Neural development of methyl-CpG-binding protein 2 null embryonic stem cells: a system for studying Rett syndrome.

Mutations in methyl-CpG-binding protein 2 (MeCP2) gene cause the neurodevelopmental disorder Rett syndrome (RTT). Here, we describe a new experimental system that efficiently elucidates the role of MeCP2 in neural development. MeCP2-null and control ES cells were generated by adenoviral conditional targeting and examined for maintenance of the undifferentiated ES cell state, neurogenesis, and gliogenesis during in vitro differentiation. In addition, dopamine release and electrophysiological features of neurons differentiated from these ES cells were examined. Loss of MeCP2 did not affect undifferentiated ES cell colony morphology and growth, or the timing or efficiency of neural stem cell differentiation into Nestin-, TuJ- or TH-positive neurons. In contrast, gliogenesis was drastically accelerated by MeCP2 deficiency. Dopamine production and release in response to a depolarizing stimulus in MeCP2-null ES-derived dopaminergic neurons was intact. However, MeCP2-null differentiated neurons showed significantly smaller voltage-dependent Na(+) currents and A-type K(+) currents, suggesting incomplete maturation. Thus, MeCP2 is not essential for maintenance of the undifferentiated ES cell state, neurogenesis, or dopaminergic function; rather, it is principally involved in inhibiting gliogenesis. Altered neuronal maturity may indirectly result from abnormal glial development and may underlie the pathogenesis of RTT. These data contribute to a better understanding of the developmental roles of MeCP2 and the pathogenesis of RTT.

Thiamylal antagonizes the inhibitory effects of dorsal column stimulation on dorsal horn activities in humans.

In humans, peripheral somatosensory information converges upon dorsal horn neurons in the spinal cord, which can be recorded from the dorsal epidural space as spinal cord potentials (SCPs) following segmental dorsal root stimulation (SS) employing epidural catheter electrodes. Antidromic action potentials and descending inhibition from the dorsolateral funiculus may contribute to SCPs following dorsal column stimulation (DCS). Effects of thiamylal (2.5-7.5 mg/kg, i.v.) on SCPs evoked by independent DCS or SS were compared with those evoked by simultaneous DCS and SS (DCS/SS). DCS- and SS-evoked SCPs recorded from the lumbar enlargement consisted of a sharp negative (N) followed by a slow positive (P) potential. Thiamylal induced dose-dependent increases in amplitude and duration of both N and P potentials evoked by DCS and SS, whether the responses were summed or evoked simultaneously. In awake subjects, N and P potentials produced by simultaneous DCS/SS were significantly smaller than the sum of independent responses. Thiamylal anesthesia antagonized this inhibition; responses to simultaneous DCS/SS were larger than the sum of independent responses. These results suggest that in wakefulness DCS inhibits dorsal horn neuron activity in the lumbar spinal cord, while thiamylal antagonizes DCS-induced inhibition in dose-dependent fashion.

Regulation of DARPP-32 phosphorylation by three distinct dopamine D1-like receptor signaling pathways in the neostriatum.

Dopamine D(1)-like receptors play a key role in dopaminergic signaling. In addition to G(s/olf)/adenylyl cyclase (AC)-coupled D(1) receptors, the presence of D(1)-like receptors coupled to G(q)/phospholipase C (PLC) has been proposed. Benzazepine D(1) receptor agonists are known to differentially activate G(s/olf)/AC and G(q)/PLC signaling. By utilizing SKF83959 and SKF83822, we investigated the D(1)-like receptor signaling cascades, which regulate DARPP-32 phosphorylation at Thr34 (the PKA-site) in mouse neostriatal slices. Treatment with SKF83959 or SKF83822 increased DARPP-32 phosphorylation. The SKF83959- and SKF83822-induced increase in DARPP-32 phosphorylation was largely, but partially, antagonized by a D(1) receptor antagonist, SCH23390, and the residual SCH23390-insensitive increase was abolished by an adenosine A(2A) receptor antagonist. In addition, the SKF83959-induced, SCH23390-sensitive increase in DARPP-32 phosphorylation was enhanced by a PLC inhibitor. Analysis in slices from D(1)R/D(2)R-DARPP-32 mice revealed that both D(1) receptor agonists regulate DARPP-32 phosphorylation in striatonigral, but not in striatopallidal, neurons. Thus, dopamine D(1)-like receptors are coupled to three signaling cascades in striatonigral neurons: (i) SCH23390-sensitive G(s/olf)/AC/PKA, (ii) adenosine A(2A) receptor-dependent G(s/olf)/AC/PKA, and (iii) G(q)/PLC signaling. Interestingly, G(q)/PLC signaling interacts with SCH23390-sensitive G(s/olf)/AC/PKA signaling, resulting in its inhibition. Three signaling cascades activated by D(1)-like receptors likely play a distinct role in dopaminergic regulation of psychomotor functions.

Activation of phospholipase C pathways by a synthetic chondroitin sulfate-E tetrasaccharide promotes neurite outgrowth of dopaminergic neurons.

In dopaminergic neurons, chondroitin sulfate (CS) proteoglycans play important roles in neuronal development and regeneration. However, due to the complexity and heterogeneity of CS, the precise structure of CS with biological activity and the molecular mechanisms underlying its influence on dopaminergic neurons are poorly understood. In this study, we investigated the ability of synthetic CS oligosaccharides and natural polysaccharides to promote the neurite outgrowth of mesencephalic dopaminergic neurons and the signaling pathways activated by CS. CS-E polysaccharide, but not CS-A, -C or -D polysaccharide, facilitated the neurite outgrowth of dopaminergic neurons at CS concentrations within the physiological range. The stimulatory effect of CS-E polysaccharide on neurite outgrowth was completely abolished by its digestion into disaccharide units with chondroitinase ABC. Similarly to CS-E polysaccharide, a synthetic tetrasaccharide displaying only the CS-E sulfation motif stimulated the neurite outgrowth of dopaminergic neurons, whereas a CS-E disaccharide or unsulfated tetrasaccharide had no effect. Analysis of the molecular mechanisms revealed that the action of the CS-E tetrasaccharide was mediated through midkine-pleiotrophin/protein tyrosine phosphatase zeta and brain-derived neurotrophic factor/tyrosine kinase B receptor pathways, followed by activation of the two intracellular phospholipase C (PLC) signaling cascades: PLC/protein kinase C and PLC/inositol 1,4,5-triphosphate/inositol 1,4,5-triphosphate receptor signaling leading to intracellular Ca(2+) concentration-dependent activation of Ca(2+)/calmodulin-dependent kinase II and calcineurin. These results indicate that a specific sulfation motif, in particular the CS-E tetrasaccharide unit, represents a key structural determinant for activation of midkine, pleiotrophin and brain-derived neurotrophic factor-mediated signaling, and is required for the neuritogenic activity of CS in dopaminergic neurons.

Effects of various K+ channel blockers on spontaneous glycine release at rat spinal neurons.

Molecular biology approaches have identified more than 70 different K+ channel genes that assemble to form diverse functional classes of K+ channels. Although functional K+ channels are present within presynaptic nerve endings, direct studies of their precise identity and function have been generally limited to large, specialized presynaptic terminals such as basket cell terminals and Calyx of Held. In the present study, therefore, we investigated the functional K+ channel subtypes on the small glycinergic nerve endings (< 1 microm diameter) projecting to spinal sacral dorsal commissural nucleus (SDCN) neurons. In the presence of TTX, whole-cell patch recording of mIPSCs was made from mechanically dispersed SDCN neurons in which functional nerve endings remain attached. Glycinergic responses were isolated by blocking glutamatergic and GABAergic inputs with CNQX, AP5 and bicuculline. The K+ channel blockers, 4-AP, TEA, delta-dendrotoxin, margatoxin, iberiotoxin, charybdotoxin and apamin, significantly increased 'spontaneous' mIPSC frequency without affecting mIPSC amplitude. The results suggest the existence of the following K+ channel subtypes on glycinergic nerve endings that are involved in regulating 'spontaneous' glycine release (mIPSCs): the Shaker-related K+ channels Kv1.1, Kv1.2, Kv1.3, Kv1.6 and Kv1.7 and the intracellular Ca2+ -sensitive K+ channels BKCa, IKCa and SKCa. Ca2+ channel blockers by themselves, including L-type (nifedipine), P/Q-type (omega-agatoxin IVA, AgTX) and N-type (omega-conotoxin GVIA, CgTX), did not alter the 'spontaneous' mIPSC frequency or amplitude, but inhibited the increase of the mIPSC frequency evoked by 4-AP, indicating the participation of L-, P/Q- and N-type Ca2+ channels regulating 'spontaneous' glycine release from the nerve terminals.

Regulation of spinophilin Ser94 phosphorylation in neostriatal neurons involves both DARPP-32-dependent and independent pathways.

Spinophilin is a protein phosphatase-1 (PP-1)- and actin-binding protein that is enriched in dendritic spines. Phosphorylation of the actin-binding domain of rat spinophilin at one or more sites by protein kinase A (PKA) inhibits actin binding. Here, we investigated the regulation of mouse spinophilin that contains only a single PKA-site (Ser94) within its actin-binding domain. In vitro phosphorylation of Ser94 resulted in the dissociation of spinophilin from actin filaments. In mouse neostriatal slices, phospho-Ser94 (p-Ser94) was dephosphorylated mainly by PP-1 and also by PP-2A. Activation of dopamine D1 receptors in striatonigral medium spiny neurons, and of adenosine A 2A receptors in striatopallidal medium spiny neurons increased, whereas activation of dopamine D2 receptors in striatopallidal neurons decreased, spinophilin Ser94 phosphorylation. In neostriatal slices from DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) knockout mice, the effects of D1, D2 and A 2A receptors were largely attenuated. Activation of NMDA receptors decreased Ser94 phosphorylation in a PP-2A-dependent, but DARPP-32-independent, manner. These results suggest that PKA-dependent phosphorylation of spinophilin at Ser94 in both striatonigral and striatopallidal neurons requires synergistic contributions from the PKA and DARPP-32/PP-1 pathways. In addition, PP-2A plays a role in Ser94 dephosphorylation in response to activation of both D2 and NMDA receptors.

Nicotine regulates DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) phosphorylation at multiple sites in neostriatal neurons.

Nicotinic acetylcholine receptors (nAChRs) regulate dopaminergic signaling in the striatum by modulating the release of neurotransmitters. We have recently reported that nicotine stimulates the release of dopamine via alpha4beta2(*) nAChRs and/or alpha7 nAChRs, leading to the regulation of DARPP-32 at Thr34, the site involved in regulation of protein phosphatase-1 (PP-1). In this study, we investigated the regulation of DARPP-32 phosphorylation at its other sites, Thr75 [cyclin-dependent kinase-5 (Cdk5) site], Ser97 (CK2 site), and Ser130 (CK1 site), that serve to modulate Thr34 phosphorylation and dephosphorylation. In neostriatal slices, nicotine (100 microM) increased phosphorylation of DARPP-32 at Ser97 and Ser130 at an early time point (30 s) and decreased phosphorylation of DARPP-32 at Thr75 at a late time point (3 min). The increase in Ser97 and Ser130 phosphorylation was mediated through the release of dopamine via activation of alpha4beta2(*) nAChRs and alpha7 nAChRs and the subsequent activation of dopamine D1 and D2 receptors. The decrease in Thr75 phosphorylation was mediated through the release of dopamine via activation of alpha4beta2(*) nAChRs and the subsequent activation of dopamine D1 receptors. These various actions of nicotine on modulatory sites of phosphorylation would be predicted to result in a synergistic increase in the state of phosphorylation of DARPP-32 at Thr34 and thus would contribute to increased dopamine D1 receptor/DARPP-32 Thr34/PP-1 signaling.

Glutamate regulation of DARPP-32 phosphorylation in neostriatal neurons involves activation of multiple signaling cascades.

Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in medium spiny neurons in the neostriatum in the integration of various neurotransmitter signaling pathways. In its Thr-34-phosphorylated form, it acts as a potent protein phosphatase-1 inhibitor, and, in its Thr-75-phosphorylated form, it acts as a cAMP-dependent kinase inhibitor. Here, we investigated glutamate-dependent signaling cascades in mouse neostriatal slices by analyzing the phosphorylation of DARPP-32 at Thr-34 and Thr-75. Treatment with glutamate (5 mM) caused a complex change in DARPP-32 Thr-34 phosphorylation. An initial rapid increase in Thr-34 phosphorylation was NMDA/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/metabotropic glutamate-5 receptor-dependent and was mediated through activation of a neuronal nitric oxide synthase/nitric oxide/cGMP/cGMP-dependent kinase signaling cascade. A subsequent decrease in phosphorylation was attributable to activation of an NMDA/AMPA receptor/Ca2+/protein phosphatase-2B signaling cascade. This decrease was followed by rephosphorylation via a pathway involving metabotropic glutamate-5 receptor/phospholipase C and extracellular receptor kinase signaling cascade. Treatment with glutamate initially decreased Thr-75 phosphorylation through activation of NMDA/AMPA receptor/Ca2+/protein phosphatase-2A signaling. Thereafter, glutamate slowly increased Thr-75 phosphorylation through activation of metabotropic glutamate-1 receptor/phospholipase C signaling. Our analysis of DARPP-32 phosphorylation in the neostriatum revealed that glutamate activates at least five different signaling cascades with different time dependencies, resulting in complex regulation of protein kinase and protein phosphatase activities.

Protective actions of various local anesthetics against the membrane dysfunction produced by in vitro ischemia in rat hippocampal CA1 neurons.

Local anesthetics block not only the Na(+) but also the K(+) and Ca(2+) channels in the mammalian neurons. It is well known that lidocaine has neuroprotective actions against the ischemic insult of neurons in the central nervous system. In order to elucidate how other local anesthetics as well as lidocaine show the neuroprotective effects against in vitro ischemic insult, intracellular recordings were made from CA1 pyramidal neurons in rat hippocampal slices. Superfusion with the medium deprived of oxygen and glucose (in vitro ischemia) produced a rapid depolarization after 5 min of exposure. When the normal medium was immediately reintroduced after the rapid depolarization, the membrane depolarized further (persistent depolarization), the neurons showed no functional recovery. Pretreatment with tetracaine, bupivacaine, procaine, lidocaine, mepivacaine, or dibucaine (10 or 300 microM) prolonged the latency of the rapid depolarization, and most of the drugs partially restored the membrane potential toward the pre-exposure level after the reintroduction. Judging from the neuroprotective actions such as the prolongation and the potential recovery by these drugs, lidocaine, bupivacaine, and dibucaine are candidates for the therapeutic use against the ischemic insult. Suppression of the regenerative Na(+) conductance is somehow involved in the neuroprotective actions of local anesthetics.

Differential regulation of dopamine D1 and D2 signaling by nicotine in neostriatal neurons.

Nicotine, acting on nicotinic acetylcholine receptors (nAChRs) expressed at pre-synaptic dopaminergic terminals, has been shown to stimulate the release of dopamine in the neostriatum. However, the molecular consequences of pre-synaptic nAChR activation in post-synaptic neostriatal neurons are not clearly understood. Here, we investigated the effect of nAChR activation on dopaminergic signaling in medium spiny neurons by measuring phosphorylated DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) at Thr34 (the PKA-site) in mouse neostriatal slices. Nicotine produced dose-dependent responses, with a low concentration (1 microm) causing a sustained decrease in DARPP-32 Thr34 phosphorylation and a high concentration (100 microm) causing a transient increase in DARPP-32 Thr34 phosphorylation. Depending on the concentration of nicotine, either dopamine D2 or D1 receptor signaling was predominantly activated. Nicotine at a low concentration (1 microm) activated dopamine D2 receptor signaling in striatopallidal/indirect pathway neurons, likely by activating alpha4beta2* nAChRs at dopaminergic terminals. Nicotine at a high concentration (100 microm) activated dopamine D1 receptor signaling in striatonigral/direct pathway neurons, likely by activating (i) alpha4beta2* nAChRs at dopaminergic terminals and (ii) alpha7 nAChRs at glutamatergic terminals, which, by stimulating the release of glutamate, activated NMDA/AMPA receptors at dopaminergic terminals. The differential effects of low and high nicotine concentrations on D2- and D1-dependent signaling pathways in striatal neurons may contribute to dose-dependent actions of this drug of abuse.

Distinct profiles of refilling of inhibitory neurotransmitters into presynaptic terminals projecting to spinal neurones in immature rats.

Corelease of glycine and GABA from the single synaptic terminal (synaptic bouton) is well accepted in immature rat spinal cord and brainstem. However, it raises the question of how glycine and GABA are accumulated in the same synaptic vesicles and coreleased. To address this issue, spontaneous miniature inhibitory postsynaptic currents (mIPSCs) and focally evoked IPSCs (eIPSCs) mediated via a single synapse were recorded from synaptic bouton preparations of the rat immature sacral dorsal commissural nucleus (SDCN) neurones by whole-cell patch recording. Focal stimulation of a single synaptic bouton revealed that three different quantal releases occur from a single synaptic bouton: i.e. pure glycine, pure GABA, and mixed. Prolonged treatment with bafilomycin A1, a vacuolar-type H+/ATPase inhibitor, to the SDCN neurone greatly suppressed frequency and amplitude of the mIPSCs. During washing out of bafilomycin A1, complete recovery in the amplitude of glycinergic mIPSCs was observed, while that of GABAergic and mixed mIPSCs was incomplete. These observations indicate that three types of vesicles coexist in single synaptic terminals, and that refilling of glycine into the synaptic vesicle predominantes over GABA after pretreatment with bafilomycin A1 in immature rats. This could be explained by the decrease in the cytosolic concentration of GABA, or by the presence of subtypes of vesicular inhibitory amino acid transporter in the synaptic vesicle membrane.

Regulation of DARPP-32 Thr75 phosphorylation by neurotensin in neostriatal neurons: involvement of glutamate signalling.

Neurotensin is a neuropeptide involved in dopaminergic signalling. We have recently reported that neurotensin stimulates the phosphorylation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) at Thr34 (PKA-site) by activating dopamine D1-type receptors in neostriatal neurons. DARPP-32 is also phosphorylated by cyclin-dependent kinase 5 on Thr75, and the phosphorylated form of DARPP-32 at Thr75 inhibits protein kinase (PKA) activity. In this study, we examined the effect of neurotensin on DARPP-32 Thr75 phosphorylation using mouse neostriatal slices. Neurotensin decreased the level of phospho-Thr75 DARPP-32 at 2 min of incubation, maximally to about 50% of control at a concentration of 1 micro m. Pretreatment with a combined neurotensin receptor type 1 (NTR1)/type 2 (NTR2) antagonist, SR142948, reduced the basal level of phospho-Thr75 DARPP-32 and abolished the ability of neurotensin to decrease DARPP-32 Thr75 phosphorylation. However, neither an NTR1 antagonist, SR48692, an NTR2 antagonist, levocabastine, nor the two combined affected the basal level and the neurotensin-mediated decrease in DARPP-32 Thr75 phosphorylation. The effect of neurotensin was abolished by tetrodotoxin (TTX) or MK801 plus CNQX, but not by SCH23390 or raclopride. These results indicate that neurotensin stimulates the release of glutamate by activating a hypothesized unidentified neurotensin receptor, resulting in the dephosphorylation of DARPP-32 at Thr75 by activating NMDA and AMPA receptors expressed at medium spiny neurons. Thus, neurotensin, by removing the inhibition of PKA by phospho-Thr75 DARPP-32, potentiates its signalling via the dopamine/D1 receptor/PKA/phospho-Thr34 DARPP-32/PP-1 cascade.

Electrical and pharmacological properties of the slow depolarization induced by Schaffer collateral stimulation in astrocytes of the rat hippocampal CA1 region.

Intracellular recordings were made from astrocytes of the hippocampal CA1 region in rat slice preparations. A single stimulus at the Schaffer collaterals with high intensities (>/=10 V), but not low intensities (<10 V), induced a slow depolarization similar to the evoked postsynaptic potentials observed in hippocampal CA1 neurons. The slow depolarization was almost abolished in the presence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM) and N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonopentanoic acid (100 microM). Tetrodotoxin (0.5 microM) or Co(2+) (2 mM) also abolished this slow depolarization. The slow depolarization reversed its polarity at -8 mV in the presence of tetraethylammonium (20 mM). These results suggest that the synaptically released glutamate (Glu) induces depolarization, which is mainly mediated by ionotropic Glu receptors, in astrocytes in the hippocampal CA1 region.

Metabotropic mGlu5 receptors regulate adenosine A2A receptor signaling.

Dopamine, by activating dopamine D1-type receptors, and adenosine, by activating adenosine A(2A) receptors, stimulate phosphorylation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000) at Thr-34. In this study, we investigated the effect of metabotropic glutamate (mGlu) receptors on DARPP-32 phosphorylation at Thr-34 in neostriatal slices. A broad-spectrum mGlu receptor agonist, trans-ACPD, and a group I mGlu receptor agonist, DHPG, stimulated DARPP-32 phosphorylation at Thr-34. Studies with mGlu receptor antagonists revealed that the effects of trans-ACPD and DHPG were mediated through activation of mGlu5 receptors. The action of mGlu5 receptors required activation of adenosine A(2A) receptors by endogenous adenosine. Conversely, the action of adenosine A(2A) receptors required activation of mGlu5 receptors by endogenous glutamate. Coactivation of mGlu5 and adenosine A(2A) receptors by exogenous agonists synergistically increased DARPP-32 phosphorylation. mGlu5 receptors did not require activation of dopamine D1-type receptors by endogenous dopamine, nor did dopamine D1-type receptors require activation of mGlu5 receptors by endogenous glutamate. DHPG potentiated the effect of forskolin, but not that of 8-bromo-cAMP, and stimulated DARPP-32 phosphorylation in the presence of the phosphodiesterase inhibitor IBMX, suggesting that mGlu5 receptors stimulate the rate of cAMP formation coupled to adenosine A(2A) receptors. The action of mGlu5 receptors was attenuated by inhibitors of extracellular signal-regulated kinase, but not by inhibitors of phospholipase C, p38, casein kinase 1, or Cdk5. The results demonstrate that mGlu5 receptors potentiate adenosine A(2A)DARPP-32 signaling by stimulating the adenosine A(2A) receptor-mediated formation of cAMP in an extracellular signal-regulated kinase-dependent manner.

Effect of methylphenidate on dopamine/DARPP signalling in adult, but not young, mice.

Methylphenidate (MPH), a dopamine uptake inhibitor, is the most commonly prescribed drug for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children. We examined the effect of MPH on dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) phosphorylation at Thr34 (PKA-site) and Thr75 (Cdk5-site) using neostriatal slices from young (14-15- and 21-22-day-old) and adult (6-8-week-old) mice. MPH increased DARPP-32 Thr34 phosphorylation and decreased Thr75 phosphorylation in slices from adult mice. The effect of MPH was blocked by a dopamine D1 antagonist, SCH23390. In slices from young mice, MPH did not affect DARPP-32 phosphorylation. As with MPH, cocaine stimulated DARPP-32 Thr34 phosphorylation in slices from adult, but not from young mice. In contrast, a dopamine D1 agonist, SKF81297, regulated DARPP-32 phosphorylation comparably in slices from young and adult mice, as did methamphetamine, a dopamine releaser. The results suggest that dopamine synthesis and the dopamine transporter are functional at dopaminergic terminals in young mice. In contrast, the lack of effect of MPH in young mice is likely attributable to immature development of the machinery that regulates vesicular dopamine release.

Postnatal changes in the overall postsynaptic currents evoked in CA1 pyramidal neurons of the rat hippocampus.

Evoked fast postsynaptic currents (fPSCs) during the postnatal development of rats (postnatal day 6-70, P6-P70) were systematically examined in hippocampal CA1 pyramidal neurons using whole-cell recordings with biocytin-filled electrodes. Focal stimulation of the stratum radiatum in the CA1 region elicited fPSCs in 80% of the neurons P6-7, 90% of P9-10, and 100% of > or =P11. In neurons P6-7, the fPSCs were exclusively inward and had multiple (on average 5.6) peaks. The fPSCs increased in amplitude with the growth of dendritic arborization, but decreased in the number of peaks. A distinct outward fPSC following the inward fPSC emerged in neurons > or =P11 and was abolished by bicuculline (50 microM). Bicuculline increased the amplitude and duration of the initial inward fPSC (fEPSC) in all age groups and characteristically recruited the polysynaptic second component of fEPSCs in neurons P11-P21. No spontaneous periodic inward current was detected in any age group after blocking GABAA receptors. The coapplication of DL-2-amino-5-phosphonopentanoic acid (AP5, 100 microM) with bicuculline did not eliminate the polysynaptic second component, but the second component was only elicited in slices in which the CA3 region was kept intact. Moreover, the bicuculline- and AP5-resistant second component was due to the burst activity of CA3 pyramidal neurons, which were excited through excitatory recurrents of the Schaffer collaterals. Plausible physiological functions of the generation of the second component in vivo were discussed.

Neurotensin regulates DARPP-32 thr34 phosphorylation in neostriatal neurons by activation of dopamine D1-type receptors.

Neurotensin modulates dopaminergic transmission in the nigrostriatal system. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cAMP-dependent protein kinase, resulting in its conversion into a potent inhibitor of protein phosphatase-1 (PP 1). Here, we examined the effect of neurotensin on DARPP-32 Thr34 phosphorylation using mouse neostriatal slices. Neurotensin stimulated DARPP-32 Thr34 phosphorylation by 4-7-fold with a K(0.5) of approximately 50 nM. The effect of neurotensin was antagonized by a combined neurotensin receptor type-1 (NTR1)/type-2 (NTR2) antagonist, SR142948. It was not antagonized by a NTR1 antagonist, SR48692 or by a NTR2 antagonist, levocabastine; neither was it antagonized by the two combined. Pretreatment with TTX or cobalt abolished the effect of neurotensin. The effect of neurotensin was antagonized by a dopamine D1 antagonist, SCH23390, and by ionotropic glutamate receptor antagonists, MK801 and CNQX. These results indicate that neurotensin stimulates the release of dopamine from nigrostriatal presynaptic terminals in an NMDA receptor- and AMPA receptor-dependent manner, leading to the increase in DARPP-32 Thr34 phosphorylation. Neurotensin stimulated the phosphorylation of Ser845 of the AMPA receptor GluR1 subunit in wild-type mice but not in DARPP-32 knockout mice. Thus, neurotensin, by stimulating the release of dopamine, activates the dopamine D1-receptor/cAMP/PKA/DARPP-32/PP 1 cascade.

Regulation of DARPP-32 dephosphorylation at PKA- and Cdk5-sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase-2A.

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+) -dependent activation of dephosphorylation by protein phosphatase-2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP-32 by different protein phosphatases.

Bupivacaine, but not tetracaine, protects against the in vitro ischemic insult of rat hippocampal CA1 neurons.

Neuroprotective actions of local anesthetics, bupivacaine and tetracaine, against the irreversible membrane dysfunction induced by in vitro ischemia were investigated. Intracellular recordings were made from hippocampal CA1 neurons in rat brain slice preparations. Oxygen and glucose deprivation (in vitro ischemia) produced a rapid depolarization after approximately 5 min of exposure. When oxygen and glucose were reintroduced, the membrane depolarized further and reached at 0 mV: the membrane showed no functional recovery (irreversible membrane dysfunction). Pretreatment with tetracaine or bupivacaine significantly prolonged the latency of rapid depolarization. Bupivacaine, but not tetracaine, restored the membrane potential after the reintroduction of oxygen and glucose. Tetracaine and bupivacaine depressed both field postsynaptic potentials and presynaptic volleys. The drugs also reduced the dV/dt of Ca(2+)-dependent spikes and the rapid rise of [Ca(2+)](i) induced by in vitro ischemia. Compared with tetracaine, bupivacaine markedly suppressed the resting K(+) conductance and the ATP-sensitive and Ca(2+)-dependent K(+) conductances. Moreover, in the presence of tetraethylammonium (TEA), a majority of CA1 neurons impaled with Cs acetate-filled electrodes showed complete or partial recovery of the membrane potential after reintroducing oxygen and glucose. These results suggest that the neuroprotective action of bupivacaine is mainly due to the suppression of the K(+) conductances.