The effect of proteolytic enzymes on the α9-nicotinic receptor-mediated response in isolated frog vestibular hair cells

Holt, Joseph C.; Lioudyno, Maria; Athas, Grace; Garcia, Meredith M.; Perin, Paola; Guth, Paul S.

doi:10.1016/s0378-5955(00)00225-2

Cited by 49 publications

(72 citation statements)

References 66 publications

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“…Similar observations have also been made in the lateral line (Bobbin et al 1985). In the frog saccule, however, ACh elicits a rapid inhibition of afferent firing that is antagonized by strychnine, d-tubocurarine (dTC), atropine, and apamin (Guth et al 1994a;Holt et al 2001;Sugai et al 1992). This inhibition is thought to be mediated by ␣9-containing nicotinic acetylcholine receptors (␣9nAChR) functionally coupled to the activation of small-conductance, calcium-dependent potassium channels (SK), similar to what is observed in acoustic hair cells (Art et al 1984;Fuchs and Murrow 1992;Nenov et al 1996).…”

Section: Introductionsupporting

confidence: 65%

“…Leopard frogs (Rana pipiens) were anesthetized (by chilling), doubly pithed, and decapitated. The labyrinth was exposed, and the nerve to either the posterior semicircular canal (SCC) or saccule was cut, thereby producing a decentralized stump estimated to contain Յ1,200 fibers (Honrubia et al 1989). The isolated nerve was delicately pulled into a suction electrode filled with artificial perilymph (AP, see following text).…”

Section: Multiunit Afferent Recordingsmentioning

confidence: 99%

“…Dissociation of frog vestibular hair cells was optimized for the purpose of observing reliable ACh responses (Holt et al 2001). The saccular macula or canal crista was dissected free from the whole labyrinth and transferred to a dish containing Hanks Balanced Salt Solution (Gibco, Life Technologies, Grand Island, NY) nominally devoid of calcium and magnesium but containing trypsin (0.05%) and EDTA (0.53 mM).…”

Section: Isolation Of Hair Cellsmentioning

confidence: 99%

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A Pharmacologically Distinct Nicotinic ACh Receptor Is Found in a Subset of Frog Semicircular Canal Hair Cells

Holt

Lioudyno

Guth

2003

Journal of Neurophysiology

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A pharmacologically distinct nicotinic ACh receptor is found in a subset of frog semicircular canal hair cells. J Neurophysiol 90: 1526 -1536, 2003; 10.1152/jn.00273.2002. Frog vestibular organs are endowed with a prominent cholinergic efferent innervation whose stimulation results in several different effects, thereby suggesting diversity in the expression of postsynaptic acetylcholine (ACh) receptors. The application of ACh can mimic efferent stimulation in producing both an inhibition and a facilitation of afferent discharge which are thought to be mediated by at least two distinct ACh receptors present on vestibular hair cells, i.e., ␣9-containing nicotinic receptors (␣9nAChR) and muscarinic receptors (mAChR), respectively. Using patch-clamp and multiunit vestibular afferent recordings, we demonstrate the presence of an additional excitatory hair cell nicotinic ACh receptor pharmacologically distinct from both ␣9nAChR and mAChR. In order of increasing potency, this distinct receptor was activated by ACh, carbachol, and particularly by the selective nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP). This DMPP-sensitive nicotinic receptor (R DMPP ) was antagonized by the classic nicotinic antagonist d-tubocurarine, but refractory to strychnine, atropine, and propylbenzilylcholine mustard, at concentrations that completely block ␣9nAChR and/or mAChR. Activation of R DMPP on application of ACh or DMPP to a subpopulation of isolated posterior semicircular canal (SCC) hair cells resulted in a large depolarization (18.0 Ϯ 1.2 mV). The current underlying this depolarization was typically small (80.1 Ϯ 21.6 pA) and showed an inward rectification starting around Ϫ45 mV. Given their respective EC 50 s (47 nM vs. 20 M), R DMPP was nearly 400 times more sensitive to ACh than ␣9nAChR and thus responded to concentrations of ACh considered too low to be effective at stimulating ␣9nAChR. Despite this remarkable sensitivity, exogenous ACh readily stimulated the mAChR in the intact posterior SCC preparation but failed to activate R DMPP unless the acetylcholinesterase inhibitor physostigmine was present, or high concentrations of ACh were used (Ͼ3 mM). In frog, R DMPP most likely underlies the rapid excitatory response seen during efferent stimulation.

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Section: Introductionsupporting

confidence: 65%

Section: Multiunit Afferent Recordingsmentioning

confidence: 99%

Section: Isolation Of Hair Cellsmentioning

confidence: 99%

See 1 more Smart Citation

A Pharmacologically Distinct Nicotinic ACh Receptor Is Found in a Subset of Frog Semicircular Canal Hair Cells

Holt

Lioudyno

Guth

2003

Journal of Neurophysiology

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“…Afferent recordings show that efferent excitation and inhibition in frog vestibular organs (Rossi et al, 1980;Sugai et al, 1991), as well as inhibition in the toadfish lagena (Locke et al, 1999), are correlated with changes in quantal rate arising from hair cells. Inhibition in the frog saccular macula, a vibratory organ, is consistent with the sequential activation of ␣9/␣10 and SK channels (Sugai et al, 1992;Holt et al, 2001;Rothlin et al, 2003). Studies done in the frog posterior crista, a more typical vestibular organ as it monitors head movements, indicate that multiple nicotinic receptors are involved, but a more precise identification has not been made (Guth et al, 1998;Holt et al, 2003).…”

Section: Introductionmentioning

confidence: 81%

Mechanisms of Efferent-Mediated Responses in the Turtle Posterior Crista

2006

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“…The vast majority of protocols currently used to dissociate neural tissues into single cells rely on mechanical trituration after incubation in proteolytic enzymes (e.g., trypsin, papain, dispase, nagarse, pronase; see Drujan and Svaetichin, 1972;Lam, 1975;Kay and Wong, 1986;Huettner and Baughman 1986;Vaughan and Fisher, 1987;Montague and Friedlander, 1989;Mody et al, 1989). Unfortunately, several studies have shown that proteolytic enzymes can alter the amplitude, kinetics, localization, and pharmacological properties of voltage-and ligand-gated ion currents; these effects have been observed when enzymes are applied not only intracellularly but also extracellularly (e.g., Rojas and Armstrong, 1971;Lee et al, 1977;Hestrin and Korenbrot, 1987;Budde et al, 1994;Shen et al, 1995;Hermann et al, 1997;Armstrong and Roberts, 1998), and even as briefly as 1-3 min (Holt et al, 2001). These results have lead to attempts to isolate cells solely by mechanical means.…”

Section: Introductionmentioning

confidence: 99%

Dissociation of retinal ganglion cells without enzymes

Hayashida

Partida

Ishida

2004

Journal of Neuroscience Methods

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We describe here methods for dissociating retinal ganglion cells from adult goldfish and rat without proteolytic enzymes, and show responses of ganglion cells isolated this way to step-wise voltage changes and fluctuating current injections. Taking advantage of the laminar organization of vertebrate retinas, photoreceptors and other cells were lifted away from the distal side of freshly isolated goldfish retinas, after contact with pieces of membrane filter. Likewise, cells were sliced away from the distal side of freshly isolated rat retinas, after these adhered to a membrane filter. The remaining portions of retina were incubated in an enzyme-free, low Ca 2+ solution, and triturated. After aliquots of the resulting cell suspension were plated, ganglion cells could be identified by dye retrogradely transported via the optic nerve. These cells showed no obvious morphological degeneration for several days of culture. Perforated-patch whole-cell recordings showed that the goldfish ganglion cells spike tonically in response to depolarizing constant current injections, that these spikes are temporally precise in response to fluctuating current injections, and that the largest voltage-gated Na + currents of these cells were larger than those of ganglion cells isolated with a neutral protease.

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The effect of proteolytic enzymes on the α9-nicotinic receptor-mediated response in isolated frog vestibular hair cells

Cited by 49 publications

References 66 publications

A Pharmacologically Distinct Nicotinic ACh Receptor Is Found in a Subset of Frog Semicircular Canal Hair Cells

A Pharmacologically Distinct Nicotinic ACh Receptor Is Found in a Subset of Frog Semicircular Canal Hair Cells

Mechanisms of Efferent-Mediated Responses in the Turtle Posterior Crista

Dissociation of retinal ganglion cells without enzymes

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