Serotonin 5‐HT1B receptor‐mediated calcium influx‐independent presynaptic inhibition of GABA release onto rat basal forebrain cholinergic neurons

Modulatory roles of serotonin (5‐HT) in GABAergic transmission onto basal forebrain cholinergic neurons were investigated, using whole‐cell patch‐clamp technique in the rat brain slices. GABA_A receptor‐mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal stimulation. Bath application of 5‐HT (0.1–300 μm ) reversibly suppressed the amplitude of evoked IPSCs in a concentration‐dependent manner. Application of a 5‐HT_1B receptor agonist, CP93129, also suppressed the evoked IPSCs, whereas a 5‐HT_1A receptor agonist, 8‐OH‐DPAT had little effect on the evoked IPSCs amplitude. In the presence of NAS‐181, a 5‐HT_1B receptor antagonist, 5‐HT‐induced suppression of evoked IPSCs was antagonised, whereas NAN‐190, a 5‐HT_1A receptor antagonist did not antagonise the 5‐HT‐induced suppression of evoked IPSCs. Bath application of 5‐HT reduced the frequency of spontaneous miniature IPSCs without changing their amplitude distribution. The effect of 5‐HT on miniature IPSCs remained unchanged when extracellular Ca²⁺ was replaced by Mg²⁺. The paired‐pulse ratio was increased by CP93129. In the presence of ω‐CgTX, the N‐type Ca²⁺ channel blocker, ω‐Aga‐TK, the P/Q‐type Ca²⁺ channel blocker, or SNX‐482, the R‐type Ca²⁺ channel blocker, 5‐HT could still inhibit the evoked IPSCs. 4‐AP, a K⁺ channel blocker, enhanced the evoked IPSCs, and CP93129 had no longer inhibitory effect in the presence of 4‐AP. CP93129 increased the number of action potentials elicited by depolarising current pulses. These results suggest that activation of presynaptic 5‐HT_1B receptors on the terminals of GABAergic afferents to basal forebrain cholinergic neurons inhibits GABA release in Ca²⁺ influx‐independent manner by modulation of K⁺ channels, leading to enhancement of neuronal activities.     

Blocking L-type calcium channels enhances long-term depression induced by low-frequency stimulation at hippocampal CA1 synapses

Specific contributions of voltage-dependent calcium channels (VDCCs) to induction of long-term depression (LTD) have not been thoroughly elucidated. The present study examined roles of T- and L-type VDCCs in N-methyl-D-aspartate (NMDA) receptor-dependent LTD induced at several different levels of synaptic activation (0.5- to 10-Hz presynaptic stimulations) at Schaffer collateral-CA1 synapses in rat hippocampal slices. Blockade of T-type VDCCs with nickel ions failed to change LTD magnitude at all levels of stimulation. However, blockade of L-type VDCCs reduced LTD in response to stimulation at 1 and 2 Hz and, conversely, enhanced LTD at a lower frequency (0.5 Hz). The enhancement of 0.5-Hz LTD under L-type VDCC blockade was shown pharmacologically to depend on NMDA receptors (NMDARs) and intracellular Ca(2+) release. Calcium imaging revealed that contribution of L-type VDCC-mediated calcium influx to the total calcium increase was greater during 0.5-Hz stimulation than during 1.0-Hz stimulation. This finding, combined with the reported suppression of NMDARs mediated by L-type VDCCs, may be relevant to the present enhancement of 0.5-Hz LTD due to L-type VDCC blockade.     

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.     

Occurrence of dentate granule cell LTP without proximal dendritic Ca2+ increase

We investigated activity-dependent calcium increases in proximal dendrites of dentate granule cells in the rat hippocampus, and its relationship with induction of LTP at perforant path synapses (PP-synapses). LTP was induced at PP-synapses by high-frequency stimulation (HFS; 100 Hz for 0.4 s), and the same HFS evoked a dendritic calcium increase in the proximal dendrite. However, bath-application of the L-type voltage-dependent calcium channel (VDCC) blocker nimodipine noticeably reduced this calcium increase without abolishing induction of LTP. This calcium increase mediated by high-threshold VDCCs is likely to be evoked by action potentials. LTP induction at PP-synapses is hence suggested to be independent of action potential-induced calcium increases in the proximal dendrite.     

Non‐classical mechanism of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor channel block by fluoxetine

Antidepressants have many targets in the central nervous system. A growing body of data demonstrates the influence of antidepressants on glutamatergic neurotransmission. In the present work, we studied the inhibition of native Ca²⁺‐permeable and Ca²⁺‐impermeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors in rat brain neurons by fluoxetine. The Ca²⁺‐impermeable AMPA receptors in CA1 hippocampal pyramidal neurons were weakly affected. The IC₅₀ value for the inhibition of Ca²⁺‐permeable AMPA receptors in giant striatal interneurons was 43 ± 7 μm . The inhibition of Ca²⁺‐permeable AMPA receptors was voltage dependent, suggesting deep binding in the pore. However, the use dependence of fluoxetine action differed markedly from that of classical AMPA receptor open‐channel blockers. Moreover, fluoxetine did not compete with other channel blockers. In contrast to fluoxetine, its membrane‐impermeant quaternary analog demonstrated all of the features of channel inhibition typical for open‐channel blockers. It is suggested that fluoxetine reaches the binding site through a hydrophobic access pathway. Such a mechanism of block is described for ligands of sodium and calcium channels, but was never found in AMPA receptors. Molecular modeling suggests binding of fluoxetine in the subunit interface; analogous binding was proposed for local anesthetics in closed sodium channels and for benzothiazepines in calcium channels.     

Developmental increase in D1‐like dopamine receptor‐mediated inhibition of glutamatergic transmission through P/Q‐type channel regulation in the basal forebrain of rats

Whole‐cell patch‐clamp recordings of non‐N‐methyl‐d ‐aspartate glutamatergic excitatory postsynaptic currents (EPSCs) were carried out from cholinergic neurons in slices of basal forebrain (BF) of developing rats aged 21–42 postnatal days to elucidate postnatal developmental change in Ca²⁺ channel subtypes involved in the transmission as well as that in dopamine D₁‐like receptor‐mediated presynaptic inhibition. The amplitude of EPSCs was inhibited by bath application of ω‐conotoxin GVIA (ω‐CgTX; 3 μm ) or ω‐agatoxin‐TK (ω‐Aga‐TK; 200 nm ) throughout the age range examined, suggesting that multiple types of Ca²⁺ channel are involved in the transmission. The EPSC fraction reduced by ω‐CgTX decreased with age, whereas that reduced by ω‐Aga‐TK increased. Inhibition of the EPSCs by a D₁‐like receptor agonist, SKF 81297 (SKF; 30 μm ) increased with age in parallel with the increase in ω‐Aga‐TK‐induced inhibition. An activator of the adenylyl cyclase (AC) pathway, forskolin (FK; 10 μm ) inhibited the EPSCs, and FK‐induced inhibition also increased with age in parallel with the increase in SKF‐induced inhibition. Throughout the age range examined, SKF showed no further inhibitory effect on the EPSCs after ω‐Aga‐TK‐ or FK‐induced effect had reached steady‐state. These findings suggest that D₁‐like receptor‐mediated presynaptic inhibition of glutamate release onto cholinergic BF neurons increases with age, and that the change is coupled with a developmental increase in the contribution of P/Q‐type Ca²⁺ channels as well as a developmental increase in AC pathway contribution.     

Insulin treatment restores glutamate (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid) receptor function in the hippocampus of diabetic rats

Type 1 diabetes is associated with cognitive dysfunction. Cognitive processing, particularly memory acquisition, depends on the regulated enhancement of expression and function of glutamate receptor subtypes in the hippocampus. Impairment of memory was been detected in rodent models of type 1 diabetes induced by streptozotocin (STZ). This study examines the functional properties of synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors and the expression of synaptic molecules that regulate glutamatergic synaptic transmission in the hippocampus of STZ‐diabetic rats. The AMPA receptor‐mediated miniature excitatory postsynaptic currents (mEPSCs) and single‐channel properties of synaptosomal AMPA receptors were examined after 4 weeks of diabetes induction. Results show that amplitude and frequency of mEPSCs recorded from CA1 pyramidal neurons were decreased in diabetic rats. In addition, the single‐channel properties of synaptic AMPA receptors from diabetic rat hippocampi were different from those of controls. These impairments in synaptic currents gated by AMPA receptors were accompanied by decreased protein levels of AMPA receptor subunit GluR1, the presynaptic protein synaptophysin, and the postsynaptic anchor protein postsynaptic density protein 95 in the hippocampus of diabetic rats. Neural cell adhesion molecule (NCAM), an extracellular matrix molecule abundantly expressed in the brain, and the polysialic acid (PSA) attached to NCAM were also downregulated in the hippocampus of diabetic rats. Insulin treatment, when initiated at the onset of diabetes induction, reduced these effects. These findings suggest that STZ‐induced diabetes may result in functional deteriorations in glutamatergic synapses in the hippocampus of rats and that these effects may be reduced by insulin treatment. © 2015 Wiley Periodicals, Inc.     

Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ‐aminobutyric acid type A receptor (GABA_AR) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABA_AR antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N‐methyl‐D‐aspartate (NMDA)‐subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA‐subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN‐93, NMDA receptor antagonist D‐APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization. © 2013 Wiley Periodicals, Inc.     

Activity‐dependent regulation of the dopamine transporter is mediated by Ca2+/calmodulin‐dependent protein kinase signaling

The mechanisms regulating expression of the dopamine transporter are poorly understood. We tested the hypothesis that neuronal activity is one of the non‐genetic determinants of dopamine transporter abundance. Sustained changes in neuronal activity caused by tetrodotoxin and 4‐aminopyridine altered the dopamine uptake and abundance of dopamine transporter and its mRNA in rat mesencephalic cultures. The altered neuronal activity caused by these two drugs is accompanied by changes in intracellular calcium concentrations and Ca²⁺/calmodulin‐dependent protein (CaM) kinase II activity in dopamine neurons. Chronic treatment with an L‐type calcium channel blocker (nifedipine) or CaM kinase inhibitor (KN93) decreased dopamine transporter‐mediated uptake and occluded the effects of tetrodotoxin and 4‐aminopyridine. These data suggest that neuronal activity can regulate dopamine transporter function and abundance via calcium/CaM kinase II signaling.     

Targeting microglia L‐type voltage‐dependent calcium channels for the treatment of central nervous system disorders

Calcium (Ca²⁺) is a ubiquitous mediator of a multitude of cellular functions in the central nervous system (CNS). Intracellular Ca²⁺ is tightly regulated by cells, including entry via plasma membrane Ca²⁺ permeable channels. Of specific interest for this review are L‐type voltage‐dependent Ca²⁺ channels (L‐VDCCs), due to their pleiotropic role in several CNS disorders. Currently, there are numerous approved drugs that target L‐VDCCs, including dihydropyridines. These drugs are safe and effective for the treatment of humans with cardiovascular disease and may also confer neuroprotection. Here, we review the potential of L‐VDCCs as a target for the treatment of CNS disorders with a focus on microglia L‐VDCCs. Microglia, the resident immune cells of the brain, have attracted recent attention for their emerging inflammatory role in several CNS diseases. Intracellular Ca²⁺ regulates microglia transition from a resting quiescent state to an “activated” immune‐effector state and is thus a valuable target for manipulation of microglia phenotype. We will review the literature on L‐VDCC expression and function in the CNS and on microglia in vitro and in vivo and explore the therapeutic landscape of L‐VDCC‐targeting agents at present and future challenges in the context of Alzheimer's disease, Parkinson's disease, Huntington's disease, neuropsychiatric diseases, and other CNS disorders.     

Presynaptic muscarinic receptors,calcium channels,and protein kinase C modulate the functional disconnection of weak inputs at polyinnervated neonatal neuromuscular synapses

We studied the relation among calcium inflows, voltage‐dependent calcium channels (VDCC), presynaptic muscarinic acetylcholine receptors (mAChRs), and protein kinase C (PKC) activity in the modulation of synapse elimination. We used intracellular recording to determine the synaptic efficacy in dually innervated endplates of the levator auris longus muscle of newborn rats during axonal competition in the postnatal synaptic elimination period. In these dual junctions, the weak nerve terminal was potentiated by partially reducing calcium entry (P/Q‐, N‐, or L‐type VDCC‐specific block or 500 μM magnesium ions), M1‐ or M4‐type selective mAChR block, or PKC block. Moreover, reducing calcium entry or blocking PKC or mAChRs results in unmasking functionally silent nerve endings that now recover neurotransmitter release. Our results show interactions between these molecules and indicate that there is a release inhibition mechanism based on an mAChR‐PKC‐VDCC intracellular cascade. When it is fully active in certain weak motor axons, it can depress ACh release and even disconnect synapses. We suggest that this mechanism plays a central role in the elimination of redundant neonatal synapses, because functional axonal withdrawal can indeed be reversed by mAChRs, VDCCs, or PKC block. © 2008 Wiley‐Liss, Inc.     

Inhibition of L-type voltage dependent calcium channels causes impairment of long-term potentiation in the hippocampal CA1 region in vivo

Long-term potentiation (LTP), in the hippocampal CA1 region is dependent on postsynaptic calcium influx. It is generally accepted that calcium influx occurs via activation of the NMDA receptor channel complex. However, studies in vitro using a high-frequency stimulus protocol (> or =200 Hz) demonstrated previously an NMDA receptor-independent form of LTP that is dependent upon activation of L-type voltage-dependent calcium channels (VDCCs). Here we have investigated a role for L-type VDCCs in LTP in vivo. Two structurally different, L-type VDCC blockers, verapamil (1, 3 and 10 mg/kg) and diltiazem (1, 10 and 20 mg/kg), depressed the induction of LTP in a dose-dependent manner. Increased activation of L-type VDCCs by Bay K 8644, an L-type agonist, however, did not enhance LTP. The NMDA receptor antagonist D-AP5 (5 and 20 mM injected i.c.v) impaired, but failed to block fully LTP in vivo. A reduced level of LTP could still be recorded following co-administration of verapamil and D-AP5. The level of LTP recorded was similar to that observed in the presence of either verapamil (10 mg/kg) or D-AP5 alone. These results suggest that activation of the NMDA receptor/channel and L-type VDCCs are involved in the induction of LTP in area CA1 in vivo. However, it appears that activation of other receptor/channels may also play a role in this form of LTP.     

Inflammation alters AMPA‐stimulated calcium responses in dorsal striatal D2 but not D1 spiny projection neurons

Neuroinflammation precedes neuronal loss in striatal neurodegenerative diseases and can be exacerbated by the release of proinflammatory molecules by microglia. These molecules can affect trafficking of AMPARs. The preferential trafficking of calcium‐permeable versus impermeable AMPARs can result in disruptions of [Ca²⁺]_i and alter cellular functions. In striatal neurodegenerative diseases, changes in [Ca²⁺]_i and L‐type voltage‐gated calcium channels (VGCCs) have been reported. Therefore, this study sought to determine whether a proinflammatory environment alters AMPA‐stimulated [Ca²⁺]_i through calcium‐permeable AMPARs and/or L‐type VGCCs in dopamine‐2‐ and dopamine‐1‐expressing striatal spiny projection neurons (D2 and D1 SPNs) in the dorsal striatum. Mice expressing the calcium indicator protein, GCaMP in D2 or D1 SPNs, were utilized for calcium imaging. Microglial activation was assessed by morphology analyses. To induce inflammation, acute mouse striatal slices were incubated with lipopolysaccharide (LPS). Here we report that LPS treatment potentiated AMPA responses only in D2 SPNs. When a nonspecific VGCC blocker was included, we observed a decrease of AMPA‐stimulated calcium fluorescence in D2 but not D1 SPNs. The remaining agonist‐induced [Ca²⁺]_i was mediated by calcium‐permeable AMPARs because the responses were completely blocked by a selective calcium‐permeable AMPAR antagonist. We used isradipine, the highly selective L‐type VGCC antagonist to determine the role of L‐type VGCCs in SPNs treated with LPS. Isradipine decreased AMPA‐stimulated responses selectively in D2 SPNs after LPS treatment. Our findings suggest that dorsal striatal D2 SPNs are specifically targeted in proinflammatory conditions and that L‐type VGCCs and calcium‐permeable AMPARs are important mediators of this effect.     

Interaction of DHPG‐LTD and synaptic‐LTD at senescent CA3‐CA1 hippocampal synapses

The susceptibility, but not the magnitude, of long‐term depression (LTD) induced by hippocampal CA3‐CA1 synaptic activity (synaptic‐LTD) increases with advanced age. In contrast, the magnitude of LTD induced by pharmacological activation of CA3‐CA1 group I metabotropic glutamate receptors (mGluRs) increases during aging. This study examined the signaling pathways involved in induction of LTD and the interaction between paired‐pulse low frequency stimulation‐induced synaptic‐LTD and group I mGluR selective agonist, (RS)‐3,5‐dihydroxyphenylglycine (DHPG, 100 µM)‐induced DHPG‐LTD in hippocampal slices obtained from aged (22–24 months) male Fischer 344 rats. Prior induction of synaptic‐LTD did not affect induction of DHPG‐LTD; however, prior induction of the DHPG‐LTD occluded synaptic‐LTD suggesting that expression of DHPG‐LTD may incorporate synaptic‐LTD mechanisms. Application of individual antagonist for the group I mGluR (AIDA), the N‐methyl‐d ‐aspartate receptor (NMDAR) (AP‐5), or L‐type voltage‐dependent Ca²⁺ channel (VDCC) (nifedipine) failed to block synaptic‐LTD and any two antagonists severely impaired synaptic‐LTD induction, indicating that activation of any two mechanisms is sufficient to induce synaptic‐LTD in aged animals. For DHPG‐LTD, AIDA blocked DHPG‐LTD and individually applied NMDAR or VDCC attenuated but did not block DHPG‐LTD, indicating that the magnitude of DHPG‐LTD depends on all three mechanisms. © 2014 Wiley Periodicals, Inc.     

Differential Ca2+-dependence of transmitter release mediated by P/Q- and N-type calcium channels at neonatal rat neuromuscular junctions

N- and P/Q-type voltage dependent calcium channels (VDCCs) mediate transmitter release at neonatal rat neuromuscular junction (NMJ). Thus the neonatal NMJ allows an examination of the coupling of different subtypes of VDCCs to the release process at a single synapse. We studied calcium dependence of transmitter release mediated by each channel by blocking with omega-conotoxin GVIA the N-type channel or with omega-agatoxin IVA the P/Q-type channel while changing the extracellular calcium concentration ([Ca2+]o). Transmitter release mediated by P/Q-type VDCCs showed steeper calcium dependence than N-type mediated release (average slope 3.6 +/- 0.09 vs. 2.6 +/- 0.03, respectively). Loading the nerve terminals with 10 microm BAPTA-AM in the extracellular solution reduced transmitter release and occluded the blocking effect of omega-conotoxin GVIA (blockade -2 +/- 9%) without affecting the action of omega-agatoxin IVA (blockade 85 +/- 4%). Both VDCC blockers were able to reduce the amount of facilitation produced by double-pulse stimulation. In these conditions facilitation was restored by increasing [Ca2+]o. The facilitation index (fi) was also reduced by loading nerve terminals with 10 microm BAPTA-AM (fi = 1.2 +/- 0.1). The control fi was 2.5 +/- 0.1. These results show that P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than were N-type VDCCs at the neonatal neuromuscular junction. This difference could be accounted for by a differential location of these channels at the release site. In addition, our results indicate that space-time overlapping of calcium domains was required for facilitation.     

Activation of the prelimbic medial prefrontal cortex induces anxiety‐like behaviors via N‐Methyl‐D‐aspartate receptor‐mediated glutamatergic neurotransmission in mice

We investigated the possible roles of the prelimbic medial prefrontal cortex (PL) in the regulation of anxiety‐like behaviors by pharmacologically activating the terminals of neuronal inputs or postsynaptic efferent neurons with a sodium channel activator veratrine. The extracellular glutamate levels were measured by in vivo microdialysis, and the behaviors were assessed with the open field (OF) test in mice simultaneously. The samples were collected every 10 min for 60 min, as basal levels of glutamate. The medium containing drugs were perfused for 30 min. The OF test was performed in the last 10 min of drug perfusion. After the drug treatments, the perfusion medium containing drugs was switched back to perfusion medium without drugs, and then samples were collected for another 90 min. The extracellular glutamate levels were significantly elevated after local perfusion of veratrine in the PL. At the same time, perfusion of veratrine in the PL produced anxiety‐like behaviors in mice. Local coperfusion of a sodium channel blocker, lamotrigine, completely diminished the veratrine‐induced elevated extracellular glutamate levels and the behavioral changes. Local coperfusion of an NMDA receptor antagonist, MK‐801, but not a non‐NMDA (AMPA/kainate) receptor antagonist, CNQX, completely diminished the behavioral changes without any effects on the veratrine‐induced elevated extracellular glutamate levels. This study demonstrates that the activation of the PL with veratrine induces anxiety‐like behaviors via NMDA receptor‐mediated glutamatergic neurotransmission in mice. © 2014 Wiley Periodicals, Inc.     

荷包牡丹碱对海马CA2区的神经毒性与P／Q型钙通道有关

目的：探讨荷包牡丹碱（BiC）对海马CA2区的神经毒性作用以及电压依赖性钙通道（VDCC）对神经毒性作用的影响。方法：BiC6μmol·L^-1刺激培养的大鼠海马组织72h，同时用N，L以及P／Q型钙通道拮抗剂分别阻断相应的钙通道，测定神经细胞摄取的碘化丙啶（PI）。结果：BiC刺激6h后，神经细胞的PI摄取首先出现在CA2区。随着BiC刺激时间的延长，摄取PI的神经细胞的分布范围由CA2区扩散到其他区域。P／Q型VDCC拮抗剂抑制神经细胞的PI摄取，然而N和L型VDCC的拮抗剂无明显抑制效果。结论：在大鼠海马的组织培养中CA2区的神经细胞对BiC的刺激最易受损。CA2区神经细胞的损伤过程中P／Q型VDCC起重要作用。     

The effects of anticonvulsant drugs on long-term potentiation (LTP) in the rat hippocampus

In hippocampal CA1 area, there are at least two forms of long-term potentiation (LTP): one is N-methyl-D-aspartate (NMDA) receptor-dependent LTP (NMDA LTP), which is induced with a 25 Hz tetanus and blocked by 50 μM 2-amino-5-phosphonovaleric acid (APV); the other is NMDA receptor-independent LTP (VDCC LTP), which is induced by 200 Hz tetanus stimulation in the presence of APV and blocked by nifedipine, a voltage-dependent Ca⁺⁺ channel (VDCC) blocker, or by the intracellular injection of 1,2-bis(2-Aminophenoxoy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The effects of anticonvulsant drugs phenobarbital, phenytoin, and valproic acid on both NMDA LTP and VDCC LTP were investigated in rat hippocampal slices. The results showed that 0.1 mg/ml valproic acid significantly altered baseline population spike amplitude by 34.6%, but the other drugs had no significant effect on the baseline population spike amplitude. Phenobarbital (0.025 mg/ml) potently blocked NMDA LTP and inhibited VDCC LTP. Phenytoin (0.02 mg/ml) had no effect on NMDA LTP but reduced VDCC LTP. Valproic acid did not inhibit VDCC LTP, but it abolished the expression of NMDA LTP in a similar manner as H-7, a nonspecific protein kinase C inhibitor. These data suggest that the anticonvulsant effects of these three drugs may be via different cellular mechanisms.     

Rapamycin prevents N‐methyl‐D‐aspartate‐induced retinal damage through an ERK‐dependent mechanism in rats

Recent studies have demonstrated that inhibition of the mammalian target of rapamycin (mTOR) protects against neuronal injury, but the mechanisms underlying this protection are not fully understood. The present study investigates whether rapamycin, an inhibitor of the mTOR pathway, protects against N‐methyl‐D ‐aspartate (NMDA)‐induced retinal neurotoxicity and whether the extracellular signal‐regulated kinase (ERK) pathway contributes to this protective effect in rats. Significant cell loss in the ganglion cell layer and a reduction in thickness of the inner plexiform layer were observed 7 days after a single intravitreal injection of NMDA (200 nmol/eye). These NMDA‐induced morphological changes were significantly reduced by rapamycin (20 nmol/eye). The number of terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling‐positive apoptotic cells had increased 6 hr after NMDA injection, an effect that was significantly attenuated by rapamycin. The ERK inhibitor U0126 (1 nmol/eye) almost completely abolished rapamycin's inhibition of NMDA‐induced apoptosis. Immunohistochemical studies showed that NMDA caused a time‐dependent increase in levels of the phosphorylated form of the ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. The increased pS6 levels were markedly decreased by rapamycin. Both NMDA and rapamycin increased the level of phosphorylated ERK (pERK) in Müller cells, and coinjection of both agents further increased pERK levels. These results suggest that rapamycin has a neuroprotective effect against NMDA‐induced retinal neurotoxicity and that this effect could be patially mediated by activation of the ERK pathway in retinal Müller cells. © 2014 Wiley Periodicals, Inc.