Long-term effects of brain-derived neurotrophic factor on the frequency of inhibitory synaptic events in the rat superficial dorsal horn

Chronic constriction injury (CCI) of rat sciatic nerve produces a specific pattern of electrophysiological changes in the superficial dorsal horn that lead to central sensitization that is associated with neuropathic pain. These changes can be recapitulated in spinal cord organotypic cultures by long term (5–6 days) exposure to brain-derived neurotrophic factor (BDNF) (200 ng/ml). Certain lines of evidence suggest that both CCI and BDNF increase excitatory synaptic drive to putative excitatory neurons while reducing that to putative inhibitory interneurons. Because BDNF slows the rate of discharge of synaptically-driven action potentials in inhibitory neurons, it should also decrease the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) throughout the superficial dorsal horn. To test this possibility, we characterized superficial dorsal horn neurons in organotypic cultures according to five electrophysiological phenotypes that included tonic, delay and irregular firing neurons. Five to 6 days of treatment with 200 ng/ml BDNF decreased sIPSC frequency in tonic and irregular neurons as might be expected if BDNF selectively decreases excitatory synaptic drive to inhibitory interneurons. The frequency of sIPSCs in delay neurons was however increased. Further analysis of the action of BDNF on tetrodotoxin-resistant miniature inhibitory postsynaptic currents (mIPSC) showed that the frequency was increased in delay neurons, unchanged in tonic neurons and decreased in irregular neurons. BDNF may thus reduce action potential frequency in those inhibitory interneurons that project to tonic and irregular neurons but not in those that project to delay neurons.     

Neuron type-specific effects of brain-derived neurotrophic factor in rat superficial dorsal horn and their relevance to 'central sensitization'

Chronic constriction injury (CCI) of the rat sciatic nerve increases the excitability of the spinal dorsal horn. This 'central sensitization' leads to pain behaviours analogous to human neuropathic pain. We have established that CCI increases excitatory synaptic drive to putative excitatory, 'delay' firing neurons in the substantia gelatinosa but attenuates that to putative inhibitory, 'tonic' firing neurons. Here, we use a defined-medium organotypic culture (DMOTC) system to investigate the long-term actions of brain-derived neurotrophic factor (BDNF) as a possible instigator of these changes. The age of the cultures and their 5–6 day exposure to BDNF paralleled the protocol used for CCI in vivo . Effects of BDNF (200 ng ml⁻¹) in DMOTC were reminiscent of those seen with CCI in vivo. These included decreased synaptic drive to 'tonic' neurons and increased synaptic drive to 'delay' neurons with only small effects on their membrane excitability. Actions of BDNF on 'delay' neurons were exclusively presynaptic and involved increased mEPSC frequency and amplitude without changes in the function of postsynaptic AMPA receptors. By contrast, BDNF exerted both pre- and postsynaptic actions on 'tonic' cells; mEPSC frequency and amplitude were decreased and the decay time constant reduced by 35%. These selective and differential actions of BDNF on excitatory and inhibitory neurons contributed to a global increase in dorsal horn network excitability as assessed by the amplitude of depolarization-induced increases in intracellular Ca²⁺. Such changes and their underlying cellular mechanisms are likely to contribute to CCI-induced 'central sensitization' and hence to the onset of neuropathic pain.     

Cannabinoid-induced presynaptic inhibition of glutamatergic EPSCs in substantia gelatinosa neurons of the rat spinal cord.

The effect of cannabinoids on excitatory transmission in the substantia gelatinosa was investigated using intracellular recording from visually identified neurons in a transverse slice preparation of the juvenile rat spinal cord. In the presence of strychnine and bicuculline, perfusion of the cannabinoid receptor agonist WIN55,212-2 reduced the frequency and the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Furthermore, the frequency of miniature EPSCs (mEPSCs) was also decreased by WIN55,212-2, whereas their amplitude was not affected. Similar effects were reproduced using the endogenous cannabinoid ligand anandamide. The effects of both agonists were blocked by the selective CB(1) receptor antagonist SR141716A. Electrical stimulation of high-threshold fibers in the dorsal root evoked a monosynaptic EPSC in lamina II neurons. In the presence of WIN55,212-2, the amplitude of the evoked EPSC (eEPSCs) was reduced, and the paired-pulse ratio was increased. The reduction of the eEPSC following CB(1) receptor activation was unlikely to have a postsynaptic origin because the response to AMPA, in the presence of 1 microM TTX, was unchanged. To investigate the specificity of this synaptic inhibition, we selectively activated the nociceptive C fibers with capsaicin, which induced a strong increase in the frequency of EPSCs. In the presence of WIN55,212-2, the response to capsaicin was diminished. In conclusion, these results strongly suggest a presynaptic location for CB(1) receptors whose activation results in inhibition of glutamate release in the spinal dorsal horn. The strong inhibitory effect of cannabinoids on C fibers may thereby contribute to the modulation of the spinal excitatory transmission, thus producing analgesia at the spinal level.     

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- N ⁶-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.     

Opioid-like actions of neuropeptide Y in rat substantia gelatinosa: Y1 suppression of inhibition and Y2 suppression of excitation

Neuropathic pain that results from injury to the peripheral or CNS responds poorly to opioid analgesics. Y1 and Y2 receptors for neuropeptide Y (NPY) may, however, serve as targets for analgesics that retain their effectiveness in neuropathic pain states. In substantia gelatinosa neurons in spinal cord slices from adult rats, we find that NPY acts via presynaptic Y2 receptors to attenuate excitatory postsynaptic currents (EPSCs) and predominantly on presynaptic Y1 receptors to attenuate glycinergic and GABAergic inhibitory postsynaptic currents (IPSCs). Because NPY attenuates the frequency of TTX-resistant miniature EPSCs and IPSCs, perturbation of the neurotransmitter release process contributes to its actions at both excitatory and inhibitory synapses. These effects, which are reminiscent of those produced by analgesic opioids, provide a cellular basis for previously documented spinal analgesic actions mediated via Y1 and Y2 receptors in neuropathic pain paradigms. They also underline the importance of suppression of inhibition in spinal analgesic mechanisms.     

Nociceptin inhibits excitatory but not inhibitory transmission to substantia gelatinosa neurones of adult rat spinal cord.

Although intrathecal administration of nociceptin, an endogenous ligand of the opioid receptor-like1 receptor, exhibits an antinociceptive effect in various pain models, cellular mechanisms underlying this action are still unknown. Here, we investigated the effects of nociceptin on excitatory and inhibitory synaptic transmission to substantia gelatinosa neurones of an adult rat spinal cord slice with an attached dorsal root by use of the blind whole-cell patch-clamp technique; this was done under the condition of a blockade of a hyperpolarising effect of nociceptin. In about 70% of the neurones examined, nociceptin (1 microM) reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) which were monosynaptically evoked by stimulating Adelta- or C-afferent fibres; the inhibition of C-fibre EPSCs (50+/-6%, n=11) was larger than that of Adelta-fibre EPSCs (30+/-5%, n=23; P<0.05). Each of the nociceptin actions was dose-dependent in a concentration range of 0.1 to 1 microM, and was largely suppressed by a selective opioid receptor-like1 receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (3 microM). Nociceptin (1 microM) also decreased miniature EPSCs frequency by 22+/-6% (n=7) while not affecting their amplitude. Responses of substantia gelatinosa neurones to bath-applied alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (10 microM) were not changed by nociceptin. Both electrically evoked and miniature inhibitory postsynaptic currents, mediated by either the GABA(A) or glycine receptor, were unaffected by nociceptin.These results indicate that nociceptin suppresses excitatory but not inhibitory synaptic transmission to substantia gelatinosa neurones through the activation of the opioid receptor-like1 receptor; this action is pre-synaptic in origin. Considering that the substantia gelatinosa is the main part of termination of Adelta- and C-fibres transmitting nociceptive information, the present finding would account for at least a part of the inhibitory action of nociceptin on pain transmission. Nociceptin could inhibit more potently slow-conducting than fast-conducting pain transmission.     

Long-term actions of BDNF on inhibitory synaptic transmission in identified neurons of the rat substantia gelatinosa

Peripheral nerve injury promotes the release of brain-derived neurotrophic factor (BDNF) from spinal microglial cells and primary afferent terminals. This induces an increase in dorsal horn excitability that contributes to "central sensitization" and to the onset of neuropathic pain. Although it is accepted that impairment of GABAergic and/or glycinergic inhibition contributes to this process, certain lines of evidence suggest that GABA release in the dorsal horn may increase after nerve injury. To resolve these contradictory findings, we exposed rat spinal cord neurons in defined-medium organotypic culture to 200 ng/ml BDNF for 6 days to mimic the change in spinal BDNF levels that accompanies peripheral nerve injury. Morphological and electrophysiological criteria and glutamic acid decarboxylase (GAD) immunohistochemistry were used to distinguish putative inhibitory tonic-islet-central neurons from putative excitatory delay-radial neurons. Whole cell recording in the presence of 1 μM tetrodotoxin showed that BDNF increased the amplitude of GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs) in both cell types. It also increased the amplitude and frequency of spontaneous, action potential-dependent IPSCs (sIPSCs) in putative excitatory neurons. By contrast, BDNF reduced sIPSC amplitude in inhibitory neurons but frequency was unchanged. This increase in inhibitory drive to excitatory neurons and decreased inhibitory drive to inhibitory neurons seems inconsistent with the observation that BDNF increases overall dorsal horn excitability. One of several explanations for this discrepancy is that the action of BDNF in the substantia gelatinosa is dominated by previously documented increases in excitatory synaptic transmission rather than by impediment of inhibitory transmission.     

Phospholipase A2 activation by melittin enhances spontaneous glutamatergic excitatory transmission in rat substantia gelatinosa neurons

In order to know a role of phospholipase A2 in modulating nociceptive transmission, the effect of a secreted phospholipase A2 activator melittin on spontaneous glutamatergic excitatory transmission was investigated in substantia gelatinosa neurons of an adult rat spinal cord slice by using the whole-cell patch-clamp technique. Bath-applied melittin at concentrations higher than 0.5 microM increased both the amplitude and the frequency of spontaneous excitatory postsynaptic current in a manner independent of tetrodotoxin; the latter effect of which was examined in detail. In 80% of the neurons examined (n = 64), melittin superfused for 3 min gradually increased spontaneous excitatory postsynaptic current frequency (by 65+/-6% at 1 microM; n = 51) in a dose-dependent manner (effective concentration for half-maximal effect = 1.1 microM). This effect subsided within 3 min after washout. The spontaneous excitatory postsynaptic current frequency increase produced by melittin was reduced by the phospholipase A2 inhibitor 4-bromophenacryl bromide (10 microM) while being unaffected by the cyclooxygenase inhibitor indomethacin (100 microM) and the lipoxygenase inhibitor nordihydroguaiaretic acid (100 microM). A similar increase in spontaneous excitatory postsynaptic current frequency was produced by exogenous arachidonic acid (50 microM); this effect was also unaffected by the cyclooxygenase or lipoxygenase inhibitor. Melittin failed to increase spontaneous excitatory postsynaptic current frequency in a nominally Ca2+-free or La3+-containing Krebs solution. We conclude that melittin increases the spontaneous release of L-glutamate to substantia gelatinosa neurons by activating secreted phospholipase A2 and increasing Ca2+ influx through voltage-gated Ca2+ channels in nerve terminals, probably with an involvement of arachidonic acid but not its metabolites produced by cyclooxygenase and lipoxygenase. Considering that the substantia gelatinosa plays an important role in regulating nociceptive transmission, it is suggested that this transmission may be positively modulated by secreted phospholipase A2 activation in the substantia gelatinosa.     

Action of neuropeptide Y on nociceptive transmission in substantia gelatinosa of the adult rat spinal dorsal horn

Effects of neuropeptide Y (NPY) on substantia gelatinosa neurons were investigated in adult rat spinal cord slices using blind whole-cell patch-clamp technique. Bath application of NPY (1 microM) induced a membrane hyperpolarization, resulting in a suppression of the dorsal root stimulation-induced action potentials in 24% of the substantia gelatinosa neurons tested. In voltage clamp mode, NPY produced an outward current dose-dependently in about one third of substantia gelatinosa neurons at the holding potential of -60 mV, which was not affected by tetrodotoxin (1 microM). The NPY-induced current was suppressed by perfusion with a Ba2+-containing external solution and a Cs2SO4 or tetraethylammonium-containing pipette solution. In addition, The NPY-induced outward currents reversed its polarity near the equilibrium potential of K+ ions (-93 mV). The response to NPY recorded with guanosine-5'-O-(2-thiodiphosphate)-beta-S (GDP-beta-S) containing pipette solution was abolished 30 min after patch formation, suggesting that the response was mediated by the G-protein-coupled receptors. Application of an NPY-Y1 selective agonist, [Leu(31), Pro(-34)]-NPY (1 microM), for 30 s also induced an outward current with a similar time course and amplitude to that induced by NPY. On the other hand, the NPY response was blocked by a simultaneous application of NPY-Y1 selective antagonist, BIBP 3226 (1 microM). No significant changes were found in amplitude and frequency of miniature excitatory postsynaptic currents and dorsal root evoked excitatory postsynaptic currents by NPY. In addition, NPY did not affect both of the miniature inhibitory postsynaptic currents and evoked inhibitory postsynaptic currents, mediated by either the GABA or glycine receptor. These findings, taken together, suggest that NPY produces an outward current in substantia gelatinosa neurons through G-protein coupled, and NPY-Y1 receptor-mediated activation of K+ channels without affecting presynaptic components. The inhibition of the synaptic transmission from the primary fibers to the substantia gelatinosa neurons is considered to contribute to the antinociceptive effects of NPY.     

Postsynaptic action mechanism of somatostatin on the membrane excitability in spinal substantia gelatinosa neurons of juvenile rats

We used tight-seal, whole-cell recording in juvenile rat spinal slices to investigate the action of somatostatin on substantia gelatinosa neurons. Bath application of somatostatin caused a robust and repeatable hyperpolarization or outward current in substantia gelatinosa neurons. Somatostatin inhibited spontaneous action potentials in subpopulation of substantia gelatinosa neurons. The amplitude of dorsal root-evoked excitatory postsynaptic currents and the frequency of spontaneous excitatory postsynaptic currents were not affected by somatostatin. The current induced by somatostatin developed almost instantaneously and did not show any time-dependent inactivation. The current-voltage relationship exhibited inward rectification. The conductance of somatostatin-sensitive current increased with the concentration of external K(+). The reversal potentials in different external K(+) concentrations were close to the K(+) equilibrium potentials. The effect of somatostatin was dose-dependent, with an EC(50) of 113 nM. The somatostatin-sensitive current was blocked by low concentration of extracellular Ba(2+) but not by glibenclamide, an inhibitor of ATP-sensitive K(+) channels. Hyperpolarization-activated cation current in a subpopulation of substantia gelatinosa neurons was not affected by somatostatin. In neurons recorded with an internal solution containing GTPgammaS, somatostatin induced outward current and hyperpolarization that did not reverse on washing. When the spontaneous induction of outward current with GTPgammaS was greatest, somatostatin did not induce any outward currents. Furthermore, intracellular dialysis of GDPbetaS, a G-protein antagonist, abolished the effect of somatostatin. In addition, SST-sensitive neurons were fewer in slices incubated with pertussis toxin than in adjacent control slices incubated without pertussis toxin.These results suggest that somatostatin decreases the postsynaptic membrane excitability of substantia gelatinosa neurons by a pertussis toxin-sensitive G-protein-mediated activation of an inwardly rectifying K(+) conductance.     

Presynaptic angiotensin II AT1 receptors enhance inhibitory and excitatory synaptic neurotransmission to motoneurons and other ventral horn neurons in neonatal rat spinal cord

In neonatal spinal cord, we previously reported that exogenous angiotensin II (ANG II) acts at postsynaptic AT1 receptors to depolarize neonatal rat spinal ventral horn neurons in vitro. This study evaluated an associated increase in synaptic activity. Patch clamp recordings revealed that 38/81 thoracolumbar (T7-L5) motoneurons responded to bath applied ANG II (0.3-1 microM; 30 s) with a prolonged (5-10 min) and reversible increase in spontaneous postsynaptic activity, selectively blockable with Losartan (n = 5) but not PD123319 (n = 5). ANG-II-induced events included both spontaneous inhibitory (IPSCs; n = 6) and excitatory postsynaptic currents (EPSCs; n = 5). While most ANG induced events were tetrodotoxin-sensitive, ANG induced a significant tetrodotoxin-resistant increase in frequency but not amplitude of miniature IPSCs (n = 7/13 cells) and EPSCs (n = 2/7 cells). In 35/77 unidentified neurons, ANG II also induced a tetrodotoxin-sensitive and prolonged increase in their spontaneous synaptic activity that featured both IPSCs (n = 5) and EPSCs (n = 4) when tested in the presence of selective amino acid receptor antagonists. When tested in the presence of tetrodotoxin, ANG II was noted to induce a significant increase in the frequency but not the amplitude of mIPSCs (n = 9) and mEPSCs (n = 8). ANG also increased spontaneous motor activity from isolated mouse lumbar ventral rootlets. Collectively, these observations support the existence of a wide pre- and postsynaptic distribution of ANG II AT1 receptors in neonatal ventral spinal cord that are capable of influencing both inhibitory and excitatory neurotransmission.     

In vivo patch-clamp analysis of IPSCs evoked in rat substantia gelatinosa neurons by cutaneous mechanical stimulation

To know a functional role of inhibitory synaptic responses in transmitting noxious and innoxious information from the periphery to the rat spinal dorsal horn, we examined inhibitory postsynaptic currents (IPSCs) elicited in substantia gelatinosa (SG) neurons by mechanical stimuli applied to the skin using the newly developed in vivo patch-clamp technique. In the majority (80%) of SG neurons examined, a brush stimulus applied to the ipsilateral hind limb produced a barrage of IPSCs that persisted during the stimulus, while a pinch stimulus evoked IPSCs only at its beginning and end. The pinch-evoked IPSCs may have been caused by a touch that occurs at the on/off time of the pinch. The evoked IPSCs were blocked by either a glycine-receptor antagonist, strychnine (4 microM), or a GABA(A)-receptor antagonist, bicuculline (20 microM). All SG neurons examined received inhibitory inputs from a wide area throughout the thigh and lower leg. When IPSCs were examined together with excitatory postsynaptic currents (EPSCs) in the same neurons, a brush evoked a persistent activity of both IPSCs and EPSCs during the stimulus while a pinch evoked such an activity of EPSCs but not IPSCs. It is suggested that innoxious mechanical stimuli activate a GABAergic or glycinergic circuitry in the spinal dorsal horn. This inhibitory transmission may play an important role in the modulation of noxious information in the SG.     

Tonic NMDA receptor-mediated current in prefrontal cortical pyramidal cells and fast-spiking interneurons

Tonically activated neuronal currents mediated by N-methyl-d-aspartate receptors (NMDARs) have been hypothesized to contribute to normal neuronal function as well as to neuronal pathology resulting from excessive activation of glutamate receptors (e.g., excitotoxicity). Whereas cortical excitatory cells are very vulnerable to excitotoxic insult, the data regarding resistance of inhibitory cells (or interneurons) are inconsistent. Types of neurons with more pronounced tonic NMDAR current potentially associated with the activation of extrasynaptic NMDARs could be expected to be more vulnerable to excessive activation by glutamate. In this study, we compared tonic activation of NMDARs in excitatory pyramidal cells and inhibitory fast-spiking interneurons in prefrontal cortical slices. We assessed tonic NMDAR current by measuring holding current shift as well as noise reduction following NMDAR blockade after removal of spontaneous glutamate release. In addition, we compared NMDAR miniature excitatory postsynaptic currents (EPSCs) in both cell types. We have demonstrated for the first time that tonic NMDAR currents are present in inhibitory fast-spiking interneurons. We found that the magnitude of tonic NMDAR current is similar in pyramidal cells and fast-spiking interneurons, and that quantal release of glutamate does not significantly impact tonic NMDAR current.     

内吗啡肽-1对成年大鼠脊髓胶状质内突触传递的抑制作用强于内吗啡肽-2(英文)

本实验采用全细胞电压钳记录方法,研究了内吗啡肽1(EM1)和内吗啡肽2(EM2)对脊髓背角胶状质神经元突触传递的抑制性作用。EM1(1μmol/L)和EM2(1μmol/L)都能够显著抑制微小兴奋性突触后电流(mEPSCs)和微小抑制性突触后电流(mIPSCs)的频率而不改变其幅值。这种抑制作用能被μ受体选择性拮抗剂βfunaltrexamine(βFNA,10μmol/L)阻断。值得注意的是,EM1对mEPSCs和mIPSCs的频率的抑制作用强于EM2。上述结果提示在脊髓胶状质,内吗啡肽通过激活突触前膜上的μ受体,抑制兴奋性和抑制性的突触传递;与EM2相比,EM1可能是脊髓水平的更强效的内源性镇痛剂。     

Dual-component miniature excitatory synaptic currents in rat hippocampal CA3 pyramidal neurons.

1. Spontaneous miniature synaptic events were studied with tight-seal whole-cell recordings from CA3 neurons maintained in the hippocampal slice from immature rats (3-15 days). CA3 neurons suffer a constant, high-frequency barrage of inhibitory synaptic input. When inhibitory postsynaptic currents were suppressed by bicuculline, a smaller contribution from excitatory synapses was revealed. 2. Addition of tetrodotoxin (TTX) removed a persistent inward current and substantially reduced the baseline noise facilitating the detection of "miniature" excitatory currents. Addition of hyperosmotic media increased the frequency of spontaneous excitatory postsynaptic currents (EPSCs). 3. Under both physiological and elevated potassium conditions, individual spontaneous miniature EPSCs (10-30 pA amplitude) were composed of components mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors as determined by their voltage dependence, time course, and sensitivity to selective antagonists. 6-Cyano-7-nitro-quinoxaline-2,3-dione (CNQX) or D-2-amino-5-phosphonovaleric acid (D-APV) shifted the amplitude distribution of miniature EPSCs to a smaller mode at both +40 mV and -40 mV. Similar to EPSCs recorded in CA1 neurons, the rise and decay times of the NMDA receptor component were slower than those of the non-NMDA component. The time course of the non-NMDA component was voltage independent. 4. In 13 of 21 neurons, no correlation existed between individual EPSC rise times and their corresponding halfwidth, peak amplitude, or decay time constant. This suggests that the large range of EPSC kinetics observed in each individual neuron was not due solely to cable attenuation of EPSCs widely distributed over the dendritic tree. Plots of the mean EPSC rise time against mean halfwidth for each cell, however, revealed a striking correlation, suggesting that in neonates, active synapses may be grouped in a restricted region of the dendritic tree and as such are subject to similar amounts of dendritic filtering. 5. The electrotonic length of CA3 neurons (L = 0.52) predicted that at this maturity the electrotonic compactness of the neuron facilitated voltage control over all but the most distal synapses. The reversal potential of the fast component of spontaneous events was close to 0 mV, whereas the reversal potential of exogenously applied kainate and NMDA was more positive. This discrepancy likely reflects a compromise of the voltage clamp by the activation of conductances distributed over the entire cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Wheat germ agglutinin enhances EPSCs in cultured postnatal rat hippocampal neurons by blocking ionotropic quisqualate receptor desensitization.

1. The effect of the lectin wheat germ agglutinin (WGA), an inhibitor of ionotropic quisqualate receptor desensitization, on both evoked and spontaneous fast excitatory postsynaptic events was examined in cultured postnatal rat hippocampal neurons with the use of whole cell recordings. 2. WGA, at 580 nM, potentiated evoked fast excitatory postsynaptic currents (EPSCs) by increasing the amplitudes by 100 +/- 27% (mean +/- SE) and the time constant of decay from 5.8 +/- 0.6 to 7.9 +/- 0.5 ms. The increases in these parameters were not accompanied by changes in the current-voltage (I-V) relationship or pharmacological profile of the fast EPSCs. 3. WGA did not alter the amplitude or time course of decay of inhibitory postsynaptic currents (IPSCs), and it did not alter neuronal input resistance or action potentials. 4. WGA increased the amplitude of spontaneous fast miniature EPSCs (MEPSCs), defined as spontaneous EPSCs recorded in the presence of tetrodotoxin, by 53 +/- 11% and increased the time required to decay to 50% of the peak amplitude by 48 +/- 23%. These changes were not associated with a change in the rate of MEPSC occurrence. 5. These results suggest that WGA augments hippocampal excitatory postsynaptic events via a postsynaptic mechanism. The results further imply that ionotropic quisqualate receptor desensitization can modulate the amplitude and time course of decay of fast excitatory synaptic events. Thus desensitization may be one factor that regulates fast excitatory synaptic transmission.     

Excitatory and inhibitory postsynaptic currents in a rat model of epileptogenic microgyria

Developmental cortical malformations are common in patients with intractable epilepsy; however, mechanisms contributing to this epileptogenesis are currently poorly understood. We previously characterized hyperexcitability in a rat model that mimics the histopathology of human 4-layered microgyria. Here we examined inhibitory and excitatory postsynaptic currents in this model to identify functional alterations that might contribute to epileptogenesis associated with microgyria. We recorded isolated whole cell excitatory postsynaptic currents and GABA(A) receptor-mediated inhibitory currents (EPSCs and IPSCs) from layer V pyramidal neurons in the region previously shown to be epileptogenic (paramicrogyral area) and in homotopic control cortex. Epileptiform-like activity could be evoked in 60% of paramicrogyral (PMG) cells by local stimulation. The peak conductance of both spontaneous and evoked IPSCs was significantly larger in all PMG cells compared with controls. This difference in amplitude was not present after blockade of ionotropic glutamatergic currents or for miniature (m)IPSCs, suggesting that it was due to the excitatory afferent activity driving inhibitory neurons. This conclusion was supported by the finding that glutamate receptor antagonist application resulted in a significantly greater reduction in spontaneous IPSC frequency in one PMG cell group (PMG(E)) compared with control cells. The frequency of both spontaneous and miniature EPSCs was significantly greater in all PMG cells, suggesting that pyramidal neurons adjacent to a microgyrus receive more excitatory input than do those in control cortex. These findings suggest that there is an increase in numbers of functional excitatory synapses on both interneurons and pyramidal cells in the PMG cortex perhaps due to hyperinnervation by cortical afferents originally destined for the microgyrus proper.     

Endomorphin-like immunoreactivity in the rat dorsal horn and inhibition of substantia gelatinosa neurons in vitro

Endomorphin 1 and 2 are two tetrapeptides recently isolated from bovine as well as human brains and proposed to be the endogenous ligand for the mu-opiate receptor. Opioid compounds expressing mu-receptor preference are generally potent analgesics. The spinal cord dorsal horn is considered to be an important site for the processing of sensory information including pain. The discovery that endomorphins produced greater analgesia in mice upon intrathecal as compared to intracerebroventricular injections raises the possibility that dorsal horn neurons may represent the anatomic site upon which endomorphins exert their analgesic effects. We report here the detection of endomorphin 2-immunuoreactive fiber-like elements in superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell patch recordings from substantia gelatinosa neurons of cervical spinal cord slices revealed two conspicuous effects of exogenously applied endomorphin 1 and 2: (i) depression of excitatory postsynaptic potentials evoked by stimulation of dorsal root entry zone, and (ii) hyperpolarization of substantia gelatinosa neurons. These effects were reversed by the selective mu-opiate receptor antagonist beta-funaltrexamine. Collectively, the detection of endomorphin-like immunoreactivity in nerve fibers of the superficial layers and the inhibitory action of endomorphins on substantia gelatinosa neurons provide further support for a potential role of these two peptides in spinal nociception.     

神经降压素对大鼠脊髓背角胶状质内突触前神经递质释放的影响

目的:研究内源性神经肽神经降压素(neurotensin,NT)在脊髓背角胶状质(substantia gelatinosa,SG)内对突触前神经递质释放的影响。方法:采用全细胞电压膜片钳记录方法,在脊髓薄片上观察NT对SG内微小兴奋性突触后电流(mEPSCs)和微小抑制性突触后电流(mIPSCs)的频率和幅值的影响。结果:(1)灌流NT(2μmol/L)对SG内神经元mEPSCs的频率和幅值均无明显影响,说明NT不影响SG内兴奋性神经递质的释放;(2)灌流NT(2μmol/L)能增加SG内神经元mIPSCs的频率,但对幅值无明显影响,即NT可引起突触前抑制性神经递质的释放增加,但对突触后神经元无明显影响。结论:NT可通过增加SG内抑制性神经递质释放的途径抑制伤害性信息的传递,从而实现镇痛效应。     

Glial glutamate transporter 1 regulates the spatial and temporal coding of glutamatergic synaptic transmission in spinal lamina II neurons

Glutamatergic synaptic transmission is a dynamic process determined by the amount of glutamate released by presynaptic sites, the clearance of glutamate in the synaptic cleft, and the properties of postsynaptic glutamate receptors. Clearance of glutamate in the synaptic cleft depends on passive diffusion and active uptake by glutamate transporters. In this study, we examined the role of glial glutamate transporter 1 (GLT-1) in spinal sensory processing. Excitatory postsynaptic currents (EPSCs) of substantia gelatinosa neurons recorded from spinal slices of young adult rats were analyzed before and after GLT-1 was pharmacologically blocked by dihydrokainic acid. Inhibition of GLT-1 prolonged the EPSC duration and the EPSC decay phase. The EPSC amplitudes were increased in neurons with weak synaptic input but decreased in neurons with strong synaptic input upon inhibition of GLT-1. We suggest that presynaptic inhibition, desensitization of postsynaptic AMPA receptors, and glutamate "spillover" contributed to the kinetic change of EPSCs induced by the blockade of GLT-1. Thus, GLT-1 is a key component in maintaining the spatial and temporal coding in signal transmission at the glutamatergic synapse in substantia gelatinosa neurons.