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Objective: To study the effect of 3-n-butylphthalide (NBP) on neuronal apoptosis in rats with cerebral infarction (CI) through the p38/mitogen-activated protein kinase (MAPK) pathway., Materials and Methods: A total of 30 rats were divided into control group (healthy rats, n=10), model group (CI rat model, n=10), and NBP group (CI rat model + intraperitoneal injection of NBP, n=10). Then, the neurological function, degree of cerebral ischemia, apoptosis of brain tissues, the protein and messenger ribonucleic acid (mRNA) expressions of p-p38 and MAPK in brain tissues were detected using the neurological score, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and Western blotting, respectively., Results: In NBP group, the neurological score was significantly lower than in model group, and the difference was statistically significant (p<0.05). The results of TTC staining revealed that the area of the white region in brain slices was significantly larger in model group than in control group, indicating the successful establishment of middle cerebral artery occlusion (MCAO) model. Compared with model group, NBP group had a smaller area and lighter color of the white region in brain slices, suggesting that NBP markedly reduces the MCAO-induced CI. The apoptosis rate in NBP group was higher than in control group (p<0.05), but lower than in model group (p<0.05), while it was higher in model group than in control group (p<0.05). The protein expressions of p38 and MAPK in NBP group were higher than in control group (p<0.05), but lower than in model group (p<0.05), while they were higher in model group than in control group (p<0.05). Moreover, the mRNA expressions of p38 and MAPK were lower in control group than in model group (p<0.05), while they were higher in model group than in NBP group (p<0.05), but there was no significant difference in the mRNA expression of p38 between NBP group and control group (p>0.05)., Conclusions: NBP alleviates neuronal apoptosis in CI by down-regulating the p38 signal and inhibiting the expression of MAPK, thereby treating CI.

We have demonstrated two-dimensional and three-dimensional transfer of B35 neuronal cells onto and within polymerized Matrigel substrates, using matrix-assisted pulsed laser evaporation-direct write (MDW). The B35 cells were transferred from a quartz ribbon to depths of up to 75 microm by systematically varying the fluence emitted from the ArF (lambda = 193 nm) laser source. MDW-transferred cells were examined using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), 4',6-diamidino-2-phenylindole (DAPI), and alpha-tubulin staining. Confocal microscopy has shown that the transferred B35 cells extended their axons outward in three dimensions within the polymerized Matrigel substrate. The B35 cells made axonal connections and formed a three-dimensional neural network within 72 h after MDW transfer. In addition, TUNEL staining demonstrated that only 3% of the B35 cells underwent apoptosis after being transferred using the MDW process. MDW and other emergent direct write processes may provide unique approaches for creating layered, heterogeneous, three-dimensional cell-seeded scaffolds for use in peripheral nerve repair.

P2X receptors are a family of ligand-gated ion channels, activated by extracellular ATP. The seven subunits cloned (P2X1-7) can assemble to form homomeric and heteromeric receptors. Peripheral neurons of neural crest origin (e.g. those in dorsal root, trigeminal, sympathetic and enteric ganglia) and placodal origin (e.g. those in nodose and petrosal ganglia) express mRNAs for multiple P2X subunits. In this review, we summarize the molecular biological, electrophysiological and immunohistochemical evidence for P2X receptor subunits in sensory, sympathetic, parasympathetic, pelvic and myenteric neurons and adrenomedullary chromaffin cells. We consider the pharmacological properties of these native P2X receptors and their physiological roles. The responses of peripheral neurons to ATP show considerable heterogeneity between cells in the same ganglia, between ganglia and between species. Nevertheless, these responses can all be accounted for by the presence of P2X2 and P2X3 subunits, giving rise to varying proportions of homomeric and heteromeric receptors. While dorsal root ganglion neurons express predominantly P2X3 and rat sympathetic neurons express mainly P2X2 receptors, nodose and guinea-pig sympathetic neurons express mixed populations of P2X2 and heteromeric P2X2/3 receptors. P2X receptors are important for synaptic transmission in enteric ganglia, although their roles in sympathetic and parasympathetic ganglia are less clear. Their presence on sensory neurons is essential for some processes including detection of filling of the urinary bladder. The regulation of P2X receptor expression in development and in pathological conditions, along with the interactions between purinergic and other signalling systems, may reveal further physiological roles for P2X receptors in autonomic and sensory ganglia.

1. Application of ATP and alpha,beta-methylene ATP (alpha beta meATP) to voltage-clamped guinea-pig pelvic neurons produced three types of inward currents. A fast-desensitizing response was present in 5% (25/660) of neurons, 70% gave slowly-desensitizing currents, and the remainder had biphasic responses. 2. Slowly-desensitizing responses were characterized pharmacologically. The response to alpha beta meATP 100 microM was 46+/-27% (range 0--100%) of that evoked by ATP 100 microM in the same cell. Cross-desensitization indicated the presence of alpha beta meATP-sensitive and -insensitive receptors. 3. The concentration-response curve for alpha beta meATP had an EC(50) of 55 microM, and a Hill coefficient of 0.99, while at the alpha beta meATP-insensitive receptor, ATP had an EC(50) of 73 microM, with a Hill coefficient of 1.78. 4. The response to alpha beta meATP was blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin and Cibacron blue. However, the alpha beta meATP-insensitive receptor was inhibited by PPADS, but not by the other two antagonists. 5. 2'- (or 3'-) O-trinitrophenyl-ATP was 10 times more potent in inhibiting responses to alpha beta meATP than to ATP (at the alpha beta meATP-insensitive receptor). 6. Lowering extracellular pH potentiated responses to alpha beta meATP and ATP, while raising pH attenuated them. 7. Co-application of Zn(2+) (3--300 microM) inhibited the responses to alpha beta meATP and ATP, with IC(50) values of 286 and 60 microM, respectively. 8. In conclusion, unlike rat and mouse pelvic ganglion neurons, which only express P2X(2) homomers, at least three distinct P2X receptors are present in guinea-pig pelvic neurons, probably homomeric P2X(2), P2X(3) and heteromeric P2X(2/3) receptors. However, some of the novel pharmacological properties observed suggest that the guinea-pig P2X receptor subtypes may differ from their rat orthologues.

Odor-induced neural activity was recorded by Ca2+ imaging in the cell body region of the Drosophila mushroom body (MB), which is the second relay of the olfactory central nervous system. The signals recorded are mainly from the cell layers on the brain surface because of the limited penetration of Ca2+-sensitive dyes. The densely packed cell bodies and their accessibility allow visualization of odor-induced population neural activity. It is revealed that odors evoke diffused neural activities in the MB. Although the signals cannot be attributed to individual neurons, patterns of the population neural activity can be analyzed. The activity pattern, but not the amplitude, of an odor-induced population response is specific for the chemical identity of an odor and its concentration. The distribution pattern of neural activity can be altered specifically by genetic manipulation of an odor binding protein and this alteration is closely associated with a behavioral defect of odor preference. These results suggest that the spatial pattern of the distributed neural activity may contribute to coding of odor information at the second relay of the olfactory system.

1. Characterization of P2X receptors on neurons of guinea-pig superior cervical ganglion (SCG) has been carried out using a whole-cell voltage-clamp technique. 2. Application of ATP and alpha,beta-methylene ATP (alphabeta-MeATP) produced fast activating inward currents, which desensitized slowly. The maximum response to alphabeta-MeATP was 36 +/- 23 % (range 0.1-100 %) of that evoked by ATP in the same cell. 3. Co-application of alphabeta-MeATP (300 microM) with ATP (300 microM) produced a response that was 97 +/- 1 % of that given by ATP alone. Following desensitization with alphabeta-MeATP, the decrease in response to ATP was equal to the absolute reduction in response to alphabeta-MeATP in the same cell. 4. The concentration-response curve for alphabeta-MeATP had an EC50 of 42 microM and a Hill coefficient of 1.17. For cells where the ratio of alphabeta-MeATP/ATP currents at 100 microM was < 0.1, the ATP concentration-response curve had an EC50 of 56 microM and a Hill coefficient of 1.95. However, in cells where the ratio was > 0.7, the curve had an EC50 of 60 microM and a Hill coefficient of 0.97. 5. The response to 100 microM alphabeta-MeATP was inhibited by 2' (or 3')-O-trinitrophenyl-ATP (TNP-ATP) with an IC50 of 70 nM. However, on cells where the ratio of alphabeta-MeATP/ATP currents was < 0.1, ATP was inhibited by TNP-ATP with an IC50 of 522 nM. 6. Immunohistochemical staining with antibodies raised against rat P2X2 and P2X3 epitopes suggested that both subunits were expressed by guinea-pig SCG neurons. 7. We conclude that varying proportions of two distinct P2X receptors coexist on the cell bodies of individual guinea-pig SCG neurons, which may correspond to homomeric P2X2 and heteromeric P2X2/3 receptors.

Characteristics of P2X receptors on neurons of the rat coeliac, mouse coeliac and mouse pelvic ganglia have been studied using the whole cell voltage-clamp technique. Fast application of ATP (100 microM) on to isolated neurons voltage clamped at -70 mV induced a slowly desensitising inward current in 96% of the cells tested. Concentration-response curves for ATP yielded EC50 values of 86 microM, 64 microM and 123 microM, for rat coeliac, mouse coeliac and mouse pelvic ganglion neurons, respectively, while alpha,beta-methylene ATP was inactive. The response to ATP was antagonised by suramin, Cibacron blue and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). The potency of ATP was increased by extracellular acidification and by co-application of micromolar concentrations of Zn2+, while raising pH decreased it. On rat coeliac ganglion neurons, the EC50 values for ATP were 35 microM and 253 microM at pH 6.8 and 8.0, respectively. On mouse coeliac and pelvic ganglion neurons, altering the pH produced comparable changes. In conclusion, our results indicate that, in contrast to the guinea-pig coeliac ganglion, the characteristics of the P2X receptors present on rat coeliac, mouse coeliac and mouse pelvic ganglia are all identical to those present on rat pelvic ganglion, i.e. they are homomeric P2X2 receptors, or heteromultimers with P2X2 being the dominant subunit.

1. The presence and characteristics of P2X receptors on neurons of the rat major pelvic ganglia (MPG) have been studied using whole cell voltage-clamp, in situ hybridization and immunohistochemistry. 2. Rapid application of ATP (100 microM) to isolated rat MPG neurons induced moderately large inward currents (0.33-5.3 nA) in 39% of cells (108/277). The response to ATP occurred very rapidly, with an increase in membrane conductance, and desensitized slowly. 3. The concentration-response curve for ATP yielded an EC50 of 58.9 microM. The agonist profile was ATP> or =2MeSATP=ATPgammaS>BzATP, while alpha,beta-MeATP, beta,gamma-MeATP, UTP and ADP were all inactive at concentrations up to 100 microM. 4. The response to ATP was antagonized by suramin (pA2=5.6), reactive blue-2 (IC50=0.7 microM) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). 5. Lowering the pH from 7.4 to 6.8 produced a marked potentiation (to 339% of control) of the responses to ATP (30 microM), while raising the pH to 8.0 attenuated the responses (to 20% of control). The EC50s for ATP were 28.8, 58.9 and 264 microM at pH 6.8, 7.4 and 8.0, respectively. 6. Co-application of ATP with Zn2+ produced a marked enhancement of the responses to ATP, with an EC50 of 9.55 microM. In the presence of Zn2+ (30 microM), the EC50 for ATP was decreased to 4.57 microM. 7. In situ hybridization revealed that the P2X receptor transcripts levels in rat MPG neurons are P2X2>P2X4>P2X1, P2X3, P2X5 and P2X6. The immunohistochemical staining revealed a small number of neurons with strong P2X2 immunoreactivity. 8. In conclusion, our results indicate that there are P2X receptors present on MPG neurons. The pharmacological characteristics of these receptors, the in situ hybridization and immunohistochemical evidence are consistent with them being of the P2X2 subtype, or heteromultimers. with P2X2 being the dominant component.

This article reports the analysis of a novel serotonin (5-HT)-sensitive leak channel. The 5-HT responses were recorded in acutely dissociated Drosophila adult and larval central nervous system (CNS) neurons by the patch-clamp method, in an attempt to establish a model preparation suitable for the genetic study of signal transduction underlying central neurotransmission. Focal perfusion or iontophoresis of 5-HT onto some patched neurons induced either an apparent inward or outward current. This apparent outward current is able to cause a strong hyperpolarization of the neuron. This article focuses on the predominant hyperpolarizing response, which is observed in a significant fraction of larger CNS neurons and in different developmental stages. The hyperpolarizing response is in fact mediated by inhibiting an inward leak current, which has a reversal potential around 0 mV. This 5-HT-sensitive leak current appears to be mediated mainly by one type of newly identified leak channel with a similar reversal potential of 0 mV and a conductance of 24 pS. In addition, it was also demonstrated that neurotransmitter-induced responses in both larval and adult Drosophila CNS neurons can be analyzed in this acutely dissociated preparation.