Reduction of GABAA receptor-mediated inhibition by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione in cultured neurons of rat brain.

The action of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on gamma-aminobutyric acid-A (GABAA) receptor-mediated currents was studied in dissociated rat midbrain and hypothalamic cultures using whole-cell recording. Spontaneous synaptic activity consisted of excitatory (EPSCs) and inhibitory postsynaptic currents (IPSCs). Bicuculline (20 microM) blocked IPSCs and increased the frequency of EPSCs. CNQX (1 microM) reduced both EPSCs and IPSCs. In the presence of 0.3 microM tetrodotoxin (TTX), CNQX (1-20 microM) blocked miniature EPSCs and reduced IPSCs. In TTX, increasing K+ (20 mM) evoked EPSCs and IPSCs in a Ca-dependent manner. CNQX (10 microM) blocked evoked EPSCs and diminished evoked IPSCs similarly as miniature IPSCs. Muscimol-(0.2-5 microM) induced currents were dose-dependently reduced by CNQX (10-50 microM). It is concluded that CNQX reduces GABAA receptor-mediated inhibition primarily by reducing the excitatory drive in the evolving network, but, in addition, has a significant blocking effect on the GABAA receptor-channel complex.     

Direct demonstration of an N-methyl-D-aspartate receptor mediated component of excitatory synaptic transmission in area CA1 of the rat hippocampus

The action of a new non-N-methyl-D-aspartate (NMDA) receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), on synaptic transmission in area CA1 of the rat hippocampus has been examined. Intracellular and extracellular recordings showed CNQX to be a potent antagonist of synaptic potentials evoked by stimulation of the Schaffer collateral-commissural fibre system. One to 2 microM CNQX was sufficient to reduce the excitatory postsynaptic potential (EPSP) by 50%. CNQX is therefore about 100 times more potent than previously available non-NMDA receptor antagonists. In the presence of CNQX, a small depolarizing potential could still be evoked. This potential was sensitive to the NMDA-receptor blocker, 2-amino-5-phosphonovaleric acid (APV), increased in size on depolarizing the neurone and also increased in size on removing Mg2+ from the perfusing medium. This residual EPSP therefore has characteristics which are consistent with its mediation via the NMDA receptor-coupled ionophore. These results indicate a dual composition of the monosynaptic excitatory potential in area CA1.     

Excitatory postsynaptic potentials in rat neocortical neurons in vitro. III. Effects of a quinoxalinedione non-NMDA receptor antagonist

1. Intracellular microelectrodes were used to obtain recordings from neurons in layer II/III of rat frontal cortex. A bipolar electrode positioned in layer IV of the neocortex was used to evoke postsynaptic potentials. Graded series of stimulation were employed to selectively activate different classes of postsynaptic responses. The sensitivity of postsynaptic potentials and iontophoretically applied neurotransmitters to the non-N-methyl-D-asparate (NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was examined. 2. As reported previously, low-intensity electrical stimulation of cortical layer IV evoked short-latency early excitatory postsynaptic potentials (eEPSPs) in layer II/III neurons. CNQX reversibly antagonized eEPSPs in a dose-dependent manner. Stimulation at intensities just subthreshold for activation of inhibitory postsynaptic potentials (IPSPs) produced long-latency (10 to 40-ms) EPSPs (late EPSPs or 1EPSPs). CNQX was effective in blocking 1EPSPs. 3. With the use of stimulus intensities at or just below threshold for evoking an action potential, complex synaptic potentials consisting of EPSP-IPSP sequences were observed. Both early, Cl(-)-dependent and late, K(+)-dependent IPSPs were reduced by CNQX. This effect was reversible on washing. This disinhibition could lead to enhanced excitability in the presence of CNQX. 4. Iontophoretic application of quisqualate produced a membrane depolarization with superimposed action potentials, whereas NMDA depolarized the membrane potential and evoked bursts of action potentials. At concentrations up to 5 microM, CNQX selectively antagonized quisqualate responses. NMDA responses were reduced by 10 microM CNQX. D-Serine (0.5-2 mM), an agonist at the glycine regulatory site on the NMDA receptor, reversed the CNQX depression of NMDA responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hypertonic saline on rat hypothalamic paraventricular nucleus parvocellular neurons in vitro

The effects of hypertonic saline on hypothalamic paraventricular nucleus (PVN) parvocellular neurons were examined using whole-cell patch-clamp technique. Under current-clamp, 50% (41/82) of parvocellular neurons were depolarized than the predicted values by hypertonic saline, and associated with increasing action potential frequency. Under voltage-clamp, unless hypertonic saline induced a shift of reverse potential to more positive values, neither mannitol nor hypertonic saline obviously increased the conductance in parvocellular neurons. Moreover, spontaneous excitatory postsynaptic currents (sEPSCs) were increased by isotonic increases in [Na⁺]_o in the parvocellular neurons. Bath application AMPA receptor antagonist CNQX or non-selective glutamate antagonist kynurenic acid almost completely blocked the sEPSCs. Extracellular application of gadolinium (Gd³⁺) blocked the hypertonic saline-induced response. These results suggested that subpopulation of PVN parvocellular neurons are selectively sensitive to NaCl. Hypertonic saline excited the PVN parvocellular neurons through Na⁺-detection and the excitatory glutamatergic synaptic input.     

Kindling-induced long-lasting changes in synaptic transmission in the basolateral amygdala.

1. Intracellular current-clamp recordings were obtained from neurons of the basolateral amygdala (BLA) in an in vitro slice preparation from control and kindled animals. Postsynaptic potentials, elicited by stimulation of the stria terminalis (ST) or lateral amygdaloid nucleus (LA), were used to investigate the role of excitatory and inhibitory amino acid transmission in kindling-induced epileptiform activity. The contributions of glutamatergic and GABAergic receptor subtypes were analyzed by use of the non-N-methyl-D-aspartate (non-NMDA) antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the NMDA antagonist DL-2-amino-5-phosphonovaleric acid (APV), and the GABAA antagonist bicuculline methiodide (BMI). 2. The synaptic waveform evoked in control neurons consisted of an excitatory postsynaptic potential (EPSP), a fast inhibitory postsynaptic potential (f-IPSP), and a slow inhibitory postsynaptic potential (s-IPSP). Stimulation of the ST or LA pathways evoked a burst-firing response in BLA neurons contralateral from the site of stimulation of kindled animals. 3. APV (50 microM) reduced, but CNQX (10 microM) completely blocked, the burst-firing response in BLA neurons from kindled animals and bicuculline-induced bursting in control neurons. 4. Kindling significantly increased the amplitude of both the slow NMDA- and the fast non-NMDA-receptor-mediated components of synaptic transmission (s- and f-EPSPs, respectively). Furthermore, the stimulus intensities required to evoke EPSPs just subthreshold for action potential generation were significantly lower in slices from kindled animals. 5. In kindled neurons no significant change was observed in the membrane input resistance and resting membrane potential or in the number of action potentials elicited in response to depolarizating current injection. 6. Kindling resulted in a pathway-specific loss of ST- and LA-evoked feedforward GABAergic synaptic transmission and of spontaneous IPSPs. In the same BLA neurons, direct GABAergic inhibition via stimulation of the LA was not affected by kindling. 7. The enhanced glutamatergic transmission was not due to disinhibition, because, in the presence of BMI (and CNQX to prevent BMI-induced bursting), the s-EPSP amplitude was still greater in kindled than in control neurons. 8. These results provide evidence that the epileptiform activity observed in BLA neurons after kindling results from an increase in excitatory NMDA- and non-NMDA-receptor-mediated glutamatergic transmission and a decrease in inhibitory gamma-aminobutyric acid (GABA)-receptor-mediated transmission; the enhanced excitatory transmission cannot be accounted for by reduced inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

GABA- and glutamate-mediated synaptic potentials in rat dorsal raphe neurons in vitro

1. Synaptic potentials were recorded with intracellular electrodes from rat dorsal raphe neurons in a slice preparation. 2. Synaptic potentials were evoked by applying electrical pulses to bipolar stimulating electrodes positioned immediately dorsal to the raphe nucleus; these arose after a latency of 0.5-5 ms and had a duration of 20-200 ms. 3. The synaptic potential was biphasic (at the resting potential) when the recording electrodes contained potassium citrate; a depolarization was followed by a hyperpolarization. The hyperpolarization reversed in polarity at -70 mV and was blocked by bicuculline. 4. The depolarizing synaptic potential was reduced to 50-90% of control by kynurenate (1-2 mM) or 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX) (10 microM) and increased in amplitude and duration by magnesium-free solution. 5. In magnesium-free solutions (with CNQX), the depolarizing synaptic potential was blocked by DL-2-amino-5-phosphonovaleric acid (APV, 50 microM). APV also blocked depolarization caused by adding N-methyl-D-aspartate (NMDA) to the superfusion solution. 6. The results indicate that raphe neurons display two synaptic potentials having a duration of 150-200 ms: one that is mediated by GABA and a second that is due to an excitatory amino acid. The component mediated by an excitatory amino acid involves, in part, a receptor of the NMDA type.     

Initiation of epileptiform activity by excitatory amino acid receptors in the disinhibited rat neocortex.

1. Intracellular recordings were obtained from neurons in layer II-III of rat frontal cortex maintained in vitro. The role of excitatory amino acid receptors in generation of picrotoxin (PTX)-induced epileptiform activity was investigated with the use of D-2-amino-5-phosphonovaleric acid (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) as selective antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, respectively. 2. Bath application of PTX resulted in a decrease in evoked inhibitory postsynaptic potentials (IPSPs) in neocortical neurons and a concomitant increase in a polysynaptic late excitatory postsynaptic potential (IEPSP). Epileptiform burst responses, termed paroxysmal depolarizing shifts (PDSs), subsequently developed. Based on response duration, two types of PDSs were identified. Long PDSs were greater than 100 ms in duration, whereas short PDSs lasted less than 50 ms. An early depolarizing potential preceded both types of epileptiform burst response. 3. The NMDA receptor antagonist D-APV reduced the peak amplitude and duration of the PDS. D-APV-insensitive portions of the PDS were greatly attenuated or abolished by CNQX. The non-NMDA antagonist also increased the latency to PDS onset and reduced its duration without affecting peak amplitude. CNQX-insensitive components of the PDS, when present, were abolished by D-APV. 4. Short-duration PDSs could be blocked by CNQX. In these neurons, increasing the stimulation strength produced epileptiform responses of reduced amplitude. 5. Under control conditions, PDS amplitude was a linear function of membrane potential, increasing with hyperpolarization and diminishing on depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitatory amino acid response in isolated spiral ganglion cells of guinea pig cochlea.

1. The physiological and pharmacological properties of excitatory amino acid (EAA)-induced responses were investigated in acutely isolated spiral ganglion cells of guinea pig by a conventional patch-clamp technique combined with a rapid drug application (Y-tube) method. 2. L-glutamate (Glu) and its agonists, quisqualate (QA) and kainate (KA), induced inward currents in a concentration-dependent manner at a holding potential (VH) of -70 mV. The values of half-maximal concentration (EC50) were 4.0 x 10(-4) M for Glu, 2.3 x 10(-5) M for QA, and 1.4 x 10(-4) for KA. The Hill coefficients were 0.96, 1.00, and 1.56 for Glu, QA, and KA, respectively. However, one of Glu agonists, N-methyl-D-aspartate (NMDA), and another excitatory amino acid, L-aspartate (Asp), did not induce any responses even in Mg2(+)-free external solution containing 10(-6) M glycine (Gly). 3. The current-voltage (I-V) relationships for the Glu-, QA-, and KA-induced responses were linear, and these reversal potentials were near 5 mV. 4. Kynurenic acid (Kyn), 6,7-dichloro-3-hydroxy-2-quinoxalinecarboxylic acid (diCl-HQC), and quinoxalinediones such as 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitro-quinoxaline-2,3-dione (DNQX) suppressed the Glu-, QA-, and KA-induced currents in a concentration-dependent manner. The inhibitory potency was in the order of DNQX = CNQX greater than diCl-HQC greater than Kyn. 5. CNQX antagonized the Glu-, QA-, and KA-induced currents without affecting the maximum responses showing no voltage-dependency, indicating the competitive inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitatory amino acid receptors involved in primary afferent-evoked polysynaptic EPSPs of substantia gelatinosa neurons in the adult rat spinal cord slice.

Intracellular recordings were made from substantia gelatinosa (SG) neurons in spinal cord slices to determine a subclass of excitatory amino acid receptors involved in polysynaptic excitatory postsynaptic potentials (EPSPs). In the majority of neurons, polysynaptic EPSPs evoked by A delta fiber were not affected by 2-amino-5-phosphonovaleric acid (APV), while all EPSPs including monosynaptic EPSPs were depressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). All spontaneous EPSPs were blocked by CNQX, while spontaneous EPSPs in a few SG neurons were attenuated by APV. These observations suggest that polysynaptic EPSPs evoked through A delta fibers are predominantly mediated by activation of the non-N-methyl-D-aspartate (non-NMDA) receptor subclass.     

电鱼小脑浦肯野细胞对急性缺氧的功能反应

目的:通过研究急性缺氧对电鱼(mormyrid electric fish)小脑浦肯野细胞(Purkinje cell,PC)的功能影响,阐明缺氧耐受动物神经元在缺氧条件下的电生理特征。方法:采用全细胞膜片钳记录法,观察急性缺氧对电鱼小脑主神经元PC膜电位、兴奋性和平行纤维(parallel fiber,PF)-PC突触传递的影响。结果:(1)短暂缺氧使电鱼小脑PC膜电位发生迅速而持久的超极化,可持续30 min以上,同时伴随自发放电频率的显著下降。谷氨酸AMPA受体阻断剂CNQX不影响PC缺氧性超极化的产生,但可阻断缺氧性超极化的持续存在;而GABAA受体阻断剂Bicuculline则完全阻断缺氧性超极化的产生,并使膜电位在缺氧开始后发生短暂的去极化。(2)缺氧使PC诱发动作电位的阈值增高,频率减低,幅值减小。(3)急性缺氧使刺激PF诱发的PC兴奋性突触后电流(excitatorypostsynaptic current,EPSC)呈现长时程增强(long term potentiation,LTP),同时使EPSC双脉冲增强现象(pair-pulsefacilitation,PPF)显著衰减。CNQX逆转了PF EPSC的缺氧性LTP,表现为长时程抑制(Long Term Depression,LTD);而Bicuculline则使PF EPSC的缺氧性LTP增强。结论:耐缺氧动物电鱼小脑神经元的缺氧反应特征与哺乳类动物显著不同,AMPA受体和GABAA受体均参与电鱼小脑PC的缺氧性超极化和PF LTP的产生,表明维持GABA能突触和谷氨酸能突触活动的适度平衡,可能是电鱼以及其他耐缺氧动物脑保护机制的关键。     

The GABA(B) receptor antagonist CGP 55845A reduces presynaptic GABA(B) actions in neocortical neurons of the rat in vitro.

Use-dependent depression of inhibitory postsynaptic potentials was investigated with intracellular recordings and the paired-pulse paradigm in rat neocortical neurons in vitro. Pairs of stimuli invariably reduced the second inhibitory postsynaptic potential-A (GABA(A) receptor-mediated inhibitory postsynaptic potential) of a pair; at interstimulus intervals of 500 ms, the amplitude of the second inhibitory postsynaptic potential-A was considerably smaller than the first (36.2 +/- 6.2%, n= 17). Decreasing the interstimulus interval reduced the second inhibitory postsynaptic potential-A further and with interstimulus intervals shorter than 330 ms the compound excitatory postsynaptic potential-inhibitory postsynaptic potential response reversed from a hyperpolarizing to a depolarizing response. The depression of the inhibitory postsynaptic potential-A exhibited a maximum at interstimulus intervals near 150 ms and recovered with a time constant of 282 +/- 96.2 ms. Elimination of excitatory transmission by the application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonovaleric acid yielded an essentially unaltered time-course of paired-pulse depression (maximum depression near 150 ms, time constant of recovery 232 +/- 98 ms). The polarity change of the compound excitatory postsynaptic potential response at shorter interstimulus intervals was abolished in the presence of CNQX and D(- )-2-amino-5-phosphonovaleric acid. CNQX and D(-)-2-amino-5-phosphonovaleric acid also reduced the apparent depolarizing shift of the reversal potential between the first and second inhibitory postsynaptic potential-A from about 6 mV to less than 2 mV. Application of the GABA(B) receptor antagonist CGP 55845A in the presence of CNQX and (-)-2-amino-5-phosphonovaleric acid abolished the inhibitory postsynaptic potential-B and paired-pulse depression. Under these conditions, the amplitude of the second inhibitory postsynaptic potential was, on average, about 90% of the first, i.e. reduced by about 10%. The second inhibitory postsynaptic potential-A was approximately constant at interstimulus intervals between 100 and 500 ms. It is concluded that paired-pulse depression of cortical inhibition is predominantly mediated by presynaptic GABA(B) receptors of GABAergic interneurons. The abolition of net inhibition at interstimulus intervals near 330 ms may facilitate spread of excitation and neuronal synchrony during repetitive cortical activation near 3 Hz. This use-dependent depression of inhibition may contribute to highly synchronized slow electroencephalogram activity during spike-and-wave or delta activity.     

Excitatory synaptic transmission mediated by NMDA and non-NMDA receptors in the superficial/middle layers of the epileptogenic human neocortex maintained in vitro.

Conventional intracellular recordings were made from regular-spiking cells located in layers II-IV to examine the involvement of excitatory amino acid receptors in synaptic transmission in epileptogenic human neocortical slices maintained in vitro. Extracellular stimuli that were below the threshold for generating action potentials evoked an excitatory postsynaptic potential (EPSP) with short latency to onset (0.8-4 ms). When suprathreshold stimuli were delivered, 95% of the neurons fired a single action potential. In 5% of the population, however, an all-or-none bursting discharge was observed. The EPSP and the bursting discharge were tested with the N-methyl-D-aspartate (NMDA) antagonist 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP, 5 microM) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 4 microM). In the presence of CNQX the peak amplitude of the EPSP was reduced by 85% and the bursting discharge was abolished completely. By contrast, CPP reduced the peak amplitude of the EPSP by 52%, attenuated the late phase of the bursting discharge and increased its threshold. These results indicate that excitatory amino acids function as excitatory transmitters in the human brain. While the involvement of non-NMDA receptors in the EPSP is in line with data from normal neocortical slices of other mammals, the participation of NMDA-mediated conductances to the EPSP appears peculiar to the epileptogenic human neocortex. This evidence, together with the contribution of NMDA and non-NMDA receptors to the all-or-none bursting discharge suggests that excitatory amino acid-mediated transmission might be modified in the epileptogenic human neocortex.     

Long-term enhancement of excitatory synaptic inputs to layer V parahippocampal neurons by low frequency stimulation in rat brain slices

Excitatory inputs to layer V neurons of the parasubiculum and medial entorhinal cortex were examined in rat brain slices with intracellular and field potential recordings. Single extracellular stimuli to layer V evoked subthreshold excitatory postsynaptic potentials (EPSPs) or a long duration (>100 ms) depolarization that sustained high frequency firing. Repetitive stimulation at low frequencies (from 1/10 s to 1/min) induced stable long-lasting decreases in the threshold for firing in individual cells or population events, and also induced stable long-lasting increases in evoked intracellular or field response amplitudes. More stimuli were required to produce the equivalent changes in threshold and amplitude in the presence of MCPG (200 microM). Smaller changes in amplitude, but equivalent changes in threshold were elicited in the presence of CPP (10 microM), or CPPG (20 microM). No changes in threshold or amplitude were detected in the presence of CNQX (10 microM), even when used in combination with picrotoxin (100 microM). EPSP facilitation was enhanced greatly by firing in postsynaptic cells. It is suggested that stable changes in excitatory inputs to layer V parahippocampal neurons involve the activation of NMDA and metabotropic glutamate receptors, but requires AMPA receptor activation and postsynaptic cell firing.     

Excitatory transmission in the basolateral amygdala.

1. Intracellular current-clamp recordings obtained from neurons of the basolateral nucleus of the amygdala (BLA) were used to characterize postsynaptic potentials elicited through stimulation of the stria terminalis (ST) or the lateral amygdala (LA). The contribution of glutamatergic receptor subtypes to excitatory postsynaptic potentials (EPSPs) were analyzed by the use of the non N-methyl-D-aspartate (non-NMDA) antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), and the NMDA antagonist, (DL)-2-amino-5-phosphonovaleric acid (APV). 2. Basic membrane properties of BLA neurons determined from membrane responses to transient current injection showed that at the mean resting membrane potential (RMP; -67.2 mV) the input resistance (RN) and time constant for membrane charging (tau) were near maximal, and that both values were reduced with membrane hyperpolarization, suggesting an intrinsic regulation of synaptic efficacy. 3. Responses to stimulation of the ST or LA consisted of an EPSP followed by either a fast inhibitory postsynaptic potential (f-IPSP) only, or by a fast- and subsequent slow-IPSP (s-IPSP). The EPSP was graded in nature, increasing in amplitude with increased stimulus intensity, and with membrane hyperpolarization after DC current injection. Spontaneous EPSPs were also observed either as discrete events or as EPSP/IPSP waveforms. 4. In physiological Mg2+ concentrations (1.2 mM), at the mean RMP, the EPSP consisted of dual, fast and slow, glutamatergic components. The fast-EPSP (f-EPSP) possessed characteristics of kainate/quisqualate receptor activation, namely, the EPSP increased in amplitude with membrane hyperpolarization, was insensitive to the NMDA receptor antagonist, APV (50 microM), and was blocked by the non-NMDA receptor antagonist, CNQX (10 microM). In contrast, the slow-EPSP (s-EPSP) decreased in amplitude with membrane hyperpolarization, was insensitive to CNQX (10 microM), and was blocked by APV (50 microM), indicating mediation by NMDA receptor activation. 5. In the presence of CNQX (10 microM), ST stimulation evoked an APV-sensitive s-EPSP. In contrast, LA stimulation evoked a f-IPSP, which when blocked by subsequent addition of bicuculline methiodide (BMI; 30 microM) revealed a temporally overlapping APV-sensitive s-EPSP. These data suggest that EPSP amplitude and duration are determined, in part, by the shunting of membrane conductance caused by a concomitant IPSP. 6. Superfusion of either CNQX or APV in BLA neurons caused membrane hyperpolarization and blockade of spontaneous EPSPs and IPSPs, suggesting that these compounds may act to block tonic excitatory amino acid (EAA) release within the nucleus, and that a degree of feed-forward inhibition occurs within the nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Single-electrode voltage-clamp analysis of the N-methyl-D-aspartate component of synaptic responses in neocortical slices from children with intractable epilepsy.

1. Synaptic transmission mediated by the N-methyl-D-aspartate (NMDA)-receptor type was studied in neocortex from children undergoing surgical treatment for intractable epilepsy. Intracellular recordings from pyramidal cells were obtained in slices of neocortical tissue by use of microelectrodes. Synaptic responses were induced by electrical stimulation and studied with current-clamp and single-electrode voltage-clamp techniques. The NMDA-receptor-mediated component of the synaptic responses was isolated by addition of 10 microM bicuculline and 30 microM 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) in the perfusion solution. 2. In the presence of bicuculline and CNQX, electrical stimulation evoked an excitatory postsynaptic potential (EPSP) in every recorded cell. The amplitude of this EPSP increased when membrane potential was depolarized with injected current. 3. All cells studied in voltage clamp were recorded with microelectrodes containing Cs+ and QX 314. To avoid contamination of the responses from voltage-dependent Ca2+ conductances, membrane potential was held at depolarized potentials until Ca2+ spiking inactivated completely. The evoked excitatory postsynaptic currents (EPSCs) measured at resting membrane potential ranged from 100 to 400 pA. The NMDA receptor-selective antagonist DL-2-amino-5-phosphonopentanoic acid (AP-5) reversibly decreased the current amplitude by 60% for 10 microM and 80% for 30 microM. 4. The current-voltage (I-V) relation showed a region of negative slope conductance between -100 and -20 mV. The largest currents (-250 to -900 pA) were recorded in the range of -45 to -20 mV and reversed between -10 and +10 mV. Removing Mg2+ from the perfusion solution decreased the negativity of the slope, which is consistent with a reduction in the voltage-dependent Mg2+ block of the NMDA-receptor channel. 5. The I-V plots obtained from cells recorded in the most abnormal tissue were averaged and compared with those from the least abnormal tissue. No significant difference was found between these two groups. The averaged plots from the youngest patients (8 and 10 mo old) and those from the oldest (5-15 yr old) patients were also compared, and the results from these two groups were not significantly different.(ABSTRACT TRUNCATED AT 400 WORDS)     

Olfactory bulb networks revealed by lateral olfactory tract stimulation in the in vitro isolated guinea-pig brain

Olfactory information processing is mediated by synaptic connections between the olfactory bulbs (OBs) and piriform-limbic cortices. Limited accessibility using common in vivo and in vitro preparations has hindered previous attempts to define these synaptic interactions. We utilized the isolated guinea-pig brain preparation to overcome these experimental limitations. Previous studies demonstrated extensive functional preservation in this preparation maintained in vitro by arterial perfusion. Field potential laminar profiles were performed with multi-channel probes in the OB following stimulation of both the lateral olfactory tract (LOT) and the anterior piriform cortex (APC). Current-source density analysis was carried out on laminar profiles to reconstruct current sinks/sources associated with intrinsic synaptic activities. LOT stimulation induced sequentially i) an antidromic population spike (at 2.66+/-0.39 ms) located in the mitral cell layer that was resistant to 100 Hz high-frequency stimulation (HFS) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM), ii) a component located in the external plexiform layer at 3.85+/-0.63 ms that was unaffected by HFS, iii) a large amplitude potential (peak amplitude at 5.84+/-0.58 ms) generated in the external plexiform layer, abolished by HFS and CNQX, but not by bicuculline (50 microM), iv) a late response (onset at 20.00+/-2.94 ms) abolished by CNQX and enhanced by bicuculline. Stimulation of the APC also induced a late potential abolished by HFS and CNQX. Both APC-evoked and late LOT-evoked responses were abolished by a transverse cut to separate OB from APC. These results demonstrate in an isolated mammalian brain preparation the presence of reciprocal synaptic interactions between the OB and piriform cortical structures.     

Glutamate mediates an excitatory influence of the paraventricular hypothalamic nucleus on the dorsal motor nucleus of the vagus

Data have shown that the paraventricular nucleus of the hypothalamus (PVN) and the dorsal motor nucleus of the vagus (DMNV) play important roles in the regulation of gastrointestinal function and eating behavior. Anatomical studies have demonstrated direct projections from the PVN to the DMNV and physiological studies showed that the DMNV mediates many of the effects of PVN stimulation and electrical current stimulation of the PVN excites a subset of DMNV neurons. The aim of this study was to characterize the role of glutamate receptors in the excitatory influence of the PVN on gut-related DMNV neurons. Using single-cell recording techniques, we determined the effects of kynurenic acid, 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), and DL-2-amino-5-phosphonopentanoic acid (DL-AP5) on the increase in firing rate due to electrical current stimulation of the PVN. In initial experiments, we studied 24 DMNV neurons excited by electrical current stimulation of the PVN. Kynurenic acid, a broad-spectrum glutamate receptor antagonist, prevented the PVN effect in 22 neurons and significantly attenuated the effect in the other cells. Nine of these neurons demonstrated an inhibition in firing rate with PVN stimulation after pretreatment with kynurenic acid. In a separate group of 12 neurons, we determined the effects of CNQX (1.2 nmol) injected into the DMNV. This AMPA receptor antagonist completely blocked the excitatory response to PVN stimulation of six DMNV neurons and significantly attenuated the response of the other six DMNV neurons. The addition of 1.2 nmol DL-AP5, a N-methyl-D-aspartate (NMDA) receptor antagonist, further attenuated the response to PVN stimulation in four of the five DMNV neurons that were still excited after CNQX treatment. The fifth neuron demonstrated PVN- induced inhibition of firing rate after treatment with CNQX and DL-AP5. In a separate group of 11 DMNV neurons excited by electrical stimulation of the PVN, DL-AP5 partially attenuated the excitatory responses of only four DMNV neurons and did not block the excitation of any cells. The mean latency (14 neurons tested) from the PVN to the DMNV was 37.71 +/- 2.40 (SE) ms. Monosynaptic action potentials and excitatory postsynaptic potentials were demonstrated in three DMNV neurons by intracellular recording. Our results indicate that glutamate released from PVN neurons projecting to the DMNV excite the gut-related vagal motor neurons by acting predominantly on the AMPA receptor. The NMDA receptor plays only a minor role in the excitatory effect.     

K-dependent inhibition in the dentate-CA3 network of guinea pig hippocampal slices.

1. The occurrence of potassium-dependent inhibitory postsynaptic potentials (K-IPSPs) in relation to burst discharges induced by 4-aminopyridine (4-AP; 30 microM) was studied in CA3, granule and hilar neurons in guinea pig hippocampal slices with the use of paired extra- and/or intracellular recording. 2. Slow small (2-5 mV) and large (up to 30 mV) K-IPSPs were observed in CA3, granule and in some hilar neurons during 4-AP applications in the presence of blockers for fast synaptic transmission, picrotoxin (50 microM), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5-10 microM). Amplitudes of K-IPSPs were linearly related to voltage, and they reversed in sign close to -100 mV, as expected for synaptic potentials generated by an increase in K-conductance. 3. In CA3 neurons, 4-AP applied in the presence of picrotoxin elicited burst discharges and K-IPSPs. CNQX blocked the burst discharge activity and increased the amplitude of K-IPSPs. 4. In granule cells, 4-AP applied in the presence of picrotoxin elicited K-IPSPs and only inconsistently small excitatory postsynaptic potentials (EPSPs). The EPSPs were blocked by CNQX, but CNQX application did not affect the K-IPSPs. However, in granule cells it could be observed that blockade of Cl-inhibition by picrotoxin in the presence of CNQX increased the amplitude of K-IPSPs. 5. In hilar neurons, 4-AP applied in the presence of picrotoxin elicited mainly burst discharges. CNQX blocked the burst discharges only in a few cells. In most hilar neurons K-IPSPs were observed at the beginning of the 4-AP effect, but subsequently K-IPSPs were replaced by burst discharges. 6. To determine the type of cells that burst in picrotoxin and 4-AP, neurons were stained intracellularly with horseradish peroxidase. Neurons stained in the granule cell layer did not burst and were morphologically identified as granule cells. Neurons stained in the hilar region burst and were nonpyramidal, nongranule cells. Bursting cells stained in the CA3 area were all pyramidal cells. 7. The hilar neurons varied considerably in size and dendritic organization. They could be classified as aspiny and spiny cells, the latter including mossy cells. 8. We conclude that K-dependent inhibition may explain the long-lasting IPSPs observed in in vivo recordings from hippocampal cells. In a hippocampal lamella, burst discharge activity of hilar neurons including presumed excitatory mossy cells is associated with inhibition of granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential activation of glutamate receptors by spontaneously released transmitter in slices of neocortex

Whole-cell recordings were made from neurons in neocortical brain slices in order to characterize excitatory synaptic currents mediated by glutamate receptors. Glutamate receptor antagonists, D-aminophosphonovalerate (D-APV) and CNQX, selectively attenuated distinct components in evoked synaptic currents, and were used to differentiate spontaneous synaptic currents mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Spontaneous excitatory synaptic currents were independent of action potentials, varied linearly with voltage, and were blocked by the non-NMDA receptor antagonist CNQX. An NMDA receptor-mediated component was not apparent in these spontaneous synaptic currents, however, when magnesium was omitted from the recording medium, fluctuations in current and sustained inward current became apparent, and these were blocked by the NMDA receptor antagonist D-APV. Based on these findings, we conclude that NMDA and non-NMDA receptors are activated differentially by transmitter released independently of action potentials.     

Persistent sodium current contributes to induced voltage oscillations in locomotor-related hb9 interneurons in the mouse spinal cord

Neurochemically induced membrane voltage oscillations and firing episodes in spinal excitatory interneurons expressing the HB9 protein (Hb9 INs) are synchronous with locomotor-like rhythmic motor outputs, suggesting that they contribute to the excitatory drive of motoneurons during locomotion. Similar to central pattern generator neurons in other systems, Hb9 INs are interconnected via electrical coupling, and their rhythmic activity does not depend on fast glutamatergic synaptic transmission. The primary objective of this study was to determine the contribution of fast excitatory and inhibitory synaptic transmission and subthreshold voltage-dependent currents to the induced membrane oscillations in Hb9 INs in the postnatal mouse spinal cord. The non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced the amplitude of voltage oscillations but did not alter their frequency. CNQX suppressed rhythmic motor activity. Blocking glycine and GABA_A receptor-mediated inhibitory synapses as well as cholinergic transmission did not change the properties of CNQX-resistant membrane oscillations. However, disinhibition triggered new episodes of slow motor bursting that were not correlated with induced locomotor-like rhythms in Hb9 INs. Our observations indicated that fast excitatory and inhibitory synaptic inputs did not control the frequency of induced rhythmic activity in Hb9 INs. We next examined the contribution of persistent sodium current (I_NaP) to subthreshold membrane oscillations in the absence of primary glutamatergic, GABAergic and glycinergic synaptic drive to Hb9 INs. Low concentrations of riluzole that blocked the slow-inactivating component of sodium current gradually suppressed the amplitude and reduced the frequency of voltage oscillations. Our finding that I_NaP regulates locomotor-related rhythmic activity in Hb9 INs independently of primary synaptic transmission supports the concept that these neurons constitute an integral component of the rhythmogenic locomotor network in the mouse spinal cord.