Structure-activity studies with car☐y- and amino-terminal fragments of neurotensin on hypothalamic neurons in vitro

The purpose of this study was to determine the structural requirements for the activity of neurotensin (NT_1–13) on preoptic/anterior hypothalamic (POAH) neurons in vitro. Standard explant culture electrophysiological techniques were employed. NT was administered to POAH cultures through the superfusion fluid, or, to the vicinity of individual neurons by pressure ejection (0.5–10 psi) from micropipettes. Computer-generated, peri-event histograms were used to quantitate neuronal responses. Pressure ejection of NT_1–13 (50 pM to 1 μM) consistently produced an excitatory effect on 30 of 42 neurons. The reamaining cells were either inhibited or unaffected. Application of the C-terminal hexapeptide, NT_8–13, but not the N-terminal octapeptide, NT_1–8 (</1 mM), produced an excitatory response in 21 of 30 neurons, but was less potent than NT_1–13. Application of an N-acetylated NT_8–13 fragment (NT_AC8–13) produced a response that was similar to that produced by NT_8–13. The excitatory effects of NT_1–13 were maintained in medium which effectively blocked synaptic transmission (0 mM Ca²⁺/12 mM Mg²⁺ 1 mM EGTA). These data indicate that the C-terminal hexapeptide, but not the N-terminal octapeptide, produces a dose-related, excitatory effect on single neurons in the POAH in vitro. The persistence of these effects in Ca²⁺-free medium supports a postsynaptic site of action for these peptides.     

l-Aspartic acid potentiates ‘slow’ inward current in cultured spinal cord neurons

Intracellular recordings (current-and two electrode voltage-clamp) were made from mouse spinal cord neurons grown dissociated in tissue culture. Neurons were bathed in elevated concentrations of calcium (Ca) and sometimes tetraethylammonium (TEA). Brief depolarizing current injections activated graded ‘after-depolarizations’ which summated to trigger prolonged all-or-none action potentials. Under voltage-clamp both of these active potentials were manifest as ‘slow’ inward current. Net inward current was observed in some neurons during 0.5–1.0 s depolarizing command steps. However, in the majority of cases the inward current was seen as large inward current tails (outward current relaxations) upon repolarization of the membrane potential to holding values. Cadmium (Cd) blocked this slow inward current, ‘after-depolarizations’ and prolonged action potentials. Applications of l-aspartic acid increased the magnitude of net inward current evoked by command steps and potentiated and prolonged inward current tails. This potentiation and prolongation of voltage-dependent inward current likely accounts for the prolonged action potentials or ‘bursting’ characteristic of responses to l-aspartic acid and related amino acids such as N-methyl-d-aspartic acid.     

L-aspartic acid induces a region of negative slope conductance in the current-voltage relationship of cultured spinal cord neurons

Individual murine spinal cord neurons were grown in dissociated tissue cultures. Using either one or two conventional intracellular microelectrodes neurons were voltage-clamped and current-voltage (I–V) curves were constructed using command pulses.l-Aspartic acid was then applied and I–V curves repeated in the presence of this excitatory amino acid.l-Aspartic acid induced a region of negative slope conductance (RNSC) in the steady-state I–V relationships of these neurons. Such a RNSC accounts for the apparent reduction of conductance observed in response to excitatory amino acids^5,7,8 and its instability likely contributes to the electrogenesis of bursting in central neurons.     

A quantitative description of excitatory amino acid neurotransmitter responses on cultured embryonic Xenopus spinal neurons

We have performed a quantitative analysis of excitatory amino acid neurotransmitter receptors on cultured embryonic Xenopus spinal neurons using the whole-cell patch-clamp technique. Neuroblasts and underlying mesodermal cells isolated from spinal regions of neural plate-stage embryos were placed into dissociated cell culture, and responses were studied soon after the appearance of neurites on embryonic neurons. Glutamate (Glu) receptors were separated into two general classes based on responses to the characteristic agonists quisqualate (Quis), kainate (Ka) and N-methyl-d-aspartate (NMDA); these were NMDA receptors (those activated by NMDA) and non-NMDA receptors (those activated by Ka and Quis). Half-maximal responses to Glu and other agonists on NMDA and non-NMDA receptors were determined from Hill analysis of dose response relations. The order of sensitivities observed was: Glu_NMDA(ED₅₀ = 5.1 μM) >Glu_non-NMDA(ED₅₀ = 28 μM), and for Glu receptor agonists, Quis (ED₅₀ = 1.5 μM) >NMDA(ED₅₀ = 41 μM) >Ka(ED₅₀ = 58 μM). The order of response amplitudes recorded at concentrations near the appropriate ED₅₀s was Glu_NMDA > Glu_non-NMDA, and Ka > NMDA > Quis. A 10-fold decrease in external [Na⁺] shifted the reversal potentials for Glu_non-NMDA, Ka, and Quis to more negative voltages. Increasing external [Ca²⁺] shifted the reversal potential for NMDA responses to more positive potentials, an observation consistent with Ca²⁺ permeation of the embryonic NMDA-activated channel. NMDA-evoked currents could not be recorded in nominally glycine (Gly)-free media. Addition of Gly to external solutions potentiated NMDA responses (ED₅₀ = 644nM). NMDA responses were blocked by dl-2-amino-5-phosphonovaleric acid (APV;ED₅₀ = 1.9 μM) and by Mg²⁺ at negative potentials. In their sensitivities to agonists and antagonists, and ionic dependences, amino acid neurotransmitter responses on embryonic Xenopus neurons closely resembled those previously observed for mature Xenopus and mammalian central neurons. The Glu_NMDA receptors present on these immature neurons were sufficiently sensitive to be activated by endogenous concentrations of extracellular Glu, suggesting a possible role for receptor activation in modulating early neural development.     

Characterization of specific, high-affinity binding sites for l-[H]glutamic acid in rat brain membranes

l-[³H]Glutamic acid binds reversibly to rat brain membranes with high affinity. Specific binding is linear with tissue concentration and has a pH optimum at neutrality. Saturation isotherms reveal anomalous kinetics of specific binding with an high affinity site with a K_D of 11 nM and a lower affinity site with a K_D of 80 nM; the Scatchard plots intercept at a common bound/free ratio. Hill plots of the complete saturation isotherms have a slope of 1.0. There are marked regional differences in the distribution of binding sites in rat brain: parietal cortex, frontal cortex, hippocampus striatum thalamus cerebellum, pons-medulla and hypothalamus. Except for a small amount of specific binding in heart, other peripheral tissues do not exhibit specific binding of l-[³H]glutamic acid. Several amino acids with neuroexcitatory effects inhibit the specific binding: l-glutamic acid l-aspartic acid and d,l-homocysteic acid d-glutamic acid and l-cysteine sulfinic acid; related amino acids without neuroexcitatory effects do not inhibit specific binding. Reputed antagonists of glutamate-induced neuronal depolarization block specific binding: α-aminoadipic acid 2-amino,4-phosphonobutyric acid glutamate diethylester. Prior kainate lesion of the neurons intrinsic to the striatum results in a 45% decrement in specific binding of l-[³H]glutamic acid whereas cortical ablation, which causes degeneration of a cortical-striatal glutamatergic projection and reduces striatal glutamate synaptosomal uptake, does not affect specific binding. These results are compatible with the interpretation that the binding of [³H]glutamic acid occurs at excitatory receptors on neurons.     

Long-lasting enhancement of synaptic excitability of CA1/subiculum neurons of the rat ventral hippocampus by vasopressin and vasopressin(4–8)

Vasopressin (VP) is axonally distributed in many brain structures, including the ventral hippocampus. Picogram quantities of VP injected into the hippocampus improve the passive avoidance response of rats, presumably by enhancing memory processes. Vasopressin is metabolized by the brain tissue into shorter peptides, such as [pGlu⁴r,Cyt⁶]VP(4–9[ and [pGIu⁴,Cyt⁶,]VP(4–8), which preserve the behavioral activity but lose the peripheral activities of the parent hormone. Using brain slices, we investigated whether VP or VP(4–8) affects excitatory postsynaptic potentials (EPSPs) and/or membrane responses to depolarization in neurons of the CA 1 /subiculum of the ventral hippocampus. The EPSPs were evoked by stimulating the stratum radiatum of the CAI field; the membrane responses were elicited by current injections. Exposure of slices for 15 min to 0.1 nM solution of these peptides resulted in an increase in the amplitude and slope of the EPSPs in 21 neurons (67%) tested. No consistent change in either the resting membrane potential or the input resistance of the neurons was observed. The peptide-induced increase in EPSPs reached a maximum 30–45 min after peptide application. In 14 of these neurons (66%), the peptide-induced increase in EPSPs remained throughout the entire 60–120 min washout period. In the remaining 7 neurons (33%), the initial increase in EPSPs amplitude was followed by a gradual decline to the pre-administration level. The increase in EPSP amplitude was often. but not always, associated with a decrease in the threshold and increase in the number of action potentials in response to depolarizing current injection. Suppression of GABA_A receptor-mediated inhibition and N-methyl-d-aspartate (NMDA) receptor-mediated excitation did not prevent the effects of VP and VP(4–8[ on the EPSP amplitude or the threshold for action potentials. The results demonstrate that 0.1 nM concentrations of these neuropeptides can elicit a long-lasting enhancement of the excitability of CA1/subiculum neurons of the ventral hippocampus to excitatory, glutamatergic synaptic input. This novel action of VP and its metabolite in the ventral hippocampus may be the physiological action, mediating the memory-enhancing effect of these peptides.     

Cardiovascular effects of microinjections of opioid agonists into the `Depressor Region' of the ventrolateral periaqueductal gray region

Microinjections of excitatory amino acids made into the ventrolateral midbrain periaqueductal gray of the rat have revealed that neurons in this region integrate a reaction characterised by quiescence, hyporeactivity, hypotension and bradycardia. Microinjections of both excitatory amino acids and opioids into the ventrolateral periaqueductal gray have shown also that it is a key central site mediating analgesia. The effects of injections of opioids into the ventrolateral periaqueductal gray on arterial pressure and heart rate or behaviour are unknown. In this study we first mapped in the rat the extent of the ventrolateral periaqueductal gray hypotensive region as revealed by microinjections of excitatory amino acids. We found that ventrolateral periaqueductal gray depressor region extended more rostrally than previously thought into the tegmentum ventrolateral to the periaqueductal gray. Subsequently we studied for the first time, the effects of microinjections of μ-, δ-, and κ-opioid agonists made into the ventrolateral periaqueductal grey depressor region. In contrast to the effects of excitatory amino acid injections, microinjections of the μ-opioid agonist ([d-Ala²,N-Me-Phe⁴,Gly-ol⁵]enkephalin) evoked hypertension and tachycardia at approximately 50% of sites. Similar to excitatory amino acid injections, microinjections of both the δ-opioid agonist ([d-Pen²,d-Pen⁵]enkephalin), and the κ-opioid agonist ((5,7,8)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide) evoked either a hypotension and bradycardia, or had no effect. These results indicate that different opiate receptor subtypes are present on a distinct population of ventrolateral periaqueductal gray neurons, or at different ventrolateral periaqueductal gray synaptic locations (pre- or post-synaptic).     

Characterization of the excitatory amino acid receptor-mediated release of [H]acetylcholine from rat striatal slices

The pharmacological nature of the interaction of excitatory amino acids with striatal cholinergic neurons was investigated in vitro. Agonists of excitatory amino acid receptors evoked the release of [³H]acetylcholine from slices of rat striatum, in the presence of magnesium (1.2 mM). Removal of magnesium from the medium markedly increased the release of [³H]acetylcholine evoked by all excitatory amino acid receptor agonists tested, with the exception of kainate. In the absence but not the presence of magnesium, a clear rank order of potency was found: N-methyl-dl-aspartate = ibotenate >l-glutamate >l-aspartate cysteate > kainate = quisqualate.The excitatory amino acid receptor mediating [³H]acetylcholine release resembles the N-methyl-d-aspartate preferring (N-type) receptor, as previously characterized electrophysiologically, according to 3 criteria: (1) rank order of potency of agonists; (2) magnesium-sensitivity; and (3) antagonism by 2-amino-5-phosphonovalerate.The release of [³H]acetylcholine evoked by N-methyl-dl-aspartate was blocked by tetrodotoxin (0.5 μM). Moreover, N-methyl-dl-aspartate failed to evoke [³H]acetylcholine release from slices of hippocampus, where cholinergic afferents, rather than interneurons, are found. These results suggest that excitatory amino acids act at receptors on the dendrites of striatal cholinergic interneurons, giving rise to action potentials and release of acetylcholine from cholinergic nerve terminals.     

Effects of neurotensin on neurons in the rat central amygdaloid nucleus in vitro

The effects of neurotensin (NT) on neurons in the central amygdaloid nucleus (ACe) were investigated in rat brain slice preparations by adding the peptide to the perfusing medium. Of 115 ACe neurons, 69 cells (60%) showed excitatory responses and 10 cells (9%) showed inhibitory responses to application of NT. The excitatory response to NT was observed in a dose-dependent manner and the threshold concentration was approximately 3 × 10⁻⁹ M. The excitatory effects of NT persisted under blockade of synaptic transmission. The NT fragment neurotensin 8–13 and the NT analogue neuromedin N showed effects similar to those of NT, whereas the NT fragment neurotensin 1–8 had no effect on ACe neurons. Of 43 neurons in the septal nucleus, 8 cells (19%) and 3 cells (7%) showed excitatory and inhibitory responses, respectively, to NT. The results suggest that NT exerts a potent excitatory effect on ACe neurons through a direct action on specific receptors, in which NT may play a role in amygdala-relevant functions.     

Streptomycin blocks the postsynaptic effects of excitatory amino acids on the vestibular system primary afferents

It has been suggested that streptomycin might be an antagonist of the glutamate receptors, and that it selectively blocks quisqualic acid receptors. We studied whether streptomycin blocks the responses to excitatory amino acid agonists on the vestibular system primary afferents, and if it allows us to differentiate between kainate (KA) and quisqualate (QA) receptor mediated responses. The experiments were performed in the axolotl (Ambystoma tigrinum). Intra- and extracellular records of the electrical activity of semicircular canal afferent fibers were obtained. Drugs were applied by pressure ejection in volumes of 20 μl in a 10 ml bath. Streptomycin (0.01–10 mM), induced a dose dependent reversible inhibition of the basal spike discharge of the afferent fibers. This coincided with a reduction in the amplitude of excitatory postsynaptic potentials (EPSP) recorded recorded intracellularly in the afferent fibers. Streptomycin also blocked the excitatory action produced by KA and QA; increasing concentrations of streptomycin produced a rightward shift in the concentration-response curves for both KA and QA. This action persisted even in a high Mg²⁺ (10 mM), low Ca²⁺ (0.09 mM) Ringer solution, indicating its postsynapsic nature. These results show that streptomycin might be a non-selective excitatory amino acid (EAA) receptor antagonist.     

Adenosine A1 receptor-mediated depression of corticostriatal and thalamostriatal glutamatergic synaptic potentials in vitro

Electrophysiological recordings in rat brain slices have been used to study the actions of adenosine on striatal neurons and striatal excitatory amino acid neurotransmission originating in the cortex or the thalamus. Adenosine had no effects on membrane properties of striatal neurons. Adenosine and the A₁ agonist N⁶-Cyclopentyl adenosine reduced EPSPs of both cortical and thalamic origin by more than 50%. Depression of EPSPs was associated with an increase in paired-pulse facilitation, suggesting a presynaptic locus of action. EPSP depression was blocked by the A₁ antagonist, 8-Cyclopentyl-1,3-dipropyl xanthine. The A₂ agonist 5′-(N-cyclopropyl)-carboxamidoadenosine had no effect on excitatory amino acid neurotransmission. The A₁ antagonist alone enhanced the synaptic component of the evoked field potential (23±12%). These results indicate that endogenous adenosine, acting via A₁ receptors, limits striatal glutamatergic neurotransmission, serving a modulatory and neuroprotective role.     

Excitatory amino acid receptor subtype agonists induce feeding in the nucleus accumbens shell in rats: opioid antagonist actions and interactions with μ-opioid agonists

Administration of μ-opioid receptor subtype agonists into the nucleus accumbens shell elicits feeding which is dependent upon the normal function of μ-, δ- and κ-opioid receptors, D₁ dopamine receptors and GABA_B receptors in the nucleus accumbens shell for its full expression. Whereas the AMPA antagonist, DNQX administered into the nucleus accumbens shell elicits a transient, though intense feeding response, feeding is elicited by excitatory amino acid agonists administered into the lateral hypothalamus. The present study examined whether excitatory amino acid agonists elicited feeding following administration into the nucleus accumbens shell of rats, whether such feeding responses were altered by opioid antagonist pretreatment, and whether such feeding responses interacted with feeding elicited by μ-opioid agonists. Both AMPA (0.25–0.5 μg) and NMDA (1 μg) in the nucleus accumbens shell significantly and dose-dependently increased food intake over 4 h. Both feeding responses were blocked by naltrexone pretreatment in the nucleus accumbens shell. The μ-opioid agonist, [D-Ala²,NMe-Phe⁴,Gly-ol⁵]-enkephalin in the nucleus accumbens shell significantly increased food intake which was significantly enhanced by AMPA cotreatment. This enhanced feeding response was in turn blocked by pretreatment with either general or μ-selective opioid antagonists. In contrast, cotreatment of NMDA and the μ-opioid agonist in the nucleus accumbens shell elicited feeding which was significantly less than that elicited by either treatment alone. These data indicate the presence of important interactions between excitatory amino acid receptors and μ-opioid receptors in the nucleus accumbens shell in mediating feeding responses in nondeprived, ad libitum-fed rats.     

Response of Schaffer collateral-CA1 pyramidal cell synapses of the hippocampus to analogues of acidic amino acids

Analogues of the putative excitatory transmitters aspartic acid and glutamic acid were tested for antagonism against stimulus-evoked activation of Schaffer collateral-CA1 pyramidal cell synapses in slices of rat hippocampus. Responses to the analogues, applied via the superfusing medium, were extracellularly recorded. The compounds examined includedd- andl-α-aminodicar☐ylic acids, diamonodicar☐ylic acids, phosphonate analogues of acidic amino acids,d andl-γ-glutamyl glycine, and the cis- and trans-isomers of piperidine 2,3-, and 2,4-dicar☐ylic acid. Many of these compounds are known to be potent and selective antagonists for excitatory amino acids and a few excitatory pathways.In this hippocampal pathway most of these analogues showed relatively low and similar potency. The most potent antagonist uncontaminated with agonist activity wasd-α-aminosuberate, with an apparent antagonist dissociation constant (K_d of 3 mM. Only 5 of the analogues, 3 of the piperidine dicar☐ylates, kainic acid, andl-α-aminopimelic acid, reduced the amplitude of the extracellularly recorded field potentials more than 30% at 0.5 mM. However, all of the others reduced the potential by more than 30% at 5 mM. Most analogues also evoked extracellular responses whoch can be attributed to depolarization of the pyramidal neurons. Agonist activity was particularly strong among the most potent analogues.These results contrast with the responses documented by others for the N-methyl-d-aspartate receptor of the dorsal-ventral root excitatory pathway of the spinal cord in which the higher homologues tested here were the most potent antagonists, and thed-isomers were more potent than thel-isomoers. It also contrasts with the response of the perforant path synapses to granule cells of the dentate gyrus in which the portion derived from the lateral entorhinal cortex is sensitive tol-2-amino-4-phosphonobutyric acid. Thus the Schaffer-CA1 pyramidal cell synaptic field utilizes a novel excitatory transmitter receptor which interacts detectably but only weakly with a variety of acidic amino acids with potent specific inhibitory action for receptors elsewhere in the central nervous system.     

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.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and α-amino 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM )-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 μM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 μM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a longlasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3± 66.1 %) and aspartate (286.3 ± 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

Kainic acid produces depolarization of CA3 pyramidal cells in the in vitro hippocampal slice

Kainic acid (KA) (10⁻⁶–10⁻⁸ M) reversibly depolarized CA3 pyramidal cells when applied topically to the apical dendritic area of these cells in the hippocampal slice. The magnitude of membrane depolarization and the time to recovery of resting membrane potential were concentration-related. Application of 10⁻⁵ M KA produced complete membrane depolarization which did not recover to baseline levels. Unlike CA3 neurons cells from the CA1 region were unaffected by KA (10⁻⁶–10⁻⁸ M). However, 10⁻⁵ M KA also proved effective in depolarizing CA1 cells.     

Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro

Glutamate is the major excitatory neurotransmitter in the retina, but excessive stimulation of its receptors leads to widespread neuronal stress and death. Both growth factors and gangliosides display important influences on responses to neuronal injury and degeneration. In this study, we have investigated the potential protective effects of two well characterized growth factors, epidermal and basic fibroblast growth factor (EGF and bFGF respectively), and the monosialoganglioside GM1, on cultured rat retinal neurons submitted to toxic levels of excitatory amino acids. Application of 1 mM glutamic acid reduced global neuronal viability by 80% when compared to control untreated cultures, whereas treatment with the glutamic acid agonist kainic acid (1 mM) led to specific, large decreases (75% reduction) in amacrine cell numbers. 24 h pretreatment with either EGF or bFGF (500 pM each) prevented the majority of excitatory amino acid-induced neuronal death, whereas similar treatment with 10⁻⁵ M GM1 did not block neuronal degeneration. These findings demonstrate that EGF and bFGF act as neuroprotective agents against retinal excitotoxicity in vitro, whereas ganglioside GM1 is not effective in this particular paradigm.     

Involvement of the glutamatergic metabotropic receptors in the regulation of glutamate uptake and extracellular excitatory amino acid levels in the striatum of chloral hydrate-anesthetized rats

The microdialysis technique was used to assess in vivo the putative functional role of metabotropic excitatory amino acid receptors in regulating extracellular levels of the excitatory amino acids glutamate and aspartate in the striatum of chloral hydrate-anesthetized rats. Addition of the metabotropic glutamate receptor antagonist (+)-α-methyl-4-carboxyphenylglycine (MCPG) (10⁻³ or 4×10⁻³ M) in the dialysis probe did not modify the basal extracellular levels of glutamate and aspartate but induced a significant dose-dependent decrease in the KCl-elicited elevation of glutamate and aspartate extracellular levels. The effect of MCPG on glutamate extracellular concentration under K⁺ stimulation was reduced by the simultaneous superfusion of the metabotropic glutamate receptor agonist (2S,3S,4S)-α-(carboxycyclopropyl)glycine (L-CCG) (10⁻³ M) which had no significant effect when tested alone. In contrast, L-CCG alone significantly potentiated the KCl-elicited elevation of aspartate extracellular concentrations but failed to modify the MCPG effect on this amino acid concentration. In a parallel series of experiments, high-affinity glutamate uptake was measured ex vivo 20 min after an in vivo injection of 10 pmol of MCPG in the striatum. MCPG was found unable to modify the glutamate uptake rate. In vitro, MCPG (1–1000 μM) again had no effect on glutamate transport rate. These data suggest that metabotropic excitatory amino acid receptors (1) may act to increase the extracellular levels of glutamate and aspartate under depolarizing conditions, and (2) may not have a major role in the regulation of high affinity glutamate uptake under basal conditions. In addition, it can be assumed that the control of glutamate and aspartate extracellular levels may involve different metabotropic receptors.     

Cells in the anteroventral cochlear nucleus are insensitive to l-glutamate and l-aspartate; excitatory synaptic responses are not blocked by d-α-aminoadipate

Auditory nerve fibers transmit signals from the cochlea to the 3 regions of the cochlear nuclear complex, the anteroventral (AVCN), posteroventral, and dorsal cochlear nucleus in the brainstem. It has been suggested that the amino acids l-aspartate and l-glutamate might serve as a neurotransmitter in auditory nerve fibers^{6–10,13,17–20}. The sensitivity of postsynaptic cells in the cochlear nuclei to these amino acids has been tested by iontophoretic techniques^4,9,10. One difficulty with these experiments is that responses were recorded only extracellularly. A second difficulty is that the concentrations needed to affect cells could not be determined. To avoid these difficulties a brain slice preparation was used to test the sensitivity of cells in the AVCN to bath applied l-glutamate and l-aspartate at concentrations ranging from 10⁻⁵ to 10⁻² M. All cells that were tested in the cochlear nuclear complex were insensitive at all concentrations used; the resting potentials and the input resistances remained unchanged and the synaptic responses to electrical stimulation of the auditory nerve were not desensitized. All cells that were tested in the hippocampus, however, depolarized in the presence of 10⁻⁴ M l-glutamate and l-aspartate. The synaptic responses to electrical stimulation of the auditory nerve were not blocked by d-α-aminoadipate, an amino acid which has been shown to block excitation of cells in the cochlear nuclei by auditory nerve fibers¹⁰. The results are not consistent with l-glutamate and l-aspartate serving as neurotransmitters in the AVCN.     

Role of opioid receptors (μ, δ1, δ2) in modulating responses of nociceptive neurons in the superficial and deeper dorsal horn of the medulla (trigeminal nucleus caudalis) in the rat

This report describes the effects of intravenously administered agonists and antagonists at μ-, δ₁- and δ₂-opioid receptors on the Aδ- and C-fiber-evoked responses of trigeminal nociceptive neurons in anesthetized rats. Extracellular single unit recordings were made from 61 nociceptive neurons (23 NS, 38 WDR) in the superficial and 37 nociceptive neurons (3 NS, 34 WDR) in the deeper dorsal horn of the medulla (trigeminal nucleus caudalis). Administration of either the δ₁-receptor agonist [

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-Pen^2,5]enkephalin (DPDPE; 0.05–2 mg/kg), the δ₂-receptor agonist [

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-Ala²,Glu⁴]deltorphin (DELT; 1–2 mg/kg) or the μ-receptor agonist [

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-Ala²,N-MePhe⁴,Gly⁵-ol]enkephalin (DAMGO; 0.05–1 mg/kg) inhibited the Aδ- and C-fiber-evoked responses of nociceptive neurons in the superficial and deeper dorsal horn. The inhibitory effect was more pronounced on the C-fiber-evoked responses than on the Aδ-fiber-evoked responses. In other neurons, DPDPE also produced facilitation, or inhibition followed by facilitation, or differential effects (inhibition of the C-fiber-evoked responses and facilitation of the Aδ-fiber-evoked responses) on the Aδ- and C-fiber-evoked responses. The effects of DPDPE were antagonized by 7-benzylidenenaltrexone (BNTX, 0.4–1 mg/kg), a δ₁-receptor antagonist, in 88% (7/8) of neurons. Naltriben (NTB, 0.7–1 mg/kg), a δ₂-receptor antagonist, antagonized the effects of both DELT and DPDPE. A smaller dose of NTB (0.3 mg/kg), which failed to reverse the effects of DPDPE in 100% (4/4) of neurons, effectively antagonized the effects of DELT in 100% (6/6) of neurons. The inhibitory action of DAMGO was completely antagonized by naloxone (0.2 mg/kg) in 100% (6/6) of neurons. The results of the present investigation suggest that: (1) μ-, δ₁- and δ₂-opioid receptors play an important role in the inhibitory modulation of the Aδ- and C-fiber-evoked responses of nociceptive neurons in the superficial and deeper dorsal horn of the medulla; (2) selective inhibition of the C-fiber-evoked responses by activation of opioid receptors may account for the opioid-mediated selective suppression of second or persistent pain as compared to first pain; and (3) NTB, in a limited dose range, can discriminate between δ₁- and δ₂-opioid receptor subtypes.