Effects of excitatory amino acid receptor antagonists and agonists on suprachiasmatic nucleus responses to retinohypothalamic tract volleys

A slice preparation of the mouse hypothalamus that includes the suprachiasmatic nuclei (SCN), the optic chiasm and the optic nerves was used for pharmacologic investigations of the nature of the receptors mediating the excitation of SCN neurons by input from the retinohypothalamic tract (RHT). Bath application of cis-2,3-piperidinedicarboxylic acid, a non-selective antagonist of excitatory amino acid receptors, reversibly blocked the postsynaptic component of the field potentials evoked in the dorsolateral SCN by stimulation of the optic nerve. The selective antagonist of N-methyl-D-aspartate receptors, 2-amino-5-phosphonovaleric acid, had no effect on SCN responses. Glutamic acid diethyl ester and 2-amino-4-phosphonobutyric acid also were without effect, but gamma-D-glutamylglycine caused a small decrease in the amplitude of the postsynaptic wave. Addition of the agonists, kainate and N-methyl-D,L-aspartate, to the superfusate also blocked the postsynaptic response. Kainate was the most potent agonist. L-Glutamate was without effect at up to 100 microM. These results indicate that postsynaptic responses in the SCN to retinohypothalamic tract volleys are mediated by a non-NMDA class of excitatory amino acid receptors.     

Excitatory amino acid-evoked membrane currents and excitatory synaptic transmission in lamprey reticulospinal neurons

The characteristics of excitatory amino acid-evoked currents and of excitatory synaptic events have been examined in lamprey Müller neurons using voltage clamp and current clamp recording techniques. Application of glutamate evoked depolarizations associated with a decrease in input resistance. The reversal potential of the responses was -35 mV. Under voltage clamp conditions, a series of excitatory amino acid agonists evoked inward currents associated with little or no increase in baseline current noise. The order of potency of the excitatory amino acid agonists was quisqualate greater than kainate greater than glutamate greater than aspartate, while N-methyl-D-aspartic acid (NMDA) was inactive. Inward currents evoked by glutamate, as well as by kainate and quisqualate were attenuated reversibly by 1 mM kynurenic acid (KYN). In contrast, glutamate-evoked currents were not affected by 100 microM D(-)-2-amino-5-phosphonovaleric acid (APV), a selective NMDA antagonist. Spontaneously occurring and stimulus-evoked excitatory postsynaptic events were antagonized reversibly by 1 mM KYN. At this concentration, KYN had no effect on membrane potential, input resistance, or excitability of the cells. In contrast, excitatory postsynaptic currents were unaffected by APV. It is concluded that both glutamate responses and excitatory synaptic transmission in lamprey Müller neurons are mediated by non-NMDA-type receptors and that these receptors are associated with ionic channels with a low elementary conductance. The combined pharmacological and biophysical characteristics of these responses are therefore different from those previously reported in other preparations. Spontaneous (but not stimulus-evoked) inhibitory synaptic events in Müller neurons were blocked reversibly by 1 mM KYN but not by 100 microM APV, suggesting that excitation of interneurons inhibitory to Müller cells was also mediated by non-NMDA receptors.     

Electrophysiological evidence for the presence of ionotropic and metabotropic excitatory amino acid receptors on dopaminergic neurons of the rat mesencephalon: an in vitro study.

The actions of the ionotropic and metabotropic excitatory amino acid agonists AMPA, quisqualate, kainate, NMDA and trans-ACDP were studied by means of intracellular electrophysiological recordings from dopaminergic neurons of rat mesencephalon in brain slices. It was observed that all these agents evoked an inward current in cells which were voltage-clamped near the resting potential (-50, -60 mV). The membrane responses produced by AMPA, kainate and quisqualate were associated with an increase of the apparent input conductance while the responses induced by NMDA and trans-ACDP were associated with a decrease in the apparent input conductance. Therefore, stimulation of ionotropic and metabotropic amino acid receptors activates inward currents in the dopaminergic cells by different mechanisms.     

Excitatory Actions of the Metabotropic Excitatory Amino Acid Receptor Agonist,trans-(+/-)-1-amino-cyclopentane-1,3-dicarboxylate (t-ACPD), on Rat Thalamic Neurons In Vivo

The metabotropic excitatory amino acid receptor agonist trans-(+/-)-1-amino-cyclopentane-1,3-dicarboxylate (t-ACPD) was applied to rat ventrobasal thalamic neurons by iontophoresis. This agonist typically evoked an excitatory response which was slower in onset and of longer duration than responses to the other excitatory amino acid agonists, N-methyl-aspartate, kainate or (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate. Responses to t-ACPD were resistant to the excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione, 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid and kynurenate. These results suggest that t-ACPD may exert its effects via the so-called 'metabotropic' excitatory amino acid receptor. The putative antagonists at this receptor, d-2-amino-4-phosphono-butyrate (d-AP4), l-2-amino-4-phosphono-butyrate (l-AP4) and l-2-amino-3-phosphono-propionate (l-AP3), were able to reduce responses to t-ACPD under certain circumstances. However, such antagonism was always accompanied by similar reductions in excitatory responses to other agonists. These non-selective effects would appear to limit the usefulness of AP4 and AP3 as antagonists of t-ACPD.     

Differences in ischemia-induced accumulation of amino acids in the cat cortex

It is well established that excitatory amino acid neurotransmitters are extensively liberated during ischemia and that they have neurotoxic properties contributing to neuronal injury. To study changes in the liberation of excitatory and other amino acids during cerebral ischemia, we measured their extracellular concentrations and related them to blood flow levels and electrophysiologic activity (electrocorticogram and auditory evoked potentials) before and for up to 2 hours after multiple cerebral vessel occlusion in 14 anesthetized cats. Blood flow levels between 0 and 43 ml/100 g/min were reached. Concentrations of the excitatory amino acid neurotransmitters increased most (aspartate 10-fold, glutamate 30-fold, and gamma-aminobutyric acid 300-fold compared with control values) below a blood flow threshold of 20 ml/100 g/min. The total power of the electrocorticogram and the amplitude of the auditory evoked potentials were affected below the same blood flow threshold. In contrast, concentrations of the nontransmitter amino acids taurine, alanine, asparagine, serine, and glutamine increased 1.5-5-fold as blood flow decreased, while concentrations of the essential amino acids phenylalanine, valine, leucine, and isoleucine did not change during cerebral ischemia. The great increases in concentrations of the excitatory amino acid neurotransmitters below a blood flow threshold close to that for functional disturbance is in accordance with the role of these amino acids in ischemic cell damage. Their release at blood flow levels compatible with cell survival and the increase in their concentrations with severity and duration of cerebral ischemia imply that excitotoxic antagonists may have potential as therapeutic agents.     

Neuropharmacological actions of kynurenic and quinolinic acids on horizontal cells of the isolated fish retina

We have studied the effects of naturally occurring metabolites of tryptophan, kynurenic and quinolinic acids, on the electrophysiological responses of retinal horizontal cells in the fish (Rutilus rutilus, the roach). Quinolinic acid usually hyperpolarizes the cells and reduces their light evoked responses (S-potentials) but on occasion, it causes a slight depolarization of the membrane potential. These actions are similar to those found with N-methyl-d-aspartate (NMDA) and our results are consistent with the proposal that it acts at NMDA binding sites. Kynurenic acid (1mM) invariably hyperpolarizes horizontal cells to their potassium Nernst equilibrium potential and, more significantly, blocks the depolarizing actions exerted on them by excitatory amino acids, such as kainic and quisqualic acids. We show that this action persists in the presence of the synaptic blocker, cobalt chloride, and thus is not mediated by chemical synaptic activity. Kynurenic acid does not reverse depolarization of horizontal cells by dopamine or γ-aminobutyric acid, thus its inhibitory effects are selective to the actions of excitatory amino acids. Neither xanthurenic acid, a close structural analogue of kynurenic acid, nor quinolinic acid are effective in blocking depolarizations by excitatory aino acids.     

Brain-derived neurotrophic factor and neurotrophin receptors modulate glutamate-induced phase shifts of the suprachiasmatic nucleus

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells. Previous work raised the possibility that brain-derived neurotrophic factor (BDNF) and its high-affinity tropomyosin-related receptor kinase may be important as modulators of this excitatory input into the SCN. In order to test this possibility, we used whole-cell patch-clamp methods to measure spontaneous excitatory currents in mouse SCN neurons. We found that the amplitude and frequency of these currents were increased by BDNF and decreased by the neurotrophin receptor inhibitor K252a. The neurotrophin also increased the magnitude of currents evoked by application of N-methyl-d-aspartate and amino-methyl proprionic acid. Next, we measured the rhythms in action potential discharge from the SCN brain slice preparation. We found that application of K252a dramatically reduced the magnitude of phase shifts of the electrical activity rhythm generated by the application of glutamate. By itself, BDNF caused phase shifts that resembled those produced by glutamate and were blocked by K252a. The results demonstrate that BDNF and neurotrophin receptors can enhance glutamatergic synaptic transmission within a subset of SCN neurons and potentiate glutamate-induced phase shifts of the circadian rhythm of neural activity in the SCN.     

Brain-derived neurotrophic factor regulation of N-methyl-D-aspartate receptor-mediated synaptic currents in suprachiasmatic nucleus neurons

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells. Previous work raises the possibility that brain-derived neurotrophic factor (BDNF) and its high-affinity receptor TrkB may be important as modulators of this excitatory input into the SCN. To test this possibility, we used whole-cell patch-clamp methods to measure excitatory currents in rat SCN neurons. These currents were evoked by electrical stimulation of the optic nerve. We found that the amplitude of the N-methyl-D-aspartate (NMDA) component of the evoked excitatory postsynaptic currents (NMDA-EPSC) was increased by application of BDNF. The neurotrophin also increased the magnitude of NMDA-evoked currents in SCN neurons. The BDNF enhancement of the NMDA-EPSC was blocked by treatment with the neurotrophin receptor antagonist K252a as well as treatment with the soluble form of the TrkB receptor engineered as an immunoadhesin (TrkB IgG). Finally, the BDNF enhancement was lost in brain slices treated with the NR2B antagonist ifenprodil. The results demonstrate that BDNF and TrkB receptors are important regulators of retinal glutamatergic synaptic transmission within the SCN.     

Excitatory amino acid receptors appear to mediate paroxysmal depolarizing shifts in rat neocortical neurons in vitro.

This study was designed to assess some of the contributions of excitatory amino acids to locally evoked responses in neurons in slices from frontal motor cortex in Sprague-Dawley rats. Intracellular recordings were obtained from 54 cortical neurons. Paroxysmal depolarization shifts (PDS) were evoked by local single pulse stimulation in cortex or in a small number of cases (n = 2) occurred spontaneously. These potentials could be abolished by application of kynurenic acid, a broad spectrum excitatory amino acid receptor antagonist. They were enhanced in Mg(2+)-free medium and could then be antagonized by application of D,L-2-amino-5-phosphonovalerate (AP5), a selective blocker of the N-methyl-D-aspartate (NMDA) receptors.     

DL-Quisqualic and L-aspartic acids activate separate excitatory conductances in cultured spinal cord neurons

Intracellular recordings were made from mouse spinal cord neurons in dissociated tissue cultures. Input conductance (G_M) of these neurons was assessed using injected constant current pulses before and during applications of excitatory amino acids. Using this technique, it was demonstrated that dl-quisqualic acid depolized and excited these neurons, as did l-aspartic acid, but the evoked change in G_M differed significantly between responses to these amino acids. In neurons where l-aspartic acid activated a voltage-dependent decrease (apparent) in G_M^6–8dl-quisqualic increased G_M by a relatively voltage-independent mechanism and resembled most closely the effects of another amino acid analogue dl-kainic acid.Quisqualic acid, as a consequence, was unable to induce regenerative spikes or burst which are characteristic of responses to a variety of excitatory amino acids^6–8 such as l-aspartic.     

Opiate antagonism of glycine-evoked membrane polarizations in cultured mouse spinal cord neurons

The effect of several opiate receptor agonists on the responses of spinal cord neurons to putative inhibitory and excitatory amino acids was studied using an in vitro model system, cultured fetal mouse spinal cord neurons, and bath application of opiates. Intracellular recordings were made from the cultured neurons with conventional voltage recording techniques or under voltage clamp conditions. The putative amino acid neurotransmitters were applied by iontophoresis or micropressure ejection.Our main finding is that the opiate agonists, morphine and levorphanol (5–100 μM), consistently depressed the responses evoked by the putative inhibitory amino acid neurotransmitters glycine and β-alanine but not GABA. Dextrorphan, the inactive isomer of levorphanol, also depressed the glycine and β-alanine responses, but higher concentrations were required. The excitatory glutamate response was unaltered by these opiates. Leucine enkephalin, an opioid peptide, had no effect on the amino acid responses in the neurons where it was also tested. The opiate antagonist naloxone (10–100 μM) did not reverse the morphine or levorphanol depression of the amino acid responses making it unlikely that opiate receptors mediate this effect.Strychnine was considerably more effective than morphine as a glycine antagonist, producing depressions at nM concentrations compared to the μM concentrations required for morphine. Preliminary studies indicate that both morphine and strychnine act in a non-competitive manner. However, additional studies will be required before the sites of action for these agents can be identified.The possible pharmacological or toxicological significance of the present work remains to be determined. Considering the high doses of opiates (μM concentrations) required to depress the glycine and β-alanine responses, it is unlikely that this action is relevant to normal therapeutic situations. However, such concentrations of opiates are often utilized in pharmacological studies and may be achieved when opiates are applied by iontophoresis. Our data indicate that consideration of the present opiate action should be made when μM concentrations or iontophoretic application of opiates are used for pharmacological studies of CNS tissue.     

Selective synaptic antagonism by atropine and alpha-aminoadipate of pulvinar and cortical afferents to the suprasylvian visual area (Clare-Bishop area)

The synaptic excitations of cells of the Clare-Bishop cortical region produced by electrical stimulation of the pulvinar and ipsilateral cortex, have been shown to be differentially antagonized by iontophoretically applied antagonists. Atropine attenuated the responses evoked by pulvinar stimulation without having an appreciable effect against either iontophoretically applied aspartate or cortically evoked responses. alpha-Aminoadipate antagonized aspartate elicited excitations and those obtained with cortical stimulation while leaving unaffected acetylcholine and pulvinar evoked responses. The results are supportive of the view that acetylcholine and aspartate, or a similar excitatory amino acid, act as synaptic transmitters of some afferents from the pulvinar and ipsilateral cerebral cortex, respectively.     

Ionophoretically applied substance P activates hamster suprachiasmatic nucleus neurons

Ionophoretic ejection of substance P (SP) activated 31% and suppressed 9% of hamster suprachiasmatic nucleus (SCN) cells in vitro. Hamster SCN cells did not demonstrate variation in sensitivity to SP across the circadian phases tested. SP modulated the response of 47% of hamster SCN cells to the excitatory amino acid (EAA) agonists glutamate and N-methyl-d-aspartate (NMDA). The results indicate that SP can alter both the spontaneous and EAA-evoked firing rate characteristics of hamster SCN neurons.     

Selective synaptic antagonism by atropine and α-aminoadipate of pulvinar and cortical afferents to the suprasylvian visual area (Clare-Bishop area)

The synaptic excitations of cells of the Clare-Bishop cortical region produced by electrical stimulation of the pulvinar and ipsilateral cortex, have been shown to be differentially antagonized by iontophoretically applied antagonists. Atropine attenuated the responses evoked by pulvinar stimulation without having an appreciable effect against either iontophoretically applied aspartate or cortically evoked responses. α-Aminoadipate antagonized aspartate elicited excitations and those obtained with cortical stimulation while leaving unaffected acetylcholine and pulvinar evoked responses. The results are supportive of the view that acetylcholine and aspartate, or a similar excitatory amino acid, act as synaptic transmitters of some afferents from the pulvinar and ipsilateral cerebral cortex, respectively.     

Field potentials in the suprachiasmatic nucleus of rat hypothalamic slice produced by optic nerve stimulation

Spatial profile of a projection from retina to the suprachiasmatic nucleus (SCN) was studied by electrophysiological method. Stimulation of contralateral optic nerve evoked the fast positive and late large negative waves in only the ventrolateral but not the dorsolateral parts of the posterior SCN. This result, in agreement with anatomical evidence suggests that the late negative wave is due to excitatory synaptic potential in the SCN and has a role for elevating the neuronal activity in the ventrolateral part of the posterior SCN daytime.     

Pharmacological characterisation of excitatory synaptic transmission in the cat red nucleus in vivo

Extracellular recordings were made from the magnocellular neurones of the red nucleus (mRN) in anaesthetised cats. A study was made of the effects of selective excitatory amino acid receptor antagonists on excitatory monosynaptic responses evoked from the sensorimotor cortex (SMC) and cerebellar interpositus nucleus (IPN). Iontophoretically applied CNQX and NBQX antagonised both SMC and IPN responses whereas,d-AP5 inhibited the SMC response but was ineffective to the IPN. At currents that selectively antagonised NMDA responses, CPPene had no effect on either SMC or IPN responses. 7-chlorokynurenate inhibited both SMC and IPN responses but required currents that antagonised both AMPA and NMDA responses and was therefore acting in a non-selective manner. Iontophoretically applied glycine was inhibitory to both agonist and synaptic responses, whilstd-serine potentiated NMDA responses but did not enhance monosynaptic responses of the SMC. However in the presence of either 7-chlorokynurenate or high currents of CNQX that reduced the SMC synaptic activation of the mRN neurones,d-serine attenuated the inhibitory action of these antagonists. It is concluded that monosynaptic responses from the SMC are mediated by both NMDA and non-NMDA receptors whereas the monosynaptic responses evoked from the IPN are mediated only by non-NMDA receptors. The lack of effect of CPPene is consistent with the postulate that two NMDA receptor subtypes are present on mRN neurones.     

Actions of excitatory amino acid acid agonists and antagonists on the primary afferents of the vestibular system of the axolotl (Ambystoma mexicanum)

In order to determine the nature of the transmitter in the synapse between hair cells and primary afferent fibers, both resting and evoked spike activity of vestibular system afferents were recorded. Excitatory amino acid agonists and antagonists were applied by micro perfusion. Excitatory amino acid agonists consistently increased the firing rate of these afferents. The rank order in potencies of the agonists tested was: kainate greater than or equal to quisqualate greater than D-aspartate greater than or equal to L-glutamate greater than or equal to L-aspartate greater than N-methyl D-aspartate. Blockade of synaptic transmission with high-Mg2+ and low-Ca2+ solutions did not seem to affect the responses to the excitatory amino acid agonists indicating their postsynaptic action. Excitatory amino acid antagonists inhibit both resting and physiologically evoked activity. The rank order of inhibitory potency was: kynurenate greater than L-glutamate diethyl ester greater than D,L-2-amino-4-phosphono-butyrate greater than D-alpha-amino adipate greater than D,L-2-amino-5-phosphonovalerate. These findings suggest that an amino acid-related compound may be the transmitter at this synapse. The relative potencies of agonists and antagonists tested provide evidence that the transmitter released from the hair cells' basal pole in the axolotl vestibular system interacts with postsynaptic kainic/quisqualic type receptors.     

The antinociceptive activity of excitatory amino acids in the rat brainstem: an anatomical and pharmacological analysis.

Rats were stereotaxically implanted with microinjection cannulae aimed at sites ranging caudally from the lower medulla and rostrally to the diencephalon and received microinjections of the excitatory amino acid: L-glutamate 30 nmol/0.5 microliters. The subsequent spontaneous behavioral response and the effect on the thermal noxious-evoked tail flick (TF) and hot plate (HP) responses was recorded. From 331 brain sites mapped with glutamate, an elevation of tail flick and hot plate response latencies was observed in 59 cases and in 34 of these sites the antinociceptive activity was preceded by a shortlasting aversion characterized by vocalization and running. The glutamate-sensitive sites at which TF and HP response latencies were elevated were exclusively distributed in the medullary reticular formation (MRF) and the mesencephalic periaqueductal gray matter (PAG). The aversive and antinociceptive activity of glutamate was dose-dependent and mimicked by the excitatory amino acid (EAA) receptor agonists N-methyl-D-aspartate + (NMDA) kainate and less so quisqualate. The EAA receptor antagonists MK-801 and AP-5, but not glutamyl-amino-methyl-sulfonic acid, antagonized in a dose-dependent fashion both the aversive and antinociceptive responses evoked from the PAG. It is suggested that NMDA receptor-linked neurons in the PAG activate both nociceptive and antinociceptive systems.     

Electrophysiological analysis of suprachiasmatic nucleus projections to the ventrolateral preoptic area in the rat

The circadian pacemaker housed in the suprachiasmatic nucleus (SCN) synchronizes daily sleep-wake cycles, presumably by modulating the sleep-wake regulatory system, including ventrolateral preoptic area (VLPO) neurons. We used whole-cell patch-clamp recording to study the projections from the SCN to the VLPO in horizontal slices of rat hypothalamus. Single-pulse stimulation of the SCN region elicited postsynaptic currents (PSCs) in 20 of 66 neurons (30%) recorded within the VLPO region as verified by intracellular biocytin labelling. At a holding potential of -60 mV, the evoked PSCs had an amplitude of 17.6 +/- 3.2 pA (SEM) and a latency of 6.3 +/- 0.5 ms (n = 10). There was a trend for simple excitatory postsynaptic currents (EPSCs) to be evoked in the VLPO cluster, simple inhibitory postsynaptic currents (IPSCs) in the extended VLPO, and a combination of EPSCs and IPSCs in both regions. IPSCs were blocked reversibly by bicuculline (10 microm, n = 11). In both the presence and absence of bicuculline, EPSCs had fast and slow components that were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 microm; n = 7), and (+/-)3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 10 microm, n = 6), respectively. Reversal potentials for the evoked IPSCs and EPSCs were consistent with mediation via GABAA and ionotropic glutamate receptors, respectively. These results suggest that the SCN region provides both inhibitory and excitatory inputs to single VLPO neurons, which are mediated, respectively, by GABAA receptors and by both non-NMDA and NMDA glutamate receptors. These projections may play important roles in conveying circadian input to systems in the preoptic area that regulate sleep and waking.     

Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro.

We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively. Inhibitory postsynaptic responses were evoked less frequently and were blocked by the GABAA receptor antagonist (-)-bicuculline methochloride. The pharmacological profiles of whole-cell calcium currents and evoked EPSCs were compared. With 50 microM cadmium chloride (Cd), whole-cell low voltage-activated (LVA) calcium currents were reduced in amplitude and high voltage-activated (HVA) calcium currents and excitatory synaptic transmission were completely blocked. This suggests that the residual calcium influx through LVA channels into the presynaptic terminal does not suffice to trigger transmitter release. A saturating concentration of omega-conotoxin GVIA (omega-CgTx) (2.5 microM) blocked one-third of whole-cell HVA calcium currents and evoked EPSCs. The dihydropyridine nifedipine (50 microM) reversibly reduced whole-cell HVA calcium currents in a voltage-dependent manner but not excitatory synaptic transmission. Cd and omega-CgTx did not alter amplitude distributions of miniature EPSCs, demonstrating that the inhibition of synaptic transmission was due to block of presynaptic calcium channels. We conclude that excitatory glutamatergic transmission in thalamic neurons in vitro was mediated mainly by HVA calcium currents, which were insensitive to omega-CgTx and nifedipine.