Amino Acid Receptor Antagonists Research Articles

SAT592 Sexually Dimorphic Modulation Of Arcuate Kisspeptin Neurons Activity In Response To NPY

Abstract Disclosure: N.D. Mansano: None. R. Frazao: None. Kisspeptin neurons are part of an intricate brain circuit that receives neuronal inputs related tonutritional status from at least three neuronal populations, including NPY/AgRP and POMC neurons of the arcuate nucleus of the hypothalamus (ARH) and cells in the ventral premammillary nucleus. Its well-known action on the HPG axis, hypothalamic kisspeptin neurons also modulate the activity of specific neurons of the paraventricular and the dorsomedial nuclei of the hypothalamus, regions involved in the regulation of appetite and energy expenditure. However, it is not completely elucidated if NPY can influence the HPG axis through the modulation of kisspeptin neuron activity. To determine the NPY effect in the hypothalamic kisspeptin neurons we performed whole-cell patch-clamp recordings. The hypothalamic slices were obtained from the adult Kiss1/hrGFP female (diestrus-stage) or male mice. Kisspeptin cells located in the arcuate nucleus of the hypothalamus (ARHkisspeptin) and at the anteroventral periventricular and rostral periventricular nuclei (AVPV/PeNkisspeptin) were recorded. On female mice, ARHkisspeptin neurons exhibited a rest membrane potential (RMP) average of -52.5±2.5 mV (-40 to -62 mV) and 1.3±0.2 GΩ input resistance (IR). NPY induced the hyperpolarization of 45% of the ARHkisspeptin neurons (n= 9 of 20 cells, out of 9 mice). ARHkisspeptin neuron’s RMP hyperpolarization was followed by a significant decrease in IR and frequency of action potentials (fAPS). In the AVPV/PeNkisspeptin neurons showed RMP average of -69.6±1.9 mV (-51 to - 88 mV) and IR of 0.8±0.09 GΩ (n=18 cells out of 9 mice). No significant NPY-induced effect was noted when recordings were performed on AVPV/PeNkisspeptin cells from female mice. To elucidate if the ARHkisspeptin neuron’s hyperpolarization depends on action potentials (APs) mediated by synaptic transmission we further evaluated NPY effects in the presence of TTX and amino acid receptor antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10μM), DL-2-amino-5-phosphonopentanoic acid (AP5, 50μM), and picrotoxin (50μM). NPY induced no effect on ARHkisspeptin neuron’s RMP, and IR in the presence of TTX and amino acid receptor antagonists. Suggesting that NPY-induced hyperpolarization of ARHkisspeptin neurons is an indirect effect. To further verify whether sex must be considered as a biological variable to study the NPY effect, we recorded ARHkisspeptin cells from male mice. ARHkisspeptin neurons recorded from male mice exhibited RPM average of -55.6±1.5 (-45 to -66 mV) and IR of 1.5±0.15 GΩ (n= 13 cells out of 10 mice). Interestingly, NPY induced no effect on RMP, IR, or fAPs in male animals. In conclusion, our results suggest that NPY-induced hyperpolarization of ARHkisspeptin cells depends on AP’s mediate synaptic transmission, as a sex-specific effect. Presentation: Saturday, June 17, 2023

Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity.

The synthesis of new amino acid-containing, cell-specific, therapeutically active polymers is presented. Amino acids served as starting material for the preparation of tailored polymers with different amino acids in the side chain. The reversible addition-fragmentation chain-transfer (RAFT) polymerization of acrylate monomers yielded polymers of narrow size distribution (Đ ≤ 1.3). In particular, glutamate (Glu)-functionalized, zwitterionic polymers revealed a high degree of cytocompatibility and cellular specificity, i.e., showing association to different cancer cell lines, but not with nontumor fibroblasts. Energy-dependent uptake mechanisms were confirmed by means of temperature-dependent cellular uptake experiments as well as localization of the polymers in cellular lysosomes determined by confocal laser scanning microscopy (CLSM). The amino acid receptor antagonist O-benzyl-l-serine (BzlSer) was chosen as an active ingredient for the design of therapeutic copolymers. RAFT copolymerization of Glu acrylate and BzlSer acrylate resulted in tailored macromolecules with distinct monomer ratios. The targeted, cytotoxic activity of copolymers was demonstrated by means of multiday in vitro cell viability assays. To this end, polymers with 25 mol % BzlSer content showed cytotoxicity against cancer cells, while leaving fibroblasts unaffected over a period of 3 days. Our results emphasize the importance of biologically derived materials to be included in synthetic polymers and the potential of zwitterionic, amino acid-derived materials for cellular targeting. Furthermore, it highlights that the fine balance between cellular specificity and unspecific cytotoxicity can be tailored by monomer ratios within a copolymer.

Direct inhibition of arcuate kisspeptin neurones by neuropeptide Y in the male and female mouse.

Adverse energy states exert a potent suppressive influence on the reproductive axis by inhibiting the pulsatile release of gonadotrophin-releasing hormone and luteinising hormone. One potential mechanism underlying this involves the metabolic-sensing pro-opiomelanocortin and agouti-related peptide/neuropeptide Y (AgRP/NPY) neuronal populations directly controlling the activity of the arcuate nucleus kisspeptin neurones comprising the gonadotrophin-releasing hormone pulse generator. Using acute brain slice electrophysiology and calcium imaging approaches in Kiss1-GFP and Kiss1-GCaMP6 mice, we investigated whether NPY and α-melanocyte-stimulating hormone provide a direct modulatory influence on the activity of arcuate kisspeptin neurones in the adult mouse. NPY was found to exert a potent suppressive influence upon the neurokinin B-evoked firing of approximately one-half of arcuate kisspeptin neurones in both sexes. This effect was blocked partially by the NPY1R antagonist BIBO 3304, whereas the NPY5R antagonist L152,804 was ineffective. NPY also suppressed the neurokinin B-evoked increase in intracellular calcium levels in the presence of tetrodotoxin and amino acid receptor antagonists, indicating that the inhibitory effects of NPY are direct on kisspeptin neurones. By contrast, no effects of α-melanocyte-stimulating hormone were found on the excitability of arcuate kisspeptin neurones. These studies provide further evidence supporting the hypothesis that AgRP/NPY neurones link energy status and luteinising hormone pulsatility by demonstrating that NPY has a direct suppressive influence upon the activity of a subpopulation of arcuate kisspeptin neurones.

Somatostatin Inhibition of Gonadotropin-Releasing Hormone Neurons in Female and Male Mice

Previous studies indicate that somatostatin regulates gonadotropin secretion. We investigated here whether somatostatin has direct effects on GnRH neurons in the adult male and female mice. Dual-labeling immunofluorescence experiments revealed the presence of somatostatin-immunoreactive fibers adjacent to GnRH neurons, and three-dimensional confocal reconstructions demonstrated apparent somatostatin fiber appositions with 50–60% of GnRH neurons located throughout the brain in both male and female mice. Perforated patch-clamp recordings from GnRH-green fluorescent protein neurons revealed that approximately 70% of GnRH neurons responded in a dose-dependent manner to 10–300 nm somatostatin with an acute membrane hyperpolarization and cessation of firing. This effect persisted in the presence of tetrodotoxin and amino acid receptor antagonists, indicating a direct postsynaptic site of action on the GnRH neuron. The identity of the somatostatin receptors underlying this action was assessed using GnRH neuron single-cell RT-PCR. Of the somatostatin receptor subtypes, the sstr2 transcript was the most prevalent and detected in both males and females. The expression of sstr2 by GnRH neurons was confirmed in the sstr2 knockout/LacZ knock-in mouse line. Electrophysiological studies demonstrated that the sstr2-selective agonist seglitide exerted acute hyperpolarizing actions on GnRH neurons identical to those of somatostatin. Together, these studies reveal somatostatin, acting through sstr2, to be one of the most potent inhibitors of electrical excitability of male and female GnRH neurons identified thus far.

Somatostatin Inhibition of Gonadotropin-Releasing Hormone Neurons in Female and Male Mice

Previous studies indicate that somatostatin regulates gonadotropin secretion. We investigated here whether somatostatin has direct effects on GnRH neurons in the adult male and female mice. Dual-labeling immunofluorescence experiments revealed the presence of somatostatin-immunoreactive fibers adjacent to GnRH neurons, and three-dimensional confocal reconstructions demonstrated apparent somatostatin fiber appositions with 50-60% of GnRH neurons located throughout the brain in both male and female mice. Perforated patch-clamp recordings from GnRH-green fluorescent protein neurons revealed that approximately 70% of GnRH neurons responded in a dose-dependent manner to 10-300 nm somatostatin with an acute membrane hyperpolarization and cessation of firing. This effect persisted in the presence of tetrodotoxin and amino acid receptor antagonists, indicating a direct postsynaptic site of action on the GnRH neuron. The identity of the somatostatin receptors underlying this action was assessed using GnRH neuron single-cell RT-PCR. Of the somatostatin receptor subtypes, the sstr2 transcript was the most prevalent and detected in both males and females. The expression of sstr2 by GnRH neurons was confirmed in the sstr2 knockout/LacZ knock-in mouse line. Electrophysiological studies demonstrated that the sstr2-selective agonist seglitide exerted acute hyperpolarizing actions on GnRH neurons identical to those of somatostatin. Together, these studies reveal somatostatin, acting through sstr2, to be one of the most potent inhibitors of electrical excitability of male and female GnRH neurons identified thus far.

Effect of excitatory amino acid, dopamine, and oxytocin receptor antagonists on noncontact penile erections and paraventricular nitric oxide production in male rats.

In male rats, noncontact erections occur concomitantly with an increase in NO2- and NO3- in the paraventricular nucleus of the hypothalamus (PVN). In the present study, both responses were reduced by the blockade of PVN excitatory amino acid receptors by dizocilpine, (+)-MK-801(1 and 5 microg), but not by 6-cyano-7-nitro-quinoxaline-2,3-dione (5 microg) or (+)-2-amino-4-phosphono-butanoic acid (5 microg). Also ineffective when injected into the PVN were the dopamine antagonists SCH 23390 (5 microg), S(+)-raclopride (10 microg), and cis-flupenthixol (10 microg), and the oxytocin antagonist d(CH2)5Tyr(Me)2-Om8-vasotocin (1 microg). However, when the last was given into the lateral ventricles, it reduced noncontact erections without modifying NO2- and NO3- increases. These results suggest that excitatory amino acid transmission increases in the PVN during noncontact erections. This may contribute to increased NO production in the PVN, and it may activate oxytocin neurons mediating this sexual response.

Excitotoxicity Plays a Role in the Death of Tyrosine Hydroxylase- Immunopositive Nigral Neurons Cultured in Serum-Free Medium

We studied the effects of different amino acid receptor antagonists and a calcium (Ca2+) channel blocker on the survival of embryonic tyrosine hydroxylase (TH)-immunopositive nigral neurons grown under serum-free culture conditions. Ventral mesencephalic neurons were cultivated for 2 or 7 days. Following serum withdrawal on day 2, some cultures were treated with different concentrations of the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine hydrogen maleate (MK-801), the competitive NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid, the competitive kainate/quisqualate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione, and the Ca2+ channel blocker flunarizine. Treatment with MK-801 or flunarizine increased the survival of TH-positive neurons after serum deprivation. These findings suggest a possible role for excitotoxicity in dopaminergic cell death which can be prevented by blocking the NMDA receptor or by inhibiting Ca2+ entry through voltage-gated channels.

Glycine stimulation of glutamate binding to chick retinal pigment epithelium.

The effect of glycine (Gly) and taurine (Tau) on the biochemical and pharmacological properties of [3H]L-glutamate ([3H] Glu) binding to membranes from primary cultures of chick retinal pigment epithelium (RPE), as well as from intact tissue during development was studied. Gly and Tau increase Bmax of [3H]Glu binding to a high affinity site (KB = 300 nM) in membranes from 16 days in vitro (immature) cultures; additionally, Gly discloses a low affinity Glu-binding site (KB = 970 nM) at this stage. In membranes from 25 days in vitro (mature) cultures, the high affinity site is no longer present and Tau has no effect on Glu-binding; Gly still stimulates binding to the low affinity site by four fold, with an EC50 = 200 microM. Pharmacological profile using specific excitatory amino acid (EAA) receptor agonists and antagonists suggests that at 16 days in vitro Glu binds preferentially to metabotropic Glu receptors (mGluRs), and at 25 days in vitro to ionotropic receptors different from neuronal ones. The stimulatory effect of Gly and Tau was also observed in intact RPE, and decreased with increasing embryonic age. Glu binding was also stimulated in membranes from chick retina, but not in those from rat brain. Results support the possibility of EAA participation in several aspects of RPE physiology, including phagocytosis and cell division.

Potassium-dependent prolonged field bursts in the dentate gyrus: Effects of extracellular calcium and amino acid receptor antagonists

The dentate gyrus in rat hippocampal slices produces spontaneous, prolonged bursts of population spikes (i.e. prolonged field bursts) when [Ca 2+] 0 is lowered (0-0.5 mM) and [K +] 0 is concurrently elevated (9–11 mM). In this investigation we examined whether the dentate gyrus could also generate spontaneous field bursts in relatively “normal” (i.e. nominal 1.3 mM) or only moderately decreased [Ca 2+] 0 (i.e. nominal 0.9 mM). In 1.3 mM [Ca 2+] 0, no prolonged field bursts occurred spontaneously in the dentate gyrus when [K +] 0 was raised as high as 12 mM. Prolonged field bursts were generated, however, when [K +] 0 was further increased to 13–15 mM. Similar bursts could be generated at [K +] 0 within the “physiological ceiling level” observed in vivo during seizure activity (i.e. 11–12 mM) if: (i) the bath [Ca 2+] was reduced to 0.9 mM; or (ii) the GABA type A-receptor antagonist bicuculline was added in the presence of “normal” (1.3 mM) [Ca 2+] 0. Adding both the N-methyl- d-aspartate and non N-methyl- d-aspartate receptor antagonists, (±)-2-amino-5-phosphonopentanoic acid (50–100 μM) and 6,7-dinitroquinoxaline-2,3-dione (50–100 μM), respectively, did not block the occurrence of the field bursts. The bursts generated in 1.3 mM [Ca 2+] 0, 12 mM [K +] 0, bicuculline, (±)-2-amino-5-phosphonopen-tanoic acid and 6,7-dinitroquinoxaline-2,3-dione could, however, be reversibly depressed or blocked if [Ca 2+] 0 was raised to 2.0 mM. These data suggest that: (i) the dentate gyrus (like CA1) can produce prolonged field bursts in “normal” [Ca 2+] 0 when [K +] 0 is elevated, although the [K +] 0 threshold is higher; (ii) the dentate gyrus can generate prolonged field bursts in response to [K +] 0 elevations within the physiological ceiling, provided [Ca 2+] 0 is moderately lowered (i.e. 0.9 mM) or GABA type A-transmission is blocked; and (iii) antagonists of excitatory amino acid-mediated neurotransmission do not block the prolonged field bursts produced under any of these conditions.

NMDA receptors mediate neuronal burst firing in rat somatosensory cortex in vivo.

Two patterns of neuronal firing, bursting and regular spiking, are observed in the somatosensory cortex of anesthetized rats. The effects of excitatory amino acid receptor agonists and antagonists on these discharge types have been examined in vivo, in order to assess their involvement in the generation of such firing patterns. The analysis of interspike interval histograms indicates that N-methyl-D-aspartate (NMDA) receptors are involved in the generation of bursting responses while non-NMDA receptors mediate the regular spiking pattern of firing in individual neurons. It is also suggested that a basal level of ongoing neuronal excitation via the activation of non-NMDA receptors is required for bursting to be evoked through NMDA receptors.

Antimyoclonic Effect of MK-801

Rat pups exhibit transient "developmental dyskinesias," such as tremor and myoclonus, that are analogous to motor immaturities of the human neonate. Myoclonic jerks in the neonatal rat may reflect a developmental imbalance of excitatory and inhibitory neurotransmission. To test this hypothesis, spontaneous myoclonic jerks of naive rat pups (n = 200) were characterized behaviorally and pharmacologically. The frequency of myoclonus was high (154 +/- 14 jerks/30 min) in the first week. The distribution of jerks included limbs (47%) (27% in forelimbs and 20% in hindlimbs), tail (30%), trunk (12%), and head (11%). Myoclonus constituted the predominant neonatal adventitious movement (81%). Myoclonic jerks were variable in intensity, focal and multi-focal more often than generalized, and occurred when nonrespiratory movements were infrequent or absent, suggesting sleep. Myoclonic frequency significantly diminished after the second week; therefore, drug effects were studied in the first 7 days. Systemic injection of the novel noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 blocked neonatal myoclonus in a dose-dependent manner (ID50 = 0.67 mg/kg; r = 0.93). The nonselective excitatory amino acid (EAA) receptor antagonist kynurenic acid was ineffective. The EAA antagonist AP4 (1 and 10 mg/kg) also reduced myoclonic jerks, but other drugs, such as the selective glycine antagonist Iso-THAO (1 and 10 mg/kg), strychnine (0.5 mg/kg), clonazepam, and diazepam (1 mg/kg), were ineffective blockers. The putative agonists quisqualic acid (1-50 mg/kg) and NMDA (1-10 mg/kg) altered myoclonus only at behaviorally toxic doses. These data suggest that EAA receptors participate in developmental myoclonus of the neonatal rat and that development myoclonus may be a useful quantitative model of functional maturity of excitatory/inhibitory synapses. The efficacy of MK-801 also should be evaluated in drug- and lesion-induced myoclonus. Recognition of the high frequency and state dependence of spontaneous myoclonic jerks in neonatal rats may be important to neonatal antiepileptic drug studies.

Excitatory amino acid receptors in the caudal ventrolateral medulla mediate a vagal cardiopulmonary reflex in the rat

The importance of the caudal ventrolateral medulla (CVLM) in mediating vagal cardiopulmonary (Bezold-Jarisch reflex) reflex activity was studied in urethane-anaesthetized rats. Unilateral electrolytic lesion of the CVLM markedly attenuated Bezold-Jarisch reflex responses (hypotension and bradycardia) elicited by intravenous injections of 5-HT. Bilateral lesion of the CVLM virtually abolished the reflex responses. Microinjection of the excitatory amino acid (EAA) receptor antagonist kynurenate (KYN), but not the inactive analogue xanthurenate, into the CVLM markedly attenuated the reflex responses to 5-HT. The N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 also markedly attenuated reflex activity. Furthermore, lesions, KYN and MK-801 all tended to elevate resting blood pressure and to reduce resting heart rate. These findings support the hypothesis that the CVLM is an important medullary locus mediating cardiovascular reflex integration and that an EAA synapse in the CVLM is important in the cardiopulmonary reflex arc.

Ventrolateral medullary neurons modulate pressor reflex to muscular contraction.

Cardiorespiratory alterations during exercise are mediated through feedback from contracting muscles and descending drive from rostral brain sites such as the posterior hypothalamus. The role of medullary sites, which process this information, was examined in this study. In anesthetized cats, muscular contraction elicited by stimulation of L7 and S1 ventral roots and electrical stimulation of sites in the posterior hypothalamus both evoked increases in arterial pressure, heart rate, and minute ventilation. The reflex increase in arterial pressure produced by muscular contraction was attenuated significantly 15-20 min after bilateral microinjections of an excitatory amino acid (EAA) receptor antagonist, kynurenic acid (KYN), into the ventrolateral medulla (VLM). The reflex increase in arterial pressure evoked by muscular contraction returned to control levels 90 min after VLM microinjections of KYN. Microinjection of KYN into the VLM had no effect on the cardiorespiratory responses to posterior hypothalamic stimulation. These findings suggest that neurons in the VLM modulate the reflex pressor response evoked by muscular contraction. This reflex may be mediated through an interaction with EAA receptors on neurons in the VLM.

Kainate receptor-mediated synaptic currents in mudpuppy inner retinal neurons reduced by D-O-phosphoserine

1. The effects of D-O-phosphoserine (DOS) were examined on proximal neurons in the superfused mudpuppy retinal-eyecup preparation by measuring their synaptically evoked whole-cell currents with the use of patch-clamp electrodes. 2. DOS reduced the light-evoked excitatory postsynaptic potentials (EPSPs) of amacrine and ganglion cells. This suppression was present even though the center responses of both ON- and OFF-bipolar cells were unaffected by DOS. 3. When recordings were done under voltage-clamp conditions. DOS diminished the magnitude of light-evoked synaptic currents associated with a reduction in synaptic conductance. 4. To determine which acidic amino acid receptor mediated the network-selective action of DOS, various glutamate agonists were tested against this excitatory amino acid receptor (EAAR) antagonist. DOS blocked the depolarizing effects of kainate (KA), but not those of N-methyl-D-aspartate (NMDA) or quisqualate (QQ). Thus DOS was a selective KA antagonist, and KA receptors appear to be the dominant EAAR subtype that mediates synaptic inputs into the inner retina of the mudpuppy.

Specific binding of L-[3H]-glutamic acid to rat substantia nigra synaptic membranes.

The specific binding of L-[3H]-glutamic acid (GLU) was investigated in synaptic membranes from rat substantia nigra. L-[3H]-GLU binding to the membrane preparations occurred in a reversible and saturable way. The specific binding was stimulated by the presence of CaCl2 and was reduced by freezing and thawing the membranes. Scatchard analysis of the saturation isotherms yielded a non-linear plot suggesting that the binding reaction does not occur through a simple bimolecular association. Assuming non-interacting binding sites, a high (KD1, 139 nM; Bmax1, 3.5 pmoles/mg protein) and a low (KD2, 667 nM; Bmax2, 15.1 pmoles/mg protein) affinity L-[3H]-GLU binding site were obtained. The kinetics of dissociation of bound L-[3H]-GLU was biphasic; the respective dissociation rate constant (k-1) being 0.20 min-1 and 0.013 min-1. A series of amino acid receptor agonists and antagonists were tested as inhibitors of L-[3H]-GLU specific binding. Quisqualic acid, L-GLU and D-alpha-aminoadipate (D-alpha-AA) were the most potent inhibitors. DL-2-amino-4-phosphonobutyrate (APB), N-Methyl-D-aspartate (NMDA) and D-GLU were moderate inhibitors, whereas diaminopimelic acid (DAPA) and glutamate diethyl ester (GDEE) exhibited the lowest relative potency. Kainic acid (KA), gamma-aminobutyric acid (GABA) and bicuculline were not able to modify at any concentration used the specific binding of L-[3H]-GLU. These data demonstrate the presence of specific GLU binding sites in synaptic structures at substantia nigra level and support the idea that excitatory amino acids may play a role in synaptic transmission in this brain region.