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Acetylcholine sensitivity in sensory neurons dissociated from the cat
petrosal ganglion
a b a ,
*
Rodrigo Varas , Julio Alcayaga , Patricio Zapata
a
´ ´ ´ ´
Laboratorio de Neurobiologıa, Departamento de Ciencias Fisiologicas, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile,
Santiago, Chile
b
´ ´
Laboratorio de Neurobiologıa, Departamento de Biologıa, Facultad de Ciencias, Universidad de Chile, Santiago, Chile
Accepted 8 August 2000
Abstract
The petrosal ganglia contain the somata of the sensory fibers of the glossopharyngeal nerves, innervating structures of the tongue, pharynx, carotid sinus and carotid body. Petrosal ganglia were excised from adult cats and their neurons were dissociated and kept in tissue culture for 7–12 days. Intracellular recordings were obtained through conventional microelectrodes. In response to depolarizing pulses, most cells (41 / 60) presented a ‘hump’ in the falling phase of their action potentials (H-type), while the remaining neurons lack such hump (F-type). The two types of cells had no differences in resting membrane potential or action potential amplitude. Acetylcholine (ACh) applied locally elicited responses in nearly two thirds of both H-type and F-type neurons tested. Most H-type neurons (17 / 19) responded with a slow long lasting depolarization, while the remaining (2) did so by generating spikes. In contrast, half of F-type neurons (6 / 12) responded with one or more spikes and the other half only with a slow depolarization. These results indicate that ACh receptors are present in the soma of many petrosal ganglion neurons subjected to tissue culture, thus supporting the idea that — under normal conditions — their peripheral sensory processes may be excited by ACh. 2000 Elsevier Science B.V. All rights reserved.
Theme: Sensory systems
Topic: Somatic and visceral afferents
Keywords: Acetylcholine; Action potential; Carotid body; Membrane potential; Petrosal ganglion; Primary sensory neuron
The petrosal ganglion (PG) contains the somata of the ACh of the somata of sensory neurons isolated from the sensory neurons that project peripherally through the PG.
glossopharyngeal nerve, divided into: the glossopharyngeal Petrosal ganglia were excised from adults cats (2.2–4.0 branch [14], containing mechanosensory and gustatory kg), of either sex, anesthetized with sodium pentobarbitone fibers from tongue and pharynx; and the carotid (sinus) (40 mg / kg) i.p. The neck was opened and both glos-nerve containing chemosensory and barosensory fibers sopharyngeal nerves were exposed. After removing the coming from carotid body and carotid sinus, respectively. tympanic bullae and the ventral walls of the jugular When the PG and its branches are excised from adult cats foramina to expose the petrosal ganglia, their central and and superfused in vitro [1], application of acetylcholine peripheral processes were cut. The ganglia were removed,
21 21
(ACh) to the ganglion elicits bursts of discharges in the placed in ice-chilled, Ca –Mg -free Hanks’ solution, carotid nerve. Such selective action of ACh on the somata minced into 15–20 pieces, and enzymatically dissociated of sensory neurons projecting to the carotid bifurcation in Hanks’ solution supplemented with 0.1% collagenase, was correlated to the intense chemosensory excitation 0.05% trypsin and 150 U / ml DNAse for 30 to 60 min. The induced by ACh applied to the carotid body [7,11]. dissociation, carried out under agitation at 388C, was Based on the above observations, the present research stopped by adding soybean trypsin inhibitor and fetal was intended to record at cellular level the responses to bovine serum to final concentrations of 0.1 mg / ml and 10%, respectively. The cell suspension was centrifuged for 15 min at 5000 rev. / min and the pellet suspended in *Corresponding author. Tel.:156-2-686-2850; fax:156-2-222-5515.
E-mail address: [email protected] (P. Zapata). HEPES-modified Ham’s F-12 nutrient mixture
plemented with 10% horse serum, 10% fetal bovine serum were clearly seen, with well-defined processes extending and nerve growth factor (15 ng / ml). The cells were plated from the soma in some cases (Fig. 1B).
into 35 mm petri-dishes previously coated with poly-L- Acetylcholine chloride (Sigma) was freshly prepared in lysine (0.1 mg / ml), and maintained at 388C in water- Hanks’ solution and delivered by conventional bath perfu-saturated, 5% CO in air atmosphere. The culture medium2 sion under gravity flow from a pipette whose tip was was changed every other day after a 3-day initial lag located at about 150mm from the cell surface. All data are during which the cultures were left undisturbed. expressed as means6S.E.M.’s. Statistical differences were
After 7–12 days, the cultures were placed on the stage assessed by two-tailed Student’s t-tests.
of an inverted microscope and superfused (1.2 ml / min, The impalement of the cultured PG neurons permitted 328C) with Hanks’ solution supplemented with 5 mM stable recordings for at least 10 min, with membrane HEPES. Neurons were impaled with 15–55 MV impe- potentials ranging from 262 to 240 mV (251.366 mV, dance glass microelectrodes filled with 3 M KCl and n560). Action potentials, evoked by brief (,5 ms) connected to a conventional system for registering mem- depolarizing currents, had amplitudes of 44–92 mV, with brane and action potentials. Recordings were displayed on overshoots of 4–40 mV (see Table 1).
an oscilloscope screen and stored on FM magnetic tape, Two configurations of action potentials were recorded after which data were digitized at 20 kHz using Axotape from cultured PG neurons. Most cells (41 / 60; 68.3%) (Axon Instruments Inc., USA) data acquisition software. presented an inflection or ‘hump’ in the falling phase of Under phase-contrast microscopy, petrosal neurons ap- the evoked action potential (Fig. 2A), as H-type neurons peared as bright round or ovoid cells lying on a back- recorded from acutely excised cat PG [4]. The remaining ground of fusiform non-neuronal cells (Fig. 1A). At higher neurons (19 / 60; 31.7%) responded with action potentials magnification, the nucleus and nucleolus of these neurons devoid of hump, as F-type (F, fast) neurons of acutely
Table 1
a
Electrical parameters of cultured cat petrosal ganglion neurons
Neuron Membrane Action potential After-hyperpolarization ACh sensitive
type potential neurons
(mV) Threshold Amplitude Duration Amplitude Duration (%)
(mV) (mV) (ms) (mV) (ms)
H 250.461.0 234.461.1 73.469.01 3.0260.19 12.160.8 67.762.4 65.5
(68.3%) (n541) (n537) (n537) (n537) (n529) (n529) (19 / 29)
F 253.161.4 222.062.4* 68.2612.1 1.3960.08* 11.160.9 14.661.5* 70.6
(31.7%) (n519) (n516) (n518) (n518) (n517) (n517) (12 / 17)
Total 251.360.8 230.661.1 71.6610.0 2.6860.10 11.760.6 47.261.5 67.4
(n560) (n553) (n555) (n555) (n546) (n546) (31 / 46)
a
Data expressed as6S.E.M.
* P,0.05 vs. H-type neurons by two-tailed Student’s t-test.
excised PG [4]. H-type and F-type neurons presented 65.5%) and F-type (12 / 17; 70.6%) cultured PG neurons. similar resting membrane potentials, action potential am- The response to ACh was either the generation of one or plitude, and after-hyperpolarization amplitude (Table 1). more spikes followed by a slow repolarization (Figs. 3A However, F-type neurons have significantly (P,0.05) and 4A, or a slow and sustained depolarization devoid of lower thresholds, shorter action potentials and much spikes (Figs. 3B and 4B).
shorter after-hyperpolarizations than H-type neurons. Nearly 90% (17 / 19) of H-type neurons responding to ACh (0.2–5 mM) evoked responses in H-type (19 / 29; ACh did so with a sustained depolarization devoid of
cat PG neurons recorded from acutely excised ganglia and projecting through the carotid nerve [4] or the glos-sopharyngeal branch [14]. Thus, based on the presence (H-type) or absence (F-type) of a hump in the falling phase of the action potential, nearly two thirds of our cultured neurons were of the H-type, while the remaining third was classifiable as F-type neurons. Both resting membrane potentials and action potential amplitudes of our cultured neurons were within the ranges of those of acutely excised ganglia [4,14].
The presence of the hump in the action potential and the number of action potentials fired during long lasting depolarizing pulses have been correlated with the sensory modality of myelinated carotid nerve fibers originated from PG neurons in the cat [4]. Thus, H-type neurons that fire a single or few spikes provide the chemosensory innervation of the carotid body, while F-type neurons and multiple spiking H-type neurons (S-type; S, slow) project barosen-sory fibers to the carotid sinus. It is noteworthy that PG neurons projecting through the glossopharyngeal branch were also classified into H- and F-types based on the action potentials evoked by peripheral nerve stimulation, but these H-type cells presented higher action potential threshold or did not reach threshold when depolarized by intracellular current pulses [14]. Thus, neurons originally providing chemosensory and barosensory carotid innerva-tion, as well as ganglion cells projecting through the Fig. 4. Responses of cultured petrosal F-type neurons to ACh. (A) glossopharyngeal branch, appear to be present in our Generation of a brief burst of spikes followed by sustained depolarization,
cultures. observed in half of such neurons (6 / 12). (B) Slow and sustained
The other studies on the membrane properties of PG depolarization, recorded from remaining neurons. ACh delivered at arrow.
neurons in vitro have been addressed to other characteris-Interrupted line, resting membrane potential. Oscillations superimposed in
B, artifact introduced by tape recorder. tics: the ionic basis of their action potentials in prepara-tions acutely excised from adult cats [9] or tissue cultures obtained from perinatal rats [16], and changes in mem-spikes (Fig. 3B), while only 10% of them (2 / 19) re- brane properties during early development in rats [6] and sponded generating action potentials (Fig. 3A). In contrast, after peripheral axotomy and reinnervation in ganglia ACh-responsive F-type neurons generated one or more excised from cats [5,10].
spikes in 50% of the cases (Fig. 4A), and the remaining We report here that a large proportion of cultured PG neurons responded with slow sustained depolarization only neurons (.65%) from adult cats are sensitive to ACh, a
(Fig. 4B). proportion similar to the ACh-sensitivity (|68%) reported
In all cases, ACh-evoked responses presented strong for rat pups PG neurons dissociated and cultured for 4 h to desensitization, recovering to full response after several 14 days [18]. We observed that both H- and F-type cultured minutes. Thus, dose–response relationships or threshold PG neurons responded to ACh. Since ACh applied to the doses could not be determined. However, when the stabili- cat PG superfused in vitro produces a specific activation of ty of recordings allowed the exposure of the cell to a those neurons projecting through the carotid nerve [1], part higher dose of ACh, the second response presented faster of the ACh-sensitive H-type neurons might correspond to depolarization, affecting neither the action potential param- chemosensory fibers innervating the carotid body, a struc-eters nor the firing pattern of cell discharges. Those cells ture known to be excited by ACh [7] and capable of ACh presenting subthreshold depolarization on a first exposure synthesis [3] and release [8]. If so, ACh sensitivity would to ACh retained their non-spiking characteristic on follow- be present in both the soma and peripheral endings of ing exposures to higher ACh doses. This may be due to a carotid chemosensory neurons. It must be noted that the cat low density of ACh receptors or to a difussion barrier PG is completely devoid of synapses [17].
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we had previously demonstrated that the carotid nerve [8] R.S. Fitzgerald, M. Shirahata, H.Y. Wang, Acetylcholine release from cat carotid bodies, Brain Res. 841 (1999) 53–61.
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[13] A. Mandelzys, E. Cooper, V.M.K. Verge, P.M. Richardson, Nerve growth factor induces functional nicotinic acetylcholine receptors on rat sensory neurons in culture, Neuroscience 37 (1990) 523–530. Acknowledgements
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afferent system, Brain Res. 814 (1998) 213–217. Commission of Scientific and Technological Research),
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