N-methyl-D,L-aspartate Research Articles

The effect of lactation on induced Fos-like immunoreactivity in the rat hypothalamic paraventricular nucleus

Lactating rats display a period of blunted hypothalamo-pituitary-adrenal (HPA) response to a variety of stressors. This hyporesponsiveness is reported to be dependent upon continuous mother–pup interactions. In this study, computer-assisted densitometric methods were used to measure levels of induced Fos-like immunoreactivity (FLI) in the hypothalamic paraventricular nucleus (PVN) of lactating and non-lactating rats. Adrenalectomy (ADX) induces elevated levels of FLI in the PVN of non-lactating rats. We have observed that, between post-partum day (pd) 4 and pd 21, the level of ADX-induced FLI in the PVN of lactating rats follows a U-shaped distribution; that the persistence of this phenomenon is dependent upon continued mother–pup interaction and that sustained mother–pup interaction beyond the end of the normal suckling period (pd 21) does not extend the period of refractoriness. We have further determined that both the non-specific neural activator Metrazole, and the glutamate agonist N-methyl- d, l-aspartate (NMA), induced smaller increases in FLI in the PVN of lactating rats compared to non-lactating cohorts, and that the suppressing effect of lactation on Metrazole-induced FLI does not extend to all brain regions. These results suggest that mechanisms responsible for the onset and maintenance of the so-called lactational stress-hyporesponsive period (LSHRP) include altered function of glutamatergic pathways. © 1997 Elsevier Science B.V. All rights reserved.

The influence of reproductive status on the stimulatory action of N-methyl-D,L-aspartate on growth hormone secretion, in vitro in rainbow trout, Oncorhynchus mykiss

The glutamate agonist, N-methyl-D,L-aspartate (NMA) stimulates the secretion of growth hormone (GH) from pituitary fragments in vitro and increases plasma GH levels in vivo in rainbow trout, Oncorhynchus mykiss (Flett et al. 1994; Holloway and Leatherland 1997a,b); however gonadal steroid hormones appear to modulate this response in experimental situations. This study examines whether steroid hormones also modulate the GH-regulatory actions of NMA during the normal reproductive cycle of rainbow trout by examining the relationship between the stage of sexual maturation and the pituitary release of GH in vitro in response to an NMA (10-8 M) challenge. NMA had no effect on mean GH release from the pituitary glands of fish that were immature (GSI <1.0), from males during early development (GSI 1.0-3.0), or from sexually mature males (with free running milt) and females (ovulated). However, NMA significantly increased GH release from pituitary glands taken from females during the early stages of gonadal growth (GSI 1.0-9.0) and from males and females sampled during the later stages of gonadal growth (males GSI 3.01-6.0; females GSI 9.01-15.0). The GH-stimulatory action of NMA in males and females progressed to a maximum effect during the late stages of gonadal growth, and disappeared in ovulated females and free running males. Moreover, in female fish, the maximal GH release in response to the NMA challenge is positively correlated with plasma 17β-estradiol levels; no such correlation was evident for plasma testosterone levels in males. Changes in the GH response to NMA during maturation while gonadal steroid levels fluctuate provides further evidence to suggest that the effects of NMA on GH secretion are intimately linked to endogenous gonadal steroid hormone levels.

Regional distribution of the locomotor pattern-generating network in the neonatal rat spinal cord.

The regional distribution of spinal cord networks producing locomotor-like, as well as non-locomotor-like, activity was studied with the use of an in vitro neonatal rat preparation. Rhythmic activity was induced by bath application of either serotonin (5-HT), acetylcholine (ACh), N-methyl-D,L-aspartate (NMA), or combined 5-HT/NMA, and was monitored via hindlimb flexor (peroneal) and extensor (tibial) electroneurograms (ENGs) or ventral root recordings. In some experiments, synchronous patterns were produced by the addition of inhibitory amino acid (IAA) receptor antagonists. Selective application of 5-HT to cervical and thoracic cord regions induced rhythmic activity in these segments but failed to evoke hindlimb ENG discharge. Exposure of the isolated lumbar region to 5-HT produced tonic activity only. Application of 5-HT to the whole cord produced locomotor-like activity in hindlimb ENGs that persisted after midsagittal section of the spinal cord from the conus to the thoracolumbar junction. In other experiments, transverse hemisection of the rostral lumbar cord during whole cord exposure to 5-HT abolished rhythmic activity in ipsilateral hindlimb ENGs, suggesting that under these conditions rhythmic activity on one side of the lumbar cord was insufficient to maintain rhythmic activity on the contralateral side. Selective application of NMA or ACh to cervical and/or thoracic cord regions evoked rhythmic activity in these supralumbar segments, as well as rhythmic, but non-locomotor-like, activity in the lumbar region. In contrast to the effect of 5-HT, both NMA and ACh evoked rhythmic activity when applied solely to the lumbar region, and the side-to-side alternation produced by whole cord ACh application was uncoupled by midsagittal lesions of the lumbar region. In the presence of IAA antagonists, the side-to-side coupling of bilaterally synchronous rhythms was maintained despite extensive midsagittal lesions leaving all but one or two segments of either cervical, thoracic, or lumbar cord bilaterally intact, and rhythmic activity could be maintained even in single isolated hemisegments. The effects of 5-HT/NMA were similar to those observed with the use of 5-HT alone, although 5-HT/NMA induced rhythmic activity in hindlimb ENGs when applied selectively to supralumbar regions. The results suggest that 1) a 5-HT-sensitive oscillatory network, capable of producing a locomotor-like pattern of activity, is distributed throughout the supralumbar region of the spinal cord and mediates descending rhythmic drive to lumbar motor centers; 2) NMA- and ACh-sensitive rhythmogenic elements are distributed throughout the spinal cord, including the lumbar region; and 3) the spinal cord contains an extensive propriospinal network of reciprocal inhibitory and excitatory connections characterized by redundantly organized side-to-side projections.

Dual-component excitatory amino acid-mediated responses in trigeminal motoneurons and their modulation by serotonin in vitro.

1. Intracellular recordings were made from guinea pig trigeminal motoneurons in brain stem slices. Monosynaptic excitatory postsynaptic potentials (EPSPs) were evoked in trigeminal motoneurons by focal stimulation of the mesencephalic nucleus of V (Mes V), a region containing cell bodies of primary afferent fibers of jaw muscle spindle and periodontal receptor origin. 2. The presence of N-methyl-D,L-aspartate (NMDA) and non-NMDA excitatory amino acid receptors (EAAs) was determined by iontophoretic application of NMDA and (+/-)-alpha-amino-3-hydroxy methylisoxazole-4-propionic acid (AMPA) from multi-barreled micropipettes. Application of either agonist in normal Mg(2+)-containing extracellular solutions produced a membrane depolarization or inward current from resting potential in current-or voltage-clamp modes, respectively. However, the voltage and current responses to NMDA and AMPA differed in their dependence on initial membrane potential. In voltage clamp between -70 and -50 mV, the peak current response to NMDA application increased whereas the response to AMPA application decreased as a function of holding potential. 3. Mes-V-induced synaptic potentials were examined for the presence of NMDA and non-NMDA components in extracellular solutions devoid of Mg2+. In the presence of DL-2-amino-5-phosphonopentanoic acid (30 microM), the peak amplitude and half-amplitude duration were decreased 52 and 36% (n = 2), respectively, compared with control. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-15 microM), a specific non-NMDA antagonist, the mean decrease in evoked synaptic potential amplitude was 55% (n = 5), but the half-amplitude duration increased by a mean of 63% (n = 5). 4. The non-NMDA component of the evoked EPSP obtained in the presence of DL-2-amino-5-phosphonovaleric acid showed a linear decrease in peak amplitude as a function of holding potential. In contrast, the NMDA EPSP component obtained in the presence of CNQX increased in peak amplitude as a function of holding potential between -70 and -40 mV. The increase in EPSP peak amplitude was abolished by removal of Mg2+ from the extracellular media. 5. Bath application of serotonin (5-HT) (50-100 microM) increased the peak NMDA and non-NMDA EPSP components by 31% (n = 10) and 23% (n = 8), respectively, while producing a mean increase in half-amplitude duration of 67 and 88%, respectively. Concomitantly, 5-HT increased input resistance by approximately 40% and produced a membrane depolarization. 6. In the presence of tetrodotoxin, 5-HT enhanced both iontophoretic NMDA and AMPA voltage responses. In cells voltage clamped between -60 and -70 mV in Mg(2+)-free solutions, iontophoretic NMDA and AMPA peak currents were enhanced 27 and 32%, respectively, by 5-HT. 7. The enhancement of the iontophoretic NMDA and non-NMDA responses was mimicked by (+/-)-1-(2,5-dimethyoxy-4-iodophenyl)-2- aminopropane HCL and blocked by 3-[2-[4-(fluorobenzoyl)-1-piperdinyl]-2,4 (1H,3H)-quinazolinedione-tartrate and N'-[(8a)-1,6-dimethylergolin-8-yl]-N,N-dimethyl-sulfamide hydrochloride, suggesting 5-HT2 receptor involvement. 8. The results demonstrate that trigeminal motoneurons possess both NMDA and non-NMDA receptors that are activated during Mes-V-induced synaptic transmission. The data suggest that EAA-mediated responses in trigeminal motoneurons are substrates for modulation by 5-HT via changes in membrane resistance and modulation of the EAA-induced synaptic current.

One-way cross-sensitization and cross-tolerance to seizure activity from cocaine to lidocaine.

The present study examined the effects of daily treatment with a subconvulsant dose (50 mg/kg) of cocaine or lidocaine on susceptibility to seizures induced by cross-injections of the same dose of the other local anesthetic, and to seizures induced by pentylenetetrazol (PTZ) or N-methyl-DL-aspartate (NMDLA) in ddY mice. Repeated administration of 50 mg/kg cocaine caused the development of sensitization to cocaine-induced seizures during an initial 3 or 4 days, followed by the development of tolerance on days 4-6. The same dose of lidocaine, however, produced little or no seizure activity following repeated administration. In contrast, when injected 24 hr after 2-4 days of cocaine treatment, 50 mg/kg lidocaine produced severe seizures. Interestingly, this cross-sensitization from cocaine to lidocaine diminished upon further cocaine treatment. In contrast, treatment with lidocaine for 2-6 days had no effect on subsequent changes in seizure susceptibility following repeated cocaine injections. Neither treatment with cocaine nor lidocaine for 2 or 5 days influenced susceptibility to seizures induced by a challenge injection of PTZ (50 mg/kg, i.p.) or NMDLA (300 mg/kg, i.p.) 24 hr after treatment. HPLC analyses revealed that the cocaine treatment paradigm used in these studies increased the levels of the polyamines, putrescine and spermidine, in mouse brain, while lidocaine treatment had no effect on cerebral polyamine levels. These results suggest that there are differences in the neural mechanisms underlying the convulsant properties of cocaine and lidocaine in ddY mice.

N-methyl-D,L-aspartate-induced growth hormone secretion in barrows: possible mechanisms of action.

Four experiments were conducted to determine mechanisms by which n-methyl-d,l-aspartate (NMA) increases serum concentrations of growth hormone (GH). Blood samples were collected from barrows every 15 min for 2 h (Exp. 1, 2, and 3) or 3 h (Exp. 4) immediately before and immediately after i.v. treatments. In Exp. 1, barrows (n = 4/treatment) received either .9% saline or 1.25, 2.5, or 5 mg of NMA/kg of BW. The change in circulating GH concentrations was greater (P < .05) for barrows receiving 2.5 mg (by 883%) or 5.0 mg of NMA/kg of BW (by 1,095%) than for those injected with saline. In Exp. 2, barrows (n = 4/treatment) received NMA (2.5 mg/kg of BW) or injections of 1.25 mg of the pure d or pure 1 isomers of NMA/kg of BW. Growth hormone concentrations increased by 177% (P < .025) after NMA treatment and by 245% (P < .01) after injection of the pure d isomer of NMA. The pure 1 isomer of NMA had no effect (P > .1) on GH concentrations. In Exp. 3, barrows received NMA (2.5 mg/kg of BW) 10 min after i.m. injection of saline (n = 7) or ketamine hydrochloride ( n = 8; 19.9 mg/kg of BW), an n-methyl-d-aspartate (NMDA) receptor antagonist. The NMA increased (P < .01) GH concentrations by 289% in saline-pretreated barrows but had no effect (P > .1) on barrows pretreated with ketamine hydrochloride. In Exp. 4, barrows (n = 4/treatment) received NMA 3 h after i.v. pretreatment with antisera to GH-releasing factor (GRF; 154 mL) or no pretreatment. Serum GH concentrations increased by 166% (P < .05) after injection of NMA in barrows receiving no pretreatment. The NMA had no effect (P > .1) on GH concentrations in barrows receiving antisera to GRF. Our results support the concept that NMDA stimulates GRF, and hence GH secretion, by activating an NMDA receptor.

The role of excitatory amino acids in pulsatile secretion of luteinizing hormone in gilts and barrows.

The relationship of excitatory amino acid (EAA) activity to LH secretion was investigated in ovariectomized crossbred prepuberal gilts (93 +/- 1 kg BW) and Yorkshire barrows (94 +/- 2 kg BW) in two experiments. In Exp. 1, eight gilts received, i.m., saline (S) or 20 mg of Ketamine (K)/kg BW, a noncompetitive EAA receptor antagonist. Within these groups, four then received 10 mg of N-methyl-DL-aspartate (NMA)/kg BW, an EAA agonist, or S i.v. Mean serum LH concentrations were similar among groups before treatment, did not change after S+S, but decreased (P < .05) by 1 h after S+NMA, 3 h after K+S, and 2 h after K+NMA. Serum cortisol concentrations did not change after S+S, but were increased (P < .05) from 30 to 90 min after S+NMA, at 120 min after K+S, and from 30 to 120 min after K+NMA. In Exp. 2, barrows received 2.5 mg of NMA/kg BW i.v. immediately after i.m. injection of S (n = 7) or 19.9 mg of K/kg BW (n = 8). Mean serum LH concentrations for the 2 h before treatment were similar among barrows, but decreased (P < .05) by 2 h after K+NMA and was not altered after S+NMA. Serum cortisol concentrations were increased at 30 min after S+NMA and from 60 to 90 min after K+NMA. We suggest that EAA both inhibit and stimulate LH secretion, with the inhibitory effects lying within the basal hypothalamus and the stimulatory effects lying within higher brain centers.

Effects of inhibitory amino acid antagonists on reciprocal inhibitory interactions during rhythmic motor activity in the in vitro neonatal rat spinal cord.

1. The role of inhibitory amino acid transmission in the coordination and generation of rhythmic motor activity was examined with the use of an in vitro neonatal rat spinal cord preparation. Before adding gamma-aminobutyric acid (GABA) or glycine receptor agonists and antagonists, rhythmic motor activity was induced by bath application of acetylcholine (ACh), N-methyl-D,L-aspartate (NMA), or serotonin (5-HT) while monitoring bilateral ankle flexor and extensor electroneurograms (ENGs). The timing of rhythmic flexor and extensor discharge was consistent with that seen during overground locomotion in 27% of 84 bath applications of these substances (n = 65 preparations). 2. Subsequent addition of the GABAA receptor agonist muscimol, the GABAB receptor agonist baclofen, or glycine, abolished rhythmic activity in 95% of the tested applications. 3. GABAB receptor blockade did not disrupt alternating patterns of ENG discharge. However, addition of the GABAA receptor antagonist bicuculline, or the glycine receptor antagonist strychnine, transformed alternating flexor-extensor and left-right activity into patterns characterized by bilaterally synchronous rhythmic activation of all hindlimb ENGs. The onset of individual ENG bursts was more abrupt following bicuculline or strychnine. Strychnine also synchronized high-frequency (4-8 Hz) packets of rhythmic discharge within ENG bursts. 4. Some preparations developed synchronous, but unstable, rhythmic activity in the presence of bicuculline or strychnine alone. However, NMA, 5-HT, or ACh was usually required in addition to these antagonists to promote sustained rhythmic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of ventral tegmental administration of GABA A, GABA B and NMDA receptor agonists on medial forebrain bundle self-stimulation

Using a conditioned discrimination ICSS paradigm, rate-current intensity functions were determined for both reward- and nonreward-associated responding for electrical self-stimulation of the MFB following intra-VTA infusion of baclofen, muscimol and N-methyl-D,L-aspartate (NMDLA). A low dose (0.064 μg/0.5 μl) of the GABA B receptor agonist, baclofen, microinjected into the VTA, ipsilateral to the lateral hypothalamic stimulating electrode, resulted in a rightward shift of the ICSS curve without significantly influencing either maximal rates of operant responding for electrical brain stimulation or nonreinforced performance levels. Increases in MFB current thresholds were also evident after infusion of higher doses of baclofen (0.128, 0.26 and 0.52 μg) into the ventral tegmentum. Intra-VTA administration of the GABA A receptor agonist, muscimol (0.006, 0.012, 0.025 and 0.05 μg) and NMDA receptor activation by NMDLA (0.5, 1, 2 and 5 μg) did not affect reward thresholds; however, the low dose of muscimol and the high dose of NMDLA elicited behavioral activation resulting in reward-unrelated performance effects. These results implicate the specific involvement of the GABA B receptor in the reward neurocircuitry of the VTA.

Response linearity and kinetics of the cat retina: the bipolar cell component of the dark-adapted electroretinogram.

The electroretinogram (ERG) of the dark-adapted cat eye in response to brief ganzfeld flashes of a wide range of intensities was recorded after intravitreal injection of n-methyl DL aspartate (NMDLA, cumulative intravitreal concentration of 1.3-3.9 mM) to suppress inner-retinal components, and after intravitreal DL or L-2-amino-4-phosphonobutyric acid (DL-APB, 1-3 mM; L-APB, 1.2 mM) and 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX, 40-60 microM), to suppress all post-receptoral neuronal responses. Rod PII, the ERG component arising from rod bipolar cells, was derived by subtracting records obtained after APB and CNQX from post-NMDLA records. When we measured the derived response at fixed times after the stimulus, we found that PII initially increased in proportion to stimulus intensity without any sign of a threshold. The leading edge of PII at early times after the stimulus, when the response was still small, was well described by V(t) = kI(t-td)5 where k is a constant, I is the intensity of the stimulus, and td is a brief delay of about 3 ms. Correspondingly, the time for the response to rise to an arbitrary small criterion voltage Vcrit was adequately fitted by tcrit = td + (Vcrit/kI)1/5. The time course of the leading edge of the PII response can be interpreted to indicate that the mechanism generating PII introduces three stages of temporal integration in addition to the three stages that are provided by the mechanism of the rod photoreceptors. This finding is consistent with the operation within the rod bipolar cell of a G-protein cascade similar to that in the rods.

Do GnRH neurons express the gene for the NMDA receptor?

Previous studies have revealed that in several animal models, N-methyl- d,l-Aspartate (NMA) stimulates LH secretion by acting at a suprapituitary site. In addition, NMDA receptor antagonists appear to block GnRH neuronal activation on the afternoon of proestrous as evidenced by the lack of c-Fos expression in the neurons and by the absence of an ovulatory LH surge. However, administration of NMA does not induce c-Fos or c-Jun expression in GnRH neurons. To better understand the effects of NMDA receptor activation on GnRH neuronal function, we examined whether GnRH neurons express the NMDA receptor in male rats, and in female rats during diestrus and proestrus, by performing double label in situ hybridization. An 35S-labeled cRNA probe for the NMDA receptor subunit (NMDAR1) was used to quantify NMDAR1 mRNA and a digoxigenin-labeled cRNA probe for GnRH was used to identify GnRH neurons. The data were quantified and expressed as grains/average cell area. In male and female rats, less than 5% of GnRH neurons expressed grain levels twice the minimum detectable level and were considered double-labeled. However, many non-GnRH neurons in the same areas as GnRH neurons expressed high levels of NMDAR1 mRNA. These results suggest that the effects of NMA on GnRH secretion are unlikely to be mediated solely by the activation of NMDA receptors on GnRH neurons. Given the widespread expression of NMDAR1 mRNA in the hypothalamus, it is possible that the stimulatory effects of NMA on GnRH neurons are indirect through activation of other neurons.

NMDA-induced burst discharge in guinea pig trigeminal motoneurons in vitro.

1. The responses of guinea pig trigeminal motoneurons (TMNs) to N-methyl-D,L-aspartate (NMA) were studied using brain stem slice preparations and whole cell patch-clamp (n = 89) or conventional microelectrode (n = 22) recording techniques. The primary goals of this study were to determine whether N-methyl-D-aspartate (NMDA) receptor activation would produce spontaneous bursting activity in TMNs and, if so, the underlying mechanisms responsible for the generation of these bursts. 2. Bath-applied NMA (100-300 microM, n = 80) in standard perfusion medium elicited depolarization, increase in apparent input resistance (Rinp), and rhythmic burst discharges (1-90 s in duration) from TMNs. These effects were blocked by the NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5, 30 microM, n = 6), but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5-10 microM, n = 10). Furthermore, the burst-inducing effect of NMA was not mimicked by the non-NMDA receptor agonists kainate (KA, 5-10 microM, n = 6) and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 5-10 microM, n = 5). 3. In tetrodotoxin (TTX) treatment conditions (n = 13), NMA elicited depolarization, an increase in apparent Rinp, and rhythmic membrane potential oscillations without action potential bursts (i.e., plateau potentials), suggesting that the effects of NMA observed in the TTX-free condition resulted from activation of postsynaptic NMDA receptors. 4. Graded depolarization of neurons (n = 20) by intracellular direct current injection generally led to a graded increase in frequency and duration of the NMA-induced bursts and plateau potentials until these rhythmic events eventually became transformed into continuous spike discharge and maintained depolarization, respectively. Removal of Mg2+ from the perfusion medium (n = 11) also turned the bursts and plateau potentials into continuous spike discharge and maintained depolarization, respectively. 5. The effects of NMA on the current-voltage (I-V) curve after a depolarizing ramp voltage-clamp command (15-20 mV/s) were examined (n = 40). Under NMA (100-300 microM) conditions, the I-V relationship exhibited a region of negative slope conductance (NSC) between -60 and -35 mV, thus making the I-V relationship N-shaped. The NSC was abolished by AP5 (30 microM, n = 8), but not by CNQX (5-10 microM, n = 6). The I-V relationship in AMPA (3-10 microM, n = 5) or KA (3-10 microM, n = 5) was almost linear between -80 and -30 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Lactation-induced deficits in NMDA receptor-mediated cortical and hippocampal activation: changes in NMDA receptor gene expression and brainstem activation

During lactation, there is an inhibition of cortical and hippocampal activation in response to N- methyl- d,l-aspartate (NMA), but not kainate, as assessed by induction of c-Fos expression. To study whether changes in NMDA receptor function may account for this inhibition, NMDA receptor subunit (NMDAR1) mRNA levels were measured by both Northern analysis and in situ hybridization. Analysis of NMDAR1 gene expression by Northern blot analysis did not reveal significant differences between cycling and lactating rats. Using in situ hybridization, NMDAR1 mRNA levels in several cortical and hippocampal areas appeared to be smaller in lactating rats, compared to cycling rats, although these differences reached significance only in the fronto-parietal cortex and piriform cortex. These subtle changes in NMDAR1 receptor subunit gene expression during lactation are not likely to account for the global lack of neuronal activation in response to NMA. However, it is possible that there may be changes in other NMDA receptor subunits that could account for the deficits in NMDA receptor activation. We also examined the activation state of afferent pathways in the brainstem that provide excitatory input to the cortex and hippocampus. During lactation, NMA induced c-Fos expression in similar areas of the brainstem as during the cycle, except in the locus coeruleus and dorsal raphe, where c-Fos expression was significantly less than that observed during the cycle. In contrast, no differences in the pattern of c-Fos expression in the brainstem in response to kainate were observed between cycling and lactating rats. The lack of NMA-induced activation of the locus coeruleus and dorsal raphe may contribute to the lack of cortical activation during lactation.

Regulation of gonadotropin-releasing hormone gene expression by the excitatory amino acids kainic acid and N-methyl-D,L-aspartate in the male rat.

The glutamate analogs N-methyl-D,L-aspartate (NMA) and kainic acid are involved in the regulation of GnRH and LH release in mammals. It has recently been reported that the increase in GnRH release induced by NMA is accompanied by an increase in GnRH mRNA levels, as measured by in situ hybridization. In the present study we assessed the effects of NMA and kainic acid on cytoplasmic mRNA levels using the more quantitative solution hybridization/RNase protection assay. To address the mechanism responsible for these mRNA changes, we also examined changes in heteronuclear RNA transcripts as a reflection of gene transcription. Adult male rats were implanted with a jugular catheter, and 1-2 days later, NMA (14 mg/kg BW), kainic acid (2 mg/kg BW), both NMA and kainic acid, or saline vehicle were injected through the cannula. Rats were killed 15 min or 1 h later by decapitation, blood samples were collected for RIA of LH, brains were removed, and the preoptic area was dissected and frozen. Cytoplasmic and nuclear RNA were extracted and assayed separately by RNase protection assay. Treatment with NMA or NMA plus kainic acid resulted in significantly elevated cytoplasmic mRNA levels 15 min and 1 h later compared to saline control values, with no differences between the two drug treatments observed. Kainic acid stimulated mRNA levels 1 h, but not 15 min after injection. Nuclear RNA transcripts were unaffected by all drug or vehicle treatments. As nuclear primary transcript levels presumably reflect GnRH gene transcription, and these levels are unaltered, the present study indicates that the regulation of GnRH gene expression by excitatory amino acids occurs at a posttranscriptional level. The increase in cytoplasmic GnRH mRNA levels also does not result from an increased translocation of the relatively large nuclear GnRH mRNA pool into the cytoplasm, because nuclear GnRH mRNA levels are also unchanged. Therefore, the elevation of cytoplasmic mRNA levels after excitatory amino acid treatment is probably due to an increase in mRNA stability.

Push-pull model of the primate photopic electroretinogram: a role for hyperpolarizing neurons in shaping the b-wave.

Existing models of the primate photopic electroretinogram (ERG) attribute the light-adapted b-wave to activity of depolarizing bipolar cells (DBCs), mediated through a release of potassium that is monitored by Müller cells. However, possible ERG contributions from OFF-bipolar cells (HBCs) and horizontal cells (HzCs) have not been explored. We examined the contribution of these hyperpolarizing second-order retinal cells to the photopic ERG of monkey by applying glutamate analogs to suppress photoreceptor transmission selectively to HBC/HzCs vs. DBCs. ERGs of Macaca monkeys were recorded at the cornea before and after intravitreal injection of drugs. Photopic responses were elicited by bright 200-220 ms flashes on a steady background of 3.3 log scotopic troland to suppress rod ERG components. 2-amino-4-phosphonobutyric acid (APB), which blocks DBC light responses, abolished the photopic b-wave and indicated that DBC activity is requisite for photopic b-wave production. However, applying cis-2,3-piperidine dicarboxylic acid (PDA) and kynurenic acid (KYN), to suppress HBCs/HzCs and third-order neurons, revealed a novel ERG response that was entirely positive and was sustained for the duration of the flash. The normally phasic b-wave was subsumed into this new response. Applying n-methyl-dl-aspartate (NMA) did not replicate the PDA+KYN effect, indicating that third-order retinal cells are not involved. This suggests that HBC/HzC activity is critical for shaping the phasic b-wave. Components attributable to depolarizing vs. hyperpolarizing cells were separated by subtracting waveforms after each drug from responses immediately before. This analysis indicated that DBCs and HBC/HzCs each can produce large but opposing field potentials that nearly cancel and that normally leave only the residual phasic b-wave response in the photopic ERG. Latency of the DBC component was 5-9 ms slower than the HBC/HzC component. However, once activated, the DBC component had a steeper slope. This resembles properties known for the two types of cone synapses in lower species, in which the sign-preserving HBC/HzC synapse has faster kinetics but probably lower gain than the slower sign-inverting G-protein coupled DBC synapse. A human patient with "unilateral cone dystrophy" was found to have a positive and sustained ERG that mimicked the monkey ERG after PDA+KYN, indicating that these novel positive photopic responses can occur naturally even without drug application. These results demonstrate that hyperpolarizing second-order neurons are important for the primate photopic ERG.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of excitatory amino acids on in vivo and in vitro gonadotropin and growth hormone secretion in testosterone‐primed immature rainbow trout, Oncorhynchus mykiss

AbstractThe present studies were conducted to evaluate the effects of excitatory amino acids (EAAs) on gonadotropin II (GtH II) and growth hormone (GH) secretion in testosteroneprimed immature rainbow trout, Oncorhynchus mykiss. Intraperitoneal injections of N‐methyl‐DL‐aspartate (NMA) elevated both plasma GtH II and GH levels. By comparison, Des Gly10 [D‐Ala6] LHRH‐ethylamide (LHRH‐A) elevated plasma GtH II levels but had no effect on plasma GH levels. The GtH II response to NMA was not evident in sexually regressing adult rainbow trout. GtH II secretion by perifused pituitary glands of testosterone‐primed immature rainbow trout was elevated following NMA or salmon GnRH analogue (sGnRH‐A) challenge. The GtH II response to NMA was eliminated in pituitaries previously exposed to either the NMA receptor antagonist, AP5, or a GnRH receptor antagonist (D‐pGlu1,D‐Phe2,D‐Trp3,6‐LHRH), suggesting that NMA operates by stimulating the secretion of GnRH rather than by direct actions on the pituitary gonadotropic cells. The results of these studies provide evidence of the presence of NMA‐specific receptors in the hypothalamo‐hypophyseal system that when activated led to the release of GtH II. L‐glutamic acid (GLU) also stimulated GtH II secretion from perifused pituitary glands. However, the response to GLU was only partly blocked by AP5, suggesting that GLU exerted some of its effect via non‐NMA type receptors, in addition to its actions on NMA receptors. The results of these studies support the contention that EAA receptor activation may play a role in regulating the onset of sexual maturation in teleosts, as it seems to do in mammals. © 1994 Wiley‐Liss, Inc.

A comparison of motor patterns induced by [formula omitted], acetylcholine and serotonin in the in vitro neonatal rat spinal cord

Using an in vitro preparation from neonatal rat spinal cord, we compared the motor patterns induced by three putative locomotion-inducing substances. N- Methyl- d,l-aspartate (NMA) induced rhythmic hindlimb nerve activity in 17 20 preparations that was characterized by: (a) side-to-side alternation, but co-activated intralimb flexor-extensor pairs in 29%; (b) bilateral co-activation of all flexors and extensors in 24%; and c) rhythmic but poorly coordinated activity in 35%. Acetylcholine induced rhythmic activity in 34 35 preparations, which in 68% of animals was characterized by side-to-side alternation of co-activated intralimb flexor-extensor pairs. Only rarely did NMA ( 2 20 trials) and acetylcholine 1 35 trials) induce sustained ENG patterns compatible with hindlimb stepping. Serotonin, however, induced rhythmic activity in 22 24 preparations that was consistent with locomotion in intact rats in 13 22 (59%). These findings demonstrate that exogenously applied neurochemicals induce a variety of in vitro motor rhythms although some substances preferentially activate specific patterns. The results also highlight the importance of monitoring flexor and extensor activity from both hindlimbs in order to distinguish locomotor-like patterns from other types of neurochemically-induced rhythms.

N-(3-Aminopropyl)-cyclohexylamine blocks facilitation by spermidine of [formula omitted]-induced seizure in mice in vivo

The facilitating or antagonizing effects of polyamine analogues on N- methyl- dl-aspartate (NMDLA)-induced seizures were investigated using mice. Intracerebroventricular injection of spermidine and spermine, but not putrescine, shortened the latency to appearance of clonic convulsion induced by subcutaneous administration of NMDLA. Injection of N-(3-aminopropyl) cyclohexylamine(APCHA) alone did not affect the NMDLA-induced seizure. However, APCHA, when administered together with spermidine, clearly antagonized the facilitating effect of spermidine on the NMDLA-induced seizure. Another cyclohexylamine derivative, trans-4-methylcyclohexylamine, did not block the effect of spermidine. APCHA also antagonized the facilitation by d-serine of NMDLA-induced seizure, although the blocking effect for d-serine was weaker than that for spermidine. APCHA should be useful as a new tool for pharmacological studies on the neuromodulatory action of polyamines.

The activity of opioid analgesics in seizure models utilizing N-methyl-dl-aspartic acid, kainic acid, bicuculline and pentylenetetrazole

Morphine, fentanyl and pethidine exhibited a biphasic dose response relationship with respect to their effects on seizure thresholds to bicuculline, pentylenetetrazole, N-methyl-DL-aspartate (NMDLA) and kainic acid in mice. The usual pattern was for low doses to be anticonvulsant and higher doses to be proconvulsant. However this pattern was reversed for fentanyl and pethidine when NMDLA was used to induce seizures. The low dose effects of all three opioid drugs was sensitive to 1 mg kg-1 naloxone in all seizure models. The responses to high doses of pethidine were unaffected or enhanced by this dose of naloxone. Naloxone reversed the effects of the higher doses of morphine and fentanyl in all models except bicuculline induced seizures.

GABAergic inactivation of the central pattern generators for locomotion in isolated neonatal rat spinal cord.

1. Experiments were performed using an isolated brainstem-spinal cord preparation from newborn rats, in order to study the GABAergic control of the spinal neuronal networks that generate locomotor rhythms in mammals. Locomotor-like activities were recorded in the ventral roots, and the various neurochemical compounds were added to the superfusion saline. 2. Bath application of GABA suppressed in a dose-dependent manner the motor activity induced by an excitatory amino acid N-methyl-D,L-aspartate (NMA). Both the GABAA agonist muscimol and the GABAB agonist baclofen mimicked the effects of GABA, since they either slowed down or stopped the rhythmic activity. 3. Experiments were performed in which the lumbar compartment was superfused separately from the brainstem. Chemical activation of the brainstem by NMA alone failed to induce locomotor-like activity. When GABAA (bicuculline) and GABAB (phaclofen) antagonists were simultaneously bath applied to the lumbar spinal cord, however, locomotor-like activity was induced. 4. The GABA uptake inhibitors nipecotic acid and guvacine suppressed the rhythmic motor pattern induced by NMA in a dose-dependent manner. The effects of nipecotic acid were reversed by bicuculline and phaclofen. 5. Bicuculline added during NMA-induced locomotor-like activity greatly increased both the frequency and amplitude of motor bursts, while phaclofen modified only the frequency. 6. The motor pattern depended on the balance between activatory and inactivatory influences, since the rhythmic patterns recorded with low doses of NMA associated with high doses of bicuculline were similar to those elicited by higher doses of NMA associated with low doses of bicuculline.(ABSTRACT TRUNCATED AT 250 WORDS)