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D W Sah

Publications and source records attributed to D W Sah.

17 recordsLinked to original sources
PubMed2001

GFRalpha3 is expressed predominantly in nociceptive sensory neurons.

Activation of the RET receptor tyrosine kinase by glial-derived neurotrophic factor family members is dependent on a family of coreceptors, GFRalpha1-4. GFRalpha3 preferentially binds the newest member of the glial-derived neurotrophic factor family of ligands, artemin. The major site of GFRalpha3 expression is in the dorsal root ganglion; however, the class of sensory neurons that expresses GFRalpha3 has not been reported previously. Using immunohistochemical methods, we show that the majority of dorsal root ganglion cells that express GFRalpha3 also express vanilloid receptor type 1, peripherin, RET, trkA and calcitonin gene-related peptide. In addition, a significant subpopulation of GFRalpha3-expressing cells also binds the lectin IB4. We demonstrate that GFRalpha3 artemin neurons are immunopositive for markers expected of nociceptors and include a subset of neurons distinct from the GDNF-responsive population. Our results indicate artemin may exert selective effects on pain sensation.

Animals↗
PubMed2000

Motoneuron differentiation of immortalized human spinal cord cell lines.

Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

Cell Differentiation↗
PubMed1999

Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

Action Potentials↗
PubMed1997

Regulation of voltage- and ligand-gated currents in rat hippocampal progenitor cells in vitro.

Growth medium and substrate regulate the proliferation and differentiation of neural progenitor cells in vitro and the expression of cell type-specific histochemical markers. An important question is whether neural progenitor cells exhibit voltage- and ligand-gated currents, features characteristic of neurons, and whether these currents are regulated differentially by growth conditions. Another issue of interest is whether passaged progenitor cells, after expansion with basic fibroblast growth factor (FGF-2), exhibit the same degree of plasticity as their primary counterparts, or whether they are more committed to a particular phenotype. In primary cultures of embryonic rat hippocampal progenitor cells, growth in proliferative conditions (FGF-2) was associated with low levels of sodium, calcium, N-methyl-D-aspartate (NMDA), and kainate currents compared with other growth conditions. After multiple passages in the continued presence of FGF-2, sodium, calcium, and NMDA, responses declined further; interestingly, kainate and gamma-aminobutyric acid (GABA) responses remained substantial. Moreover, the expression of functional channels and receptors in primary cultures of progenitor cells is up-regulated strongly by growth factors such as BDNF, and NT-3, whereas sodium and calcium currents in passaged cultures respond to such growth conditions to a lesser extent. Kainate and GABA responses were present to a significant extent in passaged cultures, independent of growth condition. We conclude that environmental cues regulate different channels and receptors in distinct ways in neural progenitor cells.

Animals↗
PubMed1997

Bipotent progenitor cell lines from the human CNS.

Human central nervous system (CNS) cell lines would substantially facilitate drug discovery and basic research by providing a readily renewable source of human neurons. We isolated clonal human CNS cell lines that had been immortalized with a tetracycline (Tc)-responsive v-myc oncogene; addition of Tc to the growth medium suppressed the oncoprotein rapidly and virtually completely, allowing differentiation to proceed. Two classes of bipotent precursor cells were immortalized: the first class had a default differentiation pathway of neurons only, and the second class had a default differentiation pathway of neurons and astrocytes. We found that after exposure to different external signals in vitro, the environment is capable of redirecting the fate of a particular cell, even in the case of the bipotent precursor cell whose default differentiation pathway was neurons only. These data suggest that extrinsic cues can prevail over intrinsic determinants in directing cell fate in the human CNS.

Astrocytes↗
PubMed1996

Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

Animals↗
PubMed1995

Human fetal central neurons in culture: voltage- and ligand-gated currents.

1. The functional properties of sodium, potassium, calcium, N-methyl-D-aspartate (NMDA), kainate, and gamma-aminobutyric acid (GABA) currents were studied in dissociated monolayer cultures of fetal human brain neurons, using the whole cell patch-clamp technique. 2. Sodium currents were characterized with respect to the following properties: current density, voltage dependence of activation, voltage dependence of inactivation, and sensitivity to tetrodotoxin (TTX). All sodium currents exhibited voltage dependencies of activation and inactivation, and sensitivities to TTX that are characteristic of the neuronal form of the sodium current. 3. At least two types of potassium current were present, resembling the delayed rectifier and fast-inactivating potassium current. These two types of potassium current were distinguishable by their different kinetics, voltage dependencies of activation and inactivation, and sensitivities to 4-aminopyridine and tetraethylammonium. 4. High-voltage-activated calcium channel currents were present and were characterized with respect to current density, voltage dependencies of activation and inactivation, and sensitivity to cadmium. Low-voltage-activated calcium channel currents were also present. 5. NMDA- and kainate-gated currents were studied with respect to current density, time course, and current-voltage relationship. Kainate currents were also characterized with respect to inhibition by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In addition, NMDA and kainate responses were compared for cortical versus cerebellar neurons. NMDA responses, which are only found in neurons, were present, confirming the neuronal phenotype suggested by the presence of the neuronal form of the sodium current. Nondesensitizing kainate currents were also present, with a half-maximally effective concentration (EC50) of approximately 200 microM for kainate; CNQX inhibited the kainate current with a half-inactivating concentration of 0.55 microM. 6. GABA-gated currents were characterized with respect to current density, time course, receptor subtype, desensitization, dose response, current-voltage relationship, ionic selectivity, pharmacology, and potentiation by the neurosteroid 5 alpha-pregnan-3 alpha-ol-11,20-dione (alfaxalone). Desensitizing GABAA currents were selective for chloride, inhibited by bicuculline and tert-butyl-bicyclophosphorothionate, and potentiated by diazepam, pentobarbital sodium, and alfaxalone. The EC50 for GABA was 15 microM.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1994

Inhibition of P-type and N-type calcium channels by dopamine receptor antagonists.

P-type Ca2+ channels in cerebellar Purkinje neurons and N-type Ca2+ channels in sympathetic neurons were found to be inhibited by D2 dopamine receptor antagonists with diverse structures, including phenothiazines (chlorpromazine and thioridazine), diphenylbutylpiperidines (fluspirilene and pimozide), butyrophenones (haloperidol and spiperone), and a piperazine (fluphenazine). Dopamine and quinpirole had no effect on P-type Ca2+ channels. In all cases, inhibition was characterized by slow onset and offset. The effects of P-type and N-type channels were very similar. Fluspirilene was the most potent of the drugs, with EC50 values of 6 microM for P-type current and 2 microM for N-type current. Block of P-type channels by fluspirilene was voltage dependent, being enhanced by depolarized holding potentials, and use dependent, being enhanced by higher stimulation frequencies. The effect of fluspirilene on the P-type Ca2+ channel current was not prevented by simultaneous exposure to the peptide toxin omega-agatoxin IVA, indicating that fluspirilene binds to a distinct site on the channel. The results suggest that N-type and P-type Ca2+ channels possess similar binding sites for dopamine receptor antagonists and that block of N-type and P-type channels is relatively weak, compared with that of some T-type and L-type Ca2+ channels.

Animals↗
PubMed1991

Ca2+ channels in rat central and peripheral neurons: high-threshold current resistant to dihydropyridine blockers and omega-conotoxin.

Block of Ca2+ channel current by dihydropyridines and by omega-conotoxin (omega-CgTx) was studied in a variety of freshly dissociated rat neurons. In most neurons, including those from dorsal root ganglia, sympathetic ganglia, spinal cord, cerebral cortex, and hippocampus, nitrendipine and omega-CgTx each blocked a fraction of the high-threshold current, but a substantial fraction of current remained even when the two blockers were applied together at saturating concentrations. An extreme case was cerebellar Purkinje neurons, in which very little current was blocked by either nitrendipine or omega-CgTx. These results demonstrate the existence in mammalian neurons of high-threshold channels that are resistant to both omega-CgTx and dihydropyridine blockers. Such channels might underlie instances of synaptic transmission and other processes that depend on Ca2+ entry but are not sensitive to these blockers.

Animals↗
PubMed1990

Different biological activities in conditioned media control the expression of a variety of neuropeptides in cultured sympathetic neurons.

An intriguing question regarding neuronal development is how neurons choose which neurotransmitter and/or peptide to express among over 40 candidates. We find that heart cell conditioned medium (CM) induces a number of neuropeptides and/or their precursor mRNAs, as well as acetylcholine, in cultured rat sympathetic neurons: substance P, somatostatin, vasoactive intestinal polypeptide, enkephalin derivatives, and cholecystokinin, but not neuropeptide Y. Different patterns of peptide induction were observed for CMs from primary cultures of heart, gut, and skin. Acetylcholine and substance P were induced most effectively by serum-free heart cell CM; enkephalin derivatives were induced most effectively by skin cell CM; and somatostatin and vasoactive intestinal polypeptide were induced equally well by all of the CMs. These observations suggest the possibility that many distinct, diffusible factors can influence the choice of transmitter and/or peptide phenotype in developing neurons.

Animals↗
PubMed1990

Neurotransmitter modulation of calcium current in rat spinal cord neurons.

The modulation of Ca2+ currents by neurotransmitters was studied in freshly dissociated rat spinal cord neurons, using the whole-cell patch-clamp technique. GABA, baclofen, adenosine, ATP, serotonin, norepinephrine, somatostatin, and dynorphin A inhibited the current through Ca2+ channels in a substantial fraction of cells, while substance P, vasoactive intestinal polypeptide, [D-ala2,d-leu5]-enkephalin, cholecystokinin-8 (sulfated), calcitonin gene-related peptide, angiotensin II, neurotensin, vasopressin, and thyrotropin-releasing hormone had no effect. In the case of baclofen, the inhibition is mediated, at least in part, by a GTP-binding protein. Suppression of Ca2+ current by neurotransmitters may represent a mechanism of presynaptic inhibition in the spinal cord.

Animals↗
PubMed1988

The ultrastructural distribution of putative nicotinic receptors on cultured neurons from the rat superior cervical ganglion.

The distribution of putative nicotinic receptors on cultured neurons from the rat superior cervical ganglion was determined by electron microscopic autoradiography using a radioactively labeled snake venom neurotoxin, toxin F. In a previous study, we demonstrated that toxin F blocks nicotinic transmission in these cultures of sympathetic neurons and in intact superior cervical ganglia. [125I]toxin F bound to two sites in these cultures: one site that was also recognized by the neuromuscular blocker, alpha-bungarotoxin, and a second site that was not. Since alpha-bungarotoxin neither blocks nicotinic transmission nor prevents the blocking effects of toxin F, the site specific to the binding of toxin F most probably represents neuronal nicotinic receptors. The total number of each of the toxin F binding sites was unaffected by culture conditions that are known to influence the extent to which these sympathetic neurons synthesize norepinephrine or acetylcholine. Autoradiographic analysis performed under saturating binding conditions (80 nM [125I]toxin F) revealed that the density of [125I]toxin F binding at synaptic membranes was about 5000 sites/micron 2, either in the absence of any competing ligand or in the presence of 2 microM alpha-bungarotoxin. In the presence of 2 microM unlabeled toxin F, there was no detectable binding at synapses. The density of these toxin F-specific sites was at least 80-fold higher at synaptic membranes than elsewhere. On the other hand, the data suggest that the toxin F binding site shared with alpha-bungarotoxin is exclusively extrasynaptic. Two micromolar alpha-bungarotoxin decreased the density of [125I]toxin F binding at non-synaptic sites by approximately two-thirds. These experiments support the hypothesis that toxin F blocks cholinergic transmission in cultures of sympathetic neurons by binding to nicotinic receptors and suggests that these receptors are highly clustered at synaptic membranes.

Animals↗
PubMed1987

Long-term blockade by toxin F of nicotinic synaptic potentials in cultured sympathetic neurons.

The effects of a recently identified blocker of neuronal nicotinic transmission, toxin F, were studied in cultured sympathetic neurons. Single principal neurons, dissociated from superior cervical ganglia of newborn rats, were grown on cardiac myocytes in microculture. The toxin blocked nicotinic synaptic potentials in these cultures but had no effect on muscarinic interactions. When toxin F was applied by addition to the perfusion medium, the concentration required for blocking most of the nicotinic potential was 40 nM, and the recovery from blockade was slow (t1/2 = 95 +/- 64 min). When the toxin was briefly applied by pressure ejection from a pipette, the concentration in the pipette necessary for blockade was 21 microM, and 30-60% of the response recovered within a few minutes while the remainder recovered slowly (t1/2 of the remainder = 105 +/- 82 min). One possible explanation for the difference in recovery time is that toxin F binds initially with low affinity to the nicotinic receptor but with time the toxin receptor complex converts to a high affinity state. The presence of dihydro-beta-erythroidine during perfusion of toxin F prevented the long-lasting blockade by the toxin, suggesting that toxin F and dihydro-beta-erythroidine act through a common binding site. The specificity, potency, and slow reversibility of the effects of toxin F make it useful as a probe for studying neuronal nicotinic receptors of cultured sympathetic neurons.

Adrenergic Fibers↗
PubMed1987

Evidence for serotonin synthesis, uptake, and release in dissociated rat sympathetic neurons in culture.

In the previous paper (Matsumoto et al., 1987), pharmacological evidence for secretion of serotonin (5-HT) by cultured sympathetic principal neurons was reported. Here, we present further evidence that 5-HT is a transmitter of some of these neurons under certain culture conditions, and can also be a "false" transmitter. Sympathetic principal neurons, dissociated from superior cervical ganglia (SCG) of newborn rats, were grown in cell culture. The presence of serotonin was demonstrated with high-performance liquid chromatography (HPLC), immunocytochemistry, and electrophysiological recording. Homogenates of cultures contained a substance that comigrated with authentic 5-HT in HPLC. A voltammogram for this substance was superimposed upon that for authentic 5-HT. When the cultures were examined with immunocytochemical staining, using the peroxidase-antiperoxidase technique, many neuronal processes contained 5-HT-like immunoreactive material. Many somata also contained 5-HT-like immunoreactivity when the neurons were grown in the presence of medium conditioned by heart cells (CM), but few somata stained above background in the absence of CM. Medium that contained a raised concentration of K+ (54 mM) or veratridine evoked Ca2+-dependent release of 5-HT, consistent with a neurotransmitter role for 5-HT in the cultures. In preliminary electrophysiological experiments on microcultures containing single sympathetic principal neurons and cardiac myocytes, a nonadrenergic excitatory (NAE) interaction was sometimes obtained after exposing the neurons to 100 microM 5-hydroxytryptophan. This interaction was sensitive to the serotonin blockers reserpine, methysergide, and gramine.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Hydroxytryptophan↗
PubMed1986

Reformation of specific synaptic connections by regenerating sensory axons in the spinal cord of the bullfrog.

The regrowth of sensory axons into the spinal cord of juvenile bullfrogs was studied after disruption of these fibers in the dorsal root. Within 9 d after the root had been frozen, regenerating sensory axons had reached the spinal cord, as revealed by labeling with horseradish peroxidase. Growth into the spinal cord, however, was much slower. Even several months after denervation, very few fibers had reestablished any of their normal longitudinal projections within the dorsal funiculus. Eventually, however, sensory axons grew across the region and into the dorsal horn. Intracellular recordings from motoneurons revealed that these axons made functional reconnections with spinal neurons. Muscle sensory axons established direct, monosynaptic inputs to motoneurons, whereas cutaneous fibers innervated these neurons polysynaptically. Moreover, sensory afferents from a particular muscle distinguished among different classes of motoneurons, just as in normal frogs. Thus, specific synaptic pathways can be reestablished by regenerating sensory axons if they can reach their appropriate target region within the spinal cord.

Animals↗
PubMed1984

Regeneration of sensory-motor synapses in the spinal cord of the bullfrog.

Sensory fibers innervating muscles in the arm of the bullfrog form specific patterns of monosynaptic connections with motoneurons in the spinal cord. We show here that these normal patterns are re-established after interruption of the second dorsal root (DR2) in tadpoles and postmetamorphic frogs. DR2 was either cut or crushed, and 2 to 8 months later the extent and specificity of regeneration were assessed anatomically and physiologically. Horseradish peroxidase labeling of DR2 showed that sensory afferents had regenerated back into the spinal cord to form local arborizations, as in the normal adult. However, few long-tract fibers in the dorsal columns regenerated. Intracellular recording from different classes of motoneurons at the level of DR2 revealed that triceps muscle sensory afferents had regenerated to form functionally appropriate synapses. As in the normal adult, stimulation of the triceps nerves elicited larger monosynaptic EPSPs in triceps motoneurons than in non-triceps motoneurons. Thus, in the central nervous system of the bullfrog, specific monosynaptic connections are re-formed within the region of local regeneration.

Animals↗