Long-term Potentiation in the Striatum is Unmasked by Removing the Voltage-dependent Magnesium Block of NMDA Receptor Channels

We have studied the effects of tetanic stimulation of the corticostriatal pathway on the amplitude of striatal excitatory synaptic potentials. Recordings were obtained from a corticostriatal slice preparation by utilizing both extracellular and intracellular techniques. Under the control condition (1.2 mM external Mg2+), excitatory postsynaptic potentials (EPSPs) evoked by cortical stimulation were reversibly blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) ionotropic glutamate receptors, while they were not affected by 30 - 50 microM 2-amino-5-phosphonovalerate (APV), an antagonist of N-methyl-d-aspartate (NMDA) glutamate receptors. In the presence of 1.2 mM external Mg2+, tetanic activation of cortical inputs produced long-term depression (LTD) of both extracellularly and intracellularly recorded synaptic potentials. When Mg2+ was removed from the external medium, EPSP amplitude and duration increased. In Mg2+-free medium, cortically evoked EPSPs revealed an APV-sensitive component; in this condition tetanic stimulation produced long-term potentiation (LTP) of synaptic transmission. Incubation of the slices in 30 - 50 microM APV blocked striatal LTP, while it did not affect LTD. In Mg2+-free medium, incubation of the slices in 10 microM CNQX did not block the expression of striatal LTP. Intrinsic membrane properties (membrane potential, input resistance and firing pattern) of striatal neurons were altered neither by tetanic stimuli inducing LTD and LTP, nor by removal of Mg2+ from the external medium. These findings show that repetitive activation of cortical inputs can induce long-term changes of synaptic transmission in the striatum. Under control conditions NMDA receptor channels are inactivated by the voltage-dependent Mg2+ block and repetitive cortical stimulation induces LTD which does not require activation of NMDA channels. Removal of external Mg2+ deinactivates these channels and reveals a component of the EPSP which is potentiated by repetitive activation. Since the striatum has been involved in memory and in the storage of motor skills, LTD and LTP of synaptic transmission in this structure may provide the cellular substrate for motor learning and underlie the physiopathology of some movement disorders.     

Synaptic interaction of individual motor neurons of the isolated frog spinal cord

The mode of synaptic transmission between single lumbar motoneurons in isolated spinal cord of frog was investigated by means of parallel penetration of two separate microelectrodes in two neighbouring motoneurons. The synaptic transmission between them was electrically mediated in 82 of 89 cases studied, which was indicated by the lack or very short latency of unitary intermotoneuronal EPSPs and by its amplitude persistence in Ca2+-free, Mn2+-containing solution. Effective electrotonic spread in either direction was demonstrated: both depo- and hyperpolarizing currents passed through an electrode in one motoneuron produced corresponding potentials in coupled motoneuron. The rise and decay of these potentials have much longer time constant as compared to time constant of both coupled motoneurons. The blockade of SD-component of an action potential in "trigger" motoneuron produced a decrease in the EPSP amplitude in the coupled motoneuron. Electrotonic synapses between motoneurons revealed no rectification. In four cases unitary intermotoneuronal EPSPs were chemically mediated as was indicated by their latencies (1.3-3.3 ms) and by their full blockade in CA2+-free, Mn2+-containing solution. The amplitude of this group of EPSPs fluctuated in accordance with binomial or Poisson statistics. In three cases double-component unitary intermotoneuronal EPSPs were recorded, first and second components of which were electrically and chemically mediated, respectively. Morphological structures which could be responsible for the generation of these three groups of unitary EPSPs are considered.     

Glutamate stimulation of tyrosine hydroxylase is mediated by NMDA receptors in the rat striatum.

We have studied the action of glutamate on striatal tyrosine hydroxylase activity and determined which type of glutamate receptors are involved. Glutamate stimulated (EC50 = 4 +/- 2 microM) the activity of tyrosine hydroxylase in slices of rat neostriatum. The selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (10 microM) blocked the stimulation; however, both the non-NMDA receptor antagonist glutamate diethyl ester (10 microM) and the general excitatory amino acid antagonist kynurenate (10 microM) had no effect. NMDA was even more potent than glutamate in stimulating tyrosine hydroxylase activity. Quisqualate (100 microM) only slightly stimulated the enzyme, and kainate had practically no effect. Omission of Mg2+ from the incubation medium potentiated the glutamate stimulation. Neither tetrodotoxin nor atropine prevented the stimulation. These results suggest that glutamate stimulates striatal tyrosine hydroxylase activity via NMDA receptors. The lack of effect of tetrodotoxin and atropine suggests that glutamate acts on NMDA receptors located on the dopaminergic nigrostriatal terminal. The stimulation may involve the entry of Ca2+ into the terminal through the NMDA receptor ionophore, since a Ca(2+)-free medium or cadmium totally blocked the stimulation of the enzyme by glutamate.     

Excitatory and inhibitory transmission from dorsal root afferents to neonate rat motoneurons in vitro

Intracellular recordings were made from antidromically identified motoneurons in neonate (12-22 days) rat transverse spinal cord slices and the transmitters and receptors probably involved in initiating the excitatory (EPSP) and inhibitory (IPSP) postsynaptic potentials were investigated. Stimulation of dorsal roots elicited in motoneurons an EPSP, an IPSP, or an EPSP followed by an IPSP. EPSPs in 70% of motoneurons had a short latency (less than or equal to 1 ms) and in the remaining cells a latency longer than 1 ms. The IPSPs had a long latency (greater than or equal to 1 ms). Short- and long-latency EPSPs were enhanced by the acidic amino acid uptake inhibitor L-aspartic acid-beta-hydroxamate (AAH) and depressed by the non-selective glutamate receptor antagonists gamma-D-glutamylglycine (DGG) and kynurenic acid. Short-latency EPSPs were suppressed by the quisqualate/kainate (QA/KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) but not by the N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino-5-phosphonovaleric acid (APV) and ketamine. Long-latency EPSPs were reduced by DNQX as well as by APV and ketamine. Superfusion of the slices with a Mg-free solution increased the EPSPs and unmasked a late, APV-sensitive component. The IPSP was reduced by the glycine antagonist strychnine as well as by APV and ketamine but resistant to DNQX. The results indicate that stimulation of dorsal roots elicited in motoneurons a monosynaptic EPSP mediated by glutamate/aspartate acting predominantly on the QA/KA subtype of glutamate receptors; an NMDA component can be unveiled in Mg-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate modulates neurotransmission in the submucosal plexus of guinea-pig small intestine.

Effects of glutamate on synaptic transmission in the submucosal plexus of guinea-pig small intestine were studied with intracellular electrophysiological recording methods. Glutamate suppressed stimulus-evoked slow excitatory postsynaptic potentials (EPSPs) and increased the amplitude of slow inhibitory postsynaptic potentials (IPSPs) in submucosal neurons. The actions of glutamate were mimicked by the group I metabotropic glutamate receptor (mGluRs) agonist DHPG, but not by the group II agonist S-4C3HPG, the group III agonist L-AP4, or selective agonists for ionotropic glutamate receptors (iGluRs). Glutamate actions were suppressed by the selective group I mGluRs antagonist S-4CPG, but not by group II and III mGluRs antagonist CPPG or iGluRs antagonists. Glutamate suppressed substance P- and 5-HT-evoked slow EPSP-like responses and potentiated norepinephrine-induced slow IPSP-like responses. The results suggest that group I mGluRs mediate glutamate-induced suppression of slow EPSPs and potentiation of slow IPSPs in S-type uniaxonal submucosal neurons.     

Modulation of EPSP amplitude during high frequency stimulation depends on the correlation between potentiation, depression and facilitation

High frequency bursts were delivered every 2 s to single group Ia fibers in anesthetized cats. The modulation of EPSP amplitude in target motoneurons was determined. Changes in EPSP amplitude during the high frequency stimulation (facilitation or depression) were correlated with those occurring 2 s after the burst (potentiation). Connections displaying high levels of potentiation exhibited more depression resulting in a net negative modulation. Negative amplitude modulation was greatest at connections on low rheobase ('small') motoneurons that develop large EPSPs. Positive modulation occurred at a subset of connections on high rheobase ('large') motoneurons that exhibit small EPSPs. We suggest that these synaptic properties permit the appropriate amount of depolarization to be delivered to the heterogeneous elements of the motoneuron pool during high frequency Ia fiber activity characteristic of the step cycle.     

Presynaptic inhibition of excitatory synaptic transmission by adenosine in rat hippocampus: analysis of unitary EPSP variance measured by whole-cell recording.

We have utilized the favorable signal-to-noise ratios provided by whole-cell recording, combined with variance analysis, to determine the pre- or postsynaptic actions of a variety of manipulations on unitary EPSPs evoked by low-intensity stimulation of afferents to CA1 pyramidal neurons in slices of hippocampus. Estimates of quantal content (mcv) were determined by calculating the ratio of the squared average unitary EPSP amplitude (determined from 150-275 responses) to the variance of these responses (M2/sigma 2), while quantal amplitudes (qcv) were estimated by calculating the ratio of the response variance to average EPSP size (sigma 2/M). Estimates of mcv were highly correlated with those determined using the method of failures (mf). With paired stimulation (50 msec interpulse interval) there was a significant facilitation of the second unitary EPSP, accompanied by an increase in mcv, but not qcv, suggesting that this facilitation was of presynaptic origin. Superfusion of hippocampal slices with various concentrations of adenosine, the A1-selective adenosine receptor agonist cyclohexyladenosine, or the Ca2+ channel blocker cadmium significantly reduced average unitary EPSP amplitudes and mcv, without significantly altering qcv, suggesting a presynaptic locus for this inhibition. The 50% effective concentration for the apparent presynaptic action of adenosine on mcv in the present study (5.7 microM; 95% confidence limits = 4.2-7.7 microM) was significantly lower than its EC50 for reducing conventional, large EPSPs (33 microM; recorded with high-resistance microelectrodes), or extracellular field EPSPs (29 microM), as previously reported by this laboratory. The glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) reduced average unitary EPSP amplitudes; in contrast to the above manipulations, it had no effect on mcv, but significantly altered qcv, which is consistent with its presumed postsynaptic mechanism of action. We conclude from these data that adenosine presynaptically reduces synaptic strength at Schaffer collateral-commissural synapses in the hippocampus by diminishing the number of quanta released, not by reducing the size of these individual quanta or postsynaptic sensitivity to excitatory neurotransmitter. These results suggest that the mechanism by which adenosine inhibits synaptic transmission in the hippocampus is similar, if not identical, to the mechanism by which it inhibits synaptic transmission at the neuromuscular junction.     

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg²⁺, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 μM ACPD and in 7 of 12 cells with 0.5 μM ACPD. The EPSP amplitudes with 5 and 0.5 μM ACPD were 142±10% (mean±S.E.M., n=19) and 130±13% (n=12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 μM ACPD were not significantly different in cells perfused with the low Ca²⁺-high Mg²⁺ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence ofd-(−)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg²⁺. Five μM ACPD potentiated monosynaptic EPSPs in 4 of 6 cells with 1 mM Mg²⁺ in the solution and in 2 of 17 cells with 4 mM Mg²⁺, and the EPSP amplitude was 123±9% (n=6) and 98±3% (n=17) of those before application of ACPD, respectively. These results suggest that activation of metabotropic glutamate receptors potentiates LC-EPSPs via mechanisms sensitive to Mg²⁺ and may work as a positive feedback mechanism at the excitatory amino acid-mediated synapses between the descending fibers and lumbar spinal motoneurons.     

Synaptic Excitation Triggers Oscillations During NMDA Receptor Activation in Rat Abducens Motoneurons

Rat abducens motoneurons were intracellularly recorded in vivo during synaptic excitation and extracellular microionophoretic application of N -methyl- d -aspartate (NMDA). Trigeminal excitatory post-synaptic potentials (EPSPs) evoked in abducens motoneurons were studied during intracellular current injection. They were not sensitive to hyperpolarization or depolarization of the membrane potential in the range of –75 mV to –55 mV using current pulse intensities between –3 nA and + 1 nA. Microionophoretic applications of aminophosphonovalerate (APV), MK801 and i.v. injections of MK801 (1 – 3 mg/kg) or ketamine (10 mg/kg) did not modify trigeminal EPSPs, suggesting that NMDA receptors are not involved in this synaptic transmission. However, microionophoretic applications of NMDA on abducens motoneurons enhanced trigeminal EPSPs and gave rise to regenerative oscillations. The co-activation of NMDA receptors and trigeminal synapses induced these oscillations. The trigeminal EPSP may delay and reset the oscillations depending on where it was evoked in the oscillatory cycle. Depolarizing current pulses intracellularly applied to abducens motoneurons could trigger a post-hyperpolarization followed by rebound depolarization during NMDA application, confirming the activation of active membrane properties. However, depolarizing current pulses could not trigger oscillations similar to those entrained by the EPSPs. The importance of the location of trigeminal synapses in relation to those of NMDA receptors in the dendritic arborization of abducens motoneurons is discussed. Our results show that the same sensory stimulus may have different post-synaptic effects on abducens motoneurons during the co-activation of NMDA receptors. A complete modification of the motor output during NMDA receptor activation strongly supports an active role of abducens motoneurons provided that NMDA receptors are physiologically activated during motor pattern generation.     

Synaptic connections of buccal mechanosensory neurons in the opisthobranch mollusc, Navanax inermis

Mechanical stimulation of various areas of the pharyngeal wall and lips can produce EPSPs and IPSPs, as well as abruptly rising impulses, in primary sensory cells. IPSP fields are generally larger than EPSP fields and these fields are distributed without obvious order around fields from which afferent spikes are evoked. Apparently monosynaptic excitatory and inhibitory contacts are formed between primary sensory neurons. These synapses are blocked by high Mg2+ indicating chemical transmission. IPSPs are inverted by Cl- injection. Excitatory inputs can be electrically far from the soma. Sensory cells form apparently monosynsptic excitatory or inhibitory contacts on motoneurons mediating pharyngeal expansion. Brief sensory excitation can initiate sustained firing within this neuronal population and sustained synaptic activity in motoneurons. Interactions of sensory neurons may be important in information processing and in generating motor paterns. These neurons serve both primary sensory and interneuronal functions.     

Activation of GABAA receptors causes presynaptic and postsynaptic inhibition at synapses between muscle spindle afferents and motoneurons in the spinal cord of bullfrogs

The functional role of GABAA receptors in inhibition of synaptic transmission between muscle spindle afferents and spinal motoneurons was studied in the isolated spinal cord of bullfrogs. Extracellular recording from the ventral root showed that activation of GABAA receptors by muscimol (primarily a GABAA receptor agonist) at 50 microM produced a 38% reduction in the amplitude of the excitatory postsynaptic potential (EPSP) evoked by stimulation of triceps muscle sensory afferents and a 66% reduction in the EPSP half-width, suggesting a large increase in the conductance of the motoneuronal membrane. Quantal analysis of unitary triceps EPSPs recorded intracellularly from motoneurons showed that muscimol reduced the quantal content of release (presynaptic inhibition). In addition, muscimol decreased the quantal size (postsynaptic inhibition) when the postsynaptic conductance change was large. Because the effects of muscimol were completely and reversibly blocked by 100 microM bicuculline (a specific GABAA receptor antagonist), both the pre- and the postsynaptic inhibition caused by muscimol were due to activation of GABAA receptors. Activation of GABAA receptors thus causes both pre- and postsynaptic inhibition of synaptic transmission between muscle afferents and spinal cord motoneurons in the frog.     

Dual-component synaptic potentials in the lamprey mediated by excitatory amino acid receptors

The synaptic mechanisms underlying amino acid-mediated excitation in the lamprey spinal cord have been investigated. Fine stimulating electrodes were used to stimulate single axons in the spinal cord and evoke unitary EPSPs in lamprey motoneurons and one type of premotor interneuron, the CC interneuron. Three types of EPSP, distinguished by their time course and sensitivity to amino acid antagonists, were seen. Fast EPSPs had a fast rise time (mean, 6.5 msec) and a short half-decay time (mean, 22.5 msec). Slow EPSPs lasted at least 200 msec, had a slow rise time (mean, 28 msec), and a long half-decay time (mean, 109 msec). The third type of unitary potential, called "mixed" EPSP, also lasted at least 200 msec, had a fast rise time (mean, 12 msec), and a long half-decay time (mean, 105 msec). Lamprey neurons were found to possess 3 types of excitatory amino acid receptor: N-methyl-D-aspartate (NMDA), kainate, and quisqualate receptors. 2-Amino-5-phosphonovaleric acid (APV) or Mg2+ blocked the depolarizations caused by N-methyl-D,L-aspartate (NMA) but not those of kainate or quisqualate. Cis-2, 3-piperidine dicarboxylic acid (PDA) blocked the depolarizations caused by NMA and kainate but not those of quisqualate. Fast EPSPs were unaffected by the bath application of APV or Mg2+ but were greatly reduced by PDA, suggesting that these EPSPs were mediated by non-NMDA, possibly kainate receptors. Both APV and Mg2+ blocked the slow EPSPs, suggesting that they were mediated by NMDA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of GABAB receptors causes presynaptic inhibition at synapses between muscle spindle afferents and motoneurons in the spinal cord of bullfrogs

Baclofen, a specific GABAB receptor agonist, was used to study the functional role of activation of GABAB receptors in synaptic transmission between muscle spindle afferents and motoneurons in the isolated spinal cord of bullfrogs. (+/-)-Baclofen (5 microM) reversibly reduced the amplitude of the excitatory postsynaptic potential (EPSP) evoked by simulation of various brachial muscle nerves and recorded extracellularly from the ventral root by 47% without shortening the falling phase of the EPSP. Neither the time course nor the amplitude of the action potentials in the sensory afferents was affected. Thus, baclofen caused synaptic inhibition without reducing either the potential change occurring in the muscle sensory afferents or the motoneuronal membrane resistance. Quantal analysis, performed using a deconvolution technique, of the monosynaptic EPSPs in brachial motoneurons evoked by activity in single triceps muscle spindle afferents showed that transmission at these synapses was quantal, and baclofen lowered the quantal content without altering the quantal size. Furthermore, quantal analysis of the electrical component of these unitary EPSPs showed that it did not fluctuate in amplitude, either in normal saline or with baclofen. The inhibition produced by activation of GABAB receptors is therefore presynaptic but is not likely to be caused by conduction failures in the sensory terminals.     

Catecholamine varicosities in cat dorsal root ganglion and spinal ventral roots

Convergence upon reticulospinal neurons which mediate disynaptic, contralateral pyramidal EPSPs to neck motoneurons has been examined in cats with contralateral pyramidal transection at the obex. Conditioning stimuli in the contralateral tectum and ipsilateral mesencephalic tegmentum produced monosynaptic facilitation of the disynaptic pyramidal EPSP, whereas facilitation evoked from the ipsilateral pyramid showed a disynaptic time course. These results show that contralateral pyramidal, tectal and ipsilateral tegmental fibers converge onto common reticulospinal neurons which have direct excitatory connections with neck motoneurons.     

Cholinergic suppression of excitatory synaptic responses in layer II of the medial entorhinal cortex

Theta-frequency (4-12 Hz) electroencephalographic activity is thought to play a role in mechanisms mediating sensory and mnemonic processing in the entorhinal cortex and hippocampus, but the effects of acetylcholine on excitatory synaptic inputs to the entorhinal cortex are not well understood. Field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the piriform (olfactory) cortex were recorded in the medial entorhinal cortex during behaviors associated with theta activity (active mobility) and were compared with those recorded during nontheta behaviors (awake immobility and slow wave sleep). Synaptic responses were smaller during behavioral activity than during awake immobility and sleep, and responses recorded during movement were largest during the negative phase of the theta rhythm. Systemic administration of cholinergic agonists reduced the amplitude of fEPSPs, and the muscarinic receptor blocker scopolamine strongly enhanced fEPSPs, suggesting that the theta-related suppression of fEPSPs is mediated in part by cholinergic inputs. The reduction in fEPSPs was investigated using in vitro intracellular recordings of EPSPs in Layer II neurons evoked by stimulation of Layer I afferents. Constant bath application of the muscarinic agonist carbachol depolarized membrane potential and suppressed EPSP amplitude in Layer II neurons. The suppression of EPSPs was not associated with a substantial change in input resistance, and could not be accounted for by a depolarization-induced reduction in driving force on the EPSP. The GABA(A) receptor-blocker bicuculline (50 microM) did not prevent the cholinergic suppression of EPSPs, suggesting that the suppression is not dependent on inhibitory mechanisms. Paired-pulse facilitation of field and intracellular EPSPs were enhanced by carbachol, indicating that the suppression is likely due to inhibition of presynaptic glutamate release. These results indicate that, in addition to well known effects on postsynaptic conductances that increase cellular excitability, cholinergic activation in the entorhinal cortex results in a strong reduction in strength of excitatory synaptic inputs from the piriform cortex.     

Somatostatin presynaptically inhibits transmitter release in feline parasympathetic ganglia

Intracellular recordings were made from neurons in cat parasympathetic ciliary ganglia in vitro. Somatostatin (30 nM-3 microM) reduced the amplitude of excitatory postsynaptic potentials (EPSPs), whereas the peptide did not affect acetylcholine (ACh)-induced depolarizations. Thus somatostatin depressed the EPSPs without changing the postsynaptic sensitivity to ACh. The inhibitory action of somatostatin on the EPSPs was passed off even in the presence of the peptide at concentrations higher than 100 nM. When paired stimuli at an interval of 50 ms were applied to preganglionic nerves, the second EPSP was facilitated, being larger in amplitude than the first one; this facilitation was reversibly inhibited in the presence of the peptide. Somatostatin reversibly reduced the frequency of spontaneous EPSPs without appreciably changing their mean amplitude. All of these results indicate that somatostatin may presynaptically reduce the amount of ACh released. The mechanism underlying this action was discussed.     

EPSP's of the motor neurons of cat masticatory muscles induced by stimulation of low-threshold infraorbital nerve fibers]

The effects of the infraorbital Aalpha afferents stimulation on the masseter motoneurons were investigated in cats under chloralose-nembutal anesthesia. It is found that stimuli 1.4-2.5 times threshold (T) evoke complex EPSPs in 69% of the investigated motoneurons, their latency being 2.1 +/- 0.2 ms (1.5-3.0 ms, n = 36), amplitude up to 30 mV and duration 9-15 ms. These EPSPs consist of two simpler responses produced by activation of two separate groups of afferent fibres. The short-latent EPSPs arise after activation of the infraorbital nerve by 1.1-1.5 T stimuli and have the same latency as the complex EPSP (1.5-3.0 ms) but a smaller amplitude (up to 2.0 mV) and shorter duration (up to 6 ms). The stability of these EPSPs during high frequency stimulation (120/c) and the development of facilitation and inhibition similar to those which appear in monosynaptic EPSPs in masseter motoneurons during stimulation of the 1a muscle afferents give reason to suggest that these EPSPs are monosynaptic. The slow rising rate indicates that they appear on distal dendrites of the motoneurons. The long-latent EPSPs arise with latency of 7-9 ms after activation of the infraorbital nerve by 1.1-1.5 T stimuli. Their amplitude reaches 1.5-2.0 mV and duration 7-9 ms. The long latency of these EPSPs in combination with low ability to repeat high frequency stimulation are consistent with their polysynaptic origin. It is suggested that the excitatory input from the lowest threshold Aalpha afferents of the infraorbital nerve to masseter motoneurons creates conditions for the development of a transient jaw closing reflex in response to light tactile stimulation of the cat's perioral region.     

G-protein-coupled GABAB receptors inhibit Ca2+ channels and modulate transmitter release in descending turtle spinal cord terminal synapsing motoneurons

Presynaptic gamma-aminobutyric acid type B receptors (GABA(B)Rs) regulate transmitter release at many central synapses by inhibiting Ca(2+) channels. However, the mechanisms by which GABA(B)Rs modulate neurotransmission at descending terminals synapsing on motoneurons in the spinal cord remain unexplored. To address this issue, we characterized the effects of baclofen, an agonist of GABA(B)Rs, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of the dorsolateral funiculus (DLF) terminals in a slice preparation from the turtle spinal cord. We found that baclofen depressed neurotransmission in a dose-dependent manner (IC(50) of approximately 2 microM). The membrane time constant of the motoneurons did not change, whereas the amplitude ratio of the evoked EPSPs in response to a paired pulse was altered in the presence of the drug, suggesting a presynaptic mechanism. Likewise, the use of N- and P/Q-type Ca(2+) channel antagonists (omega-conotoxin GVIA and omega-agatoxin IVA, respectively) also depressed EPSPs significantly. Therefore, these channels are likely involved in the Ca(2+) influx that triggers transmitter release from DLF terminals. To determine whether the N and P/Q channels were regulated by GABA(B)R activation, we analyzed the action of the toxins in the presence of baclofen. Interestingly, baclofen occluded omega-conotoxin GVIA action by approximately 50% without affecting omega-agatoxin IVA inhibition, indicating that the N-type channels are the target of GABA(B)Rs. Lastly, the mechanism underlying this effect was further assessed by inhibiting G-proteins with N-ethylmaleimide (NEM). Our data show that EPSP depression caused by baclofen was prevented by NEM, suggesting that GABA(B)Rs inhibit N-type channels via G-protein activation.     

Achyranthes bidentata polypeptides prevent apoptosis by inhibiting the glutamate current in cultured hippocampal neurons

Glutamate-induced excitotoxicity plays a critical role in the neurological impairment caused by middle cerebral artery occlusion.Achyranthes bidentata polypeptides have been shown to protect against neurological functional damage caused by middle cerebral artery occlusion,but the underlying neuroprotective mechanisms and the relationship to glutamate-induced excitotoxicity remain unclear.Therefore,in the current study,we investigated the protective effects of Achyranthes bidentata polypeptides against glutamate-induced excitotoxicity in cultured hippocampal neurons.Hippocampal neurons were treated with Mg^2+-free extracellular solution containing glutamate(300μM)for 3 hours as a model of glutamate-mediated excitotoxicity(glutamate group).In the normal group,hippocampal neurons were incubated in Mg^2+-free extracellular solution.In the Achyranthes bidentata polypeptide group,hippocampal neurons were incubated in Mg^2+-free extracellular solution containing glutamate(300μM)and Achyranthes bidentata polypeptide at different concentrations.At 24 hours after exposure to the agents,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Hoechst 33258 staining were used to assess neuronal viability and nuclear m'orphology,respectively.Caspase-3 expression and activity were evaluated using western blot assay and colorimetric enzymatic assay,respectively.At various time points after glutamate treatment,reactive oxygen species in cells were detected by H2 DCF-DA,and mitochondrial membrane potential was detected by rhodamine 123 staining.To examine the effect of Achyranthes bidentata polypeptides on glutamate receptors,electrophysiological recording was used to measure the glutamate-induced inward current in cultured hippocampal neurons.Achyranthes bidentata polypeptide decreased the percentage of apoptotic cells and reduced the changes in caspase-3 expression and activity induced by glutamate.In addition,Achyranthes bidentata polypeptide attenuated the amplitude of the glutamate-induced current.Furthermore,the glutamate-induced increase in intracellular reactive oxygen species and reduction in mitochondrial membrane potential were attenuated by Achyranthes bidentata polypeptide treatment.These findings collectively suggest that Achyranthes bidentata polypeptides exert a neuroprotective effect in cultured hippocampal neurons by suppressing the overactivation of glutamate receptors and inhibiting the caspase-3-dependent mitochondrial apoptotic pathway.All animal studies were approved by the Animal Care and Use Committee,Nantong University,China(approval No.20120216-001)on February 16,2012.     

Excitatory amino acid-induced alterations of cytoplasmic free Ca2+ in individual cerebellar granule neurons: role in neurotoxicity.

The effects of glutamate on intracellular free Ca2+, [Ca2+]i, and neurotoxicity were compared in cerebellar granule neurons in vitro. [Ca2+]i was measured with fura-2 and digital fluorescence imaging microscopy; neurotoxicity was monitored using a vital dye and colorimetric analysis. Glutamate produced dose-dependent increases in [Ca2+]i, which tended to be transient for glutamate concentrations in a range of 0.01-0.5 microM and sustained for higher levels of glutamate. The ED50 for the [Ca2+]i response to glutamate was 6 microM. The LD50 for glutamate-induced neurotoxicity was similar, i.e., 10 microM. The effect of glutamate on [Ca2+]i was greatly diminished when external Ca2+ was removed and blocked by Mg2+ or N-methyl-D-aspartate (NMDA)-type receptor antagonists. The latter conditions as well as preloading granule neurons with the intracellular Ca2+ chelator quin2 largely prevented glutamate cytotoxicity. The neurotoxic effect of glutamate required incubations with the stimulus for 10-20 min at 25 degrees C. Withdrawal of glutamate after this period was accompanied by a prolonged alteration in [Ca2+]i. Pretreatment of the cells with the ganglioside GM1 reduced this late increase in [Ca2+]i as well as the neurotoxic effects of glutamate. This indicates that glutamate-induced neurotoxicity results from a composite of diverse temporal alterations in Ca2+ homeostasis and that blunting any of these components reduces excitotoxicity.