5-HT2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D₁ receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K⁺-evoked [³H]ACh release while SCH 23390, a dopamine D₁ receptor antagonist, had no effect. [³H]ACh release was decreased by the dopamine D₂ receptor agonist LY 171555 (quinpirole) and slightly potentiated by the dopamine D₂ receptor antagonist haloperidol. The selective neurokinin NK₁ receptor agonist [Sar⁹, met(O₂)¹¹]SP also potentiated K⁺-evoked release of [³H]ACh. GR 82334, a NK₁ receptor antagonist, blocked not only the effect of [Sar⁹, met(O₂)¹¹]SP but also the release of ACh induced by the D₁ receptor agonist SKF 38393. Among the 5-HT agents studied, only the 5-HT_2A receptor antagonists ketanserin and ritanserin were able to reduce the ACh release induced by dopamine D₁ receptor stimulation. Mesulergine, a more selective 5-HT_2C antagonist, showed an intrinsic releasing effect but did not affect K⁺-evoked ACh release induced by SKF 38393. Methysergide and methiothepin, mixed 5-HT_1/2 antagonists, as well as ondansetron, a 5-HT₃ receptor antagonist, showed an intrinsic effect on ACh release, their effects being additive to that of SKF 38393. 5-HT₂ receptor agonists were ineffective. However, the 5-HT₂ agonist DOI was able to prevent the antagonism by ketanserin of the increased [³H]ACh efflux elicited by SKF 38393, suggesting a permissive role of 5-HT_2A receptors. None of the above indicated 5-HT agents was able to reduce the ACh release induced by the selective NK₁ agonist. The results suggest that 5-HT₂ receptors, probably of the 5-HT_2A subtype, modulate the release of ACh observed in slices from the rat striatum after stimulation of dopamine D₁ receptors. It seems that this serotonergic control is exerted on the interposed collaterals of substance P-containing neurons which promote ACh efflux through activation of NK₁ receptors located on cholinergic interneurons.     

I-BH[Sar,Met(O2)]-SP, a new selective ligand for the NK-1 receptor in the central nervous system

The selective agonist [Sar⁹,Met(O₂)¹¹]-SP was radioiodinated with¹²⁵I-Bolton Hunter in order to study its binding to rat brain membranes and for further comparison with¹²⁵I-BH.SP. Specific binding of¹²⁵I-BH[Sar⁹,Met(O₂¹¹]-SP was temperature-dependent, saturable and reversible. In brain homogenates,¹²⁵I-BH[Sar⁹,Met(O₂)¹¹]-SP interacted with a single class of high affinityk_d = 1.0nM) non-interacting binding sites (B_max of 15 fmol/mg protein). In the central nervous system,¹²⁵I-BH-[Sar⁹,Met(O₂)¹¹]-SP apparently labeled the same number of binding sites as¹²⁵I-BH.SP (19 fmol/mg protein). Competition studies with tachykinins, neurokinins and selective neurokinin agonists indicated that the pharmacological profile of the site labeled by¹²⁵I-BH[Sar⁹,Met(O₂¹¹]-SP is identical with that of NK-1 receptors. In dose-displacement studies made with radiolabeled SP and [Sar⁹,Met(O₂¹¹)]-SP, an excellent correlation (r = 0.96) was found for theK_i values of the different compounds tested; these findings suggest that both radioligands recognize the same receptor in rat brain. The affinity (K_i of various neurokinin-related peptides for the brain site were compared with their biological activities on various isolated organs (dog carotid artery, guinea-pig ileum, rat portal vein). NK-1 binding sites characterized in rat brain homogenates appear to be identical with those present on the dog carotid artery, a preparation known to possess exclusively the NK-1 receptor type.     

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.     

Effect of NMDA receptor antagonists on prostaglandin E2-induced hyperalgesia in conscious mice

Intrathecal (i.t.) injection of prostaglandin E₂ (PGE₂) to conscious mice produced a hyperalgesic action over a wide range of dosages with two apparent peaks at 100 pg and 10 ng per mouse, which may be mediated through EP₃ and EP₂ subtypes of the PGE receptor. In the present study, the effects of NMDA receptor antagonists on hyperalgesia induced by PGE₂ were evaluated by the hot plate test at 30 min after i.t. injection. Hyperalgesia induced by a higher dose of PGE₂ (10 ng/mouse) was relieved byd-AP5 (a competitive antagonist), 7-Cl-KynA (a glycine site antagonist), and ketamine and MK801 (non-competitive channel blockers). Intrathecal injection of butaprost (10 ng/mouse), an EP₂ agonist, induced hyperalgesia, and this hyperalgesia was blocked byd-AP5, 7-Cl-KynA, ketamine, and MK801, similar to that induced by 10 ng of PGE₂. On the other hand, hyperalgesia induced by a lower dose of PGE₂ (100 pg/mouse) was blocked byd-AP5 and 7-Cl-KynA, but not by ketamine and MK801. Intrathecal injection of sulprostone (100 pg/mouse), an EP₁ and EP₃ agonist, induced hyperalgesia, and this hyperalgesia was blocked byd-AP5 and 7-Cl-KynA, but not by ketamine and MK801, similar to that induced by 100 pg of PGE₂. These results first demonstrate that the NMDA receptor is involved in the PGE₂-induced hyperalgesia and suggest that the hyperalgesic action by lower and higher doses of PGE₂ may be mediated through EP₃ and EP₂ subtypes, respectively.     

Neurokinins inhibit low threshold inactivating K currents in capsaicin responsive DRG neurons

Neurokinins (NK) released from terminals of dorsal root ganglion (DRG) neurons may control firing of these neurons by an autofeedback mechanism. In this study we used patch clamp recording techniques to determine if NKs alter excitability of rat L4-S3 DRG neurons by modulating K⁺ currents. In capsaicin (CAPS)-responsive phasic neurons substance P (SP) lowered action potential (AP) threshold and increased the number of APs elicited by depolarizing current pulses. SP and a selective NK₂ agonist, [βAla⁸]-neurokinin A (4–10) also inhibited low threshold inactivating K⁺ currents isolated by blocking non-inactivating currents with a combination of high TEA, (−) verapamil and nifedipine. Currents recorded under these conditions were heteropodatoxin-sensitive (Kv4 blocker) and α-dendrotoxin-insensitive (Kv1.1 and Kv1.2 blocker). SP and NKA elicited a > 10 mV positive shift of the voltage dependence of activation of the low threshold currents. This effect was absent in CAPS-unresponsive neurons. The effect of SP or NKA on K⁺ currents in CAPS-responsive phasic neurons was fully reversed by an NK₂ receptor antagonist (MEN10376) but only partially reversed by a PKC inhibitor (bisindolylmaleimide). An NK₁ selective agonist ([Sar⁹, Met¹¹]-substance P) or direct activation of PKC with phorbol 12,13-dibutyrate, did not change firing in CAPS-responsive neurons, but did inhibit various types of K⁺ currents that activated over a wide range of voltages. These data suggest that the excitability of CAPS-responsive phasic afferent neurons is increased by activation of NK₂ receptors and that this is due in part to inhibition and a positive voltage shift in the activation of heteropodatoxin-sensitive Kv4 channels.     

Locomotor effects of amantadine in the mouse are not those of a typical glutamate antagonist

Summary Amantadine has been shown to displace [³H]MK 801 from its binding site on the NMDA receptor. We have therefore studied the motor effects of amantadine in normal and 24h reserpine-treated mice to determine whether the behavioural profile of this drug resembles that of other NMDA receptor antagonists (e.g. MK 801). In common with the latter, amantadine (5–40 mg/kg IP) produced a modest dose-dependent sedation in dopamineintact mice, with a reduction in locomotion and other species-typical behaviours (e.g. rearing and grooming), but with no signs of the hyperactivity, stereotypy, ataxia or loss of muscle tone commonly seen with MK 801. Amantadine (5–80 mg/kg IP) effected a small incrase in motility in akinetic reserpine-treated mice by itself, but this response was highly variable and not statistically significant. As with MK 801, amantadine significantly inhibited the locomotion induced by the selective D₂ agonist RU 24213 (5 mg/kg SC) and the mixed D₁/D₂ agonist apomorphine (0.5 mg/kg SC) in monominedepleted mice, without altering the animals' responsiveness to threshold doses of these drugs. However, amantadine did not facilitate the locomotion induced by threshold (3 mg/kg IP) or fully active doses (30 mg/kg IP) of the selective D₁ agonist SKF 38393, which distinguishes amantadine from other NMDA receptor blockers. Since the potentiation of dopamine D₁-dependent locomotion may be a major factor in the antiparkinson activity of MK 801 and other glutamate receptor antagonists the inability of amantadine to potentiate SKF 38393 in this study suggests the mechanism of its anti-akinetic activity differs, from that of conventional glutamate blocking drugs.     

Regulation of GABA release via NMDA and 5-HT1A receptors in guinea pig dentate gyrus

The regulation by N-methyl-d-aspartate (NMDA) and 5-HT_1A receptors of the endogenous γ-aminobutyric acid (GABA) release was investigated in slices of the guinea pig dentate gyrus. The release of GABA was increased in a concentration-dependent fashion by NMDA. The release of GABA evoked by NMDA was Ca²⁺-dependent, tetrodotoxin-resistant, Mg²⁺-sensitive and inhibited by MK-801, a selective non-competitive NMDA receptor antagonist. These results suggest that the NMDA receptor present on GABAergic neurons is involved in the stimulatory regulation of GABA release. The release of GABA was increased concentration-dependently by NAN-190, a 5-HT_1A receptor antagonist, but was not affected by 8-OH-DPAT, a 5-HT_1A receptor agonist. The release of GABA evoked by NAN-190 was Ca²⁺-dependent, tetrodotoxin-resistant and inhibited by 8-OH-DPAT. These results suggest that the 5-HT_1A receptor present on GABAergic neurons is involved in the inhibitory regulation of GABA release. The release of GABA evoked by NMDA from the dentate gyrus was inhibited by pretreatment with 8-OH-DPAT. The release of GABA evoked by NAN-190 was inhibited by pretreatment with MK-801. The release of GABA evoked by NMDA from the dentate gyrus was augmented by the concurrent application of NAN-190. Taken together, the results indicate that the NMDA receptor and the 5-HT_1A receptor, which are both located on GABAergic neurons in the guinea pig dentate gyrus, exert stimulatory and inhibitory regulation of neuronal GABA release, respectively.     

Effect of otilonium bromide and ibodutant on the internalization of the NK2 receptor in human colon

Background The present aim was to study the modulation of NK₂ receptor internalization by two compounds, the spasmolytic otilonium bromide (OB) endowed with NK₂ receptor antagonistic properties and the selective NK₂ receptor antagonist ibodutant. Methods Full‐thickness human colonic segments were incubated in the presence of OB (0.1–10 μmol L^?1) or ibodutant (0.001–0.1 μmol L^?1), with or without the NK₂ receptor selective agonist [βAla⁸]NKA(4–10) and then fixed in 4% paraformaldehyde. Cryosections were processed for NK₂ receptor immunohistochemical revelation. Quantitative analysis evaluated the number of the smooth muscle cells that had internalized the NK₂ receptor. Key Results Immuno‐histochemistry revealed that in basal condition, the NK₂ receptor was internalized in about 23% of total smooth muscle cells. The exposure to the selective NK₂ receptor agonist induced internalization of the receptor in more than 77% of the cells. Previous exposure to both OB or ibodutant, either alone or in the presence of the agonist, concentration‐dependently reduced the number of the cells with the internalized receptor. Conclusions & Inferences Both OB and ibodutant antagonize the internalization of the NK₂ receptor in the human colon. As NK₂ receptors are the predominant receptor mediating spasmogenic activity of tachykinins on enteric smooth muscle, we hypothesize that the antagonistic activity found for both OB and ibodutant should play a specific therapeutic role in gut diseases characterized by hypermotility.     

Long-term D1-dopamine receptor sensitization in neonatal 6-OHDA-lesioned rats is blocked by an NMDA antagonist

Repeated administration of the D₁-dopamine agonist SKF-38393 to adult rats having had dopaminergic neurons destroyed early in development results in an increasing enhancement of the behavioral response to SKF-38393 with each dose until a maximum is reached. This increased sensitivity lasts for at least 6 months. In the present study, this long-lasting change in behavioral responsiveness to repeated treatment with SKF-38393, referred to as D₁-dopamine receptor priming, was shown to be dose dependent with smaller doses requiring an increased number of administrations to produce a maximal response when compared to higher doses. In addition, priming occurred equally well when treatment intervals ranged from 1 day to 14 days. These latter data reinforced the view that activation of D₁-dopamine receptors results in a prolonged change in neural function. In subsequent experiments D₁-dopamine receptor priming was blocked by pretreatment with the NMDA-receptor antagonist MK-801. This antagonism of priming could not be attributed to a blockade of D₁-dopamine receptors by MK-801 or to the induction of interfering behaviors. Because an NMDA antagonist interfered with D₁-dopamine priming as it does with other long-term neural messages, a common requirement for these diverse forms of neuronal plasticity appears to involve activation of the NMDA receptor. This functional link between NMDA receptors and dopaminergic function and its relationship to neuronal palsticity could have relevance to the biochemical mechanism involved in learning and to symptons in central disorders during development that worsen over time, particularly those proposed to involved malfunctioning dopaminergic mechanisms.     

Effects of asenapine on prefrontal N‐methyl‐D‐aspartate receptor‐mediated transmission: Involvement of dopamine D1 receptors

Asenapine is a novel psychopharmacologic agent being developed for schizophrenia and bipolar disorder. Like clozapine, asenapine facilitates cortical dopaminergic and N‐methyl‐D ‐aspartate (NMDA) receptor‐mediated transmission in rats. The facilitation of NMDA‐induced currents in cortical pyramidal cells by clozapine is dependent on dopamine and D₁ receptor activation. Moreover, previous results show that clozapine prevents and reverses the blockade of NMDA‐induced currents and firing activity in the pyramidal cells by the noncompetitive NMDA receptor antagonist phencyclidine (PCP). Here, we investigated the effects of asenapine in these regards using intracellular electrophysiological recording in vitro. Asenapine (5 nM) significantly facilitated NMDA‐induced currents (162 ± 15% of control) in pyramidal cells of the medial prefrontal cortex (mPFC). The asenapine‐induced facilitation was blocked by the D₁ receptor antagonist SCH23390 (1 μM). Furthermore, the PCP‐induced blockade of cortical NMDA‐induced currents was effectively reversed by 5 nM asenapine. Our results demonstrate a clozapine‐like facilitation of cortical NMDA‐induced currents by asenapine that involves prefrontal dopamine and activation of D₁ receptors. Asenapine and clozapine also share the ability to reverse functional PCP‐induced hypoactivity of cortical NMDA receptors. The ability of asenapine to increase both cortical dopaminergic and NMDA receptor‐mediated glutamatergic transmission suggests that this drug may have an advantageous effect not only on positive symptoms in patients with schizophrenia, but also on negative and cognitive symptoms. Synapse 64:870–874, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of allopurinol on NMDA receptor modification following recurrent asphyxia in newborn piglets

The present study tests the hypothesis that repeated episodes of asphyxia will lead to alterations in the characteristics of the N-methyl-d-aspartate (NMDA) receptor in the brain cell membrane of newborn piglets and that pre-treatment with allopurinol, a xanthine oxidase inhibitor, will prevent these modifications. Eighteen newborn piglets were studied. Six untreated and six allopurinol treated animals were subjected to eight asphyxial episodes and compared to six normoxic, normocapneic controls. Brain cell membrane Na⁺,K⁺-ATPase activity was determined to assess membrane function. Na⁺,K⁺-ATPase activity was decreased from control following asphyxia in both the untreated and treated animals (47.7±3.2 vs. 43.0±2.2 and 41.0±5.3 μmol Pi/mg protein/h, p<0.05, respectively). ³H-MK-801 binding studies were performed to measure NMDA receptor binding characteristics. The receptor density (B_max) in the untreated asphyxia group was decreased compared to control animals (0.80±0.11 vs. 1.13±0.33, p<0.05); furthermore, the dissociation constant (K_d) was also decreased (3.8±0.7 vs. 9.2±2.2, p<0.05), indicating an increase in receptor affinity. In contrast, B_max in the allopurinol treated asphyxia group was similar to control (1.06±0.37); and K_d was higher (lower affinity) than in the untreated group (6.5±1.4, p<0.05). The data indicate that recurrent asphyxial episodes lead to alterations in NMDA receptor characteristics; and that despite cell membrane dysfunction as seen by a decrease in Na⁺,K⁺-ATPase activity, allopurinol prevents modification of NMDA receptor–ion channel binding characteristics induced by repeated episodes of asphyxia.     

Involvement of tachykinin NK1 receptors in nociceptin-induced hyperalgesia in mice

Intrathecal (i.t.) injection of nociceptin at small doses (3.0 and 30.0 fmol) produced a significant hyperalgesic response as assayed by the tail-flick test. This hyperalgesic effect peaked at 15 min following i.t. administration of nociceptin (3.0 fmol) and returned to control level within 30 min. Hyperalgesia elicited by nociceptin was inhibited dose-dependently by i.t. co-administration of tachykinin NK₁ receptor antagonists, CP-99,994 and sendide. A significant antagonistic effect of [ -Phe⁷, -His⁹] substance P (6–11), a selective antagonist for substance P, was observed against the nociceptin-induced hyperalgesia. Pretreatment with i.t. substance P antiserum and i.t. capsaicin resulted in a complete block of the reduced threshold produced by nociceptin. The NK₂ receptor antagonist, MEN-10,376 and pretreatment with neurokinin A antiserum did not alter the behavioural effect of nociceptin. The N-methyl- -aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and (−)-2-amino-5-phosphonovaleric acid ( -APV), and -N^G-nitro arginine methyl ester ( -NAME), a nitric oxide synthase inhibitor, failed to inhibit nociceptin-induced hyperalgesia. The results obtained suggest that the hyperalgesic effect of nociceptin may be mediated through tachykinin NK₁ receptors in the spinal cord.     

NMDA receptor activition by residual glutamate in glutamine preparations: a cautionary note regarding weak NMDA receptor agonists

During an investigation of excitatory amino acids on cultured embryonic Xenopus neurons, we observed that commercial preparations of glutamine had weal agonist activity on NMDA-type glutamate receptors. Threshold responses were observed at 100 μM glutamine, and the dose-response relation did not show inflection or saturation at concentrations of up to 10 mM. However, NMDA receptor activation induced by glutamine probably represented activity of residual glutamate because: (a) recrystallization of glutamine reduced residual glutamate levels (measured by HPLC analysis) and NMDA receptor activation by comparable amounts; and (b) glutamate at concentrations close to those predicted to be present in glutamine preparations elicited currents of similar amplitudes. Our data indicate that residual glutamate at levels of less than 0.05% are sufficient to confound studies of weak NMDA receptor agonists.     

Orphenadrine is an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist: binding and patch clamp studies

Summary Orphenadrine has been used as an antiparkinsonian, antispastic and analgesic drug for many years. Here we show that orphenadrine inhibits [³H]MK-801 binding to the phencyclidine (PCP) binding site of the N-methyl-D-aspartate (NMDA)-receptor in homogenates of postmortem human frontal cortex with a K_i-value of 6.0 ± 0.7 M. The NMDA receptor antagonistic effects of orphenadrine were assessed using concentration- and patch-clamp techniques on cultured superior colliculus neurones. Orphenadrine blocked open NMDA receptor channels with fast kinetics and in a strongly voltage-dependent manner. The IC₅₀-value against steady state currents at –70mV was 16.2 ± 1.6 M (n=6). Orphenadrine exhibited relatively fast, concentration-dependent open channel blocking kinetics (K_on 0.013 ± 0.002 10⁶M^–1S^–1) whereas the offset rate was concentration-independent (K_off 0.230 ± 0.004 S^–1). Calculation of the ratio K_off/K_on revealed an apparent K_d-value of 17.2 M which is nearly identical to the IC₅₀ calculated at equilibrium.     

Localization of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor subtypes, and vasopressin in the mouse hypothalamus

The hypothalamic angiotensin II (Ang II) system plays an important role in pituitary hormone release. Little is known about this system in the mouse brain. We studied the distribution of angiotensin-converting-enzyme (ACE), Ang II, Ang II receptor subtypes, and vasopressin in the hypothalamus of adult male mice. Autoradiography of binding of the ACE inhibitor [¹²⁵I]351A revealed low levels of ACE throughout the hypothalamus. Ang II- and vasopressin-immunoreactive neurons and fibers were detected in the paraventricular, accessory magnocellulary, and supraoptic nuclei, in the retrochiasmatic part of the supraoptic nucleus and in the median eminence. Autoradiography of Ang II receptors was performed using [¹²⁵I]Sar¹–Ang II binding. Ang II receptors were present in the paraventricular, suprachiasmatic, arcuate and dorsomedial nuclei, and in the median eminence. In all areas [¹²⁵I]Sar¹–Ang II binding was displaced by the AT₁ receptor antagonist losartan, indicating the presence of AT₁ receptors. In the paraventricular nucleus [¹²⁵I]Sar¹–Ang II binding was displaced by Ang II (K_i=7.6×10⁻⁹) and losartan (K_i=1.4×10⁻⁷) but also by the AT₂ receptor ligand PD 123319 (K_i=5.0×10⁻⁷). In addition, a low amount of AT₂ receptor binding was detected in the paraventricular nucleus using [¹²⁵I]CGP 42112 as radioligand, and the binding was displaced by Ang II (K_i=2.4×10⁻⁹), CGP 42112 (K_i=7.9×10⁻¹⁰), and PD 123319 (K_i=2.2×10⁻⁷). ACE, Ang II, and AT₁ as well as AT₂ receptor subtypes are present in the mouse hypothalamus. Our data are the basis for further studies on the mouse brain Ang II system.     

An NK1 receptor antagonist affects the circadian regulation of locomotor activity in golden hamsters

Substance P (SP) is a neuromodulator which may participate in the photic regulation of the circadian timing system in mammals. The biological effects of SP are mediated by interaction with specific receptors, designated as NK₁, NK₂, and NK₃. The NK₁ subtype receptor is expressed in the circadian system. Experiment 1 was designed to test whether an NK₁ antagonist mimics the effects of dark pulses. Hamsters were housed in constant lighting conditions, either constant darkness or constant light (around 250 lx), and they received an i.p. injection of either the specific NK₁ receptor antagonist, L-760,735 (5 mg/kg), or saline during the mid-subjective day, a time when dark pulses cause a phase-advance in circadian rhythm of locomotor activity. After treatment with the NK₁ antagonist, significant phase-advances of wheel-running activity rhythm were found in constant light, but not in constant darkness. Experiment 2 was designed to test the ability of the NK₁ antagonist to block the phase-delaying and/or the phase-advancing effects of light in animals kept in constant darkness. Phase-advances of locomotor activity rhythm that can normally be induced by light pulses given during the late subjective night were markedly reduced by pre-treatment with the NK₁ antagonist. By contrast, phase-delays that can be induced by lights pulses given during the early subjective night were unaffected by the NK₁ antagonist. These data support the hypothesis that SP within the circadian system may, by interacting with NK₁ receptors, modulate photic responses of the SCN pacemaker.     

Effect of up-regulation of NMDA receptors on cerebral O2 consumption and blood flow in rat

We tested the hypothesis that cerebrocortical blood flow and O₂ consumption would be proportional to an up-regulated number of functional N-methyl- -aspartate (NMDA) receptors. Previous work had shown a relationship between cerebral metabolism and NMDA receptor activity. We increased the specific binding to NMDA receptors in the cerebral cortex, from 2.2 ± 0.9 to 4.5 ± 0.8 (density units) in male Long-Evans rats by daily giving two intraperitoneal injections (30 mg/kg) of CGS-19755, an NMDA receptor inhibitor, for 7 consecutive days (discontinued for 20 h before experiment). Twelve up-regulated (CGS treated) and 12 control rats were used in this study. Under isoflurane anesthesia and after topical stimulation of the right cerebral cortex with 10⁻² M NMDA, the blood flow (¹⁴C-iodoantipyrine method) increased from 98 ± 11 ml/min/100 g in the unstimulated cortex of the control rats to 161 ± 37 ml/min/100 g in the stimulated cortex. The unstimulated value for blood flow (95 ± 7 ml/min/100 g) did not change in the upregulated group but it doubled (194 ± 69 ml/min/100 g) in the stimulated, upregulated cortex. Similarly, O₂ consumption (cryomicrospectrophotometrically determined) in normal rats increased 46%, from 9.3 ± 1 ml/min/100 g to 13.6 ± 4 after NMDA stimulation. While in the upregulated animals, O₂ consumption increased 103% from 7.9 ± 0.6 to 16 ± 6.5 after NMDA stimulation. In conclusion, NMDA receptor upregulation does not alter basal cerebrocortical blood flow or O₂ consumption but in the NMDA-stimulated cortex, the blood flow and O₂ consumption increase is dependent on the number of NMDA receptors present.     

Ethanol inhibition of NMDA mediated depolarizations is increased in the presence of Mg

The inhibitory potency of ethanol upon excitatory amino acid induced depolarizations of rat hippocampal CA1 pyramidal cells was assessed in the presence and absence of magnesium (Mg²⁺) using the grease-gap technique. Ethanol shifted theN-methyl-d-aspartate (NMDA) dose-response curves to the right in a non-parallel manner. In the presence of Mg²⁺, ethanol appeared to be a more effective NMDA antagonist (IC₅₀ 47 mM) than in the absence of Mg²⁺ (IC₅₀ 107 mM). The IC₅₀ for ethanol upon non-NMDA mediated CA1 pyramidal cell depolarizations was in excess of 170 mM. These results strongly suggest a preferential inhibitory action of ethanol against NMDA, rather than non-NMDA, mediated responses. Experiments in which ethanol and Mg²⁺ were covaried indicated that these substances act by two distinct mechanisms to antagonize the action of NMDA. These effects of ethanol, at concentrations which elicit intoxication(< 50mM) but not anesthesia, suggest that the NMDA receptor complex may play an important role in the acute effects of ethanol.     

Tachykinins cause inward current through NK1 receptors in bullfrog sensory neurons

The effects of tachykinins on primary afferent neurons of bullfrog dorsal root ganglia (DRG) were examined by using whole-cell patch-clamp methods. Neurokinin A (NKA) caused inward current (I_NKA) in a concentration-dependent manner. Concentration-response curve showed that the EC₅₀ for NKA was 6 nM. The I_NKA showed strong tachyphylaxis, when NKA was continuously applied for more than 1 min. Substance P (SP) also produced inward current with potency similar to that of NKA. Neurokinin B (NKB) was less effective in producing the inward current. The order of agonist potency was NKA = SP NKB. Spantide ([D-Arg¹, D-Trp^7,9, Leu¹¹]SP), non-selective peptide antagonist at tachykinin receptors, reduced the tachykinin-induced current. CP-99,994, a selective non-peptide antagonist for neurokinin-1 (NK₁) receptor, inhibited the inward currents produced by NKA and SP. The I_NKA was associated with decrease in K⁺ conductance. NKA suppressed both a voltage-dependent K⁺ current, the M-current (I_M), and a voltage-independent background K⁺ current, I_K(B). Intracellular dialysis with GTPγS (100 nM) or GDPβS (100 μM) depressed the I_NKA. Pre-treatment of DRG neurons with pertussis toxin (PTX) did not prevent the I_NKA. Depletion of intracellular ATP depressed the I_NKA. These results suggest that the tachykinin-induced inward current is mediated through the NK₁ receptor which mainly couples to PTX-insensitive G-protein in bullfrog primary afferent neurons.     

MK801 induces late regional increases in NMDA and kainate receptor binding in rat brain

Summary We have previously shown that a single dose of PCP produces a dose-related increase in NMDA-sensitive³H-glutamate binding in CA₁ of hippocampus 24 hours later, and some regional changes in kainate binding. Here we report that dizocilpine (MK 801) (O.1 mg/kg and 1 mg/kg), a selective agonist at the PCP receptor and a noncompetitive antagonist of NMDA, produces a similar increase in NMDA-sensitive glutamate and kainate receptor binding in hippocampus 24 hours after a dose. These observations support the conclusion that blockade of glutamate-mediated transmission at the NMDA receptor selectively increases NMDA-sensitive glutamate receptor binding in CA₁ of hippocampus and kainate binding in CA₃ and dentate gyrus at putatively delayed time points. Several additional areas outside of hippocampus also showed receptor changes at 24 hours after MK801.