Takashige Nishikawa

Evidence for GABAA receptor agonistic properties of ketamine: Convulsive and anesthetic behavioral models in mice

We examined the potentiation by ketamine of the &ggr;-aminobutyric acidA (GABAA) receptor function using convulsive and anesthetic behavioral models in adult male ddY mice. General anesthetic potencies were evaluated by a rating scale, which provided the data for anesthetic scores, loss of righting reflex, duration, and recovery time. All drugs were administered intraperitoneally. Small subanesthetic doses of ketamine did inhibit tonic seizures induced by a large dose of the GABAA receptor antagonist bicuculline (8 mg/kg). The 50% effective dose value was 15 (95% confidence limits 10–22) mg/kg. Even large anesthetic doses (100–150 mg/kg) did not suppress clonic seizures in 50% of the animals. The GABAA receptor agonist, muscimol (0.32–1.12 mg/kg), potentiated ketamine-induced anesthesia in a dose-dependent fashion (P < 0.05). Similarly, the benzodiazepine receptor agonist, diazepam (1–3 mg/kg), augmented ketamine anesthesia in a dose-dependent manner (P < 0.05). Bicuculline (2–5 mg/kg) dose-dependently antagonized ketamine-induced anesthesia (P < 0.05). Neither the benzodiazepine receptor antagonist, flumazenil (2–20 mg/kg), nor the GABA synthesis inhibitor, l-allylglycine (200 mg/kg), affected the anesthetic action of ketamine. These results suggest that ketamine has GABAA receptor agonistic properties and that ketamine-induced anesthesia is mediated, at least in part, by GABAA receptors. Implications We examined the potentiation by ketamine of the &ggr;-aminobutyric acidA receptor function using convulsive and anesthetic behavioral models in mice. Subanesthetic doses of ketamine-inhibited tonic convulsions induced by the &ggr;-aminobutyric acidA receptor antagonist bicuculline. The &ggr;-aminobutyric acidA receptor agonist, muscimol, potentiated ketamine-induced anesthesia. Bicuculline antagonized ketamine anesthesia, but the benzodiazepine receptor antagonist, flumazenil, and the &ggr;-aminobutyric acid synthesis inhibitor, l-allyglycine, did not. The effects of ketamine on the &ggr;-aminobutyric acidA receptors appear to correlate with its anesthetic actions.

Effects of ketamine on dopamine metabolism during anesthesia in discrete brain regions in mice: comparison with the effects during the recovery and subanesthetic phases

The effects of ketamine on the levels of dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT, serotonin) and their metabolites were examined in discrete brain regions in mice. A high dose of ketamine (150 mg/kg, i.p.) did not change DA metabolism in the frontal cortex, nucleus accumbens, striatum and hippocampus, but did decrease it in the brainstem during anesthesia. In contrast, during recovery from the ketamine anesthesia, the high dose increased the level of homovanillic acid (HVA) in all brain regions. A low subanesthetic dose of ketamine (30 mg/kg, i.p.) increased the concentrations of both 3,4-dihydroxyphenylacetic acid (DOPAC) and HVA only in the nucleus accumbens. The DA level was not affected by any ketamine treatment. During ketamine anesthesia, the content of 3-methoxy-4-hydroxy-phenylglycol (MHPG) was decreased in the brainstem, whereas during recovery from anesthesia, the MHPG level was increased in the frontal cortex, nucleus accumbens and brainstem. The NE content was not altered in any region by ketamine treatment. The concentration of 5-hydroxyindoleacetic acid (5-HIAA) was reduced in the frontal cortex, striatum, hippocampus and brainstem during ketamine anesthesia. The 5-HT level was unaltered in all regions except the brainstem where it was reduced. In contrast, after anesthesia, the concentrations of both 5-HT and 5-HIAA were increased in the striatum. During the subanesthetic phase, however, the levels of NE, 5-HT and their metabolites were unchanged. These neurochemical results are consistent with the electrophysiological findings that a high dose of ketamine does not change the basal firing rates of nigrostriatal DA neurons during anesthesia, while low subanesthetic doses significantly increase those of ventral tegmental DA neurons.

Effects of ovariectomy and calcium deficiency on learning and memory of eight-arm radial maze in middle-aged female rats

There is increasing evidence that estrogen and calcium ion are involved in learning and memory. In the present study, to examine the effect of estrogen deficiency and low-calcium diet on learning and memory, middle-aged female Wistar rats (50 weeks old) were fed either a low-calcium (0.02% Ca) or a normal-calcium (1.25% Ca) diet throughout the experiment. Rats were ovariectomized (OVX) or sham-operated (Sham). These animals were divided into four groups: 1) Sham group with normal-calcium diet [Sham-normal Ca group], 2) OVX group with normal-calcium diet [OVX-low Ca group], 3) Sham group with low-calcium diet [Sham-low Ca group], 4) OVX group with low-calcium diet [OVX-low Ca group]. Seventy-seven days after the OVX or Sham operation, the learning and memory abilities in the female rats were examined by using a radial maze task according to the method of Olton and Samuelson (regular trials) and using a delay-interposed task following regular trials. During regular trials and delay-interposed tasks, the OVX-low Ca group was inferior to all the other groups in accuracy of choice behavior. Both Sham-normal Ca and Sham-low Ca groups showed more accurate choices than the OVX-low Ca group, but were less accurate than the Sham-normal Ca group. In addition, there was no significant difference in locomotor activity between any of the groups. These results suggest that OVX or low-calcium diet may impair learning and memory, and that the combination of these factors impaired more markedly when the rats were tested in the eight-arm radial maze. These results may also imply the possibility that a woman in menopause or post-menopause suffers impairment of learning and/or memory when intakes low-calcium diet.

Inhibition of Na+, K+ ‐ATPase Activity by Phospholipase A2 and Several Lysophospholipids: Possible Role of Phospholipase A2 in Noradrenaline Release from Cerebral Cortical Synaptosomes

Abstract— p‐Bromophenacyl bromide (PBPB), quinacrine and indomethacin, which inhibit phospholipase A2 (PLA2; EC 3.1.1.4) activity in several tissues, caused a dose‐dependent inhibition of prelabelled [3H]noradrenaline ([3H]NA) release evoked by high concentrations of K+ from rat cerebral cortical synaptosomes. Release of prelabelled [3H]NA was caused by natural lysophosphatidic acid (LPA; 10−6‐10−5 gmL−1) and lysophosphatidylcholine (LPC; 10−6‐10−5 g mL−1) and synthetic LPA (6 × 10−6, 2 × 10−5 M) and LPC (6 × 10−6, 2 × 10−5 M), but not by natural lysophosphatidylserine (LPS; 10−5 g mL−1), lysophosphatidylethanolamine (LPE; 10−5 g mL−1) and lysophosphatidylinositol (LPI; 10−5 g mL−1). The release evoked by natural LPA and LPC could be inhibited only marginally by PBPB and quinacrine. Phosphatidic acid (PA)‐specific and phosphatidylcholine (PC)‐specific PLA2 activities from rat cerebral cortical synaptosomes were stimulated in incubation medium containing high concentrations of K+ or calcium ionophore A23187. Low concentrations of PLA2 (10−6–10−8 g mL−1, from bee venom) inhibited the synaptic membrane Na+, K+‐ATPase activity in incubation media with intracellular levels of free Ca2+. Several lysophospholipids (LPLs), metabolites of the PLA2 type, also inhibited the synaptic membrane Na+, K+‐ATPase activity in a dose‐dependent manner. The minimum effective concentrations of natural LPA, LPC, LPS, LPI and LPE were 10−6, 4·7 × 10−6, 10−5,4·7 × 10−5 and 4·7 × 10−5 g mL−1, respectively. These results suggest that PLA2 and/or its metabolites, LPLs, especially LPA and LPC, may play partial roles in the depolarization and/or release of noradrenaline through their inhibitory action on the Na+, K+‐ATPase activity in the brain.

Hyperlocomotion during Recovery from Isoflurane Anesthesia Is Associated with Increased Dopamine Turnover in the Nucleus Accumbens and Striatum in Mice

Background It was recently reported that isoflurane increases dopamine release in the striatum in rats both in vivo and in vitro, and that isoflurane inhibits uptake of dopamine in the rat brain synaptosomes. However, the functional role of these effects of isoflurane on dopamine neurons is uncertain. Dopaminergic mechanisms within the nucleus accumbens and striatum play an important role in the control of locomotor activity, and a change in dopamine turnover depends essentially on a change in impulse flow in the dopamine neurons. In this study, the effects of isoflurane on locomotor activity and on dopamine turnover were investigated in discrete brain regions in mice. Methods Mice were placed in individual airtight clear plastic chambers and spontaneously breathed isoflurane in 25% oxygen and 75% nitrogen (fresh gas flow, 4 l/min). Locomotor activity was measured with an Animex activity meter. Animals were decapitated after treatments with or without isoflurane, and the concentrations of monoamines and their metabolites in different brain areas were measured by high‐performance liquid chromatography. Results During the 10 min after the cessation of the 20‐min exposure to isoflurane, there was a significant increase in locomotor activity in animals breathing 1.5% isoflurane but not 0.7% isoflurane. This increase in locomotor activity produced by 1.5% isoflurane was abolished by a low dose of haloperidol (0.1 mg/kg), a dopamine receptor antagonist. Regional brain monoamine assays revealed that 1.5% isoflurane significantly increased the 3,4‐dihydroxyphenylacetic acid:dopamine ratio (one indicator of transmitter turnover) in the nucleus accumbens and striatum, but a concentration of 0.7% did not. This significant increase in dopamine turnover in these regions continued during 20 min after the cessation of the administration of 1.5% isoflurane. Conclusions These results suggest that isoflurane‐induced hyperlocomotion during emergence may be associated with increased dopamine turnover in the nucleus accumbens and striatum.

Inhibitory effects of group II mGluR-related drugs on memory performance in mice

The cAMP/protein kinase A signaling pathway is negatively modulated by group II metabotropic glutamate receptors (mGluRs), and the cross-talk that occurs between these receptors may modulate learning and memory. To examine the relationship among cAMP/PKA-signaling pathway activity, group II mGluRs, and learning and memory, mice were trained to perform a step-through-type passive avoidance task, and 10 min before each avoidance trial the following drugs were injected intracisternally (i.cist.): vehicle (0.05% dimethylsulfoxide); a specific group II mGluR agonist, DCG-IV (1-50 ng/mouse); a specific group II mGluR antagonist, LY341495 (10-300 ng); a selective inhibitor of cAMP-specific phosphodiesterase, rolipram (100-1000 ng); an activator of adenylyl cyclase, forskolin (25-250 ng); a specific inhibitor of PKA, H-89 (150 or 300 ng) or; an activator of protein kinase C, phorbol 12-myristate 13-acetate (PMA 200 ng). DCG-IV (25 and 50 ng) or LY341495 (150 and 300 ng) reduced the latency in the avoidance task. The reduction of latency by DCG-IV was not observed in mice coinjected with DCG-IV (50 ng) together with rolipram (500 ng) or forskolin (25 ng). Conversely, coinjection of LY341495 with 100 or 1000 ng rolipram, or with 25 or 250 ng forskolin tended to potentiate the LY341495-induced shortening of latency. In addition, the reduction of latency by DCG-IV (50 ng) was not observed in mice coinjected with DCG-IV and PMA together. However, the reduction of latency by LY341495 (300 ng) was potentiated when the drug was coadministered with PMA. These results suggest that changes in the cAMP/PKA-signaling pathway, mediated by group II mGluRs, influence memory in the passive avoidance task, and that both the excessive activation and deactivation of this pathway may induce the impairment of learning and memory.

Effect of acetaminophen, a cyclooxygenase inhibitor, on Morris water maze task performance in mice.

Although the mechanism of action of acetaminophen (AAP) is not fully understood, some studies suggest that AAP and phenacetin (PHE) are selective cyclooxygenase (COX)-3 inhibitors. To examine the participation of COX-3 in memory formation, water maze performance was studied in mice treated with AAP, PHE or other COX inhibitors. Mice received intraperitoneal injections of drugs immediately after each training session. Administration of high-dose AAP [302.3 mg/kg (IC50 for COX-2)] or PHE [179.2 mg/kg (IC50 for COX-2)] and of non-specific (indomethacin: 20 mg/kg) or specific COX-2 (NS-398: 10 mg/kg) inhibitor impaired the performance in hidden platform (HP) not visible platform (VP) tasks, whereas low-dose (15.1 mg/kg) AAP facilitated performance in HP and VP tasks. The facilitation of performance by low-dose AAP was reversed by co-administration with a 5-HT1/2 receptor antagonist (methysergide: 0.47 mg/kg). The middle-dose [69.5 mg/kg (IC50 for COX-3)] of AAP, the PHE [17.9 mg/kg (IC50 for COX-3)] and a specific COX-1 inhibitor (piroxicam: 10—20 mg/kg) did not influence performance in either task. These results suggest that the memory impairment by high-dose AAP and PHE and facilitation of performance by low-dose AAP could involve endogenous COX-2 and serotonergic neuronal activity, but not COX-3, respectively.

Effects of estradiol and progesterone on radial maze performance in middle-aged female rats fed a low-calcium diet

There is increasing evidence that ovarian steroids and calcium ions are involved in learning and memory. To examine the effect of ovarian steroids on learning and memory under a low-calcium condition, middle-aged female rats were fed either a low-calcium (0.02% Ca) or a normal-calcium (1.25% Ca) diet. All rats were ovariectomized (OVX), and these animals were divided into eight groups: 1) an OVX group with a normal-calcium diet (OVX-normal-Ca group), 2) an OVX group with 17beta-estradiol treatment and a normal-calcium diet (E2 group), 3) an OVX with progesterone treatment and a normal-calcium diet (P4 group), 4) an OVX with 17beta-estradiol and progesterone treatments and a normal-calcium diet (E2 + P4 group), 5) an OVX group with a low-calcium diet (OVX-low-Ca group), 6) an OVX group with 17beta-estradiol treatment and a low-calcium diet (LE2 group), 7) an OVX group with progesterone treatment and a low-calcium diet (LP4 group), and 8) an OVX group with 17beta-estradiol and progesterone treatments and a low-calcium diet (LE2 + LP4). Seventy-seven days after the OVX operation, the learning and memory abilities of the rats were examined by using an eight-arm radial maze task. E2 and E2 + P4 groups learned in fewer trials, and performed better in the radial maze and the working memory task than the other groups under the normal-calcium condition. Rats in the LE2 group learned in fewer trials, and performed better in the maze and working memory task than the other low-calcium groups, but in combination with progesterone under the low-calcium condition (LE2 + LP4 group), the facilitative effect of estradiol in all the tasks was inhibited. Treatment with progesterone alone did not inhibit the learning and memory task performance. These results suggest the possibility that treatment with estradiol under low-calcium conditions cannot improve impaired learning and memory when progesterone is applied simultaneously, and that the intake of adequate calcium may be necessary and effective for patients with learning and memory hypofunction receiving hormone replacement therapy.

Facilitative effect of a novel AVP fragment analog, NC-1900, on memory retention and recall in mice

In order to determine the mechanism of action of a new AVP(4-9) analog, NC-1900, on memory processes, memory retention and retrieval tests were conducted in a step-through passive avoidance (PA) task in mice. The administration of NC-1900 facilitated memory retention and retrieval in the PA task through vasopressin1A (V1A) receptors but not V2 receptors. The effect of NC-1900 on memory retention test performance appeared to be due to activation of the protein kinase C (PKC) signaling pathway via V1A receptors; however, the modulation of PKC was not essential for the facilitative effect of the new peptide in the retrieval test. The facilitation of memory retrieval by NC-1900 may also be mediated by other non-PKC-dependent signaling pathways, such as the phospholipase C-inositol trisphosphate pathway.

Effect of morphine on Na+, K+ -ATPase from homogenate of synaptosomes and of synaptic membrane of rat cerebral cortex

Effects of morphine on noradrenaline release from rat cerebrocortical synaptosomes and on the Na+,K(+)-ATPase activity in homogenates of synaptosomes and of synaptic membranes were examined. Both morphine (10(-3)-10(-5) M) and methionine-enkephalin (M-Enk; 10(-5) M) inhibited the enhanced [3H]noradrenaline [( 3H]NA) release evoked by high concentrations of K+ from synaptosomes and these inhibitory actions were antagonized by naloxone (10(-4), 10(-5) M). Morphine (10(-3)-10(-5) M) and M-Enk (10(-5) M) stimulated the Na+,K(+)-ATPase activity in homogenates of synaptosomes but not of synaptic membranes in the incubation medium containing 2.2 X 10(-6)-4.7 X 10(-7) M free Ca2+ and these stimulatory effects were antagonized by naloxone. In homogenates of synaptic membranes, the same concentrations of morphine and M-Enk stimulated the Na+,K(+)-ATPase activity suppressed by FeCl2 (5 X 10(-7) M) but not by CuCl2 nor ZnCl2, and these stimulatory effects were antagonized by naloxone. Significant levels of Fe2+ were liberated from synaptosomes during the preparation of synaptic membrane using distilled water. These results suggest that both morphine and M-Enk stimulate the suppressed Na+,K(+)-ATPase activity by interacting with Fe2+ at opioid receptor sites, and they may play a role in the suppression of membrane depolarization and/or the release of NA through their stimulatory action on the Na+,K(+)-ATPase activity probably suppressed by Fe2+ in the rat cerebral cortex.