP was expressed together with a fluorescent reporter in neural progenitor cells of the dentate gyrus of juvenile mice by retroviral delivery. Neuronal progeny was analyzed several days post infection (dpi). Amyloidogenic cleavage products of hAPP such as the ß-C terminal fragment (ß-CTF) induced a substantial reduction in glutamatergic connectivity at 21 dpi, at which time new GCs undergo active growth and synaptogenesis. Interestingly, this effect was transient, since the strength of glutamatergic inputs was normal by 35 dpi. This delay in glutamatergic synaptogenesis was paralleled by a decrease in dendritic length with no changes in spine density, consistent with a protracted dendritic development without alterations in synapse formation. Finally, similar defects in newborn GC development were observed by overexpression of α-CTF, a non-amyloidogenic cleavage product of hAPP. These results indicate that hAPP can elicit protracted dendritic development independently of the amyloidogenic processing pathway.

Functional and morphological adaptation of medial prefrontal corticotropin releasing factor receptor 1-expressing neurons in male mice following chronic ethanol exposure.

Chronic ethanol dependence and withdrawal activate corticotropin releasing factor (CRF)-containing GABAergic neurons in the medial prefrontal cortex (mPFC), which tightly regulate glutamatergic pyramidal neurons. Using male CRF1:GFP reporter mice, we recently reported that CRF1-expressing (mPFCCRF1+) neurons predominantly comprise mPFC prelimbic layer 2/3 pyramidal neurons, undergo profound adaptations following chronic ethanol exposure, and regulate anxiety and conditioned rewarding effects of ethanol. To explore the effects of acute and chronic ethanol exposure on glutamate transmission, the impact of chronic alcohol on spine density and morphology, as well as persistent changes in dendritic-related gene expression, we employed whole-cell patch-clamp electrophysiology, diOlistic labeling for dendritic spine analysis, and dendritic gene expression analysis to further characterize mPFCCRF1+ and mPFCCRF1- prelimbic layer 2/3 pyramidal neurons. We found increased glutamate release in mPFCCRF1+ neurons with ethanol dependence, which recovered following withdrawal. In contrast, we did not observe significant changes in glutamate transmission in neighboring mPFCCRF1- neurons. Acute application of 44 mM ethanol significantly reduced glutamate release onto mPFCCRF1+ neurons, which was observed across all treatment groups. However, this sensitivity to acute ethanol was only evident in mPFCCRF1- neurons during withdrawal. In line with alterations in glutamate transmission, we observed a decrease in total spine density in mPFCCRF1+ neurons during dependence, which recovered following withdrawal, while again no changes were observed in mPFCCRF- neurons. Given the observed decreases in mPFCCRF1+ stubby spines during withdrawal, we then identified persistent changes at the dendritic gene expression level in mPFCCRF1+ neurons following withdrawal that may underlie these structural adaptations. Together, these findings highlight the varying responses of mPFCCRF1+ and mPFCCRF1- cell-types to acute and chronic ethanol exposure, as well as withdrawal, revealing specific functional, morphological, and molecular adaptations that may underlie vulnerability to ethanol and the lasting effects of ethanol dependence.

ZCCHC17 knockdown phenocopies Alzheimer's disease-related loss of synaptic proteins and hyperexcitability.

ZCCHC17 is a master regulator of synaptic gene expression and has recently been shown to play a role in splicing of neuronal mRNA. We previously showed that ZCCHC17 protein declines in Alzheimer's disease (AD) brain tissue before there is significant gliosis and neuronal loss, that ZCCHC17 loss partially replicates observed splicing abnormalities in AD brain tissue, and that maintenance of ZCCHC17 levels is predicted to support cognitive resilience in AD. Here, we assessed the functional consequences of reduced ZCCHC17 expression in primary cortical neuronal cultures using siRNA knockdown. Consistent with its previously identified role in synaptic gene expression, loss of ZCCHC17 led to loss of synaptic protein expression. Patch recording of neurons shows that ZCCHC17 loss significantly disrupted the excitation/inhibition balance of neurotransmission, and favored excitatory-dominant synaptic activity as measured by an increase in spontaneous excitatory post synaptic currents and action potential firing rate, and a decrease in spontaneous inhibitory post synaptic currents. These findings are consistent with the hyperexcitable phenotype seen in AD animal models and in patients. We are the first to assess the functional consequences of ZCCHC17 knockdown in neurons and conclude that ZCCHC17 loss partially phenocopies AD-related loss of synaptic proteins and hyperexcitability.

HIV-1 envelope protein gp120 modulation of glutamate effects on cortical neuronal synapses: implications for HIV-1-associated neuropathogenesis.

Despite the introduction of combined antiretroviral therapy (cART) HIV-1 virus persists in the brain in a latent or restricted manner and viral proteins, such as gp120, continue to play a significant disease-inciting role. Gp120 is known to interact with N-methyl-D-aspartate (NMDA) receptors (NMDARs) resulting in neuronal injury. Glutamate is the main excitatory neurotransmitter in the brain and plays an important role in cognitive function and dysregulation of excitatory synaptic transmission impairs neurocognition. It is our hypothesis that gp120 may alter synaptic function via modulating glutamate function from a physiological molecule to a pathophysiological substance. To test this hypothesis, we studied the modulatory effects of gp120 and glutamate on NMDAR-mediated spontaneous excitatory postsynaptic current (sEPSCNMDAR) and dynamic dendritic spine changes in rat cortical neuronal cultures. Our results revealed that gp120 and glutamate each, at low concentrations, had no significant effects on sEPSCNMDAR and dendritic spines, but increased sEPSCNMDAR frequency, decreased numbers of dendritic spines when tested in combination. The observed effects were blocked by either a CXCR4 blocker or an NMDAR antagonist, indicating the involvements of chemokine receptor CXCR4 and NMDARs in gp120 modulation of glutamate effects. These results may imply a potential mechanism for HIV-1-associated neuropathogenesis in the cART era.

Abolished ketamine effects on the spontaneous excitatory postsynaptic current of medial prefrontal cortex neurons in GluN2D knockout mice.

Ketamine, a non-competitive antagonist of the N-methyl-D-aspartate receptor (NMDAR), generates a rapidly-acting antidepressant effect. It exerts psychomimetic effects, yet demands a further investigation of its mechanism. Previous research showed that ketamine did no longer promote hyperlocomotion in GluN2D knockout (KO) mice, which is a subunit of NMDAR. In the present study, we tested whether GluN2D-containing NMDARs participate in the physiological changes in the medial prefrontal cortex (mPFC) triggered by ketamine. Sub-anesthetic dose of ketamine (25 mg/kg) elevated the frequency of spontaneous excitatory postsynaptic currents (sEPSC) in wild-type (WT) mice, but not in GluN2D KO mice, 1 h after the injection. The amplitude of sEPSC and paired-pulse ratio (PPR) were unaltered by ketamine in both WT and GluN2D KO mice. These findings suggest that GluN2D-containing NMDARs might play a role in the ketamine-mediated changes in glutamatergic neurons in mPFC and, presumably, in ketamine-induced hyperlocomotion.

Restoring tripartite glutamatergic synapses: A potential therapy for mood and cognitive deficits in Gulf War illness.

Gulf War illness is associated with a combination of exposure to war-related chemical agents and traumatic stress. Currently, there are no effective treatments, and the pathophysiology remains elusive. Neurological problems are among the most commonly reported symptoms. In this study, we investigated the glutamatergic system in the hippocampi of mice exposed to war-related chemical agents and stress. Mice developed Gulf War illness-like symptoms, including mood deficits, cognitive impairments, and fatigue. They exhibited the following pathological changes in hippocampi: elevated extracellular glutamate levels, impaired glutamatergic synapses, astrocyte atrophy, loss of interneurons, and decreased neurogenesis. LDN/OSU-215111 is a small-molecule that can strengthen the structure and function of both the astrocytic processes and the glutamatergic synapses that together form the tripartite synapses. We found that LDN/OSU-215111 effectively prevented the development of mood and cognitive deficits in mice when treatment was implemented immediately following the exposure. Moreover, when symptoms were already present, LDN/OSU-215111 still significantly ameliorated these deficits; impressively, benefits were sustained one month after treatment cessation, indicating disease modification. LDN/OSU-215111 effectively normalized hippocampal pathological changes. Overall, this study provides strong evidence that restoration of tripartite glutamatergic synapses by LDN/OSU-215111 is a potential therapy for Gulf War illness.

Complete Disruption of Autism-Susceptibility Genes by Gene Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons.

Autism spectrum disorder (ASD) is phenotypically and genetically heterogeneous. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of ten ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NGN2)-directed induction of iPSCs allowed production of excitatory neurons, and mutant proteins were not detectable. RNA sequencing revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory postsynaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2-, and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.

Endomorphin-2 Decreases Excitatory Synaptic Transmission in the Spinal Ventral Horn of the Rat.

Motor impairment is one of the serious side-effects of morphine, which is an exogenous agonist of the µ-opioid receptor (MOR) as well as a widely used analgesic drug in clinical practice for chronic pain treatment. Endomorphins (EMs, including EM-1 and EM-2), the most effective and specific endogenous agonists of the MOR, exert more potent analgesia in acute and neuropathic pain than other opiates, such as morphine. Although EMs had fewer side-effects comparing to other opiates, motor impairment was still one unwanted reaction which limited its clinical application. In order to prevent and treat the motor impairment, it is critical to reveal the neural mechanisms underlying such locomotion disorder. The purpose of the present study was to reveal the neural mechanisms underlying the effects of EM-2 on the activity of motoneurons in the spinal ventral horn. First, we examine the distribution of EM-2-immunoreactive (IR) primary afferent fibers and their synaptic connections with the motoneurons innervating the skeletal muscles of the lower limb revealed by sciatic nerve retrograde tracing. The results showed that EM-2-IR fibers and terminals were sparsely observed in lamina IX and they formed symmetric synaptic connections with the motoneurons within lamina IX of the spinal ventral horn. Then, whole-cell patch-clamp technique was used to observe the effects of EM-2 on the spontaneous excitatory postsynaptic current (sEPSC) of motoneurons in lamina IX. The results showed that EM-2 could decrease both the frequency and amplitude of the sEPSC of the motoneurons in lamina IX, which was reversed by the MOR antagonist CTOP. These results indicate that EM-2-IR fibers originated from primary afferent fibers form symmetric synaptic connections with motoneurons innervating skeletal muscles of the lower limbs in lamina IX of the spinal ventral horn and EM-2 might exert inhibitory effects on the activities of these motoneurons through both presynaptic and postsynaptic mechanisms.

Effect of interaction between acute administration of morphine and cannabinoid compounds on spontaneous excitatory and inhibitory postsynaptic currents of magnocellular neurons of supraoptic nucleus.

OBJECTIVES: Opioids and cannabinoids are two important compounds that have been shown to influence the activity of magnocellular neurons (MCNs) of supraoptic nucleus (SON). The interaction between opioidergic and cannabinoidergic systems in various structures of the brain and spinal cord is now well established, but not in the MCNs of SON. MATERIALS AND METHODS: In this study, whole cell patch clamp recording of neurons in rat brain slice was used to investigate the effect of acute morphine and cannabinoid administration on spontaneous inhibitory and excitatory spostsynaptic currents (sIPSCs and sEPSCs) in MCNs. RESULTS: Bath application of morphine produced an increase in sEPSCs frequency and a decrease in sIPSCs frequency. In contrast, bath application of URB597 (fatty acid amide hydrolase (FAAH) inhibitor) produced a decrease in sEPSCs frequency but an increase in sIPSCs frequency. WIN55212-2 (cannabinoid receptor agonist) decreased both sIPSCs and sEPSCs frequencies of MCNs. Co-application of morphine and URB597 attenuated the effect of morphine on MCNs. CONCLUSION: Taken together, these data indicated that at the cellular level, pharmacological augmentation of endocannabinoids could attenuate morphine effects on MCNs.

Developmental change and sexual difference in synaptic modulation produced by oxytocin in rat substantia gelatinosa neurons.

We have previously reported that oxytocin produces an inward current at a holding potential of -70 mV without a change in glutamatergic excitatory transmission in adult male rat spinal lamina II (substantia gelatinosa; SG) neurons that play a pivotal role in regulating nociceptive transmission. Oxytocin also enhanced GABAergic and glycinergic spontaneous inhibitory transmissions in a manner sensitive to a voltage-gated Na+-channel blocker tetrodotoxin. These actions were mediated by oxytocin-receptor activation. Such a result was different from that obtained by other investigators in young male rat superficial dorsal horn neurons in which an oxytocin-receptor agonist enhanced glutamatergic and GABAergic but not glycinergic spontaneous transmissions. In order to know a developmental change and also sexual difference in the actions of oxytocin, we examined its effect on spontaneous synaptic transmission in adult female and young male rat SG neurons by using the whole-cell patch-clamp technique in spinal cord slices. In adult female rats, oxytocin produced an inward current at -70 mV without a change in excitatory transmission. GABAergic and glycinergic transmissions were enhanced by oxytocin, the duration of which enhancement was much shorter than in adult male rats. In young (11-21 postnatal days) male rats, oxytocin produced not only an inward but also outward current at -70 mV, and presynaptically inhibited or facilitated excitatory transmission, depending on the neurons tested; both GABAergic and glycinergic transmissions were enhanced by oxytocin. The inhibitory transmission enhancements in adult female and young male rats were sensitive to tetrodotoxin. Although the data may not be enough to be estimated, it is suggested that synaptic modulation by oxytocin in SG neurons, i.e., cellular mechanism for its antinociceptive action, exhibits a developmental change and sexual difference.

The comparison of the effects of acute and repeated morphine administration on fast synaptic transmission in magnocellular neurons of supraoptic nucleus, plasma vasopressin levels, and urine volume of male rats.

The activity of the magnocellular neurons (MCNs) of supraoptic nucleus (SON) is regulated by a variety of excitatory and inhibitory inputs. Opioids are one of the important compounds that affect these inputs at SON synapses. In this study, whole-cell patch clamp recording of SON neurons was used to investigate the effect of acute and repeated morphine administration on spontaneous inhibitory and excitatory post synaptic currents (sIPSCs and sEPSCs) in MCNs. While acute bath application of morphine to brain slice of intact rat produced an increase in sEPSCs frequency and a decrease in sIPSCs frequency, repeated in-vivo administration of morphine produced opposite effect. Moreover, repetitive i.c.v. administration of morphine for three consecutive days caused significant increase in urine volume, but had no significant alteration in water consumption compared to control group. The increase in urine volume was consistent with a significant decrease in plasma arginine vasopressin (AVP) levels after repetitive i.p. morphine administration. The results suggest that acute administration of morphine stimulates whereas repeated administration of morphine inhibits the MCNs. Morphine-induced MCN inhibition could result in diminished plasma AVP levels and eventually an increase in urine volume of rats.

Dopamine D2 receptor desensitization by dopamine or corticotropin releasing factor in ventral tegmental area neurons is associated with increased glutamate release.

Neurons of the ventral tegmental area (VTA) are the source of dopaminergic (DAergic) input to important brain regions related to addiction. Prolonged exposure of these VTA neurons to moderate concentrations of dopamine (DA) causes a time-dependent decrease in DA-induced inhibition, a complex desensitization called DA inhibition reversal (DIR). DIR is mediated by conventional protein kinase C (cPKC) through concurrent stimulation of D2 and D1-like DA receptors, or by D2 stimulation concurrent with activation of some Gq-linked receptors. Corticotropin releasing factor (CRF) acts via Gq, and can modulate glutamater neurotransmission in the VTA. In the present study, we used brain slice electrophysiology to characterize the interaction of DA, glutamate antagonists, and CRF agonists in the induction and maintenance of DIR in the VTA. Glutamate receptor antagonists blocked induction but not maintenance of DIR. Putative blockers of neurotransmitter release and store-operated calcium channels blocked and reversed DIR. CRF and the CRF agonist urocortin reversed inhibition produced by the D2 agonist quinpirole, consistent with our earlier work indicating that Gq activation reverses quinpirole-mediated inhibition. In whole cell recordings, the combination of urocortin and quinpirole, but not either agent alone, increased spontaneous excitatory postsynaptic currents (sEPSCs) in VTA neurons. Likewise, the combination of a D1-like receptor agonist and quinpirole, but not either agent alone, increased sEPSCs in VTA neurons. In summary, desensitization of D2 receptors induced by dopamine or CRF on DAergic VTA neurons is associated with increased glutamatergic signaling in the VTA.

Synaptic plasticity in glutamatergic and GABAergic neurotransmission following chronic memantine treatment in an in vitro model of limbic epileptogenesis.

Chronic N-methyl-D-aspartate receptor (NMDAR) blockade with high affinity competitive and uncompetitive antagonists can lead to seizure exacerbation, presumably due to an imbalance in glutamatergic and GABAergic transmission. Acute administration of the moderate affinity NMDAR antagonist memantine in vivo has been associated with pro- and anticonvulsive properties. Chronic treatment with memantine can exacerbate seizures. Therefore, we hypothesized that chronic memantine treatment would increase glutamatergic and decrease GABAergic transmission, similar to high affinity competitive and uncompetitive antagonists. To test this hypothesis, organotypic hippocampal slice culture were treated for 17-21 days with memantine and then subjected to electrophysiological recordings. Whole-cell recordings from dentate granule cells revealed that chronic memantine treatment slightly, but significantly increased sEPSC frequency, mEPSC amplitude and mEPSC charge transfer, consistent with minimally increased glutamatergic transmission. Chronic memantine treatment also increased both sIPSC and mIPSC frequency and amplitude, suggestive of increased GABAergic transmission. Results suggest that a simple imbalance between glutamatergic and GABAergic neurotransmission may not underlie memantine's ictogenic properties. That said, glutamatergic and GABAergic transmission were assayed independently of one another in the current study. More complex interactions between glutamatergic and GABAergic transmission may prevail under conditions of intact circuitry.

Dual effects of L-DOPA on nigral dopaminergic neurons.

L-DOPA (Levodopa) remains the gold standard for the treatment of motor symptoms of Parkinson's disease (PD), despite indications that the drug may have detrimental effects in cell culture. Classically, l-DOPA increases the production of dopamine (DA) in nigral dopaminergic neurons, while paradoxically inhibiting the firing of these neurons due to activation of D2 autoreceptors by extracellularly released DA. Using a combination of electrophysiology and calcium microfluorometry in brain slices, we have identified a novel effect of L-DOPA on dopaminergic neurons when D2 receptors were blocked. Under these conditions, L-DOPA (0.03-3 mM) evoked an excitatory effect consisting of two components. The 'early' component observed during and immediately after application of the drug, was associated with increased firing, membrane depolarization and inward current. This excitatory response was strongly attenuated by CNQX (10 µM), pointing to the involvement of TOPA quinone, an auto-oxidation product of L-DOPA and a potent activator of AMPA/kainate receptors. The 'late' phase of excitation persisted >30 min after brief L-DOPA application and was not mediated by ionotropic glutamate receptors, nor by D1, α1-adrenergic, mGluR1 or GABAB receptors. It was eliminated by carbidopa, demonstrating its dependence on conversion of L-DOPA to DA. Exogenous DA (50 µM) also evoked a glutamate-receptor independent increase in firing and an inward current when D2 receptors were blocked. In voltage-clamped neurons, both L-DOPA and DA produced a long-lasting increase in [Ca(2+)]i which was unaffected by block of ionotropic glutamate receptors. These results demonstrate that L-DOPA has dual, inhibitory and excitatory, effects on nigral dopaminergic neurons, and suggest that the excitation and calcium rise may have long-lasting consequences for the activity and survival of these neurons when the expression or function of D2 receptors is impaired.

Reactive oxygen species enhance excitatory synaptic transmission in rat spinal dorsal horn neurons by activating TRPA1 and TRPV1 channels.

Central neuropathic pain (CNP) in the spinal cord, such as chronic pain after spinal cord injury (SCI), is an incurable ailment. However, little is known about the spinal cord mechanisms underlying CNP. Recently, reactive oxygen species (ROS) have been recognized to play an important role in CNP of the spinal cord. However, it is unclear how ROS affect synaptic transmission in the dorsal horn of the spinal cord. To clarify how ROS impact on synaptic transmission, we investigated the effects of ROS on synaptic transmission in rat spinal cord substantia gelatinosa (SG) neurons using whole-cell patch-clamp recordings. Administration of tert-butyl hydroperoxide (t-BOOH), an ROS donor, into the spinal cord markedly increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in SG neurons. This t-BOOH-induced enhancement was not suppressed by the Na(+) channel blocker tetrodotoxin. However, in the presence of a non-N-methyl-D-aspartate glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, t-BOOH did not generate any sEPSCs. Furthermore, in the presence of a transient receptor potential ankyrin 1 (TRPA1) channel antagonist (HC-030031) or a transient receptor potential vanilloid 1 (TRPV1) channel antagonist (capsazepine or AMG9810), the t-BOOH-induced increase in the frequency of sEPSCs was inhibited. These results indicate that ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons through TRPA1 and TRPV1 channel activation. Excessive activation of these ion channels by ROS may induce central sensitization in the spinal cord and result in chronic pain such as that following SCI.

Regional and cell-type-specific effects of DAMGO on striatal D1 and D2 dopamine receptor-expressing medium-sized spiny neurons.

The striatum can be divided into the DLS (dorsolateral striatum) and the VMS (ventromedial striatum), which includes NAcC (nucleus accumbens core) and NAcS (nucleus accumbens shell). Here, we examined differences in electrophysiological properties of MSSNs (medium-sized spiny neurons) based on their location, expression of DA (dopamine) D1/D2 receptors and responses to the µ-opioid receptor agonist, DAMGO {[D-Ala(2)-MePhe(4)-Gly(ol)(5)]enkephalin}. The main differences in morphological and biophysical membrane properties occurred among striatal sub-regions. MSSNs in the DLS were larger, had higher membrane capacitances and lower Rin (input resistances) compared with cells in the VMS. RMPs (resting membrane potentials) were similar among regions except for D2 cells in the NAcC, which displayed a significantly more depolarized RMP. In contrast, differences in frequency of spontaneous excitatory synaptic inputs were more prominent between cell types, with D2 cells receiving significantly more excitatory inputs than D1 cells, particularly in the VMS. Inhibitory inputs were not different between D1 and D2 cells. However, MSSNs in the VMS received more inhibitory inputs than those in the DLS. Acute application of DAMGO reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents, but the effect was greater in the VMS, in particular in the NAcS, where excitatory currents from D2 cells and inhibitory currents from D1 cells were inhibited by the largest amount. DAMGO also increased cellular excitability in the VMS, as shown by reduced threshold for evoking APs (action potentials). Together the present findings help elucidate the regional and cell-type-specific substrate of opioid actions in the striatum and point to the VMS as a critical mediator of DAMGO effects.