Dopamine and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) plays an obligatory role in most of the actions of dopamine. In resting neostriatal slices, cyclin-dependent kinase 5 (Cdk5) phosphorylates DARPP-32 at Thr-75, thereby reducing the efficacy of dopaminergic signaling. We report here that dopamine, in slices, and acute cocaine, in whole animals, decreases the state of phosphorylation of striatal DARPP-32 at Thr-75 and thereby removes this inhibitory constraint. This effect of dopamine is achieved through dopamine D1 receptor-mediated activation of cAMP-dependent protein kinase (PKA). The activated PKA, by decreasing the state of phosphorylation of DARPP-32-Thr-75, deinhibits itself. Dopamine D2 receptor stimulation has the opposite effect. The ability of activated PKA to reduce the state of phosphorylation of DARPP-32-Thr-75 is apparently attributable to increased protein phosphatase-2A activity, with Cdk5 being unaffected. Together, these results indicate that via positive feedback mechanisms, Cdk5 signaling and PKA signaling are mutually antagonistic.D opamine and cAMP-regulated phosphoprotein of M r 32,000 (DARPP-32) is a cytosolic protein that is selectively enriched in neostriatal medium spiny neurons (1, 2). When DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) on a single threonine residue, Thr-34, it is converted into a potent inhibitor of protein phosphatase-1 (PP-1) (3). Dopamine and numerous other neurotransmitters have been shown to regulate the phosphorylation͞dephosphorylation of DARPP-32 at Thr-34 in neostriatum, thereby altering the activity of PP-1 and regulating the phosphorylation state and activity of many downstream physiological effectors, including various neurotransmitter receptors and voltage-gated ion channels (4). Mice lacking DARPP-32 exhibit profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication, demonstrating the importance of the DARPP-32͞PP-1 signaling cascade in mediating the actions of dopamine, agents that affect dopamine signaling, and other neurotransmitters that act on neostriatal neurons (4, 5).We have recently reported that DARPP-32 is phosphorylated by cyclin-dependent kinase 5 (Cdk5), both in vitro and in neostriatal neurons (6). In vitro, phospho-Thr-75 DARPP-32 inhibits PKA by a competitive mechanism. In vivo, reduction of phospho-Thr-75 DARPP-32 in neostriatal slices, either by the Cdk5 inhibitor roscovitine or by the use of genetically altered mice (p35 Ϫ/Ϫ mice), results in increased biochemical and physiological responses to dopamine (6). These results demonstrated that PKA activity in the neostriatum is regulated by the state of phosphorylation of DARPP-32 at Thr-75. Thus, DARPP-32 is a bifunctional signal transduction molecule that controls the activities of PP-1 and PKA through the phosphorylation of Thr-34 and Thr-75, respectively.Apart from the effect of Cdk5, nothing has been known about the signaling mechanisms involved in the regulation of ...

The actions of opioid receptor agonists on synaptic transmission in substantia gelatinosa (SG) neurones in adult (6‐ to 10‐week‐old) rat spinal cord slices were examined by use of the blind whole‐cell patch‐clamp technique. Both the μ‐receptor agonist DAMGO (1 μM) and the δ‐receptor agonist DPDPE (1 μM) reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) which were monosynaptically evoked by stimulating Aδ afferent fibres. Both also decreased the frequency of miniature EPSCs without affecting their amplitude. In contrast, the κ‐receptor agonist U‐69593 (1 μM) had little effect on the evoked and miniature EPSCs. The effects of DAMGO and DPDPE were not seen in the presence of the μ‐receptor antagonist CTAP (1 μM) and the δ‐receptor antagonist naltrindole (1 μM), respectively. Neither DAMGO nor DPDPE at 1 μM affected the responses of SG neurones to bath‐applied AMPA (10 μM). Evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by either the GABAA or the glycine receptor, were unaffected by the μ‐, δ‐ and κ‐receptor agonists. Similar results were also obtained in SG neurones in young adult (3‐ to 4‐week‐old) rat spinal cord slices. These results indicate that opioids suppress excitatory but not inhibitory synaptic transmission, possibly through the activation of μ‐ and δ‐ but not κ‐receptors in adult rat spinal cord SG neurones; these actions are presynaptic in origin. Such an action of opioids may be a possible mechanism for the antinociception produced by their intrathecal administration.

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP-32 (dopamine-and cAMP-regulated phosphoprotein of M r 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP-32 at Thr34 by cAMP-dependent protein kinase, and antagonized by phosphorylation of DARPP-32 at Thr75 by cyclin-dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP-32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca 2+ -dependent activation of the Ca 2+ -/calmodulin-dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca 2+ DARPP-32 (dopamine-and cAMP-regulated phosphoprotein of M r 32 kDa) is a cytosolic protein that is selectively enriched in medium spiny neurons in the neostriatum . When DARPP-32 is phosphorylated by cAMP-dependent protein kinase (PKA) on Thr34, it is converted into a potent inhibitor of protein phosphatase-1 (PP-1) (Hemmings et al. 1984b). DARPP-32 Thr34 phosphorylation leads to an increase in the state of phosphorylation of downstream PP-1 substrates, including various neurotransmitter receptors and voltage-gated ion channels . We have recently reported that DARPP-32 is phosphorylated at Thr75 by cyclin-dependent kinase 5 (Cdk5) in vitro and in neostriatal neurons (Bibb et al. 1999). DARPP-32 phosphorylated at Thr75 inhibits PKA activity and thereby reduces the efficacy of dopamine signaling. Thus, DARPP-32 is a bifunctional signal transduction molecule that controls activities of PP-1 and PKA through the phosphorylation of Thr34 and Thr75, respectively. Mice lacking DARPP-32 exhibit profound deficits in their molecular, electrophysiological and behavioral responses to dopamine, drugs of abuse and antipsychotic medication, demonstrating the importance of DARPP-32 in most of the actions of dopamine (Fienberg et al. 1998;Fienberg and Greengard 2000). Received September 11, 2002; revised manuscript received February 6, 2002; accepted February 7, 2002. Address correspondence and reprint requests to Dr Akinori Nishi, Department of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. E-mail: nishia@med.kurume-u.ac.jp Abbreviations used: AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionate; cAMP, cyclic AMP; Cdk5, cyclin-dependent kinase 5; DARPP-32, dopamine-and cAMP-regulated phosphoprotein of M r 32 kDa; PAGE, polyacrylamide gel electrophoresis; PKA, cAMPdependent protein kinase; PMSF, phenylmethylsulfonyl fluoride; PP-1, protein phosphatase-1; PP-2A, protein phosphatase-2A; SDS, sodium dodecyl sulfate.

Intracellular recordings were made to investigate the mechanism, site, and ionic basis of generation of the rapid depolarization induced by superfusion with ischemia-simulating medium in hippocampal CA1 pyramidal neurons of rat tissue slices. Superfusion with ischemia-simulating medium produced a rapid depolarization after approximately 6 min of exposure. When oxygen and glucose were reintroduced, the membrane potential did not repolarize but depolarized further, reaching 0 mV approximately 5 min after reintroduction. Simultaneous recordings of changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and membrane potential recorded from 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2- amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester (Fura-2/AM) loaded slices revealed a rapid increase in [Ca2+]i in all CA1 layers corresponding to the rapid depolarization of the soma membrane. The result suggests that the rapid depolarization is generated not only in the soma but also in the apical and basal dendrites. Application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-4-phosphonobutyric acid, and DL-2-amino-3-phosphonopropionic acid or bicuculline did not affect the amplitude and the maximal slope. Reduction in the concentration of extracellular Ca2+ or addition of CNQX or DL-2-amino-5-phosphonopentanoic acid delayed the onset of the rapid depolarization. The amplitude of the rapid depolarization recorded with Cs acetate electrodes in tetraethylammonium-containing medium had a linear relationship to the membrane potential between -50 and 20 mV. The reversal potential was shifted in the hyperpolarizing direction by a decrease in either [Na+]o or [Ca2+]o, whereas the reversal potential was shifted in the depolarizing direction by a decrease in [Cl-]o or using CsCl electrodes. An increase or decrease in [K+]o did not affect the reversal potential. These results indicate that the rapid depolarization is Na+, Ca2+, and Cl- dependent. The lack of effects of changes in [K+]o is probably due to the accumulation of interstitial K+ before generating the rapid depolarization. Prolonged application of ouabain (30 microM) caused an initial small hyperpolarization, a subsequent slow depolarization, and a rapid depolarization. In summary, the present study has demonstrated that the rapid depolarization is voltage-independent and is probably due to a nonselective increase in permeability to all participating ions, which may occur only in pathological conditions. The underlying conductance change is primarily the result of inhibition of Na,K-ATPase activity in the recorded neuron.

SUMMARY1. The effects of hypoxia on the rat hippocampal CAI neurones in tissue slices of the rat brain were studied in vitro by intracellular recording.2. In response to superfusion of a hypoxic medium equilibrated with 95 % N2-5 % C02, a majority of the neurones showed a hyperpolarization of5-15 mV in amplitude and 4-12 min in duration. The hyperpolarization was, in turn, followed by a slow depolarization which within 20 min of hypoxic exposure reached a plateau level of about 25 mV above the pre-hypoxic resting potential. Both the initial hyperpolarization and subsequent depolarization were associated with a reduction in membrane resistance. 3. The hyperpolarization reversed in polarity at a membrane potential of -83 mV. There was an almost linear relationship between amplitude of the hyperpolarization and membrane potential. The hyperpolarization was markedly enhanced in potassium-free media and was depressed in high-potassium solutions.4. The hyperpolarization was not significantly affected by low-chloride or lowsodium medium or by solution containing tetraethylammonium (10 mM), 4-aminopyridine (1-5 mM) or caesium (3 mM). Moreover, intracellular injection of ethyleneglycol-bis-(fi-aminoethylether)N,N-tetraacetic acid (EGTA) did not alter the hyperpolarization. On the other hand, barium (0 5 mM)-containing medium reduced the amplitude of the hyperpolarization by 20-40 %.5. Superfusion of ouabain (5-7 puM)-containing medium in normoxic conditions produced hyperpolarizing and depolarizing responses similar to those elicited by hypoxic exposure. The slow depolarization was also mimicked by elevation of the extracellular potassium concentration to 10-20 mm.6. Evoked i.p.s.p.s were abolished within 4 min of hypoxic exposure while evoked e.p.s.p.s were maintained for about 20 min of hypoxic superfusion. Soma spikes of the neurones elicited by a depolarizing pulse were also well preserved. Their threshold was, however, raised, concomitant with a decrease in the peak amplitude.7. When the slice was reoxygenated after 20-40 min of hypoxic exposure, the neurones immediately began to repolarize and showed a transient hyperpolarization of 5-10 mV in amplitude and 1-2 min in duration. The membrane potential, input t To whom all correspondence and reprint requests should be sent.5-2 N. FUJIWARA AND OTHERS resistance and action potential returned to the pre-hypoxic levels after 15-20 min of reoxygenation. The amplitude of the reoxygenation-induced hyperpolarization was not significantly changed when the membrane was hyperpolarized or depolarized. The hyperpolarization was eliminated by potassium-free medium or solution containing ouabain (1 ,tM).8. In a minority of the neurones the slow depolarization was suddenly followed by a rapid depolarization, after which the neurones showed no functional recovery. Such an abrupt and irreversible depolarization appeared when the slow depolarization reached membrane potentials of -30 to -40 mV.9. The results suggest that hypoxia-induced hyperpolarization is due to an increase in voltag...