Excitatory neurotransmitters in the lateral habenula and pedunculopontine nucleus of rat modulate limbic seizures induced by pilocarpine.

The involvement of the excitatory neurotransmitter system in the lateral habenula and pedunculopontine nucleus in the initiation and propagation of limbic seizures induced by pilocarpine has been investigated in the rat. Limbic seizures occur in animals following bilateral microinjection into the lateral habenula of N-methyl-D-aspartate (NMDA) (5 and 12.5 nmol) or kainate (100 and 200 pmol), 15 min prior to a subconvulsant dose of pilocarpine (150 mg/kg, i.p.). In the absence of pilocarpine NMDA (5 and 12.5 nmol) or kainate (100 and 200 pmol), injected focally into the lateral habenula or pedunculopontine nucleus, produced sniffing, grooming and tremor but no electrographic or behavioural seizures. Limbic seizures also occur after a subconvulsant dose of pilocarpine when it is preceded by injection of NMDA (5 and 12.5 nmol) or kainate (50, 100 and 200 pmol) into the pedunculopontine nucleus. Behavioural and electrographic signs of limbic seizures following pilocarpine (380 mg/kg, i.p.) were attenuated or completely antagonized by focal injection into the lateral habenula of the NMDA antagonist, 2-amino-7-phosphonoheptanoate (AP7) (10 and 50 pmol) or kainate antagonist, gamma-D-glutamylaminomethylsulphonate (GAMS) (20 nmol). In addition, AP7 (0.05, 0.1 and 1.0 nmol) or GAMS (40 nmol) injected into the pedunculopontine nucleus suppressed limbic seizures induced by i.p. administration of pilocarpine (380 mg/kg). The relative efficacy of NMDA and non-NMDA receptor antagonists revealed that the selective NMDA antagonist, AP7, was more potent in its anticonvulsant activity in comparison to GAMS, a non-NMDA receptor antagonist.     

Differential effect of peripheral glutamate (NMDA,non-NMDA) receptor antagonists on bee venom-induced spontaneous nociception and sensitization

This study aimed to investigate the role of peripheral N-methyl-d-aspartate (NMDA) and non-NMDA receptor on (1). spontaneous nociception and (2). on sensitization induced by subcutaneous (s.c.) injection of bee venom (0.2mg/50 micro l) in rats. Peripheral s.c. administration of the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5), the non-competitive NMDA receptor channel blocker MK-801, and the competitive non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were performed before (pre-treatment) and after (post-treatment) bee venom-induced inflammation. Pre-treatment with AP5 (10mM, 50 micro l) and both pre-treatment and post-treatment with MK-801 (2mM, 50 micro l) into the same area of the bee venom injection site markedly reduced the bee venom-increased spontaneous responses of wide-dynamic range (WDR) neuron of the spinal cord. Post-treatment with the same dose of AP5 as well as pre-treatment and post-treatment with CNQX (5mM, 50 micro l) did not produce any inhibitory effects. Additionally, the role of peripheral NMDA and non-NMDA receptors on bee venom-induced mechanical allodynia and hyperalgesia were investigated and assessed by the paw withdrawal reflex to the innocuous and noxious mechanical stimulation. Peripheral administration of AP5, but not CNQX, reduced mechanical allodynia and hyperalgesia. The data suggest that the peripheral NMDA receptor, but not non-NMDA receptor, plays a pivotal role in the bee venom-induced persistent nociception and hyperexcitability.     

Intrathecally administered big dynorphin,a prodynorphin-derived peptide,produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanism

Intrathecal (i.t.) administration of big dynorphin (1-10 fmol), a prodynorphin-derived peptide consisting of dynorphin A and dynorphin B, to mice produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 5-15 min after an injection. Dynorphin A produced a similar response, though the doses required were higher (0.1-30 pmol) whereas dynorphin B was practically inactive even at 1000 pmol. The behavior induced by big dynorphin (3 fmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125-2 mg/kg) and also dose-dependently, by i.t. co-administration of D(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.25-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 pmol), an inhibitor of the NMDA receptor ion-channel complex interacting with the NR2B subunit and the polyamine recognition site. On the other hand, naloxone, an opioid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe(7),D-His(9)]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10376, a tachykinin NK2 receptor antagonist, had no effect. These results suggest that big dynorphin-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the NR2B subunit and/or the polyamine recognition site but not on the glycine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms or tachykinin receptors in the mouse spinal cord.     

Differential roles of NMDA and non-NMDA receptor activation in induction and maintenance of thermal hyperalgesia in rats with painful peripheral mononeuropathy

Central activation of excitatory amino acid receptors has been implicated in neuropathic pain following nerve injury. In a rat model of painful peripheral mononeuropathy, we compared the effects of non-competitive NMDA receptor antagonists (MK 801 and HA966) and a non-NMDA receptor antagonist (CNQX) on induction and maintenance of thermal hyperalgesia induced by chronic constrictive injury (CCI) of the rat common sciatic nerve. Thermal hyperalgesia to radiant heat was assessed by using a foot-withdrawal test and NMDA/non-NMDA receptor antagonists were administered intrathecally onto the lumbar spinal cord before and after nerve injury. Four daily single treatments with 20 nmol HA966 or CNQX beginning 15 min prior to nerve ligation (pre-injury treatment), reliably reduced thermal hyperalgesia in CCI rats on days 3, 5, 7 and 10 after nerve ligation. Thermal hyperalgesia was also reduced in CCI rats receiving a single post-injury treatment with HA966 (20 or 80 nmol) or MK 801 (5 or 20 nmol) on day 3 after nerve ligation when thermal hyperalgesia was well developed. In contrast, a single post-injury CNQX (20 or 80 nmol) treatment failed to reduce thermal hyperalgesia or to potentiate effects of HA966 or MK 801 (5 or 20 nmol) on thermal hyperalgesia in CCI rats. Moreover, multiple post-injury CNQX treatments utilizing the same dose regime as employed for the pre-injury treatment attenuated thermal hyperalgesia but only when the treatment began 1 or 24 h (but not 72 h) after nerve ligation.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMDA and quisqualate modulation of visceral nociception in the rat

The effects of acid (NMDA; 100 fmol-1 nmol) or quisqualic acid (QA; 10 pmol-10 nmol) on visceromotor and pressor responses to noxious colorectal distention (CRD; 40 mmHg, 20 s duration, interstimulus interval: 4 min) were studied in awake rats. Lesser doses of NMDA (100 fmol - 1 pmol) administered intrathecally (i.t.) to the lumbar spinal cord produced a dose-dependent facilitation of visceromotor as well as pressor responses to CRD (maximum with 1 pmol NMDA at 1 min). The greatest dose tested (1 nmol) attenuated these responses (maximum at 1 min) and also produced a caudally-directed biting and scratching behavior accompanied by vocalizations. NMDA did not produce any of the above effects when administered i.t. to the thoracic spinal cord. I.t. pretreatment with the NMDA receptor antagonist, -2-amino-5-phosphonovaleric acid ( -APV; 1 pmol), which produced no change in baseline activity or control responses, blocked all NMDA-produced effects in a reversible manner. QA produced dose-dependent inhibitory effects on visceromotor as well as pressor responses to noxious CRD when given i.t. to the lumbar spinal cord but not on administration to the thoracic spinal cord. Three nmol QA produced maximum inhibition at 2 min after administration and also produced caudally-directed biting and scratching. All of the QA-produced effects were reversibly blocked by i.t. pretreatment with the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX; 3 nmol), which produced no change in baseline activity or control responses. We also examined the effects of NMDA and QA on responses to graded intensities of CRD. One pmol NMDA selectively facilitated visceromotor responses to CRD at distention pressures of 40 and 80 mmHg but not at 20 mmHg. In contrast, 3 nmol QA inhibited visceromotor responses to CRD at all intensities tested. In summary, these data suggest that activation of NMDA and non-NMDA receptors in the spinal cord differentially modulates visceral nociceptive input. Spinal segmental NMDA receptor activation produces selective facilitation of visceral nociceptive processing at noxious intensities of stimulation and may thereby contribute to central mechanisms underlying visceral hyperalgesia.     

Modulation of GABA-mediated Synaptic Potentials by Glutamatergic Agonists in Neonatal CA3 Rat Hippocampal Neurons

Intracellular recordings were made from slices of adult and neonatal hippocampal neurons. During the first 2 weeks of life the majority of pyramidal cells exhibited spontaneous gamma-aminobutyric acid (GABA)-mediated synaptic potentials, which were depolarizing at birth and became hyperpolarizing by the end of the first postnatal week. These synaptic potentials were reduced in frequency or blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist d(-)2-amino-5-phosphonovalerate (AP-5, 50 microM) (13/15 cells). The non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 - 10 microM) abolished the GABA-mediated synaptic potentials in all the cells tested (n=12), Superfusion of l-glutamate (up to 100 microM) increased the frequency of both depolarizing and hyperpolarizing GABA-mediated synaptic potentials. This effect was reduced by AP-5 or dl-2-amino-7-phosphonoheptanoate (AP-7, 50 microM) and fully blocked by concomitant application of AP-5 (50 microM) and CNQX (5 - 10 microM). NMDA (0.5 - 2 microM) increased the frequency of the GABA-mediated synaptic potentials. These effects were blocked by AP-5 (50 microM) and by bicuculline (10 microM). Quisqualate (100 - 300 nM), (RS)-alpha-amino-3-hydroxy-5-methyl-4-izopropionate (AMPA, 100 - 300 nM) and kainate (100 nM) also increased the frequency of the GABA-mediated synaptic potentials. These effects were blocked by CNQX (5 - 10 microM) and by bicuculline (10 microM) but not by AP-5 (50 microM). In the presence of tetrodotoxin (TTX, 1 microM), quisqualate (up to 300 nM), AMPA (up to 500 nM) and kainate (100 nM) had no effect on membrane potential or input resistance. In conclusion, our experiments suggest that, in early postnatal life, NMDA and non-NMDA receptors located on GABAergic interneurons modulate GABAergic synaptic potentials.     

Cardiorespiratory responses to glutamatergic antagonists in the caudal ventrolateral medulla of rats.

The role of caudal ventrolateral medullary (CVLM) depressor neurons in influencing arterial pressure and ventilation as well as the baroreflex control of arterial pressure was investigated, and the part played by excitatory N-methyl-D-aspartate (NMDA) and non-NMDA receptors in mediating the responses was determined. In urethane-anesthetized, spontaneously breathing rats unilateral microinjections into the caudal depressor area of the broad-band glutamatergic antagonist kynurenic acid (KYN, 5 nmol or 1.58 nmol), or NMDA antagonist 2-amino-5-phosphonovaleric acid (2-APV, 2.7 nmol), or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 0.257 nmol) caused a respiratory arrest within 4 min and the animals had to be artificially ventilated. Respiratory frequency increased on injecting KYN and CNQX while it did not change significantly with 2-APV. Apnea resulted from progressive decrease in tidal volume. During the apnea ventilation with 5% CO2 did not revive breathing. Mean arterial pressure (MAP) increased significantly with KYN and 2-APV injections but not with CNQX. The baroreflex decrease of MAP, elicited by left or right aortic depressor nerve stimulation, was significantly reduced or abolished after bilateral microinjections of all 3 antagonists. Ventilation as well as the baroreflex usually recovered after 1-1.5 h. Microinjections of the same doses of antagonists into the facial nucleus, as well as application of KYN (25 nmol) to the ventral medullary surface above the hypoglossal rootlets, had no significant effect. The results support previous findings that the CVLM neurons of the rat inhibit sympathetic neurons providing the vasomotor tone, and that an intact CVLM is obligatory for mediating the baroreflex decrease of arterial pressure. The results also indicate that: (1) the CVLM is essential for sustaining ventilation in the rat; (2) only NMDA receptors are involved in maintaining baseline blood pressure while both NMDA and non-NMDA receptors mediate the baroreceptor depressor reflex; and (3) both NMDA and non-NMDA receptor activation is necessary to sustain ventilation.     

Intrathecal high-dose morphine induces spinally-mediated behavioral responses through NMDA receptors

Previous research has demonstrated that intrathecal i.t. morphine in a dose of 60.0 nmol into the spinal subarachnoid space of mice can evoke nociceptive behavioral responses consisting of a severe hindlimb scratching directed toward the flank followed by biting/licking of the hindpaw. The present study was undertaken to examine the involvement of spinal N-methyl-D-aspartate (NMDA) and opioid receptors on the behavioral responses evoked by high-dose i.t. morphine. Pretreatment with naloxone, an opioid receptor antagonist (1.0 and 4.0 mg/kg, s.c.), failed to reverse the morphine-evoked behavioral response, suggesting that the morphine effect is not mediated through the opioid receptors in the spinal cord. The morphine-induced behavior was dose-dependently inhibited by i.t. co-administration of the competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphonovaleric acid (D-APV) (6.25-50.0 pmol) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (3.125-25.0 pmol). The characteristic behavior was also reduced by co-administration of (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801) (74.1-250 pmol), an NMDA ion-channel blocker. Ifenprodil, a competitive antagonist of the polyamine recognition site of NMDA receptor ion channel complex, produced a dose-related inhibitory effect on the behavioral response to i.t. morphine with less potency than the competitive and non-competitive antagonists examined. High doses of (+)-HA-966, a glycine/NMDA antagonist, induced a dose-dependent inhibition of morphine-induced response. The effective dose of i.t. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, needed to reduce the morphine-induced response, was approximately 10-fold greater than that of D-APV. These results suggest that spinal NMDA receptors, but not non-NMDA receptors, may be largely involved in elicitation of the behavioral episode following i.t. injection of morphine in mice.     

The gain of the baroreflex bradycardia is reduced by microinjection of NMDA receptor antagonists into the nucleus tractus solitarii of awake rats

The baroreflex activation with phenylephrine infusion produces a bradycardic response. In the present study, the role of NMDA receptors in the nucleus tractus solitarii (NTS) in the processing of the parasympathetic component of the baroreflex was evaluated using acid phosphonivaleric (AP-5), a selective NMDA receptor antagonist. Baroreflex activation was performed before and after bilateral microinjection of AP-5 into the intermediate commissural NTS (0.5 mm lateral to the midline). Microinjection of the vehicle (saline, 0.9%) or a dose of 2 nmol/50 nl of AP-5 into the NTS produced no effect on the gain of the baroreflex while a dose of 10 nmol/50 nl of AP-5 produced a significant reduction in the gain of the baroreflex 2 min after microinjection [-1.43+/-0.22 vs. -0. 43+/-0.03 bpm/mmHg, (n=6)], with a return to control levels 10 min after the microinjections. The dose of 10 nmol/50 nl was selective for NMDA receptors considering that the cardiovascular responses to microinjection of AMPA (0.05 pmol/50 nl), a non-NMDA receptor agonist, were not affected by this dose of AP-5 and the responses to microinjection of NMDA (2 nmol/50 nl) were blocked. The data show that the bradycardic response to baroreflex activation was blocked by AP-5 microinjected into the NTS, indicating that the neurotransmission of the parasympathetic component of the baroreflex is mediated by NMDA receptors in the NTS.     

Roles of central glutamate, acetylcholine and CGRP receptors in gastrointestinal afferent inputs to vagal preganglionic neurones

It is unknown which neurotransmitter receptors are involved in the transfer of sensory information from the digestive tract to the brainstem. We examined the potential involvement of central glutamate, acetylcholine, and CGRP receptors in vagal pathways arising from gut chemo- and mechanosensitive afferents. Unitary recordings were made from 21 vagal preganglionic efferent neurones. Neuronal discharge showed either excitation or inhibition in response to oesophageal balloon distension (1-2 ml), gastric distension (40-60 ml in whole stomach or 20 ml in proximal stomach), cholecystokinin-8s (100 pmol close intra-arterially) and bradykinin (18 nmol close intra-arterially). Effects of glutamatergic non-NMDA, muscarinic M1, and CGRP1 receptor antagonism on efferent responses were investigated using CNQX (75-155 nmol i.c.v.), pirenzepine (2.5-5.0 micromol/kg i.v.), and hCGRP8-37 (3.2-6.4 nmol i.c.v.), respectively. CNQX, pirenzepine, and hCGRP8-37, respectively, altered efferent responses in 65%, 23% and 41% of neurones. When both CNQX and hCGRP8-37 were administered, a further 58% of responses were reduced. CNQX and hCGRP8-37 reduced a proportion of efferent responses to all stimuli whereas pirenzepine selectively reduced only efferent responses to gastric distension. We conclude that central CGRP1 and non-NMDA receptors are involved in mediating a range of upper gastrointestinal mechano- and chemo-sensitive afferent inputs onto vagal efferents. M1 receptors, on the other hand, are selectively involved in neurotransmission from gastric mechanoreceptors.     

Differential blockade of early and late components of acoustic startle following intrathecal infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) ord,l-2-amino-5-phosphonovaleric acid (AP-5)

The present study investigated the individual contributions of spinal cord N-methyl-d-aspartate (NMDA) and non-NMDA receptors to the acoustic startle reflex in rats. The first experiment measured whole body acoustic startle before and after intrathecal infusion of various doses of either the NMDA receptor antagonist,d,l-2-amino-5-phosphonovaleric acid (AP-5), or the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Both compounds depressed startle in a dose-dependent fashion with similar potencies. A second experiment measured startle electromyographically (EMG) in the quadriceps femoris muscle complex in the hindlimbs during auditory stimulation to characterize the effects of these two compounds on the early ( 8ms) or late ( 15ms) EMG components of the startle response. CNQX preferentially blocked the early EMG component of startle, whereas AP-5 preferentially blocked the late component. These results suggest that the acoustic startle reflex involves an early EMG component mediated by spinal non-NMDA receptors, and a late EMG component mediated by spinal NMDA receptors.     

Differential blockade of early and late components of acoustic startle following intrathecal infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D,L-2-amino-5-phosphonovaleric acid (AP-5)

The present study investigated the individual contributions of spinal cord N-methyl-D-aspartate (NMDA) and non-NMDA receptors to the acoustic startle reflex in rats. The first experiment measured whole body acoustic startle before and after intrathecal infusion of various doses of either the NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (AP-5), or the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Both compounds depressed startle in a dose-dependent fashion with similar potencies. A second experiment measured startle electromyographically (EMG) in the quadriceps femoris muscle complex in the hindlimbs during auditory stimulation to characterized the effects of these two compounds on the early (approximately 8 ms) or late (approximately 15 ms) EMG components of the startle response. CNQX preferentially blocked the early EMG component of startle, whereas AP-5 preferentially blocked the late component. These results suggest that the acoustic startle reflex involves an early EMG component mediated by spinal non-NMDA receptors, and a late EMG component mediated by spinal NMDA receptors.     

Depressor responses to L-proline microinjected into the rat ventrolateral medulla are mediated by ionotropic excitatory amino acid receptors

The essential amino acid L-proline produces a depressor response when microinjected into the caudal ventrolateral medulla (CVLM) of anesthetized rats. L-proline may activate some excitatory amino acid (EAA) receptors. The present study tested this hypothesis by investigating the effects of two ionotropic excitatory amino acid receptor antagonists on the depressor response to L-proline in the CVLM: the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the N-methyl-D-aspartate (NMDA) receptor-selective antagonist MK801. Urethane-anesthetized rats received arterial catheters and their ventrolateral medulla surface was exposed. Injections of the antagonists CNQX and MK801 (2 mM, 68 nl in each case) into the CVLM completely blocked depressor responses to subsequent administration of AMPA (2 pmol/34 nl) and NMDA (2 pmol/34 nl), respectively. The depressor response to L-proline (3.4 nmol/34 nl) was strongly inhibited by prior injection of CNQX (2 mM, 68 nl) and significantly attenuated by prior injection of a high dose (20 mM, 68 nl), but not a low dose (2 mM, 68 nl), of MK801. The results indicate that the depressor response to L-proline in the CVLM includes mechanisms of ionotropic excitatory amino acid receptors.     

Selective blockade of non-NMDA receptors does not block rapidly triggered glutamate-induced neuronal death

The quinoxalinedione, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), has been introduced as a relatively selective antagonist of non-N-methyl-D-aspartate (non-NMDA) glutamate receptors. We studied the ability of CNQX to block excitatory amino acid-induced neurotoxicity in murine cortical cell cultures. 100 microM CNQX blocked the acute neuronal swelling induced by 500 microM kainate, but it also attenuated the swelling and degeneration induced by 500 microM NMDA. Addition of 1 mM glycine to the CNQX eliminated antagonism of NMDA toxicity, while preserving antagonism of the neuronal degeneration induced by kainate or AMPA. This selective non-NMDA antagonist combination of CNQX plus glycine substantially attenuated the acute neuronal swelling induced by brief exposure to 500 microM glutamate, but had little effect on subsequent late degeneration, supporting the conclusion that rapidly triggered glutamate-induced cortical neuronal death is predominantly mediated by NMDA receptors.     

Oxytocin microinjected into dorsal motor nucleus of the vagus excites gallbladder motility via NMDA receptor-NO-cGMP pathway

Our recent study indicated that, in the dorsal motor nucleus of the vagus (DMV), the N-methyl-D-aspartic acid (NMDA) receptor-nitric oxide (NO)-cGMP pathway participated in the regulation of gallbladder motility in rabbits. Oxytocin (OT) is involved as a neurotransmitter in autonomic regulation. The aim of the present experiments is to investigate the effect of OT microinjected into DMV on the gallbladder motility and the involvement of NMDA receptor-NO-cGMP pathway. A frog bladder connected with transducer was inserted into the gallbladder to record the gallbladder pressure. Microinjection of OT (10-50 nmol/L, 100 nl) dose dependently increased the strength of gallbladder phasic contraction. The excitatory effect of OT (10 nmol/L, 100 nl) was completely abolished by atosiban (10 mmol/L, 100 nl), the specific OT receptor antagonist, but was not influenced by [deamino-Pen(1), O-Me-Tyr(2),Arg(8)]-vasopressin (10 mmol/L, 100 nl), the V(1) receptor antagonist. Pretreatment of ketamine (10 mmol/L, 100 nl), the NMDA receptor antagonist, suppressed the gallbladder motor response to OT; but pretreatment of 6-Cyaon-7-Nitroquinoxaline-2,3-(1H,4H)-Dione (CNQX; 10 mmol/L, 100 nl), the non-NMDA receptor antagonist, did not affect it. Pretreatment of L-NAME (10 mmol/L, 100 nl), the nitric oxide synthase (NOS) inhibitor, or methyl blue (10 mmol/L, 100 nl), the guanylyl cyclase inhibitor, inhibited the excitatory effect of OT on gallbladder motility. Hence, we deduced that the microinjection of OT into the DMV enhanced the gallbladder motility through binding specific OT receptors and activating the NMDA receptor-NO-cGMP pathway.     

Parabrachial complex glutamate receptors modulate the cardiorespiratory response evoked from hypothalamic defense area

To characterize the possible role of glutamate in the interaction between Hypothalamic Defense Area (HDA) and Parabrachial complex (PBc) nuclei, cardiorespiratory changes were analyzed in response to electrical stimulation of the HDA (1 ms pulses, 30-50 μA given at 100 Hz for 5s) before and after the microinjection of the nonspecific glutamate receptor antagonist kynurenic acid (50 nl, 5 nmol), NMDA receptor antagonist MK-801 (50 nl, 50 nmol), non-NMDA receptor antagonist CNQX (50 nl, 50 nmol) or metabotropic glutamate receptor antagonist MCPG (50 nl, 5 nmol) within the PBc. HDA stimulation evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (p<0.001) due to a decrease in expiratory time (p<0.01). The respiratory response was accompanied by a pressor (p<0.001) and a tachycardic response (p<0.001). Kynurenic acid within the lateral parabrachial region (lPB) abolished the tachycardia (p<0.001) and decreased the magnitude of blood pressure response (p<0.001) to HDA stimulation. Similarly, the magnitude of the tachycardia and the pressor response was decreased after the microinjection of MK-801 (p<0.01 and p<0.001, respectively) and CNQX (p<0.05 in both cases) into the lPB. Kynurenic acid microinjection in this region produced an inhibition of the tachypnea (p<0.001) to HDA stimulation but the respiratory response persisted unchanged after MK-801 or CNQX microinjection into the lPB. Kynurenic acid within the medial parabrachial region (mPB) abolished the tachycardia (p<0.01) and decreased the magnitude of the pressor response (p<0.001) to HDA stimulation. MK-801 and CNQX microinjection in this region decreased the magnitude of the tachycardia (p<0.05, in both cases) and pressor response (p<0.05, in both cases). The respiratory response evoked by HDA stimulation was not changed after the microinjection of kynurenic acid, MK-801 or CNQX within the mPB. No changes were observed in the cardiorespiratory response evoked to HDA stimulation after MCPG microinjection within lPB and mPB. These results indicate that glutamate PBc receptors are involved in the cardiorespiratory response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.     

Effects of intracerebroventricular dizocilpine (MK801) on dehydration-induced dipsogenic responses, plasma vasopressin and c-fos expression in the rat forebrain

This study determined the interaction between glutamate receptors and dehydration-induced drinking, vasopressin (AVP) release, plasma osmolality and c-fos expression in the brain of conscious rats. The NMDA receptor antagonist dizocilpine (100 nmol infused into the cerebral ventricles) suppressed drinking following either 22 h water deprivation or intragastric injection of hypertonic saline (1.5 M), attenuated the increased plasma vasopressin induced by dehydration, but had no effects on peripheral hyperosmolality caused by either water deprivation or injections of hypertonic saline. Dizocilpine had no inhibitory effects on feeding after 24 h food deprivation. Dizocilpine also suppressed c-fos expression induced by dehydration in the median preoptic nucleus (MPN), the supraoptic and paraventricular nuclei (SON and PVN), but did not influence c-fos expression in the subfornical organ (SFO). The non-NMDA receptor antagonists CNQX (400 nmol) or DNQX (60 nmol) affected neither the animals' drinking nor c-fos expression induced by dehydration. Double staining showed that suppression of c-fos expression following dizocilpine occurred in the NMDA R1 receptor containing neurons in the hypothalamus. These results suggest that the NMDA-type glutamate receptors may be involved in dehydration induced dipsogenic and neuroendocrinological responses. They complement our earlier findings that dizocilpine also attenuated drinking and c-fos expression following intraventricular infusions of angiotensin II.     

Effects of NMDA and non-NMDA receptor antagonists on the medullary inspiratory neuronal activity during spontaneous augmented breaths in anesthetized rats

To elucidate whether there is a difference between the effects of iontophoretically applied N-methyl-

Full-size image

-aspartate (NMDA) and non-NMDA receptor antagonists on the activity of inspiratory neurons during spontaneous augmented breaths, extracellular single unit recording of inspiratory neurons (I-augmenting, I-decrementing and I-other) was performed in pentobarbital anesthetized rats. The spontaneous augmented breath was divided into two different phases; the first phase (phase I) resembled a normal inspiration, but the second phase (phase II) consisted of a marked increase in diaphragm electromyogram activity. The mean firing frequency of I-aug type neurons was significantly decreased after 50 nA application of both

Full-size image

-2-amino-5-phosphonopentanoic acid (AP-5) (NMDA receptor antagonist) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (non-NMDA receptor antagonist). The mean firing frequency of both I-dec and I-other neurons was significantly decreased by both AP-5 and CNQX applications (70 nA). After AP-5 application, relative changes in the discharge rates during inspiratory phases I and II of spontaneous augmented breaths were significantly suppressed in all types of neurons, but CNQX application had no significant effect on the response changes during phase II. In all cell types of neurons, a significant difference between the iontophoretic AP-5 and CNQX applications in the relative mean firing rate was observed. These results suggested that activation of the NMDA receptor-induced neurotransmission can modify the discharge rate of medullary inspiratory neurons, irrespective of the cell types, during the inspiratory phase II of spontaneous augmented breaths, but that non-NMDA receptor blockade may not significantly influence their discharge rate.     

Excitatory amino acid pathways in brain-stimulation reward

A range of agonists and antagonists active at different glutamate/aspartate (Glu/Asp) receptor subtypes were injected into rat ventral tegmental (VTA) sites downstream from self-stimulation electrodes in the medial forebrain bundle. Control injections were made into the contralateral tegmentum. Variable-interval (VI 10 s) self-stimulation was not significantly affected by a specific antagonist of N-methyl-D-aspartate (NMDA)-type receptors (D,L-2-amino-5-phosphonovaleric acid (2-AP5), 10 and 50 nmol). Broad-spectrum excitatory amino acid (EAA) antagonists viz cis-2,3-piperidine dicarboxylate (cPDA) (10 and 50 nmol), gamma-D-glutamylaminomethyl sulphonic acid (GAMS) (10 nmol) and p-chlorobenzoyl-2,3-piperazine dicarboxylic acid (pCB PzDA) (2.0 and 10 nmol), active at kainate, quisqualate, as well as NMDA receptors, all produced significant depression of responding when injected into the ipsilateral, but not the contralateral, tegmentum. Compounds inhibiting Glu/Asp reuptake had variable effects: strong depression with dihydrokainic acid (7.5 nmol), or no significant effect (L-threo-3-hydroxyaspartic acid, 2.0 and 10 nmol). The receptor agonist, NMDA (10 nmol), depressed responding regardless of injection side; kainic and responding regardless of injection side; kainic and quisqualic acid elicited myoclonic and other non-specific responses in preliminary tests, and were not examined further; enhanced responding was not seen. The side-specific blockade of responding by non-NMDA antagonists indicates the existence of non-NMDA EAA terminals in the VTA, signalling the receipt of hypothalamic brain-stimulation reward. Caudally directed EAA projections terminating on A10 dopamine cell bodies may account for depression of self-stimulation by EAA antagonists.     

Selective enhancement of non-NMDA receptor-mediated responses following induction of long-term potentiation in entorhinal cortex

The contribution of NMDA receptors to the expression of long-term potentiation (LTP) is controversial. In entorhinal cortex (EC) previous studies reported either that LTP was exclusively expressed through NMDA receptors or that both NMDA and non-NMDA receptors were involved in LTP expression. To reexamine this issue, horizontal entorhinal cortical slices were prepared from adult rats and electrical stimulation was delivered in layer II/III, while field potential recordings were made in layer III. In the standard condition (2.5 mM Mg(++)), LTP was reliably induced by theta burst stimulation, but was blocked by 100 microM D-AP5, an NMDA receptor antagonist. This corroborates previous reports that NMDA receptor activation is required for induction of EC LTP. The field potential response was not affected by D-AP5, but completely blocked by 10 microM CNQX, a non-NMDA receptor antagonist. This indicates that the expression of LTP is mediated by non-NMDA receptors in the standard condition. LTP of NMDA receptor-mediated responses was tested by comparing NMDA responses before and after applying theta burst stimulation in medium containing low magnesium (0.4-1 mM). Theta burst stimulation induced 43.2+/-9.7% increase of non-NMDA responses (i.e., AP5-insensitive fast component) but 5.6+/-9.0% decrease of the NMDA receptor component (AP5-sensitive slow component). These results indicate that activation of NMDA receptors is critical for induction of LTP, but LTP expression is mediated by non-NMDA receptors in EC under these experimental conditions.