Effects of N-methyl-D-aspartate glutamate receptor antagonists on oscillatory signal propagation in the guinea-pig accessory olfactory bulb slice: characterization by optical, field potential and patch clamp recordings

To characterize the role of N-methyl-d-aspartate glutamate receptors in oscillations induced by a single electrical stimulation of the vomeronasal nerve layer, optical, field potential and patch clamp recordings were carried out in guinea-pig accessory olfactory bulb slice preparations. Bath application of the N-methyl-D-aspartate receptor antagonists, 2-amino-5-phosphonovaleric acid or MK-801, produced an increase in frequency of oscillating waves (oscillation) in external plexiform and mitral cell layers. The removal of Mg2+ from perfusate abolished oscillations, while subsequent application of 2-amino-5-phosphonovaleric acid or MK-801 restored oscillations. Vomeronasal nerve layer-evoked postsynaptic currents were analyzed by whole-cell clamp recordings from mitral and granule cells. A long-lasting excitatory postsynaptic current and periodic inhibitory postsynaptic currents, which were superimposed on the long excitatory postsynaptic current, were observed in mitral cells. The frequency of the periodic inhibitory postsynaptic currents correlated with the frequency of oscillations observed in the optical and field potential recordings. Furthermore, periodic inhibitory postsynaptic currents were blocked by puff application of bicuculline to the external plexiform layer/mitral cell layer, where mitral cells make dendrodendritic synapses with granule cells. In addition, puff application of the non-N-methyl-D-aspartate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, to the external plexiform layer/mitral cell layer suppressed an early phase of periodic inhibitory postsynaptic currents (membrane oscillation), whereas 2-amino-5-phosphonovaleric acid suppressed the late phase of periodic inhibitory postsynaptic currents. These data indicate that periodic excitatory postsynaptic currents of granule cells induce relevantly periodic inhibitory postsynaptic currents in mitral cells via dendrodendritic synapses and suggest that feedback inhibition regulates generation of oscillation via activation of non-N-methyl-d-aspartate glutamate receptors and gradual attenuation of oscillation via activation of N-methyl-D-aspartate receptors on granule cells.     

Excitatory amino acid receptors in the parallel fibre pathway in rat cerebellar slices

The grease-gap technique was used on young rat cerebellar slices to study the synaptic pharmacology of the parallel fibre pathway. Electrical stimulation of the parallel fibres produced a characteristic response in Purkinje cells: a sharp negative (N) potential, representing the population action potential and underlying parallel fibre EPSP, followed by a slow positive (P) wave, the population inhibitory postsynaptic potential (IPSP). In the presence of 1.2 mM Mg2+, D-2-amino-5-phosphonovalerate (APV, 30 microM) had no effect but both potentials could be inhibited by 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX, 10 microM). Removal of Mg2+ had no effect on the N-potential but enhanced the P-wave in an APV-sensitive fashion, particularly when CNQX was present. The results provide further evidence that glutamate is the parallel fibre transmitter and suggest that its acts only on non-NMDA (non-N-methyl-D-aspartate) receptors at synapses with Purkinje cells but on both NMDA and non-NMDA receptors at synapses with inhibitory interneurones. At the latter synapses, the NMDA system is likely to be brought into operation in an activity-dependent manner.     

Whole-cell patch-clamp recordings of an NMDA receptor-mediated synaptic current in rat hippocampal slices

Whole-cell patch-clamp recordings and pharmacological techniques have been used to obtain low noise recordings of 2 excitatory postsynaptic synaptic currents (termed EPSCA and EPSCB) evoked by stimulation of the Schaffer collateral-commissural pathway in rat hippocampal slices. EPSCA was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and EPSCB was blocked by D-2-amino-5-phosphonovalerate (APV), indicating their mediation by non-N-methyl-D-aspartate (non-NMDA) and NMDA receptors, respectively. EPSCB has a slower time-course than EPSCA and its current-voltage relationship was highly non-linear with a region of negative slope conductance from -35 to -100 mV. These properties of EPSCA and EPSCB can explain their differing participation in synaptic transmission in this pathway.     

Acousticolateral and visual processing and their interaction in the torus semicircularis of the trout, Salmo gairdneri

The hypothesis that synaptic transmission between the auditory nerve and the cochlear nucleus is mediated by an excitatory amino acid acting through N-methyl-D-aspartate (NMDA) receptors was examined in an in vitro preparation of the chicken brainstem. The ability of various bath-applied excitatory amino acid receptor antagonists to inhibit synaptically-evoked responses was assessed by recording field potentials from nucleus magnocellularis (NM) following electrical stimulation of the cochlear nerve. Antagonists that selectively block responses mediated by NMDA receptors, such as D-alpha-aminoadipate and 2-amino-5-phosphonovalerate, were without effect on evoked transmission in NM. In contrast, antagonists that additionally act on non-NMDA receptors, such as cis-2,3-piperidine dicarboxylate and gamma-D-glutamylglycine, reversibly suppressed transmission. The results indicate that (1) transmission in the chicken auditory system is mediated by non-NMDA receptors, and (2) a substance(s) chemically akin to aspartate and glutamate may be the transmitter used by the auditory nerve in NM.     

Differential expression of NMDA receptors in serotonergic and/or GABAergic neurons in the midbrain periaqueductal gray of the mouse

N-methyl-d-aspartate (NMDA) receptors expressed in the midbrain periaqueductal gray (PAG) exert various physiological functions. The PAG contains various neurotransmitter phenotypes, which include GABAergic neurons and serotonergic neurons. In the present experiments, we made tight-seal whole-cell recordings from GABAergic and/or serotonergic neurons in mouse PAG slices and analyzed NMDA and non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation. The NMDA/non-NMDA ratio of EPSC amplitude was high and the decay time course of NMDA-EPSC was slow in non-serotonergic/GABAergic neurons. In contrast, serotonergic neurons exhibited a low NMDA/non-NMDA ratio and a fast decay time course of NMDA-EPSC. Peripheral nerve ligation-induced chronic pain was associated with an increased NMDA/non-NMDA ratio in serotonergic neurons. Additionally, single-cell real-time RT-PCR analysis showed that peripheral nerve ligation up-regulated NR2B subunit expression in non-serotonergic/non-GABAergic neurons. Such changes in NMDA receptor expression in the PAG result in an alteration of the descending modulation of nociception, which might be an underlying mechanism for peripheral nerve injury-evoked persistent pain. Finally, the expression of NMDA receptors seems differentially regulated among neurons of different neurotransmitter phenotypes in the PAG.     

Analysis of synaptic events in the mouse accessory olfactory bulb with current source-density techniques.

The accessory olfactory bulb of the mouse was studied by current source-density analysis of field potentials to determine the laminar and temporal distribution of synaptic currents evoked by electrical stimulation of the vomeronasal organ. The one-dimensional current source-density analysis revealed two major spatially and temporally distinct inward membrane currents (sinks): one in the glomerular layer and the other in the external plexiform layer. The glomerular layer sink preceded the external plexiform layer sink by a mean of 5.5 ms. Local infusions of the broad-spectrum excitatory amino acid antagonist, kynurenate, into the accessory olfactory bulb blocked the external plexiform layer sink without an obvious effect on the glomerular layer sink. The selective non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione produced a dose-dependent blockade of the external plexiform layer sink, whereas the selective N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate was without effect. These results, taken together with the cytoarchitecture of the accessory olfactory bulb, suggest that the glomerular layer sink results mainly from synaptic excitation evoked in the glomerular dendritic branches of mitral cells by the vomeronasal afferent fibres and the external plexiform layer sink mainly from non-N-methyl-D-aspartate receptor-mediated synaptic excitation in the peripheral processes of granule cells via the mitral to granule cell dendrodendritic synapse.     

Actions of excitatory amino acid antagonists on synaptic potentials of layer II/III neurons of the cat's visual cortex

Summary Actions of excitatory amino acid (EAA) antagonists on the responses of cells in layers II/III and IV of the cat's visual cortex to stimulation of layer VI and the underlying white matter were studied in slice preparations. Antagonists used were 2-amino-5-phosphonovalerate (APV), a selective antagonist for the N-methyl-D-aspartate (NMDA) type of EAA receptors, and kynurenate, a broadspectrum antagonist for the three types of EAA receptors. In extracellular recordings it was demonstrated that most of the layer II/III cells were sensitive to APV, while the great majority of the layer IV cells were not, By contrast, kynurenate suppressed the responses completely in both layers. Excitatory post-synaptic potentials (EPSPs) evoked by stimulation of layer VI and the while matter were recorded intracellularly from layer II/III neurons. To determine whether the EPSPs were elicited mono- or polysynaptically, the synaptic delay for each EPSP was calculated from a pair of onset latencies of EPSPs evoked by stimulation of the two sites. Forty-two percent of the layer II/III cells were classified as having monosynaptic EPSPs. In 60% of these monosynaptic cells, the rising slope of the EPSPs was reduced by APV while in the other 40%, it was not. In the former (APV-sensitive cells), subtraction of the APV-sensitive component from the total EPSP indicated that the onset latency of the NMDA receptor-mediated component was roughly equal to that of the non-NMDA component. In the latter (APV-resistant cells), only the slowly-decaying component was in part mediated by NMDA receptors. The conduction velocities of the afferent fibers innervating APV-resistant cells were slower than those of the APV-sensitive cells, suggesting that both types of cells are innervated by different types of afferents. The polysynaptic EPSPs of almost all layer II/III cells were sensitive to APV. The subtraction method indicated that the NMDA component had about the same magnitude as the non-NMDA components. When the slices were superfused by a Mg²⁺-free solution, the EPSPs were potentiated dramatically, but this potentiation was reduced to the control level during the administration of APV. Similarly, APV-sensitive components were potentiated during the administration of bicuculline, a selective antagonist for gamma-aminobutyric acid receptors of A type. These results suggest that NMDA receptors participate, at varying degrees, in excitatory synaptic transmission at most layer II/III cells in the cat's visual cortex, and their actions appear to be regulated by intracortical inhibition.     

Excitatory amino acid-mediated components of synaptically evoked input from dorsal roots to deep dorsal horn neurons in the rat spinal cord slice

The participation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the responses of deep dorsal horn neurons to single shock stimulation of dorsal roots was investigated using current- and voltage-clamp techniques. In the presence of Mg2+, superfusion of rat spinal slices with 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX), a potent antagonist of non-NMDA receptors, reversibly blocks fast excitatory synaptic responses elicited by low-frequency stimulation of dorsal roots and to a greater extent the responses to quisqualate than to kainate or NMDA. The synaptic response elicited in a zero-Mg2+ medium is less sensitive to CNQX. The CNQX-resistant component is however abolished by D-APV, a selective antagonist of NMDA receptor. Under voltage-clamp, the excitatory postsynaptic currents also showed an initial fast (CNQX-sensitive) and a late slow (2-amino-5-phosphonovalerate (APV)-sensitive, Mg2+-sensitive) component, both of which had similar thresholds but differed in their latency, time-to-peak and duration. These results support the concept that both non-NMDA and NMDA receptor channels are present in a majority of deep dorsal horn neurons and could be simultaneously activated by transmitter released from stimulated primary afferents.     

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)     

Participation of excitatory amino acid receptors in the slow excitatory synaptic transmission in the rat spinal dorsal horn in vitro

The participation of excitatory amino acid (EAA) receptors in the responses of deep dorsal horn neurons to repetitive stimulation of dorsal roots was investigated using a spinal slice preparation and current-clamp and voltage-clamp techniques. Using EAA receptor and substance P (SP) receptor antagonists and current-clamp, slow excitatory synaptic response evoked by 10-20 Hz stimulation consisted of two depolarizing components: an initial component lasting 1-5 s and a late-one of 1-3 min duration. The initial and late components of the slow excitatory postsynaptic currents (EPSCs) can also be distinguished on the basis of their voltage-dependence and sensitivity to Mg2+ ions, D-2-amino-5-phosphonovalerate (D-APV) and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX). In the presence of Mg2+, the initial component of the slow EPSC increased with membrane hyperpolarization, whereas the late component decreased. In a zero-Mg2+ medium, the initial component was potentiated, but the late component was reduced, or unchanged. CNQX reduced the initial component. In a zero-Mg2+ solution, or at membrane potentials positive to -55 mV in 1 mM Mg2+, D-APV reduced or even abolished the initial component, whereas the late component was not modified by D-APV. We propose that slow excitatory synaptic response evoked in deep dorsal horn neurons by repetitive stimulation of primary afferents has two components, an initial transient component that requires activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, and a late longer-lasting peptidergic component that has been already described (Brain Res., 290 (1984) 336-341.     

The kainate/quisqualate receptor antagonist, CNQX, blocks the fast component of spontaneous epileptiform activity in organotypic cultures of rat hippocampus

Intracellular recordings were made from CA1 pyramidal neurones of hippocampus maintained in organotypic culture. Both spontaneous interictal and ictal epileptiform activity was observed. CNQX, an antagonist at kainate/quisqualate but not at N-methyl-D-aspartate (NMDA)-sensitive excitatory amino acid receptors depressed but did not abolish spontaneous epileptiform activity. Addition of the specific NMDA receptor antagonist D-2-amino-5-phosphonovalerate (D-APV) abolished the remaining activity. Similar effects were observed on electrically evoked excitatory post synaptic potentials (EPSPs). This suggests a role for endogenous excitatory amino acids acting at both kainate/quisqualate and NMDA sensitive excitatory amino acid receptors in the generation and maintainance of epileptiform activity within these organotypic cultures.     

The involvement of N-methyl-d-aspartate (NMDA) and non-NMDA receptors in the responsiveness of rat spinal neurons with input from the chronically inflamed ankle

Unilateral adjuvant inflammation was induced at the rat ankle 2 or 20 days before an evaluation of the contribution of N-methyl-d-aspartate (NMDA) and non-NMDA receptors to the processing of nociceptive information by wide dynamic range neurons in the spinal cord. Microionophoretic application of either the NMDA receptor antagonists ketamine and DL-2-amino-5-phosphonovalerate (AP5) or the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduced the responses to innocuous and noxious mechanical stimulation of the inflamed ankle. The pattern of these effects was comparable to that in rats with acute inflammation suggesting that non-NMDA and NMDA receptors are similarly involved in acute, prolonged acute and chronic inflammation-evoked activity.     

Induction of long-term potentiation without participation of N-methyl-D-aspartate receptors in kitten visual cortex.

1. Intracellular recording was made from layer II-III cells in slice preparations of kitten (30-40 days old) visual cortex. Low-frequency (0.1 Hz) stimulation of white matter (WM) usually evoked an excitatory postsynaptic potential (EPSP) followed by an inhibitory postsynaptic potential (IPSP). The postsynaptic potentials (PSPs) showed strong dependence on stimulus frequency. Early component of EPSP and IPSP evoked by weak stimulation both decreased monotonically at frequencies greater than 0.5-1 Hz. Strong stimulation similarly depressed the early EPSP at higher frequencies (greater than 2 Hz) and replaced the IPSP with a late EPSP, which had a maximum amplitude in the stimulus frequency range of 2-5 Hz. 2. Very weak WM stimulation sometimes evoked EPSPs in isolation from IPSPs. The falling phase of the EPSP revealed voltage dependence characteristic to the responses mediated by N-methyl-D-aspartate (NMDA) receptors and was depressed by application of an NMDA antagonist DL-2-amino-5-phosphonovalerate (APV), whereas the rising phase of the EPSP was insensitive to APV. 3. The early EPSPs followed by IPSPs were insensitive to APV but were replaced with a slow depolarizing potential by application of a non-NMDA antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX), indicating that the early EPSP is mediated by non-NMDA receptors. The slow depolarization was mediated by NMDA receptors because it was depressed by membrane hyperpolarization or addition of APV. 4. The late EPSP evoked by higher-frequency stimulation was abolished by APV, indicating that it is mediated by NMDA receptors, which are located either on the recorded cell or on presynaptic cells to the recorded cells. 5. Long-term potentiation (LTP) of EPSPs was examined in cells perfused with solutions containing 1 microM bicuculline methiodide (BIM), a gamma-aminobutyric acid (GABA) antagonist. WM was stimulated at 2 Hz for 15 min as a conditioning stimulus to induce LTP, and the resultant changes were tested by low-frequency (0.1 Hz) stimulation of WM. 6. LTP of early EPSPs occurred in more than one-half of the cells (8/13) after strong conditioning stimulation. The rising slope of the EPSP was increased 1.6 times on average. 7. To test involvement of NMDA receptors in the induction of LTP in the early EPSP, the effect of conditioning stimulation was studied in a solution containing 100 microM APV, which was sufficient to block completely synaptic transmission mediated by NMDA receptors. LTP occurred in the same frequency and magnitude as in control solution.     

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.     

Synaptic activation of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors in the mossy fibre pathway in adult and immature rat cerebellar slices

The participation of excitatory amino acid receptors in mossy fibre-granule cell synapses in lobule VIa of adult and immature rat cerebellar slices was investigated using an extracellular grease-gap technique. For the immature slices, the age selected (14 days after birth) was one at which the sensitivity of granule cells to exogenous N-methyl-D-aspartate is much higher than in the adult. The principal synaptic potentials observed after low-frequency electrical stimulation of the white matter resembled closely those found to be centred in the granule cell layer in field potential studies in the cat in vivo. They comprised a short latency negative potential, a slow negative wave and, in the adult, a further late negative wave. In the adult, with 1.2 mM Mg2+ in the perfusing solution, none of these potentials was significantly affected by the N-methyl-D-aspartate antagonist, 2-amino-5-phosphonovalerate, but they were all markedly inhibited by the broad spectrum antagonist, kynurenate, and, more potently, by the selective non-N-methyl-D-aspartate receptor blocker, 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline. After removal of Mg2+, which has a blocking action on the ion channels associated with N-methyl-D-aspartate receptors, the size of all the potentials increased. The increase in the short latency potential was insensitive to 2-amino-5-phosphonovalerate but a component of the slow negative wave (and of the late negative wave) was reduced back to control levels by the antagonist. Application of 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (10 microM) in Mg2+-free solution revealed, in near isolation, a slow wave (latency to peak, 28 ms) which could be abolished by 2-amino-5-phosphonovalerate. In the immature slices, bathed in normal (Mg2+-containing) medium, 2-amino-5-phosphonovalerate caused a small reduction in the short latency potential and inhibited a component of the slow negative wave which could, again, be observed in relative isolation after perfusion of 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline. Removal of Mg2+ increased the amplitudes of the short latency potential and the slow negative wave in a manner which was sensitive to 2-amino-5-phosphonovalerate and increased the size of the slow, 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline-resistant wave. It is concluded that glutamate is likely to be the transmitter released by mossy fibres, at least those innervating lobule VIa.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excitatory and inhibitory responses of Purkinje cells, in the rat cerebellum in vivo, induced by excitatory amino acids

Responses of rat cerebellar Purkinje cells to iontophoretically administered excitatory amino acids have been studied in vivo. Responses to N-methyl-D-aspartate (NMDA) were either biphasic (excitation followed by inhibition) or purely inhibitory and were antagonized by the selective NMDA-receptor antagonist, 2-amino-5-phosphonovalerate. Quisqualate and kainate either excited or induced biphasic responses, in these neurones, which were only reduced by amino acid antagonists that acted at non-NMDA receptors. The excitatory amino acid-induced inhibitions were also antagonized by the selective gamma-aminobutyric acid (GABA) antagonist, picrotoxin, suggesting that they were indirectly mediated via GABAergic inhibitory interneurones, which could be excited via NMDA and non-NMDA receptors.     

Involvement of non-NMDA receptors in picrotoxin-induced epileptiform activity in the hippocampus

The ability of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to suppress picrotoxin-induced epileptiform burst activity was examined. Intracellular recordings were obtained from hippocampal CA1 and CA3 pyramidal neurons maintained in vitro. Bath application of CNQX (5 microM) significantly reduced or abolished evoked paroxysmal depolarizing shifts (PDSs) in all CA1 and CA3 neurons tested. In cells where a CNQX-insensitive component in the PDS was manifest, this remaining activity was abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (20 microM), suggesting the existence of a NMDA-mediated synaptic potential. Our results indicate that non-NMDA receptor antagonists are capable of markedly reducing picrotoxin-induced epileptiform activity and that these receptors play an important role in generation of PDSs.     

Long-term potentiation in slices of kitten visual cortex and the effects of NMDA receptor blockade.

1. A slice preparation was used to study layer III field potentials (FPs) evoked by electrical stimulation of the white matter-layer VI border and their potentiation by patterned stimuli. 2. The dependence of the FP on recording position was investigated. The maximum field was recorded in layer III at a position radial to the site of stimulation. Because this negative FP reflects an excitatory synaptic current sink, this site was chosen for all subsequent experiments. 3. Under normal recording conditions, components of the layer III FP with latencies greater than 3 ms were completely abolished by kynurenate but unaffected by 2-amino-5-phosphonovalerate (AP5), indicating that this potential reflects the activation of non-NMDA excitatory amino acid receptors. 4. Addition of the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline methiodide (BMI) broadened the field potential and revealed an AP5-sensitive component. By filling the recording pipette with BMI, it was possible to substantially reduce inhibition locally around the recording site while avoiding stimulus-driven and spontaneous epileptiform activity. 5. Tetanic stimulation elicited a long-term potentiation (LTP) of the FP in 14 of 17 experiments when the BMI-filled pipette method was used. 6. Addition of 100 microM D,L-AP5 significantly reduced the average probability and magnitude of LTP. Nonetheless, in 2 of 8 experiments, significant LTP was observed after a tetanus in the presence of AP5. Control experiments confirmed that this concentration of AP5 was sufficient to maximally block cortical NMDA receptors. 7. We conclude that LTP of layer III field potentials can be reliably elicited, provided that GABAA-receptor mediated inhibition is blocked locally at the site of recording and that NMDA receptors are recruited during the conditioning stimulation. However, activation of NMDA receptors is apparently not an obligatory step for the induction of use-dependent increases in synaptic strength in the kitten striate cortex.     

Respiratory activation of the genioglossus muscle involves both non-NMDA and NMDA glutamate receptors at the hypoglossal motor nucleus in vivo

Brainstem respiratory neurons innervate the hypoglossal motor nucleus which in turn transmits this respiratory drive signal to the genioglossus muscle of the tongue. The mechanism of this transmission is important to help maintain an open airspace for effective breathing, and is thought to rely almost exclusively on non-N-methyl-d-aspartate (non-NMDA) glutamate receptor activation during respiration. However those studies were performed in slices of medulla from neonatal animals in vitro which may have led to an underestimation of the contribution of NMDA glutamate receptors that may normally operate in intact preparations. The current study tests the hypothesis that both NMDA and non-NMDA receptors contribute to respiratory drive transmission at the hypoglossal motor nucleus in vivo. Experiments were performed in urethane-anesthetized and tracheotomized adult Wistar rats in which vagus nerves were either intact or sectioned. In the presence of augmented genioglossus activity produced by vagotomy, microdialysis perfusion of either an NMDA receptor antagonist (D-2-amino-5-phosphonovaleric acid, 0.001-10 mM) or a non-NMDA receptor antagonist (6-cyano-7-nitroquinoxaline-2, 3-dione disodium salt, 0.001-1 mM) to the hypoglossal motor nucleus reduced respiratory-related genioglossus activity in a dose-dependent manner (P < 0.001) indicating that both NMDA and non-NMDA glutamate receptors are necessary for transmission of the respiratory drive signal to genioglossus muscle in vivo. Similar effects were observed in the vagus nerve intact rats. Further experiments demonstrated that each delivered antagonist had effects that were specific to its respective receptor. Regression analysis also revealed that the activity of both NMDA and non-NMDA receptors at the hypoglossal motor nucleus is related to levels of the prevailing respiratory drive. These results show that both NMDA and non-NMDA glutamate receptors at the hypoglossal motor nucleus are involved in transmission of the respiratory drive signal to genioglossus muscle in vivo.     

In vivo blockade of N-methyl-D-aspartate receptors induces rapid trafficking of NR2B subunits away from synapses and out of spines and terminals in adult cortex

We investigated the role of in vivo synaptic activity upon trafficking of the N-methyl-D-aspartate (NMDA) receptor subunit, NR2B, at mature synapses by electron microscopic immunocytochemistry. In vivo blockade of NMDA receptors was achieved by applying the NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV), onto the cortical surface of one hemisphere of anesthetized adult rats. Inactive L-2-amino-5-phosphonovalerate (L-APV) was applied to the contralateral hemisphere for within-animal control and to assess basal level of NR2B subunits at synapses. Within 30 min of D-APV treatment, we observed a decrease in the number of layer I axo-spinous asymmetric synapses that are positively immuno-labeled for the NR2B subunits. This decrease was paralleled by reductions in the absolute number of immuno-gold particles found at these synapses. The decrease of NR2B labeling was detectable in all five animals examined. Significant reductions were seen not only at post-synaptic densities, but also within the cytoplasm of spines and axon terminals. The data demonstrate that blockade of NMDA receptors induces trafficking of NR2B subunits out of synaptic membranes, spines, and terminals. This is in sharp contrast to a previous observation that NR2A subunits move into spines and axon terminals following in vivo blockade with D-APV. These findings point to yet unknown, NMDA receptor activity-dependent mechanisms that separately regulate the localization of NR2A and NR2B subunits at synapses.