Monosynaptic Excitatory Postsynaptic Currents Research Articles

Heterogeneous electrophysiological and morphological properties of neurons in the mouse medial amygdala in vitro

Neurons in the medial nucleus of the amygdala (MeA) play a key role in the innate maternal, reproductive, defensive, and social behaviors. However, it is unclear how activation of the vomeronasal system leads to the behavioral outputs that are associated with pheromones. Here, we characterized the electrophysiological and morphological properties of MeA neurons using whole-cell recordings in mice slice preparations. Biocytin labeling revealed that MeA neurons possessed bipolar to multipolar cell bodies and dendritic fields covering projection areas from the accessory olfactory bulb. In 70% of recorded MeA neurons, monosynaptic excitatory postsynaptic currents (EPSCs) were evoked from the accessory olfactory bulb afferent in which the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate component was dominant and was rarely followed by the N-methyl-d-aspartic acid component. Norepinephrine increased the frequency of spontaneous inhibitory postsynaptic currents in some neurons, whereas α-methyl-5-hydroxytryptamine increased spontaneous EPSCs in other neurons. Morphologically and physiologically, heterogeneous MeA neurons appear likely to produce multiplex outputs of instinctive behaviors.

Tumor suppressor menin mediates peripheral nerve injury-induced neuropathic pain through potentiating synaptic plasticity

Synaptic plasticity is a crucial step in the development of central sensitization in the pathogenesis of neuropathic hyperalgesia. Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, possesses the property of synaptogenesis which plays an essential role in neuronal activity. We tested the contributing role of spinal menin in peripheral nerve injury-induced neuropathic hypersensitivity through modulating neuronal synaptic plasticity. After approval by the Institutional Animal Care and Use Committee, nociceptive responses were detected with von Frey filaments and thermal plate after spared nerve injury in C57BL/6 mice who were treated with either intrathecal antisense oligonucleotide of MEN1 (ASO) or vehicle. Extracellular spontaneous discharge frequency, field excitatory postsynaptic potential (fEPSP), and monosynaptic excitatory postsynaptic currents (EPSCs) were measured electrophysiologically. Intrathecal ASO alleviated nerve injury-induced mechanical and thermal hypersensitivity. Upregulated spinal menin after nerve injury colocalized with NeuN in the superficial laminae; genetic knockdown of spinal menin reduced nerve injury induced in vivo spontaneous activity and instantaneous frequency and in vitro field potentials; ASO decreased the frequency and amplitude of monosynaptic EPSCs, and reduced synaptic strength and total charge. Collectively, these findings highlight the role of upregulated neuronal menin in the spinal cord in potentiating spinal synaptic plasticity in peripheral nerve injury-induced neuropathic hypersensitivity.

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Up-regulation of Cavβ3 Subunit in Primary Sensory Neurons Increases Voltage-activated Ca2+ Channel Activity and Nociceptive Input in Neuropathic Pain

High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), β (Cavβ), and α(2)δ subunits. The Cavβ subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavβ subunits in primary sensory neurons. In this study, we found that Cavβ(3) and Cavβ(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavβ(3), but not Cavβ(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavβ(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavβ(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavβ(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavβ(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.

Input-specific excitation of olfactory cortex microcircuits

Every higher-order association cortex receives a variety of synaptic signals from different regions of the brain. How these cortical networks are capable of differentially responding to these various extrinsic synaptic inputs remains unclear. To address this issue, we studied how the basolateral amygdala (BLA) and the anterior piriform cortex (aPC) were functionally connected to the association olfactory cortex, the posterior piriform cortex (pPC). We infected the BLA and aPC with adeno-associated virus expressing channelrhodopsin-2-Venus fusion protein (ChR2-AAV) and recorded the excitatory postsynaptic currents (EPSC) resulting from photostimulation of either BLA or aPC axons in the major classes of excitatory and inhibitory neurons of the pPC. We found that BLA and aPC axons evoked monosynaptic EPSCs in every type of pPC neuron, but each fiber system preferentially targeted one excitatory and one inhibitory neuronal subtype. BLA fibers were most strongly connected to deep pyramidal cells (DP) and fast-spiking interneurons (FS), while aPC axons formed the strongest synaptic connections with DPs and irregular-spiking interneurons (IR). Overall, our findings show that the pPC differentially responds to amygdaloid versus cortical inputs by utilizing distinct local microcircuits, each defined by one predominant interneuronal subtype: FS for the BLA and IR for the aPC. It would thus seem that preferential excitation of a single neuronal class could be sufficient for the pPC to generate unique electrophysiological outputs in response to divergent synaptic input sources.

A Major Role for Intracortical Circuits in the Strength and Tuning of Odor-Evoked Excitation in Olfactory Cortex

In primary sensory cortices, there are two main sources of excitation: afferent sensory input relayed from the periphery and recurrent intracortical input. Untangling the functional roles of these two excitatory pathways is fundamental for understanding how cortical neurons process sensory stimuli. Odor representations in the primary olfactory (piriform) cortex depend on excitatory sensory afferents from the olfactory bulb. However, piriform cortex pyramidal cells also receive dense intracortical excitatory connections, and the relative contribution of these two pathways to odor responses is unclear. Using a combination of in vivo whole-cell voltage-clamp recording and selective synaptic silencing, we show that the recruitment of intracortical input, rather than olfactory bulb input, largely determines the strength of odor-evoked excitatory synaptic transmission in rat piriform cortical neurons. Furthermore, we find that intracortical synapses dominate odor-evoked excitatory transmission in broadly tuned neurons, whereas bulbar synapses dominate excitatory synaptic responses in more narrowly tuned neurons.

Nitric Oxide Inhibits Nociceptive Transmission by Differentially Regulating Glutamate and Glycine Release to Spinal Dorsal Horn Neurons

Nitric oxide (NO) is involved in many physiological functions, but its role in pain signaling remains uncertain. Surprisingly, little is known about how endogenous NO affects excitatory and inhibitory synaptic transmission at the spinal level. Here we determined how NO affects excitatory and inhibitory synaptic inputs to dorsal horn neurons using whole-cell recordings in rat spinal cord slices. The NO precursor L-arginine or the NO donor SNAP significantly increased the frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (IPSCs) of lamina II neurons. However, neither L-arginine nor SNAP had any effect on GABAergic IPSCs. L-arginine and SNAP significantly reduced the amplitude of monosynaptic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root with an increase in paired-pulse ratio. Inhibition of the soluble guanylyl cyclase abolished the effect of L-arginine on glycinergic IPSCs but not on evoked monosynaptic EPSCs. Also, inhibition of protein kinase G blocked the increase in glycinergic sIPSCs by the cGMP analog 8-bromo-cGMP. The inhibitory effects of L-arginine on evoked EPSCs and high voltage-activated Ca(2+) channels expressed in HEK293 cells and dorsal root ganglion neurons were abolished by blocking the S-nitrosylation reaction with N-ethylmaleimide. Intrathecal injection of L-arginine and SNAP significantly increased mechanical nociceptive thresholds. Our findings suggest that spinal endogenous NO enhances inhibitory glycinergic input to dorsal horn neurons through sGC-cGMP-protein kinase G. Furthermore, NO reduces glutamate release from primary afferent terminals through S-nitrosylation of voltage-activated Ca(2+) channels. Both of these actions probably contribute to inhibition of nociceptive transmission by NO at the spinal level.

Differential effects of mGluR7 and mGluR8 activation on pain-related synaptic activity in the amygdala

Pain-related plasticity in the laterocapsular division of the central nucleus of the amygdala (CeLC) depends on the activation of group I metabotropic glutamate receptors (mGluRs) whereas groups II and III mGluRs generally serve inhibitory functions. Recent evidence suggests differential roles of group III subtypes mGluR7 (pain enhancing) and mGluR8 (pain inhibiting) in the amygdala (Palazzo et al., 2008). Here we addressed the underlying synaptic mechanisms of mGluR7 and mGluR8 function in the CeLC under normal conditions and in an arthritis pain model. Using patch-clamp recordings in rat brain slices, we measured monosynaptic excitatory post-synaptic currents (EPSCs), mono- and polysynaptic inhibitory synaptic currents (IPSCs), and synaptically evoked action potentials (E-S coupling) in CeLC neurons. Synaptic responses were evoked by electrical stimulation in the basolateral amygdala (BLA). A selective mGluR8 agonist (DCPG) inhibited evoked EPSCs and synaptic spiking more potently in slices from arthritic rats than in slices from normal rats. In contrast, a selective mGluR7 agonist (AMN082) increased EPSCs and E-S coupling in slices from normal rats but not in the pain model. The effects of AMN082 and DCPG were blocked by a group III antagonist (MAP4). AMN082 increased frequency, but not amplitude, of spontaneous EPSCs but had no effect on miniature EPSCs (in TTX). DCPG decreased frequency, but not amplitude, of spontaneous and miniature EPSCs. The data suggest that mGluR8 acts presynaptically to inhibit excitatory transmission whereas the facilitatory effects of mGluR7 are indirect through action potential-dependent network action. AMN082 decreased evoked IPSCs and frequency, but not amplitude, of spontaneous and miniature IPSCs in slices from normal rats. DCPG had no effect on inhibitory transmission. The results suggest that presynaptic mGluR7 inhibits inhibitory synaptic transmission to gate glutamatergic transmission to CeLC neurons under normal conditions but not in pain. Presynaptic mGluR8 inhibits pain-related enhanced excitatory transmission in the CeLC.

MGluR1, but not mGluR5, activates feed-forward inhibition in the medial prefrontal cortex to impair decision making

Cognitive flexibility depends on the integrity of the prefrontal cortex (PFC). We showed previously that impaired decision making in pain results from amygdala-driven inhibition of medial PFC neurons, but the underlying mechanisms remain to be determined. Using whole cell patch clamp in rat brain slices and a cognitive behavioral task, we tested the hypothesis that group I metabotropic glutamate receptors (mGluRs) activate feed-forward inhibition to decrease excitability and output function of PFC pyramidal cells, thus impairing decision making. Polysynaptic inhibitory postsynaptic currents (IPSCs) and monosynaptic excitatory postsynaptic currents (EPSCs) were evoked in layer V pyramidal cells by stimulating presumed amygdala afferents. An mGluR1/5 agonist [(S)-3,5-dihydroxyphenylglycine, DHPG] increased synaptic inhibition more strongly than excitatory transmission. The facilitatory effects were blocked by an mGluR1 [(S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid, LY367385], but not mGluR5, antagonist, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine. IPSCs were blocked by bicuculline and decreased by 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX). Facilitation of synaptic inhibition by DHPG was glutamate driven because it was blocked by NBQX. DHPG increased frequency but not amplitude of spontaneous IPSCs; consistent with action potential-dependent synaptic inhibition, tetrodotoxin (TTX) prevented the facilitatory effects. DHPG decreased synaptically evoked spikes (E-S coupling) and depolarization-induced spiking [frequency-current (f-I) relationship]. This effect was indirect, resulting from glutamate-driven synaptic inhibition, because it persisted when a G protein blocker was included in the pipette but was blocked by GABA(A) receptor antagonists and NBQX. In contrast, DHPG increased E-S coupling and f-I relationships in mPFC interneurons through a presynaptic action, further supporting the concept of feed-forward inhibition. DHPG also impaired the ability of the animals to switch strategies in a decision-making task; bicuculline restored normal decision making, whereas a GABA(A) receptor agonist (muscimol) mimicked the decision-making deficit. The results show that mGluR1 activates feed-forward inhibition of PFC pyramidal cells to impair cognitive functions.

Kv1.3 channels regulate synaptic transmission in the nucleus of solitary tract

The voltage-gated K(+) channel Kv1.3 has been reported to regulate transmitter release in select central and peripheral neurons. In this study, we evaluated its role at the synapse between visceral sensory afferents and secondary neurons in the nucleus of the solitary tract (NTS). We identified mRNA and protein for Kv1.3 in rat nodose ganglia using RT-PCR and Western blot analysis. In immunohistochemical experiments, anti-Kv1.3 immunoreactivity was very strong in internal organelles in the soma of nodose neurons with a weaker distribution near the plasma membrane. Anti-Kv1.3 was also identified in the axonal branches that project centrally, including their presynaptic terminals in the medial and commissural NTS. In current-clamp experiments, margatoxin (MgTx), a high-affinity blocker of Kv1.3, produced an increase in action potential duration in C-type but not A- or Ah-type neurons. To evaluate the role of Kv1.3 at the presynaptic terminal, we examined the effect of MgTx on tract evoked monosynaptic excitatory postsynaptic currents (EPSCs) in brain slices of the NTS. MgTx increased the amplitude of evoked EPSCs in a subset of neurons, with the major increase occurring during the first stimuli in a 20-Hz train. These data, together with the results from somal recordings, support the hypothesis that Kv1.3 regulates the duration of the action potential in the presynaptic terminal of C fibers, limiting transmitter release to the postsynaptic cell.

Functional Plasticity of Group II Metabotropic Glutamate Receptors in Regulating Spinal Excitatory and Inhibitory Synaptic Input in Neuropathic Pain

Metabotropic glutamate receptors (mGluRs) are involved in the modulation of synaptic transmission and plasticity. Group II mGluRs in the spinal cord regulate glutamatergic input, but their functional changes in neuropathic pain are not clear. In this study, we determined the plasticity of spinal group II mGluRs in controlling excitatory and inhibitory synaptic transmission and nociception in neuropathic pain. Neuropathic pain was induced by spinal nerve ligation in rats, and whole-cell voltage-clamp recordings of glutamatergic excitatory postsynaptic currents (EPSCs) and spontaneous and miniature GABAergic and glycinergic inhibitory postsynaptic currents (sIPSCs and mIPSCs, respectively) were performed in spinal cord slices. The specific group II mGluR agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) had a similar inhibitory effect on monosynaptic EPSCs evoked from the dorsal root in sham and nerve-injured rats. However, DCG-IV produced a greater inhibitory effect on evoked polysynaptic EPSCs and the frequency of spontaneous EPSCs in nerve-injured rats than in control rats. Although DCG-IV similarly reduced the frequency of GABAergic sIPSCs and mIPSCs in both groups, it distinctly inhibited the frequency of glycinergic sIPSCs and mIPSCs only in nerve-injured rats. The DCG-IV effect was blocked by the group II mGluR antagonist but not by the N-methyl-D-aspartate receptor antagonist. Strikingly, intrathecal injection of DCG-IV dose-dependently attenuated allodynia and hyperalgesia in nerve-injured rats but produced hyperalgesia in control rats. Our study provides new information that nerve injury up-regulates group II mGluRs present on glutamatergic and glycinergic interneurons in the spinal cord. Activation of group II mGluRs reduces neuropathic pain probably by attenuating glutamatergic and glycinergic input to spinal dorsal horn neurons.

Spinal hyperpolarization-activated cyclic nucleotide-gated cation channels at primary afferent terminals contribute to chronic pain

Hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN channels) have large influences upon neuronal excitability. However, the participation of spinal HCN channels in chronic pain states, where pathological conditions are related to altered neuronal excitability, has not been clarified. Intraperitoneally (i.p.) or intrathecally (i.t.) administered ZD7288, a selective blocker of Ih channels, reduced thermal and mechanical hypersensitivity in mice under neuropathic conditions induced by the partial ligation of the sciatic nerve, while no analgesic effect was observed in naïve animals. Moreover, in the mouse formalin test, ZD7288 (i.p. and i.t.) reduced the licking/biting behavior observed during the second phase without affecting the first phase. To further explore the pain-modulatory action of spinal HCN channels, whole-cell patch clamp recordings were made from the visually identified substantia gelatinosa neurons in adult mouse spinal cord slices with an attached dorsal root, and A-fiber- and/or C-fiber-mediated monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the L4 or L5 dorsal root using a suction electrode. Bath-applied ZD7288 reduced A-fiber- and C-fiber-mediated monosynaptic EPSCs more preferentially in slices prepared from mice after peripheral nerve injury. In addition, ZD7288 reduced the frequency of miniature EPSCs without affecting their amplitude in cells receiving monosynaptic afferent inputs, indicating that it inhibits EPSCs via presynaptic mechanisms. The present behavioral and electrophysiological data suggest that spinal HCN channels, most likely at the primary afferent terminals, contribute to the maintenance of chronic pain.

The role of main intercalated nucleus of the mouse amygdala

Event Abstract Back to Event The role of main intercalated nucleus of the mouse amygdala M. Manko1*, R. Geracitano1 and M. Capogna1 1 Anatomical Neuropharmacology Unit, United Kingdom Intercalated cell masses (ITC) of the amygdala are clusters of GABAergic cells that surround the basolateral complex of the amygdala (BLA). There is growing evidence suggesting that ITCs contributes to fear expression and memory1. Our study aims to characterize for the first time the main intercalated nucleus (Im), the largest of the ITCs cluster, located ventrally to the BLA. We studied in acute mice slices the electrophysiological responses of anatomically identified neurons of the Im and the effects of dopamine, since this neuromodulator gates fear learning2. We found that the Im cluster consists of medium- sized, spiny neurons with bipolar dendritic organization and with axon that project to the majority of other amygdala nuclei and the intermediate capsula (IC). Whole cell patch clamp recordings (n=50) show passive and active membrane responses consistent with those previously detected in other clusters of ITCs3,4. We also functionally studied Im cells’ inputs/outputs. Monosynaptic excitatory postsynaptic currents were observed when we stimulated extracellularly the Im (n=6/6), the intermediate capsula (n=2/6), the external capsula (EC, n=3/6), or the BLA (n=2/6). An occlusion test indicated that fibres recruited by stimulating Im and IC, or Im and EC are distinct. Monosynaptic inhibitory postsynaptic currents were always observed after stimulation of the Im (n=6/6), but also of the IC (n=2/7) or EC (n=3/7), indicating functional connectivity among ITC clusters. Furthermore, dopamine (30 microMol) significantly hyperpolarised and decreased the firing frequency of Im cells (n=11) in a reversible manner. Thus, we propose that the Im cells, and not only mITCs, may be a key neuronal substrate of fear extinction.

Pain-related increase of excitatory transmission and decrease of inhibitory transmission in the central nucleus of the amygdala are mediated by mGluR1.

Neuroplasticity in the central nucleus of the amygdala (CeA), particularly its latero-capsular division (CeLC), is an important contributor to the emotional-affective aspects of pain. Previous studies showed synaptic plasticity of excitatory transmission to the CeLC in different pain models, but pain-related changes of inhibitory transmission remain to be determined. The CeLC receives convergent excitatory inputs from the parabrachial nucleus in the brainstem and from the basolateral amygdala (BLA). In addition, feedforward inhibition of CeA neurons is driven by glutamatergic projections from the BLA area to a cluster of GABAergic neurons in the intercalated cell masses (ITC). Using patch-clamp in rat brain slices we measured monosynaptic excitatory postsynaptic currents (EPSCs) and polysynaptic inhibitory currents (IPSCs) that were evoked by electrical stimulation in the BLA. In brain slices from arthritic rats, input-output functions of excitatory synaptic transmission were enhanced whereas inhibitory synaptic transmission was decreased compared to control slices from normal untreated rats. A non-NMDA receptor antagonist (NBQX) blocked the EPSCs and reduced the IPSCs, suggesting that non-NMDA receptors mediate excitatory transmission and also contribute to glutamate-driven feed-forward inhibition of CeLC neurons. IPSCs were blocked by a GABAA receptor antagonist (bicuculline). Bicuculline increased EPSCs under normal conditions but not in slices from arthritic rats, which indicates a loss of GABAergic control of excitatory transmission. A metabotropic glutamate receptor subtype 1 (mGluR1) antagonist (LY367385) reversed both the increase of excitatory transmission and the decrease of inhibitory transmission in the arthritis pain model but had no effect on basal synaptic transmission in control slices from normal rats. The inhibitory effect of LY367385 on excitatory transmission was blocked by bicuculline suggesting the involvement of a GABAergic mechanism. An mGluR5 antagonist (MTEP) inhibited both excitatory and inhibitory transmission in slices from normal and from arthritic rats. The analysis of spontaneous and miniature EPSCs and IPSCs showed that mGluR1 acted presynaptically whereas mGluR5 had postsynaptic effects. In conclusion, mGluR1 rather than mGluR5 can account for the pain-related changes of excitatory and inhibitory synaptic transmission in the CeLC through a mechanism that involves inhibition of inhibitory transmission (disinhibition).

N-methyl-D-aspartate-dependent long-term potentiation of excitatory transmission in trigeminal subnucleus oralis

This study for the first time demonstrates early developmental changes of passive/active membrane properties, and long-term potentiation (LTP) of excitatory synaptic transmission at spinal trigeminal subnucleus caudalis (Vc)-to-oralis (Vo) synapses. During postnatal development, the probability of Vo neurons with monosynaptic excitatory postsynaptic currents (EPSCs) upon Vc stimulation significantly increased, whereas the input resistances of Vo neurons and the latencies of monosynaptic EPSCs significantly decreased. Application of a 'pairing' protocol that comprises 2 Hz-conditioning stimulation of Vc with postsynaptic depolarization of Vo neuron to +30 mV generated LTP of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated monosynaptic EPSC amplitude in more than 70% of Vo neurons. The induction of LTP required the activation of N-methyl-D-aspartate receptor, but its magnitudes had correlation neither with postnatal ages nor with baseline EPSC amplitudes.

Regulation of cholinergic phenotype in developing neurons.

Specification of neurotransmitter phenotype is critical for neural circuit development and is influenced by intrinsic and extrinsic factors. Recent findings in rat hypothalamus in vitro suggest the role of neurotransmitter glutamate in the regulation of cholinergic phenotype. Here we extended our previous studies on the mechanisms of glutamate-dependent regulation of cholinergic phenotypic properties in hypothalamic neurons. Using immunocytochemistry, electrophysiology, and calcium imaging, we demonstrate that hypothalamic expression of choline acetyltransferase (the cholinergic marker) and responsiveness of neurons to acetylcholine (ACh) receptor agonists increase during chronic administration of an N-methyl-D-aspartate receptor (NMDAR) blocker, MK-801, in developing rats in vivo and genetic and pharmacological inactivation of NMDARs in mouse and rat developing neuronal cultures. In hypothalamic cultures, an inactivation of NMDA receptors also induces ACh-dependent synaptic activity, as do inactivations of PKA, ERK/MAPK, CREB, and NF-kappaB, which are known to be regulated by NMDA receptors. Interestingly, the increase in cholinergic properties in developing neurons that is induced by NMDAR blockade is prevented by the blockade of ACh receptors, suggesting that function of ACh receptor is required for the cholinergic up-regulation. Using dual recording of monosynaptic excitatory postsynaptic currents, we further demonstrate that chronic inactivation of ionotropic glutamate receptors induces the cholinergic phenotype in a subset of glutamatergic neurons. The phenotypic switch is partial as ACh and glutamate are coreleased. The results suggest that developing neurons may not only coexpress multiple transmitter phenotypes, but can also change the phenotypes following changes in signaling in neuronal circuits.

Optical and electrophysiological recordings of corticospinal synaptic activity and its developmental change in in vitro rat slice co-cultures

Electrophysiological recordings and optical imaging with a fast voltage-sensitive dye (di-4-ANNEPS) were used to directly examine the spatiotemporal properties of in vitro corticospinal synapses formed in co-cultures of cerebral cortex and spinal cord slices. Whole cell recordings from spinal cord cells showed both monosynaptic and polysynaptic excitatory postsynaptic currents (EPSCs) in response to stimulation of corticospinal axons. Monosynaptic EPSCs and excitatory postsynaptic potentials (EPSPs) were isolated in artificial cerebrospinal fluid containing high concentrations of divalent cations. Optical imaging and extracellular recordings were done simultaneously. Both EPSPs and optically recorded excitatory postsynaptic potentials (optEPSPs) lasted 300–500 ms and were almost always positive. The major component of these long-lasting potentials was blocked by ifenprodil, a specific antagonist of the NR2B subunit-containing N-methyl- d-aspartate receptor (NMDAR). The spatial distribution of corticospinal optEPSPs paralleled that of the corticospinal field excitatory postsynaptic potentials (fEPSPs), suggesting that positive fEPSP amplitude is a reliable indicator of the distribution of corticospinal synapses. Corticospinal optEPSPs spread into the ventrolateral region by 6–7 days in vitro (DIV), but were restricted to the dorsomedial area by 11–13 DIV, suggesting synapses were eliminated from the ventrolateral side of the spinal cord. After the recordings were complete, corticospinal fibers were often anterogradely labeled with biocytin to assess the relation between presynaptic fiber distribution and the optical signals (optically-recorded presynaptic fiber volley (opt-prevolley) and optEPSP). The distributions of the opt-prevolleys and optEPSPs correlated well with the distribution of presynaptic fibers, suggesting the opt-prevolley reflects corticospinal fiber activity and that the fibers made synapses relatively evenly along their axons. The NR2B-mediated component of the corticospinal synaptic response declined during the interval between 6 and 7 DIV and 11–13 DIV, suggesting that a shift in the NMDAR subtype from NR2B to something else (perhaps NR2A) may be involved in regulating developmental plasticity in the rat spinal cord and the process of corticospinal synapse elimination.

Differential activation of protein kinases in the dorsal horn in vitro of normal and inflamed rats by group I metabotropic glutamate receptor subtypes

Group I metabotropic glutamate receptors (mGluRs) contribute to spinal sensitization and synaptic plasticity but the underlying mechanisms are unknown. Here, group I mGluR modulation of evoked monosynaptic excitatory postsynaptic currents (EPSCs) in substantia gelatinosa (SG) neurones in vitro was investigated in juvenile rats. In addition, the role of group I mGluRs in dorsal horn neuronal Fos expression was determined in tetrodotoxin (TTX)-treated in vitro spinal cords of naïve rats and those with Complete Freund's Adjuvant (CFA) peripheral inflammation. In the majority of SG neurones, ( S)-3,5-Dihydroxyphenylglycine (DHPG) reduced EPSCs and this effect was inhibited by the mGluR 5 antagonist 2-Methyl-6-(phenylethynyl)-pyridine (MPEP). Data for paired-pulse and spontaneous miniature excitatory postsynaptic currents (mEPSCs) suggest mGluR 5 acts presynaptically to reduce transmitter release. DHPG-induced reduction of EPSC amplitude operated via PKC, but not ERK, signalling cascade. In the dorsal horn of naïve but not CFA rats, DHPG increased Fos expression and this was reduced by MPEP and both PKC and ERK inhibitors. In the CFA group, basal Fos expression was reduced by MPEP and the kinase inhibitors. These data infer a role for mGluR 5 in acute modulation of nociceptive synaptic efficacy within the dorsal horn and postsynaptic activation of transcription factors such as Fos that are implicated in activity-dependent neuroplastic adaptation. These actions are achieved by differential activation of PKC- and ERK-dependent transduction pathways.

GABAergic and Pyramidal Neurons of Deep Cortical Layers Directly Receive and Differently Integrate Callosal Input

We studied the involvement of deep cortical layer neurons in processing callosal information in the rat. We observed with electron microscopy that both parvalbumin (PV)-labeled profiles and unlabeled dendritic spines of deep cortical layer neurons receive synapses from the contralateral hemisphere. Stimulation of callosal fibers elicited monosynaptic excitatory postsynaptic currents in both layer VI pyramidal neurons and gamma-aminobutyric acidergic (GABAergic) interneurons immunopositive for the vesicular GABA transporter and PV. Pyramidal cells had intrinsic electrophysiological properties and synaptic responses with slow kinetics and a robust N-metyhl-D-aspartate (NMDA) component. In contrast, GABAergic interneurons had intrinsic membrane properties and synaptic responses with faster kinetics and a less pronounced NMDA component. Consistent with these results, the temporal integration of callosal input was effective over a significantly longer time window in pyramidal neurons compared with GABAergic interneurons. Interestingly, callosal stimulation did not evoke feedforward inhibition in all GABAergic interneurons and in the majority of pyramidal neurons tested. Furthermore, retrogradely labeled layer VI pyramidal neurons of the contralateral cortex responded monosynaptically to callosal stimulation, suggesting interconnectivity between callosally projecting neurons. The data show that pyramidal neurons and GABAergic interneurons of deep cortical layers receive interhemispheric information directly and have properties supporting their distinct roles.