248
Brain Research, 533 (1990) 248-254 Elsevier
BRES 16049
Neural nicotinic acetylcholine responses in sensory neurons from postnatal rat Nikolaus J. Sucher, Toni P.O. Cheng and Stuart A. Lipton Department of Neurology, The Children's Hospital, and the Program in Neuroscience, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Accepted 29 May 1990) Key words: Neuronal bungarotoxin; Nicotine; Dorsal root ganglion; Receptor
The whole-cell configuration of the patch-clamp technique was used to study nicotinic acetylcholine (ACh) responses in freshly dissociated dorsal root ganglion (DRG) cells from postnatal rat. At negative holding potentials with physiological solutions in the bath and the pipette, ACh (20/aM), nicotine (5/~M) or DMPP (20 #M) activated inward currents in 51% of the cells. Average current density was higher in 1-month-old compared to newborn animals. Nicotinic agonist-induced currents were unaffected by atropine (10/tM) but reversibly blocked by hexamethonium (20/aM). Although labeling with fluorescent a-bungarotoxin (BGT) demonstrated the presence of toxin binding sites on DRG cells, DMPP-induced inward currents were unaffected by micromolar BGT. Neuronal bungarotoxin (100 nM), in contrast, led to a largely irreversible block of the nicotinic responses. These results show that postnatal DRG cells express functional nicotinic acetylcholine receptors (nAChR) of a neuronal type. INTRODUCTION With the exception of dorsal root ganglion ( D R G ) or spinal ganglion cells, functional nicotinic acetylcholine receptors ( n A C h R ) have been unequivocally identified on the cell surface of all neural crest-derived neurons is. It is not clear whether D R G neurons are cholinoceptive although radioactive nicotine and a-bungarotoxin label the axoplasm of the central and the peripheral processes as well as the cell body of D R G neurons28"31. Moreover, nicotinic receptors have been localized in a subpopulation of rat D R G neurons by immunohistochemical labeling using monoclonal antibodies35. Early electrophysiological findings indicated that m a m m a l i a n D R G neurons did not respond to application of acetylcholine 17. However, recent preliminary evidence suggested the presence of functional n A C h R s in a subpopulation of chick D R G n e u r o n s 3. We used the patch-clamp technique to clarify whether functional n A C h R s are present on the cell body of freshly dissociated D R G neurons from postnatal rats. The snake venom-derived neurotoxins a-bungarotoxin (BGT) and neuronal bungarotoxin (NBT) have been shown to distinguish between skeletal muscle and neuronal types of n A C h R s , respectively24. Neuronal n A C h R s are thought to be composed of at least 2 different subunits, designated as ct and fl, forming a nicotinic receptor gene
family33'37. Sensitivity of n A C h R s to blockage by NBT has been found to be d e p e n d e n t on their specific subunit composition2'8'9't3'36. Therefore, we used these toxins to further characterize the nicotinic receptors found on D R G cells.
MATERIALS AND METHODS Dissociation and cell culture The DRGs were dissected from 2-30-day-old postnatal rats and rinsed several times with Hanks' saline (composition in mM: NaCI, 137; NaHCO 3, 1; Na2HPO4, 0.34; KCI, 5.36; KH2PO4, 0.44; CaC12, 1.25; MgSO4, 0.5; MgC12, 0.5; N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid (HEPES)-NaOH, 5; dextrose, 22.2; Phenol Red, 0.001% v/v; adjusted to pH 7.2 with 0.3 M NaOH). Next, the DRGs were trimmed to remove excessive nerve fibers and connective tissue, digested with an enzyme mixture (3/A/ml papain and 0.1 mg/ml collagenase) for 30 min in 2 successive episodes, and then triturated with a Pasteur pipette to dissociate the DRG cells. Dissociated DRG cells were then cultured in Earle's minimum essential growth medium (Gibco, #1090) containing 10% (v/v) heat-inactivated fetal calf serum, 5% (v/v) rat serum, glutamine 2 mM, gentamicin 1 /~g/ml, dextrose 16 mM and 0.7% (w/v) methylcellulose. The cultures were kept for a minimum of 3 h in a humidified chamber with 5% CO2/95% air at 37 °C before they were used for electrophysiological studies or a-bungarotoxin labeling. a-Bungarotoxin labeling Freshly dissociated DRG cell cultures were fixed for 15 min with 4% paraformaldehyde in 0.2 M phosphate buffer at pH 7.3, and then treated with 1 M phosphate-buffered glycine to neutralize unreactive fixative. After repeatedly rinsing with phosphate-buffered saline (PBS), the fixed cultures were incubated with phy-
Correspondence: S.A. Lipton, Department of Neurology, The Children's Hospital, Enders Building, Room 350, 300 Longwood Avenue, Boston, MA 02115, U.S.A. 0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
249 coerythrine-a-bungarotoxin conjugate (1.5 gg/ml) for 1 h. For controls the cultures were preincubated with 200/~M acetylcholine, 100/~M nicotine or 2.5/~M unlabeled a-bungarotoxin in PBS prior to labeling with the fluorescent marker. The labeled cultures and the controls were wet-mounted for fluorescence light microscopy, using a Zeiss-IM 35 inverted microscope equipped with a 63 × neofluor objective.
Electrophysiological recordings Patch-clamp recordings1°As were performed 1'2°'2~ at room temperature 3-7 h after plating. Patch pipettes were pulled in a 2-stage process on a BB-CH puller, coated with Sylgard, and fire-polished in a microforge. The electrode resistance measured in the bath was 1-4 MI2. An indifferent Ag/AgCl electrode was connected to the bathing fluid via an agarose bridge with 2 M KCI. Whole-cell recordings were made only from spatially compact cells (lacking processes) to ensure an adequate voltage clamp. Membrane capacitance and input resistance were monitored. Series resistance values were less than 10 MI2 and, if necessary, electronically corrected with the series resistance compensation circuit of an EPC-7 patch clamp amplifier (List Electronic, Darmstadt, ER.G.). Monitoring of the activation of voltage-gated sodium currents by depolarizing voltage steps was used to check the quality of the whole-cell voltage-clamp before recording drug-induced responses.
Data acquisition and analysis The membrane currents were recorded with a patch clamp amplifier, digitized with a 12-bit, 125-kHz analog to digital converter (Model DT2782 DMA: Data Translation), and viewed both on an analog oscilloscope (Model 5111A, Tektronix) and on a digital display (Model 1345A, Hewlett-Packard). The sampling rate was varied from 100 gs to 4 ms, and the signals were filtered at 0.5-5 kHz (Model 4302, Ithaco, with a Bessel frequency cut-off characteristic of 48 dB/octave). Holding and command potentials were generated by a PDP-11/23 computer (Digital Equipment Corporation) and a digital-to-analog converter (Cheshire Data). Data were stored on a 30 megabyte Winchester disk (Data System Design, U.S.A.).
RESULTS Using the whole-cell configuration of the patch-clamp technique 15, electrophysiological recordings were obtained from 47 dorsal root ganglion cells. Freshly dissociated D R G cells were uniformly round, had a smooth, phase-bright appearance, and had no or only scarce neuritic processes. The soma diameter of the cells varied between 18 and 50/~m. The average size of the D R G cells was 26.5 + 5 . 5 / t m (mean + S.D., n = 17) and 37.4 + 8.2 /~m (mean + S.D.; n = 10) in P2-5- (postnatal days 2-5) and 1-month-old (P30) animals, respectively. U n d e r current-clamp mode and with a NaCI solution in the bath and KC1 in the pipette (see Materials and Methods section), the cells m a i n t a i n e d a resting membrane potential o f - 6 3 . 0 + 6.1 m V ( m e a n + S.D.; n = 5). Input resistance values of the D R G cells recorded with the patch-pipettes were 914 + 271 MS"2 (mean + S.E.M.; n = 18) when the patch pipette contained the CsCI/TEA-CI solution and 331 + 105 MI2 (mean + S.E.M.; n = 11) with the KCI solution. Average membrane capacitance was 1.11 + 0.14~F/cm 2 in P2/3 (mean + S.D.; n = 9) cells and 1.36 + 0.23/~F/cm2 in P30 (mean + S.D.; n = 8) cells. Additional recordings obtained from 42 cells cultured for 1 day or longer yielded essentially similar results compared to those reported here for freshly dissociated cells. The cells showed outgrowth of neurites within one day in culture. The younger cells (P2) grew more extensive neurites compared to P30 cells.
Solutions Prior to each experiment, the culture medium was replaced by Hank's saline (see above) containing 2.5 mM calcium. A stainlesssteel insert was placed into the culture dish to limit the fluid volume to approximately 100/~1. The cells were continuously superfused at a rate of ~0.8 ml/min. For whole-cellrecording the pipettes were filled with a KCI solution containing in mM: KCI, 140; MgCl2, 2; CaCl2, 1; ethyleneglycol-bis-(fl-aminoethylether)tetra-acetate(EGTA), 1.5-3; HEPES-NaOH, 10; adjusted to pH 7.2 with 0.3 M NaOH. In some experiments the KCI was replaced by 120 mM CsCI and 20 mM tetraethylammonium (TEA)CI to block potassium currents 2°. The internal free calcium concentration of the solutions was calculated to be 4 × 10 -7 M for 1 mM CaCI2 with 1.5 mM EGTA and 1.6 x 10 -7 M for 1 mM CaCl2 with 2.25 mM EGTA4.
Drugs Drugs were dissolved in Hanks' saline and applied by pressure ejection through micropipettes (5-10 am tip diameter) positioned at a distance of about 10-20 /~m from the cell under study. The concentrations of the drugs applied by pressure ejection are those in the micropipette and represent an upper limit for the actual concentration attained at the cell membrane due to dilution with the bathing fluid upon ejection. BGT and NBT, dissolved in Hanks' saline, were superfused. For superfusion of the toxins, and subsequent washout, the solutions were switched with a dead time of approximately 1 min1AS, Fluorescently labeled a-bungarotoxin was obtained from Molecular Probes (Eugene, OR). Acetylcholine, hexamethonium, nicotine, and dimethylphenylpiperazinium (DMPP) were obtained from Sigma Chemical Co (St. Louis, MO). Neuronal bungarotoxin (NBT) and unlabeled a-bungarotoxin (BGT) were a kind gift of Dr. R. Loring, Northeastern University, Boston.
Effects of nicotinic agonists and antagonists on whole-cell currents Responses to nicotinic agonists were obtained in 51% of all D R G cells tested (n = 47). Acetylcholine (20 # M ) induced inward currents between - 5 and - 4 0 p A at a holding potential VH = - 6 0 mV. A C h - i n d u c e d currents were unaffected by the muscarinic antagonist atropine (10/~M) when present in the superfusion medium. Low micromolar doses of nicotine (5/~M) and D M P P (20/~M), a selective agonist at n A C h R s , induced inward currents of similar size when compared to A C h in single cells. Full d o s e - r e s p o n s e curves were not obtained, however. Agonist-induced inward currents were reversibly blocked by concomitant administration of the ganglionic blocking drug h e x a m e t h o n i u m (20 /~M). Typical examples of responses to these drugs are shown in Fig. 1. The percentage of cells with nicotinic responses was found to be similar, when recordings made from the younger animals (P2/3; 60% responding, n = 10) were compared to those in the older animals (P30, 70% responding, n = 7). H o w e v e r , the average current density was increased more t h a n 2-fold when the responses in P 2 - 7 cells were compared to P30 D R G cells
250
A
20 p M DMPP
B .
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20 IJM ACh + 10 MM ATR
+ 20 MMhex~mQthonium
contro]~
20 pA gB3 ms
~
2O pA gB3 ms
Fig. ]. Nicotinic agonists induce inward currents in freshly dissociated DRG cells from postnatal rat (P4 and P5). A: whole-cell recordings were obtained with a patch-pipette from a freshly dissociated DRG cell from rat (P4) voltage-clamped at -60 inV. Acetylcholine (20/~M) and nicotine (5/~M) induced inward currents at physiological holding potentials. The ACh-response is unaffected by the presence of the muscafinic antagonist atropine (ATR; ]0/~M). The drugs were dissolved in Hanks' saline and microperfused onto the cells from a glass pipette by pressure ejection for the time indicated by the dashed line above the traces. The pipette contained a CsC] internal solution (see Materials and Methods section for composition). B: responses of another freshly dissociated DRG cell from rat (PS) to the nicotinic agonist DMPP (20 #M). The response was completely and reversibly blocked by concomitant administration of the ganglionic blocking drug hexamethonium (20/~M). The cell was voltage-clamped at -80 inV. External and internal solutions same as in A. A small part of the control trace exceeded the range of the analog-to-digital converter at this gain and was slightly clipped.
20 MM OMPP (--- Hanks (control) (-- 5 min o(BGT
Brain Research, 533 (1990) 248-254 Elsevier
BRES 16049
Neural nicotinic acetylcholine responses in sensory neurons from postnatal rat Nikolaus J. Sucher, Toni P.O. Cheng and Stuart A. Lipton Department of Neurology, The Children's Hospital, and the Program in Neuroscience, Harvard Medical School, Boston, MA 02115 (U.S.A.) (Accepted 29 May 1990) Key words: Neuronal bungarotoxin; Nicotine; Dorsal root ganglion; Receptor
The whole-cell configuration of the patch-clamp technique was used to study nicotinic acetylcholine (ACh) responses in freshly dissociated dorsal root ganglion (DRG) cells from postnatal rat. At negative holding potentials with physiological solutions in the bath and the pipette, ACh (20/aM), nicotine (5/~M) or DMPP (20 #M) activated inward currents in 51% of the cells. Average current density was higher in 1-month-old compared to newborn animals. Nicotinic agonist-induced currents were unaffected by atropine (10/tM) but reversibly blocked by hexamethonium (20/aM). Although labeling with fluorescent a-bungarotoxin (BGT) demonstrated the presence of toxin binding sites on DRG cells, DMPP-induced inward currents were unaffected by micromolar BGT. Neuronal bungarotoxin (100 nM), in contrast, led to a largely irreversible block of the nicotinic responses. These results show that postnatal DRG cells express functional nicotinic acetylcholine receptors (nAChR) of a neuronal type. INTRODUCTION With the exception of dorsal root ganglion ( D R G ) or spinal ganglion cells, functional nicotinic acetylcholine receptors ( n A C h R ) have been unequivocally identified on the cell surface of all neural crest-derived neurons is. It is not clear whether D R G neurons are cholinoceptive although radioactive nicotine and a-bungarotoxin label the axoplasm of the central and the peripheral processes as well as the cell body of D R G neurons28"31. Moreover, nicotinic receptors have been localized in a subpopulation of rat D R G neurons by immunohistochemical labeling using monoclonal antibodies35. Early electrophysiological findings indicated that m a m m a l i a n D R G neurons did not respond to application of acetylcholine 17. However, recent preliminary evidence suggested the presence of functional n A C h R s in a subpopulation of chick D R G n e u r o n s 3. We used the patch-clamp technique to clarify whether functional n A C h R s are present on the cell body of freshly dissociated D R G neurons from postnatal rats. The snake venom-derived neurotoxins a-bungarotoxin (BGT) and neuronal bungarotoxin (NBT) have been shown to distinguish between skeletal muscle and neuronal types of n A C h R s , respectively24. Neuronal n A C h R s are thought to be composed of at least 2 different subunits, designated as ct and fl, forming a nicotinic receptor gene
family33'37. Sensitivity of n A C h R s to blockage by NBT has been found to be d e p e n d e n t on their specific subunit composition2'8'9't3'36. Therefore, we used these toxins to further characterize the nicotinic receptors found on D R G cells.
MATERIALS AND METHODS Dissociation and cell culture The DRGs were dissected from 2-30-day-old postnatal rats and rinsed several times with Hanks' saline (composition in mM: NaCI, 137; NaHCO 3, 1; Na2HPO4, 0.34; KCI, 5.36; KH2PO4, 0.44; CaC12, 1.25; MgSO4, 0.5; MgC12, 0.5; N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid (HEPES)-NaOH, 5; dextrose, 22.2; Phenol Red, 0.001% v/v; adjusted to pH 7.2 with 0.3 M NaOH). Next, the DRGs were trimmed to remove excessive nerve fibers and connective tissue, digested with an enzyme mixture (3/A/ml papain and 0.1 mg/ml collagenase) for 30 min in 2 successive episodes, and then triturated with a Pasteur pipette to dissociate the DRG cells. Dissociated DRG cells were then cultured in Earle's minimum essential growth medium (Gibco, #1090) containing 10% (v/v) heat-inactivated fetal calf serum, 5% (v/v) rat serum, glutamine 2 mM, gentamicin 1 /~g/ml, dextrose 16 mM and 0.7% (w/v) methylcellulose. The cultures were kept for a minimum of 3 h in a humidified chamber with 5% CO2/95% air at 37 °C before they were used for electrophysiological studies or a-bungarotoxin labeling. a-Bungarotoxin labeling Freshly dissociated DRG cell cultures were fixed for 15 min with 4% paraformaldehyde in 0.2 M phosphate buffer at pH 7.3, and then treated with 1 M phosphate-buffered glycine to neutralize unreactive fixative. After repeatedly rinsing with phosphate-buffered saline (PBS), the fixed cultures were incubated with phy-
Correspondence: S.A. Lipton, Department of Neurology, The Children's Hospital, Enders Building, Room 350, 300 Longwood Avenue, Boston, MA 02115, U.S.A. 0006-8993/90/$03.50 (~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
249 coerythrine-a-bungarotoxin conjugate (1.5 gg/ml) for 1 h. For controls the cultures were preincubated with 200/~M acetylcholine, 100/~M nicotine or 2.5/~M unlabeled a-bungarotoxin in PBS prior to labeling with the fluorescent marker. The labeled cultures and the controls were wet-mounted for fluorescence light microscopy, using a Zeiss-IM 35 inverted microscope equipped with a 63 × neofluor objective.
Electrophysiological recordings Patch-clamp recordings1°As were performed 1'2°'2~ at room temperature 3-7 h after plating. Patch pipettes were pulled in a 2-stage process on a BB-CH puller, coated with Sylgard, and fire-polished in a microforge. The electrode resistance measured in the bath was 1-4 MI2. An indifferent Ag/AgCl electrode was connected to the bathing fluid via an agarose bridge with 2 M KCI. Whole-cell recordings were made only from spatially compact cells (lacking processes) to ensure an adequate voltage clamp. Membrane capacitance and input resistance were monitored. Series resistance values were less than 10 MI2 and, if necessary, electronically corrected with the series resistance compensation circuit of an EPC-7 patch clamp amplifier (List Electronic, Darmstadt, ER.G.). Monitoring of the activation of voltage-gated sodium currents by depolarizing voltage steps was used to check the quality of the whole-cell voltage-clamp before recording drug-induced responses.
Data acquisition and analysis The membrane currents were recorded with a patch clamp amplifier, digitized with a 12-bit, 125-kHz analog to digital converter (Model DT2782 DMA: Data Translation), and viewed both on an analog oscilloscope (Model 5111A, Tektronix) and on a digital display (Model 1345A, Hewlett-Packard). The sampling rate was varied from 100 gs to 4 ms, and the signals were filtered at 0.5-5 kHz (Model 4302, Ithaco, with a Bessel frequency cut-off characteristic of 48 dB/octave). Holding and command potentials were generated by a PDP-11/23 computer (Digital Equipment Corporation) and a digital-to-analog converter (Cheshire Data). Data were stored on a 30 megabyte Winchester disk (Data System Design, U.S.A.).
RESULTS Using the whole-cell configuration of the patch-clamp technique 15, electrophysiological recordings were obtained from 47 dorsal root ganglion cells. Freshly dissociated D R G cells were uniformly round, had a smooth, phase-bright appearance, and had no or only scarce neuritic processes. The soma diameter of the cells varied between 18 and 50/~m. The average size of the D R G cells was 26.5 + 5 . 5 / t m (mean + S.D., n = 17) and 37.4 + 8.2 /~m (mean + S.D.; n = 10) in P2-5- (postnatal days 2-5) and 1-month-old (P30) animals, respectively. U n d e r current-clamp mode and with a NaCI solution in the bath and KC1 in the pipette (see Materials and Methods section), the cells m a i n t a i n e d a resting membrane potential o f - 6 3 . 0 + 6.1 m V ( m e a n + S.D.; n = 5). Input resistance values of the D R G cells recorded with the patch-pipettes were 914 + 271 MS"2 (mean + S.E.M.; n = 18) when the patch pipette contained the CsCI/TEA-CI solution and 331 + 105 MI2 (mean + S.E.M.; n = 11) with the KCI solution. Average membrane capacitance was 1.11 + 0.14~F/cm 2 in P2/3 (mean + S.D.; n = 9) cells and 1.36 + 0.23/~F/cm2 in P30 (mean + S.D.; n = 8) cells. Additional recordings obtained from 42 cells cultured for 1 day or longer yielded essentially similar results compared to those reported here for freshly dissociated cells. The cells showed outgrowth of neurites within one day in culture. The younger cells (P2) grew more extensive neurites compared to P30 cells.
Solutions Prior to each experiment, the culture medium was replaced by Hank's saline (see above) containing 2.5 mM calcium. A stainlesssteel insert was placed into the culture dish to limit the fluid volume to approximately 100/~1. The cells were continuously superfused at a rate of ~0.8 ml/min. For whole-cellrecording the pipettes were filled with a KCI solution containing in mM: KCI, 140; MgCl2, 2; CaCl2, 1; ethyleneglycol-bis-(fl-aminoethylether)tetra-acetate(EGTA), 1.5-3; HEPES-NaOH, 10; adjusted to pH 7.2 with 0.3 M NaOH. In some experiments the KCI was replaced by 120 mM CsCI and 20 mM tetraethylammonium (TEA)CI to block potassium currents 2°. The internal free calcium concentration of the solutions was calculated to be 4 × 10 -7 M for 1 mM CaCI2 with 1.5 mM EGTA and 1.6 x 10 -7 M for 1 mM CaCl2 with 2.25 mM EGTA4.
Drugs Drugs were dissolved in Hanks' saline and applied by pressure ejection through micropipettes (5-10 am tip diameter) positioned at a distance of about 10-20 /~m from the cell under study. The concentrations of the drugs applied by pressure ejection are those in the micropipette and represent an upper limit for the actual concentration attained at the cell membrane due to dilution with the bathing fluid upon ejection. BGT and NBT, dissolved in Hanks' saline, were superfused. For superfusion of the toxins, and subsequent washout, the solutions were switched with a dead time of approximately 1 min1AS, Fluorescently labeled a-bungarotoxin was obtained from Molecular Probes (Eugene, OR). Acetylcholine, hexamethonium, nicotine, and dimethylphenylpiperazinium (DMPP) were obtained from Sigma Chemical Co (St. Louis, MO). Neuronal bungarotoxin (NBT) and unlabeled a-bungarotoxin (BGT) were a kind gift of Dr. R. Loring, Northeastern University, Boston.
Effects of nicotinic agonists and antagonists on whole-cell currents Responses to nicotinic agonists were obtained in 51% of all D R G cells tested (n = 47). Acetylcholine (20 # M ) induced inward currents between - 5 and - 4 0 p A at a holding potential VH = - 6 0 mV. A C h - i n d u c e d currents were unaffected by the muscarinic antagonist atropine (10/~M) when present in the superfusion medium. Low micromolar doses of nicotine (5/~M) and D M P P (20/~M), a selective agonist at n A C h R s , induced inward currents of similar size when compared to A C h in single cells. Full d o s e - r e s p o n s e curves were not obtained, however. Agonist-induced inward currents were reversibly blocked by concomitant administration of the ganglionic blocking drug h e x a m e t h o n i u m (20 /~M). Typical examples of responses to these drugs are shown in Fig. 1. The percentage of cells with nicotinic responses was found to be similar, when recordings made from the younger animals (P2/3; 60% responding, n = 10) were compared to those in the older animals (P30, 70% responding, n = 7). H o w e v e r , the average current density was increased more t h a n 2-fold when the responses in P 2 - 7 cells were compared to P30 D R G cells
250
A
20 p M DMPP
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20 IJM ACh + 10 MM ATR
+ 20 MMhex~mQthonium
contro]~
20 pA gB3 ms
~
2O pA gB3 ms
Fig. ]. Nicotinic agonists induce inward currents in freshly dissociated DRG cells from postnatal rat (P4 and P5). A: whole-cell recordings were obtained with a patch-pipette from a freshly dissociated DRG cell from rat (P4) voltage-clamped at -60 inV. Acetylcholine (20/~M) and nicotine (5/~M) induced inward currents at physiological holding potentials. The ACh-response is unaffected by the presence of the muscafinic antagonist atropine (ATR; ]0/~M). The drugs were dissolved in Hanks' saline and microperfused onto the cells from a glass pipette by pressure ejection for the time indicated by the dashed line above the traces. The pipette contained a CsC] internal solution (see Materials and Methods section for composition). B: responses of another freshly dissociated DRG cell from rat (PS) to the nicotinic agonist DMPP (20 #M). The response was completely and reversibly blocked by concomitant administration of the ganglionic blocking drug hexamethonium (20/~M). The cell was voltage-clamped at -80 inV. External and internal solutions same as in A. A small part of the control trace exceeded the range of the analog-to-digital converter at this gain and was slightly clipped.
20 MM OMPP (--- Hanks (control) (-- 5 min o(BGT
