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Colin A Nurse - One of the best experts on this subject based on the ideXlab platform.

  • carotid body chemoreceptors in Dissociated Cell Culture
    Microscopy Research and Technique, 2002
    Co-Authors: Colin A Nurse, Ian M Fearon
    Abstract:

    Carotid body (CB) glomus or type 1 Cells act as peripheral chemoreceptors which detect changes in arterial PO2, PCO2, and pH and help maintain homeostasis via the reflex control of ventilation. Over the last ∼12 years significant progress has been made towards understanding chemotransduction mechanisms using freshly isolated or Cultured type 1 Cells. The latter preparation allows several powerful experimental manipulations (e.g., co-Culture with sensory neurons) resulting in significant advances in our understanding of CB chemoreception. Here, we review several properties of type 1 Cells after several days to weeks in Culture. Typically, Cultured type 1 Cells grow in monolayer clusters enveloped by glial-like, type II, or sustentacular Cells, which are immunopositive for the glial marker, glial fibrillary acid protein (GFAP). These Cells can undergo DNA synthesis, evidenced by uptake of bromodeoxyuridine (BrdU), and show a limited capacity for Cell division. Mitosis and survival of type 1 Cells can be regulated by oxygen tension and/or growth factors (e.g., bFGF, insulin). In the rat, type 1 Cells are immunopositive for several monoaminergic markers, including tyrosine hydroxylase (TH), dopamine transporter (DAT), and 5-HT. They also express cholinergic markers (e.g., vesicular acetylcholine transporter; VAChT), the highly conserved synaptic vesicle protein (SV2), and gap junctional proteins including Connexin 32 (Cx32). Moreover, in long-term Culture (∼2 weeks) they retain expression of O2-sensitive, TASK-1-like, and Ca2+-dependent (BK), K+ channels as revealed by immunocytochemistry or RT-PCR analysis of mRNA extracted from type 1 clusters after removal from the Culture surface. Microsc. Res. Tech. 59:249–255, 2002. © 2002 Wiley-Liss, Inc.

  • electrophysiological characterization of 5 ht receptors on rat petrosal neurons in Dissociated Cell Culture
    Brain Research, 1999
    Co-Authors: Huijun Zhong, Min Zhang, Colin A Nurse
    Abstract:

    Abstract The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT). Here, we combined rapid 5-HT application with patch clamp, whole-Cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), Cultured from 7–12 day-old rat pups. In responsive Cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in ∼43% of the neurons (53/123); however, in a minority population (∼6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease. In the former group, 5-HT produced a transient inward current (I5-HT) in neurons voltage-clamped near the resting potential (∼-60 mV); the effect was mimicked by the 5-HT3 receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT3 receptors. Further, I5-HT was selectively inhibited by the 5-HT3 receptor-specific antagonist MDL72222 (1–10 μM), but was unaffected by either 5-HT1/5-HT2 receptor antagonist, spiperone, or by 5-HT2 receptor-specific antagonist, ketanserin (50–100 μM). I5-HT displayed moderate inward rectification and had a mean reversal potential (±S.E.M.) of −4.3±6.6 mV (n=6). Application of 5-HT (dose range: 0.1–100 μM) produced a dose–response curve that was fitted by the Hill equation with EC50=∼3.4 μM and Hill coefficient=∼1.6 (n=8). The activation phase of I5-HT (10 μM 5-HT at −60 mV) was well fitted by a single exponential with mean (±S.E.M.) time constant of 45±30 ms (n=6). The desensitization phase of I5-HT was best fitted by a single exponential with mean (±S.E.M.) time constant of 660±167 ms (n=6). Fluctuation analysis yielded an apparent mean single-channel conductance (±S.E.M) of 2.7±1.5 pS (n=4) at −60 mV. In the minority (∼6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT2 receptor antagonist, ketanserin (50 μM). Taken together, these results suggest that 5-HT3 receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets.

  • nicotinic acetylcholine sensitivity of rat petrosal sensory neurons in Dissociated Cell Culture
    Brain Research, 1997
    Co-Authors: Huijun Zhong, Colin A Nurse
    Abstract:

    Abstract Using whole-Cell, patch-clamp techniques we investigated acetylcholine (ACh) sensitivity of Dissociated sensory neurons from rat petrosal ganglia after 4 h–14 days in vitro. In approx. 68% of petrosal neurons (PN; n =109) ACh, applied by fast perfusion or pressure ejection from a `puffer' pipette, caused a rapid depolarization associated with a conductance increase. Under voltage clamp near the resting potential (approx. −60 mV), ACh induced a hexamethonium-sensitive, inward current ( I ACh ), mimicked by nicotine application, suggesting the presence of neuronal nicotinic acetylcholine receptors (nAChR). The reversal potential of I ACh occurred near 0 mV ( n =4), a region where the I-V curve displayed a prominent rectification. The dose-response relation for I ACh versus ACh concentration was fitted by the Hill equation with EC 50 =approx. 33.9 μ M and Hill coefficient=approx. 1.6. The activation phase of I ACh was well fitted by a single exponential with mean (±S.E.M.) time constant of 102±82 ms ( n =6); the desensitization phase of I ACh was best fitted by the sum of two exponentials, with time constant of 870±210 ms ( n =6) and 8576±1435 ms (at −70 mV). Fluctuation analysis yielded an apparent single-channel conductance of 21.6±10 pS (mean±S.E.M.; n =4). These data indicate that a major subpopulation of sensory neurons in visceral petrosal ganglia of the rat express nAChR. Thus, if similar receptors are present on corresponding nerve terminals, they could mediate fast afferent excitation in response to ACh released at peripheral targets, e.g., the chemosensory carotid body.

Huijun Zhong - One of the best experts on this subject based on the ideXlab platform.

  • electrophysiological characterization of 5 ht receptors on rat petrosal neurons in Dissociated Cell Culture
    Brain Research, 1999
    Co-Authors: Huijun Zhong, Min Zhang, Colin A Nurse
    Abstract:

    Abstract The petrosal ganglion supplies chemoafferent pathways via the glossopharyngeal (IXth) nerve to peripheral targets which release various neurotransmitters including serotonin (5-HT). Here, we combined rapid 5-HT application with patch clamp, whole-Cell recording to investigate whether 5-HT receptors are expressed on isolated petrosal neurons (PN), Cultured from 7–12 day-old rat pups. In responsive Cells, the dominant effect of 5-HT was a rapid depolarization associated with a conductance increase in ∼43% of the neurons (53/123); however, in a minority population (∼6%; 8/123), 5-HT caused membrane depolarization associated with a conductance decrease. In the former group, 5-HT produced a transient inward current (I5-HT) in neurons voltage-clamped near the resting potential (∼-60 mV); the effect was mimicked by the 5-HT3 receptor-specific agonist, 2-methyl-5-HT, suggesting it was mediated by 5-HT3 receptors. Further, I5-HT was selectively inhibited by the 5-HT3 receptor-specific antagonist MDL72222 (1–10 μM), but was unaffected by either 5-HT1/5-HT2 receptor antagonist, spiperone, or by 5-HT2 receptor-specific antagonist, ketanserin (50–100 μM). I5-HT displayed moderate inward rectification and had a mean reversal potential (±S.E.M.) of −4.3±6.6 mV (n=6). Application of 5-HT (dose range: 0.1–100 μM) produced a dose–response curve that was fitted by the Hill equation with EC50=∼3.4 μM and Hill coefficient=∼1.6 (n=8). The activation phase of I5-HT (10 μM 5-HT at −60 mV) was well fitted by a single exponential with mean (±S.E.M.) time constant of 45±30 ms (n=6). The desensitization phase of I5-HT was best fitted by a single exponential with mean (±S.E.M.) time constant of 660±167 ms (n=6). Fluctuation analysis yielded an apparent mean single-channel conductance (±S.E.M) of 2.7±1.5 pS (n=4) at −60 mV. In the minority (∼6%) population of neurons which responded to 5-HT with a conductance decrease, the depolarization was blocked by the 5-HT2 receptor antagonist, ketanserin (50 μM). Taken together, these results suggest that 5-HT3 receptors are the major subtype expressed by rat petrosal neurons, and therefore are candidates for facilitating chemoafferent excitation in response to 5-HT released from peripheral targets.

  • nicotinic acetylcholine sensitivity of rat petrosal sensory neurons in Dissociated Cell Culture
    Brain Research, 1997
    Co-Authors: Huijun Zhong, Colin A Nurse
    Abstract:

    Abstract Using whole-Cell, patch-clamp techniques we investigated acetylcholine (ACh) sensitivity of Dissociated sensory neurons from rat petrosal ganglia after 4 h–14 days in vitro. In approx. 68% of petrosal neurons (PN; n =109) ACh, applied by fast perfusion or pressure ejection from a `puffer' pipette, caused a rapid depolarization associated with a conductance increase. Under voltage clamp near the resting potential (approx. −60 mV), ACh induced a hexamethonium-sensitive, inward current ( I ACh ), mimicked by nicotine application, suggesting the presence of neuronal nicotinic acetylcholine receptors (nAChR). The reversal potential of I ACh occurred near 0 mV ( n =4), a region where the I-V curve displayed a prominent rectification. The dose-response relation for I ACh versus ACh concentration was fitted by the Hill equation with EC 50 =approx. 33.9 μ M and Hill coefficient=approx. 1.6. The activation phase of I ACh was well fitted by a single exponential with mean (±S.E.M.) time constant of 102±82 ms ( n =6); the desensitization phase of I ACh was best fitted by the sum of two exponentials, with time constant of 870±210 ms ( n =6) and 8576±1435 ms (at −70 mV). Fluctuation analysis yielded an apparent single-channel conductance of 21.6±10 pS (mean±S.E.M.; n =4). These data indicate that a major subpopulation of sensory neurons in visceral petrosal ganglia of the rat express nAChR. Thus, if similar receptors are present on corresponding nerve terminals, they could mediate fast afferent excitation in response to ACh released at peripheral targets, e.g., the chemosensory carotid body.

L R Da Silva - One of the best experts on this subject based on the ideXlab platform.

  • omega conotoxin sensitivity and presynaptic inhibition of glutamatergic sensory neurotransmission in vitro
    The Journal of Neuroscience, 1994
    Co-Authors: Wilhelm Grüner, L R Da Silva
    Abstract:

    Synaptic transmission between embryonic chick dorsal root ganglion (DRG) neurons and spinal cord neurons was studied in Dissociated Cell Culture. Stimulation of DRG neurons evoked monosynaptic and polysynaptic excitatory responses in the spinal neurons. These responses could be reversibly blocked by application of 6-cyano-7- nitroquinoxaline-2,3-dione (a selective non-NMDA receptor antagonist) and irreversibly eliminated through the presynaptic action of omega- conotoxin GVIA (a selective N-type calcium channel antagonist). As N- type calcium channels in DRG neuron somata are targets for modulation via GABAB receptors, we tested the role of these receptors as regulators of synaptic transmission. Baclofen (a selective GABAB receptor agonist) reversibly inhibited synaptic transmission via a presynaptic, pertussis toxin-sensitive mechanism; CGP 35348 (a selective GABAB receptor antagonist) blocked the actions of baclofen. Taken together, these results demonstrate that N-type calcium channels play a dominant role in glutamatergic sensory neurotransmission. They suggest, in addition, that modulation of N-channel activity may underlie, at least in part, presynaptic inhibition of synaptic transmission between DRG neurons and their targets in the intact spinal cord.

Theodore J Price - One of the best experts on this subject based on the ideXlab platform.

  • pharmacological target focused transcriptomic analysis of native versus Cultured human and mouse dorsal root ganglia
    Pain, 2020
    Co-Authors: Andi Wangzhou, Lisa A Mcilvried, Candler Paige, Paulino Barraganiglesias, Stephanie Shiers, Ayesha Ahmad, Carolyn A Guzman, Robert W Gereau, Gregory Dussor, Theodore J Price
    Abstract:

    : Dorsal root ganglion (DRG) neurons detect sensory inputs and are crucial for pain processing. They are often studied in vitro as Dissociated Cell Cultures with the assumption that this reasonably represents in vivo conditions. However, to our knowledge, no study has directly compared genome-wide transcriptomes of DRG tissue in vivo versus in vitro, or between laboratories and culturing protocols. Comparing RNA sequencing-based transcriptomes of native to Cultured (4 days in vitro) human or mouse DRG, we found that the overall expression levels of many ion channels and GPCRs specifically expressed in neurons are markedly lower although still expressed in Culture. This suggests that most pharmacological targets expressed in vivo are present under the condition of Dissociated Cell Culture, but with changes in expression levels. The reduced relative expression for neuronal genes in human DRG Cultures is likely accounted for by increased expression of genes in fibroblast-like and other proliferating Cells, consistent with their mitotic status in these Cultures. We found that the expression of a subset of genes typically expressed in neurons increased in human and mouse DRG Cultures relative to the intact ganglion, including genes associated with nerve injury or inflammation in preclinical models such as BDNF, MMP9, GAL, and ATF3. We also found a striking upregulation of a number of inflammation-associated genes in DRG Cultures, although many were different between mouse and human. Our findings suggest an injury-like phenotype in DRG Cultures that has important implications for the use of this model system for pain drug discovery.

Tobias M. Boeckers - One of the best experts on this subject based on the ideXlab platform.

  • Synaptogenesis of hippocampal neurons in primary Cell Culture
    Cell and Tissue Research, 2009
    Co-Authors: Andreas Grabrucker, Bianca Vaida, Jürgen Bockmann, Tobias M. Boeckers
    Abstract:

    Hippocampal neurons in Dissociated Cell Culture are one of the most extensively used model systems in the field of molecular and Cellular neurobiology. Only limited data are however available on the normal time frame of synaptogenesis, synapse number and ultrastructure of excitatory synapses during early development in Culture. Therefore, we analyzed the synaptic ultrastructure and morphology and the localization of presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) marker proteins in Cultures established from rat embryos at embryonic day 19, after 3, 7, 10, 14, and 21 days in Culture. First excitatory synapses were identified at day 7 with a clearly defined postsynaptic density and presynaptically localized synaptic vesicles. Mature synapses on dendritic spines were seen from day 10 onward, and the number of synapses steeply increased in the third week. Fenestrated or multiple synapses were found after 14 or 21 days, respectively. So-called dense-core vesicles, responsible for the transport of proteins to the active zone of the presynaptic specialization, were seen on cultivation day 3 and 7 and could be detected in axons and especially in the presynaptic subcompartments. The expression and localization of the presynaptic protein Bassoon and of the postsynaptic molecule ProSAP1/Shank2 was found to correlate nicely with the ultrastructural results. This regular pattern of development and maturation of excitatory synapses in hippocampal Culture starting from day 7 in Culture should ease the comparison of synapse number and morphology of synaptic contacts in this widely used model system.