Different spatial expressions of c-Fos in the nucleus of the solitary tract following taste stimulation with sodium, potassium, and ammonium ions in rats.

Cation-specific epithelial receptors on the tongue have been well demonstrated. However, active regions along the nucleus of the solitary tract (NST) for cations Na(+), K(+), NH4(+) are still unclear, even though the best responses of NST neurons to taste stimuli vary depending on the cell. In the present study, the spatial distribution patterns of cation-specific active regions in the NST are investigated. The tongues of urethane-anesthetized Sprague-Dawley rats (n = 25) were stimulated with artificial saliva (control), 0.5 M NaCl, 1.0 M NaCl, 0.5 M KCl, and 0.3 M NH(4) Cl. Then, the three-dimensional positions of c-Fos-like-immunoreactive (cFLI) cells in the NST were generated. The spatial distributions of cFLI cells in the NST were compared among five taste stimulations. cFLI cells were observed throughout the NST, irrespective of the stimulus; however, the intermediate-medial central regions of the NST had higher numbers of cFLI cells than the other regions in all taste stimulations. Analysis of images revealed that the activated regions in the NST differed significantly depending on the cations. The intermediate-dorsal-central region and the caudal-ventral region were activated by a 0.5 M concentration of sodium, the rostral-ventral region and the intermediate-dorsal/ventral region were activated by a 1.0 M concentration of sodium, the intermediate-dorsal/ventral region was activated by potassium ions, and the rostral-ventral region and the intermediate-ventral central region were activated by ammonium ions. These results suggest that the responses of NST cells to cation salt ions are regulated differentially.

Peripheral NMDA Receptors Mediate Antidromic Nerve Stimulation-Induced Tactile Hypersensitivity in the Rat.

We investigated the role of peripheral NMDA receptors (NMDARs) in antidromic nerve stimulation-induced tactile hypersensitivity outside the skin area innervated by stimulated nerve. Tetanic electrical stimulation (ES) of the decentralized L5 spinal nerve, which induced enlargement of plasma extravasation, resulted in tactile hypersensitivity in the L4 plantar dermatome of the hind-paw. When intraplantar (i.pl.) injection was administered into the L4 dermatome before ES, NMDAR and group-I metabotropic Glu receptor (mGluR) antagonists and group-II mGluR agonist but not AMPA/kainate receptor antagonist prevented ES-induced hypersensitivity. I.pl. injection of PKA or PKC inhibitors also prevented ES-induced hypersensitivity. When the same injections were administered after establishment of ES-induced hypersensitivity, hypersensitivity was partially reduced by NMDAR antagonist only. In naïve animals, i.pl. Glu injection into the L4 dermatome induced tactile hypersensitivity, which was blocked by NMDAR antagonist and PKA and PKC inhibitors. These results suggest that the peripheral release of Glu, induced by antidromic nerve stimulation, leads to the expansion of tactile hypersensitive skin probably via nociceptor sensitization spread due to the diffusion of Glu into the skin near the release site. In addition, intracellular PKA- and PKC-dependent mechanisms mediated mainly by NMDAR activation are involved in Glu-induced nociceptor sensitization and subsequent hypersensitivity.

Modulation of Spinal GABAergic Inhibition and Mechanical Hypersensitivity following Chronic Compression of Dorsal Root Ganglion in the Rat.

Chronic compression of dorsal root ganglion (CCD) results in neuropathic pain. We investigated the role of spinal GABA in CCD-induced pain using rats with unilateral CCD. A stereological analysis revealed that the proportion of GABA-immunoreactive neurons to total neurons at L4/5 laminae I-III on the injured side decreased in the early phase of CCD (post-CCD week 1) and then returned to the sham-control level in the late phase (post-CCD week 18). In the early phase, the rats showed an increase in both mechanical sensitivity of the hind paw and spinal WDR neuronal excitability on the injured side, and such increase was suppressed by spinally applied muscimol (GABA-A agonist, 5 nmol) and baclofen (GABA-B agonist, 25 nmol), indicating the reduced spinal GABAergic inhibition involved. In the late phase, the CCD-induced increase in mechanical sensitivity and neuronal excitability returned to pre-CCD levels, and such recovered responses were enhanced by spinally applied bicuculline (GABA-A antagonist, 15 nmol) and CGP52432 (GABA-B antagonist, 15 nmol), indicating the regained spinal GABAergic inhibition involved. In conclusion, the alteration of spinal GABAergic inhibition following CCD and leading to a gradual reduction over time of CCD-induced mechanical hypersensitivity is most likely due to changes in GABA content in spinal GABA neurons.

Changes in cytokine expression after electroacupuncture in neuropathic rats.

The production of proinflammatory cytokines including interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) plays a key role in chronic pain such as neuropathic pain. We investigated changes in cytokine expression in injured peripheral nerves and dorsal root ganglia (DRG) following electroacupuncture (EA) treatment. Neuropathic pain was induced by peripheral nerve injury to the left hind limb of Sprague-Dawley rats under pentobarbital anesthesia. Two weeks later, the nerve-injured rats were treated by EA for 10 minutes. The expression levels of IL-1ß, IL-6, and TNF-α in peripheral nerves and DRG of neuropathic rats were significantly increased in nerve-injured rats. However, after EA, the cytokine expression levels were noticeably decreased in peripheral nerves and DRG. These results suggest that EA stimulation can reduce the levels of proinflamtory cytokines elevated after nerve injury.

Modulation of N-type Ca²âº currents by moxonidine via imidazoline I1 receptor activation in rat superior cervical ganglion neurons.

Moxonidine, an imidazoline deriviatives, suppress the vasopressor sympathetic outflow to produce hypotension. This effect has been known to be mediated in part by suppressing sympathetic outflow via acting imidazoline I(1) receptors (IR(1)) at postganglionic sympathetic neurons. But, the cellular mechanism of IR(1)-induced inhibition of noradrenaline (NA) release is still unknown. We therefore, investigated the effect of IR(1) activation on voltage-dependent Ca(2+) channels which is known to play an pivotal role in regulating NA in rat superior cervical ganglion (SCG) neurons, using the conventional whole-cell patch-clamp method. In the presence of rauwolscine (3 µΜ), which blocks α(2)-adrenoceptor (R(α2)), moxonidine inhibited voltage-dependent Ca(2+) current (I(Ca)) by about 30%. This moxonidine-induced inhibition was almost completely prevented by efaroxan (10 µΜ) which blocks IR(1) as well as R(α2). In addition, ω-conotoxin (CgTx) GVIA (1 µΜ) occluded moxonidine-induced inhibition of I(Ca), but, moxonidine-induced I(Ca) inhibition was not affected by pertussis toxin (PTX) nor shows any characteristics of voltage-dependent inhibition. These data suggest that moxonidine inhibit voltage-dependent N-type Ca(2+) current (I(Ca-N)) via activating IR(1). Finally, moxonidine significantly decreased the frequency of AP firing in a partially reversible manner. This inhibition of AP firing was almost completely occluded in the presence of ω-CgTx. Taken together, our results suggest that activation of IR(1) in SCG neurons reduced I(Ca-N) in a PTX-and voltage-insensitive pathway, and this inhibition attenuated repetitive AP firing in SCG neurons.

Inhibition of hexokinase leads to neuroprotection against excitotoxicity in organotypic hippocampal slice culture.

During seizures, glucose concentrations are high in the hippocampus. Mitochondrial hexokinase (HK) catalyzes the first essential step of glucose metabolism and directly couples extramitochondrial glycolysis to intramitochondrial oxidative phosphorylation. The neuroprotective effects of an HK inhibitor, 3-bromopyruvate (3-BrPA), on kainic acid (KA)-induced excitotoxic injury were investigated. Hippocampal slices were prepared from hippocampi of 6-8-day-old rats using a tissue chopper and placed on a membrane insert. After a treatment with KA (5 µM) for 15 hr, neuronal death was quantified by propidium iodide (PI), cresol violet, and TUNEL staining. KA-induced cell death was significantly prevented by 30 µM 3-BrPA treatment. According to Western blots, the expression level of phospho-Akt increased after 3-BrPA treatment. The induction of long-term potentiation (LTP) at 48 hr after 3-BrPA treatment tended to increase in the CA1 area compared with the KA-only group, but the difference was not significant. Blocking the PI3 kinase/Akt pathway using LY294002 reversed the neuroprotective effect of 3-BrPA. These results suggest that inhibition of HK may play a protective role against neuronal death in KA-induced excitotoxic injury.

Intracellular acidification evoked by moderate extracellular acidosis attenuates transient receptor potential V1 (TRPV1) channel activity in rat dorsal root ganglion neurons.

Transient receptor potential V1 (TRPV1) has been suggested to play an important role in detecting decreases in extracellular pH (pH(o)). Results from recent in vivo studies, however, have suggested that TRPV1 channels play less of a role in sensing a moderately acidic pH(o) (6.0 < pH < 7.0) than predicted from the in vitro experiments. A clear explanation for this discrepancy between the in vitro and in vivo data has not yet been provided. We report here that intracellular acidification induced by a moderately low pH(o) (6.4) almost completely inhibited the effect of extracellular acidosis on TRPV1 activity. In our experiments, sodium acetate (20 mm), which was used to induce intracellular acidosis, attenuated the capsaicin-evoked TRPV1 current (I(CAP)) in a reversible manner in whole-cell patch-clamp mode and shifted the concentration-response curve to the right. Likewise, the concentration-response curve was significantly shifted to the right by lowering the pH of the pipette solution from 7.2 to 6.5. In addition, application of an acidic bath solution (pH 6.4) to the intracellular side also significantly suppressed I(CAP) in inside-out patch mode. In cell-attached patch mode, the single-channel activity of i(CAP) was significantly attenuated by intracellular acidosis that was induced by a decrease in pH(o) (6.4). These results suggested that intracellular acidification induced by a low pH(o) inhibited TRPV1 activity. When studied in perforated patch mode or by acidifying the intracellular pipette solution, potentiation or activation of TRPV1 by extracellular acidosis (pH 6.4) at 37 °C was almost completely inhibited. Likewise, enhancement of neuronal excitability by a moderately acidic pH(o) (6.4) at a physiological temperature (37 °C) was attenuated by lowering the pH of the pipette solution to 6.5 or using perforated patch mode. Taken together, these results suggest that extracellular acidosis of moderate intensity may not significantly modulate TRPV1 activity in physiological conditions at which intracellular pH can be readily affected by pH(o), and this phenomenon is due to attenuation of TRPV1 channel activity by low-pH(o)-induced intracellular acidification.

Modulation of N-type calcium currents by presynaptic imidazoline receptor activation in rat superior cervical ganglion neurons.

Presynaptic imidazoline receptors (R(i-pre)) are found in the sympathetic axon terminals of animal and human cardiovascular systems, and they regulate blood pressure by modulating the release of peripheral noradrenaline (NA). The cellular mechanism of R(i-pre)-induced inhibition of NA release is unknown. We, therefore, investigated the effect of R(i-pre) activation on voltage-dependent Ca(2+) channels in rat superior cervical ganglion (SCG) neurons, using the conventional whole-cell patch-clamp method. Cirazoline (30 µM), an R(i-pre) agonist as well as an α-adrenoceptor (R(α)) agonist, decreased Ca(2+) currents (I(Ca)) by about 50% in a voltage-dependent manner with prepulse facilitation. In the presence of low-dose rauwolscine (3 µM), which blocks the α(2)-adrenoceptor (R(α2)), cirazoline still inhibited I(Ca) by about 30%, but prepulse facilitation was significantly attenuated. This inhibitory action of cirazoline was almost completely prevented by high-dose rauwolscine (30 µM), which blocks R(i-pre) as well as R(α2). In addition, pretreatment with LY320135 (10 µM), another R(i-pre) antagonist, in combination with low-dose rauwolscine (3 µM), also blocked the R(α2)-resistant effect of cirazoline. Addition of guanosine-5-O-(2-thiodiphosphate) (2 mm) to the internal solutions significantly attenuated the action of cirazoline. However, pertussis toxin (500 ng ml(1)) did not significantly influence the inhibitory effect of cirazoline. Moreover, cirazoline (30 µM) suppressed M current in SCG neurons cultured overnight. Finally, omega-conotoxin (omega-CgTx) GVIA (1 µM) obstructed cirazoline-induced current inhibition, and cirazoline (30 µM) significantly decreased the frequency of action potential firing in a partly reversible manner. This cirazoline-induced inhibition of action potential firing was almost completely occluded in the presence of omega-CgTx. Taken together, our results suggest that activation of R(i-pre) in SCG neurons reduced N-type I(Ca) in a pertussis toxin- and voltage-insensitive pathway, and this inhibition attenuated repetitive action potential firing in SCG neurons.

Modulation of neuropathic pain by galanin and neuropeptide Y at the level of the medulla in rats.

This study was conducted to examine the role of galanin and neuropeptide Y (NPY) in the modulation of neuropathic pain at the level of the medulla. Under pentobarbital anesthesia, Sprague-Dawley rats were subjected to neuropathic surgery. Intracisternal injections of galanin and NPY were performed 2 weeks after nerve injury and mechanical allodynia was monitored. In an electrophysiological experiment, rats were reanesthetized with urethane and the responses of gracile nucleus neurons to mechanical stimulation were observed. Galanin and NPY were applied microiontophoretically. Intracisternally administered NPY reduced neuropathic pain behaviors in a dose-dependent manner. High doses of galanin inhibited neuropathic pain behaviors. Iontophoretically ejected galanin and NPY inhibited responses of gracile nucleus neurons to mechanical stimulation. These results suggest that galanin and NPY play a role in modulating neuropathic pain in the gracile nucleus of the medulla.

Anti-oxidant effect of ascorbic and dehydroascorbic acids in hippocampal slice culture.

Ascorbic acid (AA) and dehydroascorbic acid (DHA) have been shown to have protective effects as anti-oxidants in experimental neurological disorder models such as stroke, ischemia, and epileptic seizures. The present study was conducted to examine the protective effects of AA and DHA on kainic acid (KA) neurotoxicity using organotypic hippocampal slice cultures. After 12h KA treatment, significant delayed neuronal death was detected in the CA3, but not the CA1, region. Pretreatment with intermediate doses of AA and DHA significantly prevented cell death and inhibited reactive oxygen species (ROS) level, and mitochondrial dysfunction in the CA3 region. In contrast, pretreatment with low or high doses of AA or DHA was not effective. These data suggest that pretreatment with both AA and DHA has dose-dependent neuroprotective effects on KA-induced neuronal injury through inhibiting ROS generation and mitochondrial dysfunction.

Repetitive motor cortex stimulation reinforces the pain modulation circuits of peripheral neuropathic pain.

Recent evidence indicates that motor cortex stimulation (MCS) is a potentially effective treatment for chronic neuropathic pain. However, the neural mechanisms underlying the attenuated hyperalgesia after MCS are not completely understood. In this study, we investigated the neural mechanism of the effects of MCS using an animal model of neuropathic pain. After 10 daily sessions of MCS, repetitive MCS reduced mechanical allodynia and contributed to neuronal changes in the anterior cingulate cortex (ACC). Interestingly, inhibition of protein kinase M zeta (PKMζ), a regulator of synaptic plasticity, in the ACC blocked the effects of repetitive MCS. Histological and molecular studies showed a significantly increased level of glial fibrillary acidic protein (GFAP) expression in the ACC after peripheral neuropathy, and neither MCS treatment nor ZIP administration affected this increase. These results suggest that repetitive MCS can attenuate the mechanical allodynia in neuropathic pain, and that the activation of PKMζ in the ACC may play a role in the modulation of neuropathic pain via MCS.

Activation of spinal GABA receptors attenuates chronic central neuropathic pain after spinal cord injury.

In this study, we investigated the role of the spinal GABAergic system in central neuropathic painlike outcomes following spinal cord injury (SCI) produced by a spinal hemitransection at T13 of the rat. After SCI, mechanical allodynia develops bilaterally in both hind paws of the rat, lasting longer than 40 days, as evidenced by an increase in paw withdrawal frequency in response to a weak von Frey filament. In naive rats, intrathecal (i.t.) administration in the lumbar spinal cord of GABAA and GABAB receptor antagonists, bicuculline (1-5 microg) and phaclofen (0.1-5 microg), respectively, causes a dose-dependent increase in the magnitude of mechanical allodynia. The SCI-induced mechanical allodynia in both hind-paws is attenuated by i.t. administration in the lumbar spinal cord of GABAA or GABAB receptor agonists, muscimol (1 microg) or baclofen (0.5 microg), respectively. In electrophysiological experiments, rats with SCI show a bilateral increase in hyperexcitability in response to natural stimuli in wide dynamic range (WDR) neurons in the lumbar spinal dorsal horn. The topical application of muscimol (1 microg) or baclofen (0.5 microg) onto the lumbar cord surface reduce the SCIinduced increased responsiveness of WDR neurons. Inhibitory effects of muscimol and baclofen on both the behavioral mechanical allodynia and the hyperexcitability in WDR neuron with SCI compared to controls, were antagonized by pre-treatment of bicuculline (10 microg) and phaclofen (5 microg), respectively. This study provides behavioral and electrophysiological evidence for the important role of the loss of spinal inhibitory tone, mediated by activation of both GABAA and GABAB receptors, in the development of central neuropathic pain following SCI.

Involvement of peripherally released substance P and calcitonin gene-related peptide in mediating mechanical hyperalgesia in a traumatic neuropathy model of the rat.

We hypothesized that neuropeptides released from the peripheral terminals of primary afferents play an important role in mechanical hyperalgesia after peripheral nerve injury. Nerve injury was performed on rats with lumbar 5 spinal nerve lesion (L5 SNL), which was preceded by L5 dorsal rhizotomy (L5 DR) to avoid the potential central effects induced by L5 SNL through the L5 dorsal root. L5 DR produced a short-lasting (<6 days) decrease in paw withdrawal threshold (PWT) while the following L5 SNL produced a persistent (>42 days) PWT decrease. When intraplantar injection to the affected hind paw was given immediately before L5 SNL, antagonists for both neurokinin 1 (NK1) and calcitonin gene-related peptide 1 (CGRP1) receptors delayed the onset of the PWT decrease for 2-4 days. However, when the same injection was given after L5 SNL, CGRP1, but not NK1, receptor antagonist reversed the decreased PWT for 105 min. It is suggested that peripherally released neuropeptides contribute to the generation of neuropathic pain, with substance P and CGRP contributing to its induction phase, but only CGRP to its maintenance phase.

Attenuation of mechanical hyperalgesia following spinal cord injury by administration of antibodies to nerve growth factor in the rat.

Spinal cord injury (SCI) often leads to central pain syndrome including hyperalgesia to mechanical stimulation. Since there is evidence that nerve growth factor (NGF) contributes to pain-related behaviors, we wished to determine if anti-NGF might inhibit abnormal somatosensory behaviors that develop following SCI in rats. SCI was performed in male Sprague-Dawley rats by T13 spinal hemisection. After spinal hemisection, animals were untreated or treated daily with anti-NGF or saline intraperitoneally for 10 days. In groups of both hemisection only and hemisection with saline treatment, mechanical hyperalgesia developed in both hindlimbs, as evidenced by a decrease in paw withdrawal thresholds. Mechanical responsiveness of wide dynamic range (WDR) neurons on both sides of spinal cord also increased. The anti-NGF treated group demonstrated significant suppression of both mechanical hyperalgesia and increased WDR neuronal responsiveness. These results indicate that anti-NGF prevents the development of abnormal somatosensory behavior and suggest a potential pre-emptive analgesic treatment for central pain.

Highly pure and expandable PSA-NCAM-positive neural precursors from human ESC and iPSC-derived neural rosettes.

Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPC(PSA-NCAM+)) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPC(PSA-NCAM+) extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80∼98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPC(PSA-NCAM+) showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro. When transplanted into the rat striatum, hNPC(PSA-NCAM+) differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPC(PSA-NCAM+) compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.

Modification of cortical excitability in neuropathic rats: a voltage-sensitive dye study.

Recent advances in optical imaging techniques have made it possible to monitor neural activity and provided powerful tools to reveal the spatiotemporal patterns of neural activity. We used optical imaging to determine whether nerve injury affects excitability of the sensory cortex. Male Sprague-Dawley rats were subjected to neuropathic surgery consisting of a tight ligation and transection of the left tibial and sural nerves while under pentobarbital anesthesia. The rats were reanesthetized with urethane two weeks post-operatively, and the exposed cortex surfaces were stained with a voltage-sensitive dye (di-2-ANEPEQ). After electrical stimulation of the receptive field, optical signals from the cerebral cortex were recorded using an optical imaging system. Increased optical intensity and an enlarged area of activation were observed in the cerebral cortex of neuropathic rats during electrical stimulation compared to normal or sham-operated rats. Higher electric stimulation resulted in more intensity and a larger area of activation in neuropathic rats. These results suggest that cortical excitability, resulting from peripheral stimulation, may be affected by nerve injury, which indicates a degree of neural plasticity.

The role of uninjured C-afferents and injured afferents in the generation of mechanical hypersensitivity after partial peripheral nerve injury in the rat.

This study was performed to determine which of uninjured lumbar 4 (L4) C-afferents and injured L5 afferents was important for the generation of mechanical hypersensitivity following L5 spinal nerve ligation-and-cut (SNLC, modified spinal nerve ligation) in the rat. The mechanical hypersensitivity established following L5 SNLC was completely abolished 6 weeks after local capsaicin treatment of the sciatic nerve or L4 spinal nerve. At this stage, a substantial number of capsaicin-sensitive C-afferents were eliminated without any loss of A-afferents in the L4 spinal segment, suggesting that the capsaicin-sensitive L4 C-afferents are a major contributor to L5 SNLC-produced mechanical hypersensitivity. The peripheral terminals of L4 C-afferents are active in maintaining mechanical hypersensitivity, even long after L5 SNLC. When capsaicin-sensitive L4 C-afferents were previously eliminated, the induction of L5 SNLC-produced hypersensitivity was partly prevented. Thus, capsaicin-sensitive L4 C-afferents are crucial for the induction and maintenance of mechanical hypersensitivity in the L5 SNLC model. Also, when capsaicin-sensitive L4 C-afferents were previously eliminated, L5 SNLC still produced a partial mechanical hypersensitivity for a 1- to 2-week maintenance period with a several-day delay. This mild hypersensitivity was prevented by the previous L5 dorsal rhizotomy, implying an involvement of inputs from injured L5 afferents in the maintenance of hypersensitivity at the earlier stage. The results suggest that uninjured C-afferents, most likely C-polymodal nociceptors, are necessary for the induction and maintenance of neuropathic pain, and that afferent inputs, presumably from injured Abeta-fibers, also contribute to the maintenance at an earlier stage.

Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells.

To induce differentiation of embryonic stem cells (ESCs) into specialized cell types for therapeutic purposes, it may be desirable to combine genetic manipulation and appropriate differentiation signals. We studied the induction of dopaminergic (DA) neurons from mouse ESCs by overexpressing the transcription factor Nurr1 and coculturing with PA6 stromal cells. Nurr1-expressing ESCs (N2 and N5) differentiated into a higher number of neurons (approximately twofold) than the naïve ESCs (D3). In addition, N2/N5-derived cells contained a significantly higher proportion (>50%) of tyrosine hydroxylase (TH)+ neurons than D3 (<30%) and an even greater proportion of TH+ neurons (approximately 90%) when treated with the signaling molecules sonic hedgehog, fibroblast growth factor 8, and ascorbic acid. N2/N5-derived cells express much higher levels of DA markers (e.g., TH, dopamine transporter, aromatic amino acid decarboxylase, and G protein-regulated inwardly rectifying K+ channel 2) and produce and release a higher level of dopamine, compared with D3-derived cells. Furthermore, the majority of generated neurons exhibited electrophysiological properties characteristic of midbrain DA neurons. Finally, transplantation experiments showed efficient in vivo integration/generation of TH+ neurons after implantation into mouse striatum. Taken together, our results show that the combination of genetic manipulation(s) and in vitro cell differentiation conditions offers a reliable and effective induction of DA neurons from ESCs and may pave the way for future cell transplantation therapy in Parkinson's disease.

Inhibition of carbachol-evoked oscillatory currents by the NO donor sodium nitroprusside in guinea-pig ileal myocytes.

The effect of sodium nitroprusside (SNP) on carbachol (CCh)-evoked inward cationic current (Icat) oscillations in guinea-pig ileal longitudinal myocytes was investigated using the whole-cell patch-clamp technique and permeabilized longitudinal muscle strips. SNP (10 microm) completely inhibited I(cat) oscillations evoked by 1 microm CCh. 1H-(1,2,4) Oxadiazole [4,3-a] quinoxaline-1-one (ODQ; 1 microm) almost completely prevented the inhibitory effect of SNP on Icat oscillations. 8-Bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP; 30 microm) in the pipette solution completely abolished Icat oscillations. However, a pipette solution containing Rp-8-Br-cGMP (30 microm) almost completely abolished the inhibitory effect of SNP on Icat oscillations. When the intracellular calcium concentration ([Ca2+]i) was held at a resting level using BAPTA (10 mm) and Ca2+ (4.6 microm) in the pipette solution, CCh (1 microm) evoked only the sustained component of Icat without any oscillations and SNP did not affect the current. A high concentration of inositol 1,4,5-trisphosphate (IP3; 30 microm) in the patch pipette solutions significantly reduced the inhibitory effect of SNP (10 microm) on Icat oscillations. SNP significantly inhibited the Ca2+ release evoked by either CCh or IP3 but not by caffeine in permeabilized preparations of longitudinal muscle strips. These results suggest that the inhibitory effects of SNP on Icat oscillations are mediated, in part, by functional modulation of the IP3 receptor, and not by the inhibition of cationic channels themselves or by muscarinic receptors in the plasma membrane. This inhibition seems to be mediated by an increased cGMP concentration in a protein kinase G-dependent manner.