Takeaki Miyamae

Postsynaptic diacylglycerol lipase α mediates retrograde endocannabinoid suppression of inhibition in mouse prefrontal cortex

Non‐Technical Summary  In multiple brain regions, endogenous cannabinoids suppress inhibitory synaptic transmission; however, the biochemical/molecular pathways for endocannabinoid synthesis are poorly understood. Endocannabinoid signalling may be crucial for microcircuit function in the prefrontal cortex (PFC), a cortical region involved in complex behaviours. However, endocannabinoid signalling remains largely unexplored in the PFC. Using enzymatic inhibitors, we show that modulation of inhibitory synaptic transmission in PFC neurons is mediated by the endocannabinoid 2‐arachidonoylglycerol synthesized postsynaptically. Interestingly, diacylglycerol lipase (DAGL), the 2‐arachidonoylglycerol synthesis enzyme, has two isoforms: DAGLα and DAGLβ. Studying PFC neurons from DAGLα−/−, DAGLβ−/− and wild‐type mice, we show that only DAGLα is involved in the suppression of inhibitory transmission in the PFC.

l-DOPA induces Ca2+-dependent and tetrodotoxin-sensitive release of endogenous glutamate from rat striatal slices

L-DOPA (10-1000 microM) concentration-dependently released glutamate (Glu) from superfused rat striatal slices. D-DOPA and dopamine (300 microM) produced no effects. The L-DOPA-induced release of Glu was not affected by 3-hydroxybenzylhydrazine (20 microM), an L-aromatic amino acid decarboxylase inhibitor. L-DOPA methyl ester (200 microM), a selective L-DOPA antagonist, antagonized the effect of L-DOPA in a competitive manner. Ca2+ deprivation and tetrodotoxin decreased L-DOPA (300 microM)-induced release of Glu. These findings indicate that L-DOPA induces a transmitter-like release of Glu via activation of a recognition site for itself.

Is DOPA a neurotransmitter

Historically, 3,4-dihydroxyphenylalanine (DOPA) has been considered to be an inert amino acid that alleviates the symptoms of Parkinsons disease by its conversion to dopamine via the enzyme aromatic L-amino acid decarboxylase. In contrast to this generally accepted idea, we propose that DOPA itself is a neurotransmitter and/or neuromodulator in addition to being a precursor of dopamine. Several criteria such as synthesis, metabolism, active transport, existence, physiological release, competitive antagonism and physiological or pharmacological responses must be satisfied before a compound is accepted as a neurotransmitter. Recent evidence suggests that DOPA fulfills these criteria in its involvement in baroreflex neurotransmission.

δ Opioid receptor mediates phospholipase C activation via Gi in Xenopus oocytes

Cloned mouse δ‐subtype opioid receptor (DOR1) was expressed in Xenopus oocytes to study the signal transduction. Opioid δ‐agonists evoked a calcium‐dependent chloride current in oocytes injected with mRNA derived from DOR1, together with that from the α subunit of Gi1. The δ‐agonist‐induced current was blocked by naltrindol, a δ‐specific antagonist. The δ‐agonist evoked no or very weak currents in oocytes with the α subunit of Gq or Go. These findings indicate the functional coupling between the opioid δ‐receptor and phospholipase C through an activation of Gi.

Non-effective dose of exogenously applied L-dopa itself stereoselectively potentiates postsynaptic D2 receptor-mediated locomotor activities of conscious rats.

We attempted to clarify whether or not under inhibition of central dopa decarboxylase non-effective i.p. doses of L-dopa potentiate D2 receptor-mediated locomotor activities without conversion to dopamine in normal and i.v.t. 6-hydroxydopamine-treated rats. In normal rats, only the highest dose of quinpirole, a selective D2 agonist, at ranges used (0.01-1 mg/kg, s.c.) slightly increased the total counts of locomotor activities for 140 min after injection. A simultaneously injected non-effective dose of L-DOPA (30 mg/kg) potentiated locomotor activities by quinpirole (0.1 and 1 mg/kg). L-dopa potentiated quinpirole (1 mg/kg)-induced locomotor activities 90 to 140 min after the injection with marked increase in basal release of L-DOPA without increase in dopamine release simultaneously monitored during striatal microdialysis, compared to quinpirole alone. D-dopa (30 mg/kg) produced no potentiation. In 6-hydroxydopamine-treated rats, a non-effective dose of L-dopa (10 mg/kg) also potentiated quinpirole (0.3 mg/kg)-induced locomotor activities. L-dopa acting on a recognition site for itself stereoselectively potentiates postsynaptic D2 receptor-mediated locomotor activities of rats.

Protein phosphatases 1 and 2A are both required for long-term depression and associated dephosphorylation of cAMP response element binding protein in hippocampal area CA1 in vivo.

Evidence shows that the serine/threonine protein phosphatase 1 (PP1) plays a critical role in synaptic plasticity and memory. Little is known about the contribution of the serine/threonine phosphatase 1 (PP2A) to synaptic plasticity. Both protein phosphatases can target the transcription factor cAMP response element binding protein (CREB), whose phosphorylation at Ser133, we previously found, was downregulated during long‐term depression (LTD) of glutamatergic transmission in area CA1 of the adult hippocampus in vivo. Other work from our group showed that the activity of PP2A, as well as that of PP1, is increased after LTD induction in area CA1 in vivo. We therefore investigated here whether both protein phosphatases are necessary for LTD in area CA1, and whether they both are involved in the LTD‐associated modification of CREB. We found that inhibition of either PP1 or PP2A interferes with the establishment of LTD. Furthermore, inhibition of either enzyme alone abrogated the LTD‐associated dephosphorylation of CREB. Interestingly, inhibition of PP1 disrupted CREB dephosphosphorylation rapidly after LTD‐inducing stimulation, whereas inhibition of PP2A did not blunt the CREB modification until a later time point. Thus, both PP1 and PP2A regulate CREB during LTD in area CA1, although possibly through different signaling pathways. Our results demonstrate that PP2A, similar to PP1, plays an essential role in the molecular events that underlie LTD at glutamatergic synapses in hippocampal area CA1 in vivo. We propose that one of the mechanisms through which these protein phosphatases may contribute to the prolonged maintenance of LTD is through the regulation of CREB.

Transmitter-like 3,4-dihydroxyphenylalanine is tonically released by nicotine in striata of conscious rats

Microdialysis and high performance liquid chromatography with an electrochemical detector were applied to compare the characteristics of nicotine-evoked release of endogenous 3,4-dihydroxyphenylalanine (DOPA) from striata of conscious rats and those of the release of dopamine (DA). Dialysates were collected every 20 min 3-8 h after the start of perfusion. Nicotine was perfused for 20 min through a probe. (+/-)-Nicotine (100-300 microM) constantly and repeatedly released DOPA and DA over a similar time course in a dose-dependent manner. The ratio of the DOPA and DA release evoked was approximately 1:3. The (+/-)-nicotine (200 microM)-induced DOPA release was mecamylamine (500 microM)-sensitive, tetrodotoxin (100 nM)-sensitive and Ca2+ (removal plus 12.5 mM Mg2+ addition)-dependent. The (+)-isomer produced no DOPA release. These characteristics of DOPA release were almost the same as those of DA release. Furthermore, mecamylamine alone inhibited the basal release of DOPA but not of DA. Nicotine released stereoselectively endogenous DOPA via nicotinic acetylcholine receptors from striata of freely moving rats in a manner similar to transmitter DA. These acetylcholine receptors function tonically for the release of DOPA. These findings are further support for our proposal that DOPA is an endogenous neuroactive substance.

Evidence forl-DOPA relevant to modulation of sympathetic activity in the rostral ventrolateral medulla of rats

By microdialysis in the rostral ventrolateral medulla (RVLM) of anesthetized rats, the spontaneous L-3,4-dihydroxyphenylalanine (DOPA) release was partially Ca(2+)-dependent and tetrodotoxin-sensitive and was markedly reduced by alpha-methyl-p-tyrosine (alpha-MPT; 200 mg/kg, i.p.). K+ (50 mM) Ca(2+)-dependently evoked L-DOPA. By microinjections into unilateral RVLM, L-DOPA (30-300 ng) produced dose-dependent hypertension and tachycardia similarly in rats untreated, treated with i.p. 3-hydroxybenzylhydrazine, a central DOPA decarboxylase inhibitor, or with i.v.t. 6-hydroxydopamine. These responses were antagonized by L-DOPA methyl ester (1.5 micrograms), a competitive L-DOPA antagonist. D-DOPA, dopamine, noradrenaline or adrenaline (300 ng) produced no effect. Furthermore, L-DOPA methyl ester alone microinjected into bilateral RVLM (2 micrograms x 2) produced prolonged hypotension and bradycardia, which were abolished by alpha-MPT. These data suggest that L-DOPA is relevant to modulation of sympathetic activity in the rat RVLM.

Opioid μ- and κ-receptor mediate phospholipase C activation through Gi1 in Xenopus oocytes

Abstract In the Xenopus oocytes expressing μ or κ-opioid receptors, agonist-induced currents were observed only when the oocyte was coinjected with Gi1 α RNA and pretreated with K-252a, a potent inhibitor of protein kinases. The evoked currents were abolished by i intracellular injection of EGTA or inositol 1,4,5-trisphosphate and the current-voltage relationship revealed that they are mediated through typical calcium-dependent chloride channels. These findings suggest that the μ- and κ-receptors mediate phospholipase C activation through Gi1 α, and that these receptor mechanisms including downstream signalings might be inhibited by phosphorylation in vivo in the Xenopus oocyte.

Some interactions of L-DOPA and its related compounds with glutamate receptors

L-DOPA is probably a transmitter and/or modulator in the central nervous system (1). L-DOPA methyl ester (DOPA ME) is a competitive L-DOPA antagonist. However, it remains to be clarified whether there exist L-DOPAergic receptors. In Xenopus laevis oocytes injected with rat brain poly(A)+ RNA, L-DOPA induced small inward currents with ED50 of 2.2 mM at a holding potential of -70 mV. The currents were abolished by kynurenic acid or CNQX. Similar L-DOPA-currents were seen in oocytes co-injected with AMPA receptors, GluRs1,2,3 and 4. In brain membrane preparations, L-DOPA inhibited specific binding of [3H]-AMPA with IC50 of 260 microM. This inhibition was not modified by 200 microM ascorbic acid, an antioxidant. L-DOPA did not inhibit binding of [3H]-ligands of MK-801, kainate, DCKA and CGP39653. DOPA ME and L-DOPA cyclohexyl ester, a novel, potent and competitive antagonist (2), inhibited specific binding of [3H]-MK-801 with respective IC50 of 1 and 0.68 mM, but elicited no effect on that of the other [3H]-ligands. With low affinities, L-DOPA acts on AMPA receptors, while competitive antagonists act on NMDA ion channel domain. L-DOPAergic agonist and antagonist may not interact on ionotropic glutamate receptors. DOPA ME-sensitive L-DOPA recognition sites (1) seem to differ from glutamate receptors.