Pituitary adenylate cyclase-activating polypeptide is up-regulated in cortical pyramidal cells after focal ischemia and protects neurons from mild hypoxic/ischemic damage

The protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP-selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)-dependent up-regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2-regulated Ser112 of pro-apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short-term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK-dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1–5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Akt-activating factors and conferred ischemic tolerance to neurons. Thus, ischemia-induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1.     

Up-regulation of L-type high voltage-gated calcium channel subunits by sustained exposure to 1,4- and 1,5-benzodiazepines in cerebrocortical neurons

The aim of this study is to examine how sustained exposure to two 1,4-benzodiazepines (BZDs) with different action period, diazepam and brotizolam, and a 1,5-BZD, clobazam, affects L-type high voltage-gated calcium channel (HVCC) functions and its mechanisms using primary cultures of mouse cerebral cortical neurons. The sustained exposure to these three BZDs increased [⁴⁵Ca²⁺] influx, which was due to the enhanced [⁴⁵Ca²⁺] entry through L-type HVCCs but not through of Cav2.1 and Cav2.2. Increase in [³H]diltiazem binding after the exposure to these three BZDs was due to the increase in the binding sites of [³H]diltiazem. Western blot analysis showed increase of Cav1.2 and Cav1.3 in association with the increased expression of α2/δ1 subunit. Similar changes in [³H]diltiazem binding and L-type HVCC subunit expression were found in the cerebral cortex from mouse with BZD physical dependence. These results indicate that BZDs examined here have the potential to increase L-type HVCC functions mediated via the enhanced expression of not only Cav1.2 and Cav1.3 but also α2/δ1 subunit after their sustained exposure, which may participate in the development of physical dependence by these BZDs.     

Quantitative analysis of glutamatergic and GABAergic neurons expressing 5-HT(2A) receptors in human and monkey prefrontal cortex

5-hydroxytryptamine (5-HT) or serotonin 2A receptors play an important role in modulation of prefrontal cortex (PFC) activity and have been implicated in the physiopathology of psychiatric disorders. There is no quantitative information on the percentage of glutamatergic and GABAergic cells that express 5-HT(2A) receptors in human and monkey PFC. We have used double in situ hybridization to quantify the mRNA co-localization of 5-HT(2A) receptor with the glutamatergic transporter vesicular glutamate transporter 1, and with the GABAergic marker glutamic acid decarboxylase 65/67 and in parvalbumin and calbindin GABAergic cell populations. Our results show that nearly every glutamatergic cell (86-100%) in layers II-V expressed 5-HT(2A) receptor mRNA in both species. This percentage was lower in layer VI (13-31%). In contrast, not all the GABAergic interneurons (13-46%) expressed 5-HT(2A) receptor mRNA. This receptor was expressed in 45-69% of parvalbumin and in 61-87% of calbindin positive cells. These results indicate that, while the majority of glutamatergic neurons can be sensitive to 5-HT action via 5-HT(2A) receptors, this modulation occurs only in a limited population of GABAergic interneurons and provides new neuroanatomical information about the role played by serotonin through 5-HT(2A) receptors in the PFC and on the sites of action for drugs such as antipsychotics and antidepressants used in treatment of psychiatric disorders.     

Distribution of sphingosine kinase activity and mRNA in rodent brain

Sphingosine-1-phosphate (S1P) is a lipid mediator that exerts multiple cellular functions through activation of a subfamily of G-protein-coupled receptors. Although there is evidence that S1P plays a role in the developing and adult CNS, little is known about the ability of brain parenchyma to synthesize this lipid. We have therefore analyzed the brain distribution of the enzymatic activity of the S1P synthesizing enzyme, sphingosine kinase (SPHK) [EC:2.7.1.91], as well as mRNA distribution for one of the two isoforms of this enzyme, sphingosine kinase 2. SPHK activity, measured by the conversion of [(3)H]sphingosine to [(3)H]S1P, is highest in cerebellum, followed by cortex and brainstem. Lowest activities were found in striatum and hippocampus. Sensitivity to 0.1% Triton-X suggests that this activity is accounted for by SPHK2. RT-PCR and in situ hybridization studies show that mRNA for this isoform has a distribution similar to that of SPHK activity. In vivo and in vitro ischemia increase SPHK activity and SPHK2 mRNA levels. These results indicate that SPHK2 is the predominant S1P-synthesizing isoform in normal brain parenchyma. Its heterogeneous distribution, in particular laminar distribution in cortex, suggests a neuronal localization and a possible role in cortical and cerebellar functions, in normal as well as ischemic brain.     

Group I metabotropic glutamate receptor-dependent TRPC channel trafficking in hippocampal neurons

The group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elicited two phases of synchronized neuronal (epileptiform) discharges in hippocampal slices: an initial phase of short duration discharges followed by a phase of prolonged discharges. We assessed the involvement of transient receptor potential canonical (TRPC) channels in these responses. Pre-treatment of hippocampal slices with TRPC channel blockers, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365) or 2-aminoethoxydiphenyl borate, did not affect the short epileptiform discharges but blocked the prolonged epileptiform discharges. SKF96365 suppressed ongoing DHPG-induced prolonged epileptiform discharges. Western blot analysis showed that the total TRPC4 or TRPC5 proteins in hippocampal slices were unchanged following DHPG. DHPG increased TRPC4 and TRPC5 in the cytoplasmic compartment and decreased these proteins in the plasma membrane. Translocation of TRPC4 and TRPC5 was suppressed when the epileptiform discharges were blocked by ionotropic glutamate receptor blockers. Translocation of TRPC4 and TRPC5 was also prevented in slices from phospholipase C (PLC) beta1 knockout mice, even when synchronized discharges were elicited by the convulsant 4-aminopyridine. The results suggest that TRPC channels are involved in generating DHPG-induced prolonged epileptiform discharges. This function of TRPC channels is associated with a neuronal activity- and PLCbeta1-dependent translocation of TRPC4 and TRPC5 proteins from the plasmalemma to the cytoplasmic compartment.     

Serotoninergic modulation of GABAergic synaptic transmission in developing rat CA3 pyramidal neurons

Serotoninergic modulation of GABAergic mIPSCs was investigated in immature (postnatal 12–16-days old) rat CA3 pyramidal neurons using a conventional whole-cell patch clamp technique. Serotonin or 5-hydroxytryptamine (5-HT) (10 μmol/L) transiently and explosively increased mIPSC frequency with a small increase in the current amplitude. However, 5-HT did not affect the GABA-induced postsynaptic currents, indicating that 5-HT acts presynaptically to facilitate the probability of spontaneous GABA release. The 5-HT action on GABAergic mIPSC frequency was completely blocked by 100 nmol/L MDL72222, a selective 5-HT₃ receptor antagonist, and mimicked by mCPBG, a selective 5-HT₃ receptor agonist. The 5-HT action on GABAergic mIPSC frequency was completely occluded either in the presence of 200 μmol/L Cd²⁺ or in the Na⁺-free external solution, suggesting that the 5-HT₃ receptor-mediated facilitation of mIPSC frequency requires a Ca²⁺influx passing through voltage-dependent Ca²⁺channels from the extracellular space, and that presynaptic 5-HT₃ receptors are less permeable to Ca²⁺. The 5-HT action on mIPSC frequency in the absence or presence of extracellular Na⁺ gradually increased with postnatal development. Such a developmental change in the 5-HT₃ receptor-mediated facilitation of GABAergic transmission would play important roles in the regulation of excitability as well as development in CA3 pyramidal neurons.     

Postnatal morphological changes in neurons and glial cells in neocortex of mice developing at the background of serotonin deficit

It has been shown that deficit of serotonin during embryogenesis in rodents is accompanied by changes of morphological characteristics of neurons and glial cells at the period of postnatal development. A characteristic peculiarity of these changes is cell vacuolization that is of different expression in various cortical layers. In the experimental animals as compared with control ones, neurons of all neocortex layers have changed nuclei and a reduced volume of the cytoplasm. In neurons of upper layers, nuclei and cytoplasm contain occasional small vacuoles. In deep layers, vacuolization both of nuclei and of the cytoplasm is expressed to the much greater degree and vacuoles of large size are predominant. Results of immunocytochemical study have shown that in animals developing at the background of serotonin deficit there takes place a delay of the rates of formation and differentiation of astrocytic glia.     

Predominant Neuritic Pathology Induced by Viral Overexpression of <Emphasis Type="Bold">α</Emphasis>-Synuclein in Cell Culture

1. Alpha-synuclein is known to play an important role in the pathogenesis of Parkinson’s disease (PD). The pathogenicity of α-synuclein is related to its ability to form intraneuronal inclusions. The inclusions, which are found in brains of patients with PD and diffuse Lewy body disease consist partially of C-terminally truncated α-synuclein. This α-synuclein species has an increased ability to form aggregates compared to full length α-synuclein. 2. We have used an adeno-associated virus (AAV) vector system to overexpress either C-terminally truncated or full length α-synuclein containing the A53T mutation, which have both been identified in brains of familial PD patients and transgenic mouse models. Dissociated mesencephalic neurons, cerebellar granule neurons, and organotypic midbrain slice cultures were infected with AAV containing the transgene under the control of the cytomegalovirus promoter. 3. We demonstrate that viral overexpression of α-synuclein(A53T) leads to the formation of distorted neurites, intraneuritic swellings, and granular perikaryal deposits in cultured neurons. Our results indicate that these cell culture models may represent an early phase of PD reflecting pathologic neuritic alterations before significant neuronal cell loss occurs.     

海马神经元的瞬间外向钾电流

瞬间外向钾电流(IA)具有快速激活和失活等特征,是动作电位复极化早期外向钾离子电流的主要成分,广泛分布在海马神经元,树突处尤为突出.该电流通过减慢去极化速度和延缓动作电位的产生等作用,调节突触的输入和动作电位的反向传播,从而在信号整合及突触可塑性等过程中扮演重要角色.很多人类疾病,如癫痫性疾病等,和海马神经元的IA电流有关.