471
Journal of Physiology (1992), 455, pp. 471-485 With 7 figures Printed in Great Britain
MUSCARINE INCREASES CATION CONDUCTANCE AND DECREASES POTASSIUM CONDUCTANCE IN RAT LOCUS COERULEUS NEURONES BY K.-Z. SHEN AND R. A. NORTH From the Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, USA
(Received 14 October 1991) SUMMARY
1. Whole-cell patch-clamp recordings were made from rat locus coeruleus neurones in slices of brain tissue in vitro. Muscarine (30 IBM) caused an inward current of about 100 pA in neurones voltage clamped at -60 mV. 2. In about 75 % of cells, the current elicited by muscarine was independent of potential in the range -60 to - 120 mV and had no associated conductance change. 3. In about 25 % of cells, the current became smaller with hyperpolarization, was associated with a decreased conductance, and reversed polarity between -100 and - 140 mV. The reversal potential changed with the logarithm of the extracellular potassium concentration. Barium and caesium blocked inward rectification and also prevented reversal of the muscarine current. 4. When potassium ions of the intracellular and extracellular solutions were replaced by caesium, the current evoked by muscarine became smaller with depolarization at reversed polarity at + 9 mV. This current was associated with an increase in conductance, and was greatly reduced when the extracellular sodium concentration was reduced to 20 mM. 5. The results could be quantitatively accounted for by a model in which muscarine both increases a voltage-independent cation conductance and decreases the inward rectifier potassium conductance.
INTRODUCTION
The noradrenaline-containing cells of the nucleus locus coeruleus are important in setting the level of attention that a mammal pays to its outside environment (AstonJones, 1985). Among the afferent inputs are cholinergic fibres from the pedunculopontine and laterodorsal tegmental nuclei (Woolf & Butcher, 1989). Exogenous acetylcholine and other cholinergic agonists excite locus coeruleus neurones through both nicotinic (Egan & North, 1986) and muscarinic (Guyenet & Aghajanian, 1979; Egan & North, 1985) receptors. The nicotinic excitation results from a cation conductance increase as for nicotinic receptors in the periphery (Egan & North, 1986) but the ionic mechanism associated with muscarinic receptor activation is not known. MS 9810 16
PHY 455
472
K. -Z. SHEN AND R. A. NORTH
Muscarinic actions of acetylcholine on vertebrate neurones vary among cells, being inhibitory in some regions and excitatory in others (North, 1989; McCormick, 1989). Direct inhibitory actions of muscarinic agonists result from an increase in membrane potassium conductance, whereas the excitatory effects generally result from a decrease in potassium conductance (Weight & Votava, 1970; Krnjevic, Pumain & Renaud, 1971). Several different potassium currents can be affected, even in the same cell (e.g. Cassel & McLachlan 1987; Dutar & Nicoll, 1988); these include an inwardly rectifying potassium current ('K IR)S a 'leakage' or background current, a calciumactivated potassium current, and the M-current (reviewed by North, 1989). Excitation by muscarine can also involve an increase in sodium conductance in some bullfrog autonomic ganglion cells (Kuba & Koketsu, 1978), although this action is more commonly seen in smooth muscles, glandular tissues and frog oocytes (see North, 1989). In previous work on the locus coeruleus, the effective concentrations of muscarine were determined, and the muscarinic receptor was shown to be relatively insensitive to pirenzepine (Egan & North, 1985). The present work was carried out with the purpose of identifying the ionic mechanism of the muscarinic depolarization. METHODS
Whole-cell tight-seal recordings were made from locus coeruleus neurones in tissue slices from the pons of adult rats. Rats were killed by exsanguination under halothane anaesthesia, and the slice containing the locus coeruleus was prepared essentially as described by Williams, North, Shefner, Nishi & Egan (1984) except that horizontal rather than coronal slices were used in most experiments. Slices (300 ,um) were cut in a vibratome in cold physiological saline, and then placed on a supporting net in the recording chamber. The slice was completely submerged in a flowing (15 ml/min) solution at 37 'C. The solution, equilibrated with 95% 02 and 5% CO2, normally comprised (mM): NaCl, 126; KCl, 6-5; CaCl2, 24; MgC12, 1-2; NaH2PO4, 1-2; NaHCO3, 19; glucose, 11. The relatively high potassium concentration was used so that potassium currents could be more easily reversed (Williams, North & Tokimasa, 1988). TTX (1 ,UM) was added in an effort to block voltage-dependent sodium currents, and to reduce transmitter release from spontaneously firing cells in the slice. Solutions with low chloride concentration were made by substituting sodium chloride with sodium isethionate. Solutions with low sodium concentrations were made by substituting sodium chloride with Tris hydrochloride. The recording methods, brain slice chamber, and dissecting microscope used were similar to those used for previous studies with intracellular recording (Williams et al. 1984). Recordings were made without visualization of individual neurones and without any cleaning of the slice surface. Pipettes contained (mM): potassium gluconate, 125; NaCl, 15; MgCl2, 2; EGTA, 11; HEPES, 10; ATP, 1-5 or 0; GTP, 0-2; and had resistances of 2-5 MCI. The pH of the internal solution was adjusted to 7 3-7A4 with KOH, bringing the intracellular potassium concentration to 145 mm. When caesium gluconate was substituted for potassium gluconate, CsOH (20 mM) was used to adjust the pH. Membrane currents were recorded with an Axopatch-IB amplifier. Series resistance was compensated partially by the amplifier; if uncorrected, the maximal error in measurement of membrane currents of 1 nA would be 5 mV. The current caused by muscarine was always considerably less than this. The voltage dependence of the current evoked by muscarine was measured in three ways. First, muscarine was applied while holding the membrane potential at a pre-set level (-60 to 0 mV) throughout the application and wash-out. Second, the current was measured at the termination of a voltage-clamp step (250-400 ms duration), a series of which were applied before, during and after muscarine application. Third, a voltage ramp from - 140 to + 20 mV (20 mV/s) was applied before, during and after muscarine application. The results of the second and third methods were not significantly different and have usually been pooled. Conductances are usually expressed as 20 mV chord conductances; e.g. the conductance at -70 mV was measured from the difference in currents at -60 and -80 mV. Values of membrane potential given have been corrected for the liquid junction potential that is present between the
M 4SCARILNE EXCITATION IN LC
473
pipette solution (potassium gluconate) and the bath solution prior to making a gigaseal (usually pipette 10 mY negative to bath). The tips of several pipettes (diameter of each was 300 yum) were positioned close to the slice surface within 500 pim from the cell under study. Aluscarine was applied in most experiments by changing the solution issuing from the pipette to one that contained muscarine. RESULTS
General observations Current oscillations Most neurones (80%) showed rhythmic oscillations of membrane current. These occurred at a frequency of 0 3-4 Hz and had amplitudes ranging from 25 to 100 pA. They were reversibly blocked by calcium-free, 10 mM-magnesium solution, which revealed the resting (zero-current) potential to be about -50 mV. The oscillations were observed even at hyperpolarized potentials, and were enhanced by substitution of barium for calcium. Similar properties have been described in a proportion of locus coeruleus neurones impaled with conventional intracellular microelectrodes (Williams et al. 1984); the oscillations were attributed to dendritic calcium action potentials. Spontaneous inward synaptic currents were seen in most neurones but not studied in detail. Inward rectification The current-voltage plot in the potential range negative to the resting potential showed inward rectification. In 6W5 mM-potassium the current-voltage relation had two relatively linear regions, between -60 and -80 mV (conductance at -70 mV was 9 1 + 0 8 nS, mean + S.E.M., n = 45) and negative to -100 mY (conductance at -10o mY was 176+ 13 nS). The inflexion point in the current-voltage relation between these two regions shifted negatively in low (2 5, 4-5 mm) and positively in high (10 5 mM) potassium concentrations. These observations are similar to those found with conventional microelectrodes (Williams et al. 1988). Potassium equilibrium potential The opioid agonist Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO, 60-300 nM), as well as somatostatin (300 nM) and noradrenaline (100 ,aM), evoked an outward current in all neurones tested. This current reversed polarity at -936+24 (n = 11; 6-5 mMpotassium). The conductance increase was 2-8+ 05 nS at -60 mYT and 5 6 + 2 2 nS at - 120 mV (n = 5). These results are also similar to previous findings with conventional intracellular microelectrodes (Williams, Egan & North, 1982; Williams et al. 1988; Inoue, Nakajima & Nakajima, 1988).
Effect of muscarine at - 60 mV Muscarine (30 uM) elicited an inward current at -60 mV. When applied by the flow-pipe method (see Methods) the inward current reached its peak (118 + 11 pA, n = 40) within 10-15 s, declined slightly during continued application (2-6 min), and reversed during 2-3 min when the application was discontinued (Fig. 1). The frequency of the spontaneous oscillations in membrane current was increased by muscarine, and this effect reversed more slowly than the inward current when I
6-2
K. -Z. SHEN AND R. A. NORTH
474
muscarine was washed out (Fig. 1B). Calcium-free solutions were used to test whether muscarine might be acting by releasing other transmitters. Within 5 min of superfusion with a solution containing no calcium (and 10 mM-magnesium) the membrane oscillations disappeared (see Williams et al. 1984), but the current caused Muscarine
B
A
Muscarine
-60 mV
10mV
-80
-100
< 100 pA
1~in 200 pA
1 min
Fig. 1. Depolarization and inward currents caused by muscarine. A, muscarine depolarizes a neurone at its resting potential (-52 mV) and causes discharge of calcium action potentials. Spikes are truncated. Lower trace shows membrane current induced by second application of muscarine to the same cell, voltage clamped at -60 mV. Thick baseline trace in this and other figures results from unclamped current oscillations (see text). Larger downward deflections are spontaneous synaptic currents. TTX (1 uM) present. B, in this cell, as in most (see text), the inward current caused by muscarine became smaller with membrane hyperpolarization but did not reverse polarity.
by muscarine was unaffected (control: 116+11 pA; calcium-free solution: 133 + 15 pA; n = 14). In calcium-free solutions, the decline of the current during the application of muscarine was more marked than in control solutions, but this was not studied in detail. Cadmium (200 or 300 ftM) also had no effect on the peak current. The effective concentrations of muscarine were similar to those required to excite locus coeruleus neurones, measured with extracellular recording (Egan & North, 1985). Therefore, 30 /,tM-muscarine was used in most experiments. (In one group of five cells, the currents evoked by 3, 10, 30 and 100 /tM-muscarine were 68 + 13-7, 95 + 14-8, 130 + 15 6 and 160 + 19-2 pA.) In 100 nM-pirenzepine, muscarine induced a current that was 35 + 2 8 % of control (n = 5), and in 30 nm the muscarine effect was 54*7 + 7-2 % of control (n = 3) (see also Egan & North, 1985). Tetraethylammonium (TEA) blocked the current induced by muscarine. The inhibition was well fitted by a logistic function of Hill coefficient 0-83 and halfmaximal block at 1-2 mM-TEA (concentrations of 0 1, 0 3, 1, 3 and 10 mm were applied; n = 3-6).
MUSCARINE EXCITATION IN LC A
475
B
Control Muscarine
Control Muscarine Wash
Wash
-60 mV -80
-60 mV -80 -100
-100
-120
-120
C
300 pA
D
Potential (mV) -140 -100
Potential (mV) -140 -100
-60 0
-60 0
1-
CL
G)
7
0.
-750
-750
c
-1500 E
F
200
200 Potential (mV) -140 -40 r . . . w
I-C
C.
(AQ
.)
0
lwww
J-200
a, C.,
CL hc-
3
J -200
Fig. 2. Two types of response to muscarine distinguished by voltage dependence; nonreversing (A, C and E) and reversing (B, D and F) current. A and B show current in response to hyperpolarizing voltage commands (150 ms) to -80, -100 and -120 mV (holding potential, -60 mV). In A, the muscarine current showed no reversal, but in B the current was inward at -80 mV and outward at -100 mV. Potassium concentration 6-5 mm in both cases. C and D show the current-voltage relation for two neurones before, during and after muscarine. The currents were recorded in response to a voltage command from a hyperpolarized potential to -60 mV (at 20 mV/s). In D the * and * show the currents recorded in the same neurone at the end of 150 ms steps (the method illustrated in B). E and F show the average current evoked by muscarine at different potentials for neurones of the non-reversing (E) and reversing (F) types. These currents were measured
4K.-Z. SHEN AND
476
R.
A. NORTH
Ionic mechanism of current induced by muscarine Potassium conductance decrease Hyperpolarization to potentials more negative than -80 mV reduced the amplitude of the muscarine-induced current in some cells and had little effect in others. Therefore. cells were arbitrarily divided into two groups, according to whether the current induced by muscarine reversed polarity at a potential less negative than 140 mV. In the majority of neurones (73 of 99), the current evoked by muscarine showed little or no decrease with membrane hyperpolarization to 140 mV (Figs lB and 2A, C and E). These cells had relatively little inward rectification prior to the application of muscarine (conductance was 741 + 0 3 nS at -70 mV and 11 7 + 0 7 nS at -110 mV, n = 46) (Fig. 2C). The inward current was accompanied by either no change or a small decrease in membrane conductance at 110 mV (0 48 + 0 20 nS, n = 46) (Fig. 2A and C). Increasing the potassium concentration also had little or no effect on the current evoked by muscarine at -60 mV (2 5 mM: 82 + 5 7 pA, n = 6; 6 5 mm: 102 + 11 pA. n = 7: 10 5 mm: 102 + 7-4 pA, n = 7). The changes in potassium concentration did not significantly affect the current-voltage relation for muscarine in the potential range -60 to -120 mV, and neither caesium (3 mM) nor barium (100 AtM) changed the current evoked by muscarine. These experiments indicate that the inward current does not result from a reduction in potassium conductance. In the minority of neurones (26 of 99), the current became smaller with hyperpolarization and reversed polarity at 109 + 4 6 mV (n = 12, from ramp voltage commands) or 107 + 2 7 mV (n = 14, from step protocols) (Fig. 2B, D and F). This reversal potential was always more negative than the potassium equilibrium potential (measured from reversal of outward current caused by opioid agonist DAMGO). These neurones had more prominent inward rectification (conductance was 7-9+0-4 nS at -70 mY and 1841 + 1P6 nS at -110 mV, n = 14) and muscarine decreased membrane conductance by 4 1 +0-8 nS at -110 mV (n = 11) (Fig. 2B and D). The reversal potential changed with the extracellular potassium concentration as expected from the Nernst equation (Fig. 3A and B). Caesium (3 mM) or barium (100 Aum) prevented the inward rectification in control conditions (see Williams et al. 1988) and also prevented the reversal of the muscarine current (Fig. 3C and D); the current-voltage plots before and after muscarine became almost parallel, as for the majority of cells. These experiments suggest that potassium conductance decrease contributes more to the inward current in this group of neurones, and that the main potassium current reduced by muscarine is IK, IR. -
-
-
-
-
Cation conductance increase The amplitude of the current induced by muscarine (30 [m) was markedly reduced in solutions with low sodium concentration (control, 126 + 13 pA; in 20 mM-sodium, 28 + 5 pA, n = 29, at -60 mV) (Fig. 4). A similar reduction was seen in the current evoked by quisqualate (3 /tM) (from 529+85 to 199+30 pA at -60 mV, n = 11). from experiments of the type illustrated in A and B. Data from twenty-eight neurones in E (except 26 at -50 mV and 20 at -40 mV) and from twelve neurones in F (except 10 at -50 mVt and 10 at -40 mV). Potassium concentration, 6-5 mM.
MUSCARINE EXCITATION IN LC
477
In the majority of neurones, which showed no current reversal at hyperpolarized potentials, the inward current at all potentials was strongly reduced by low sodium concentration (Fig. 4A and C). In the minority of neurones, which showed current reversal at hyperpolarized potentials, the low sodium concentration (20 mM) caused
A 600
B
10-5
>
E
300
65
0
625
-70
0. cn
A)
-100 -120 5 Potential (mV)
50
0) 0
-120
30
3 10 K concentration
(mM)
D In barium (1 00 ,uM)
C
200
200
0
0
-200
Journal of Physiology (1992), 455, pp. 471-485 With 7 figures Printed in Great Britain
MUSCARINE INCREASES CATION CONDUCTANCE AND DECREASES POTASSIUM CONDUCTANCE IN RAT LOCUS COERULEUS NEURONES BY K.-Z. SHEN AND R. A. NORTH From the Vollum Institute, Oregon Health Sciences University, Portland, OR 97201, USA
(Received 14 October 1991) SUMMARY
1. Whole-cell patch-clamp recordings were made from rat locus coeruleus neurones in slices of brain tissue in vitro. Muscarine (30 IBM) caused an inward current of about 100 pA in neurones voltage clamped at -60 mV. 2. In about 75 % of cells, the current elicited by muscarine was independent of potential in the range -60 to - 120 mV and had no associated conductance change. 3. In about 25 % of cells, the current became smaller with hyperpolarization, was associated with a decreased conductance, and reversed polarity between -100 and - 140 mV. The reversal potential changed with the logarithm of the extracellular potassium concentration. Barium and caesium blocked inward rectification and also prevented reversal of the muscarine current. 4. When potassium ions of the intracellular and extracellular solutions were replaced by caesium, the current evoked by muscarine became smaller with depolarization at reversed polarity at + 9 mV. This current was associated with an increase in conductance, and was greatly reduced when the extracellular sodium concentration was reduced to 20 mM. 5. The results could be quantitatively accounted for by a model in which muscarine both increases a voltage-independent cation conductance and decreases the inward rectifier potassium conductance.
INTRODUCTION
The noradrenaline-containing cells of the nucleus locus coeruleus are important in setting the level of attention that a mammal pays to its outside environment (AstonJones, 1985). Among the afferent inputs are cholinergic fibres from the pedunculopontine and laterodorsal tegmental nuclei (Woolf & Butcher, 1989). Exogenous acetylcholine and other cholinergic agonists excite locus coeruleus neurones through both nicotinic (Egan & North, 1986) and muscarinic (Guyenet & Aghajanian, 1979; Egan & North, 1985) receptors. The nicotinic excitation results from a cation conductance increase as for nicotinic receptors in the periphery (Egan & North, 1986) but the ionic mechanism associated with muscarinic receptor activation is not known. MS 9810 16
PHY 455
472
K. -Z. SHEN AND R. A. NORTH
Muscarinic actions of acetylcholine on vertebrate neurones vary among cells, being inhibitory in some regions and excitatory in others (North, 1989; McCormick, 1989). Direct inhibitory actions of muscarinic agonists result from an increase in membrane potassium conductance, whereas the excitatory effects generally result from a decrease in potassium conductance (Weight & Votava, 1970; Krnjevic, Pumain & Renaud, 1971). Several different potassium currents can be affected, even in the same cell (e.g. Cassel & McLachlan 1987; Dutar & Nicoll, 1988); these include an inwardly rectifying potassium current ('K IR)S a 'leakage' or background current, a calciumactivated potassium current, and the M-current (reviewed by North, 1989). Excitation by muscarine can also involve an increase in sodium conductance in some bullfrog autonomic ganglion cells (Kuba & Koketsu, 1978), although this action is more commonly seen in smooth muscles, glandular tissues and frog oocytes (see North, 1989). In previous work on the locus coeruleus, the effective concentrations of muscarine were determined, and the muscarinic receptor was shown to be relatively insensitive to pirenzepine (Egan & North, 1985). The present work was carried out with the purpose of identifying the ionic mechanism of the muscarinic depolarization. METHODS
Whole-cell tight-seal recordings were made from locus coeruleus neurones in tissue slices from the pons of adult rats. Rats were killed by exsanguination under halothane anaesthesia, and the slice containing the locus coeruleus was prepared essentially as described by Williams, North, Shefner, Nishi & Egan (1984) except that horizontal rather than coronal slices were used in most experiments. Slices (300 ,um) were cut in a vibratome in cold physiological saline, and then placed on a supporting net in the recording chamber. The slice was completely submerged in a flowing (15 ml/min) solution at 37 'C. The solution, equilibrated with 95% 02 and 5% CO2, normally comprised (mM): NaCl, 126; KCl, 6-5; CaCl2, 24; MgC12, 1-2; NaH2PO4, 1-2; NaHCO3, 19; glucose, 11. The relatively high potassium concentration was used so that potassium currents could be more easily reversed (Williams, North & Tokimasa, 1988). TTX (1 ,UM) was added in an effort to block voltage-dependent sodium currents, and to reduce transmitter release from spontaneously firing cells in the slice. Solutions with low chloride concentration were made by substituting sodium chloride with sodium isethionate. Solutions with low sodium concentrations were made by substituting sodium chloride with Tris hydrochloride. The recording methods, brain slice chamber, and dissecting microscope used were similar to those used for previous studies with intracellular recording (Williams et al. 1984). Recordings were made without visualization of individual neurones and without any cleaning of the slice surface. Pipettes contained (mM): potassium gluconate, 125; NaCl, 15; MgCl2, 2; EGTA, 11; HEPES, 10; ATP, 1-5 or 0; GTP, 0-2; and had resistances of 2-5 MCI. The pH of the internal solution was adjusted to 7 3-7A4 with KOH, bringing the intracellular potassium concentration to 145 mm. When caesium gluconate was substituted for potassium gluconate, CsOH (20 mM) was used to adjust the pH. Membrane currents were recorded with an Axopatch-IB amplifier. Series resistance was compensated partially by the amplifier; if uncorrected, the maximal error in measurement of membrane currents of 1 nA would be 5 mV. The current caused by muscarine was always considerably less than this. The voltage dependence of the current evoked by muscarine was measured in three ways. First, muscarine was applied while holding the membrane potential at a pre-set level (-60 to 0 mV) throughout the application and wash-out. Second, the current was measured at the termination of a voltage-clamp step (250-400 ms duration), a series of which were applied before, during and after muscarine application. Third, a voltage ramp from - 140 to + 20 mV (20 mV/s) was applied before, during and after muscarine application. The results of the second and third methods were not significantly different and have usually been pooled. Conductances are usually expressed as 20 mV chord conductances; e.g. the conductance at -70 mV was measured from the difference in currents at -60 and -80 mV. Values of membrane potential given have been corrected for the liquid junction potential that is present between the
M 4SCARILNE EXCITATION IN LC
473
pipette solution (potassium gluconate) and the bath solution prior to making a gigaseal (usually pipette 10 mY negative to bath). The tips of several pipettes (diameter of each was 300 yum) were positioned close to the slice surface within 500 pim from the cell under study. Aluscarine was applied in most experiments by changing the solution issuing from the pipette to one that contained muscarine. RESULTS
General observations Current oscillations Most neurones (80%) showed rhythmic oscillations of membrane current. These occurred at a frequency of 0 3-4 Hz and had amplitudes ranging from 25 to 100 pA. They were reversibly blocked by calcium-free, 10 mM-magnesium solution, which revealed the resting (zero-current) potential to be about -50 mV. The oscillations were observed even at hyperpolarized potentials, and were enhanced by substitution of barium for calcium. Similar properties have been described in a proportion of locus coeruleus neurones impaled with conventional intracellular microelectrodes (Williams et al. 1984); the oscillations were attributed to dendritic calcium action potentials. Spontaneous inward synaptic currents were seen in most neurones but not studied in detail. Inward rectification The current-voltage plot in the potential range negative to the resting potential showed inward rectification. In 6W5 mM-potassium the current-voltage relation had two relatively linear regions, between -60 and -80 mV (conductance at -70 mV was 9 1 + 0 8 nS, mean + S.E.M., n = 45) and negative to -100 mY (conductance at -10o mY was 176+ 13 nS). The inflexion point in the current-voltage relation between these two regions shifted negatively in low (2 5, 4-5 mm) and positively in high (10 5 mM) potassium concentrations. These observations are similar to those found with conventional microelectrodes (Williams et al. 1988). Potassium equilibrium potential The opioid agonist Tyr-D-Ala-Gly-MePhe-Gly-ol (DAMGO, 60-300 nM), as well as somatostatin (300 nM) and noradrenaline (100 ,aM), evoked an outward current in all neurones tested. This current reversed polarity at -936+24 (n = 11; 6-5 mMpotassium). The conductance increase was 2-8+ 05 nS at -60 mYT and 5 6 + 2 2 nS at - 120 mV (n = 5). These results are also similar to previous findings with conventional intracellular microelectrodes (Williams, Egan & North, 1982; Williams et al. 1988; Inoue, Nakajima & Nakajima, 1988).
Effect of muscarine at - 60 mV Muscarine (30 uM) elicited an inward current at -60 mV. When applied by the flow-pipe method (see Methods) the inward current reached its peak (118 + 11 pA, n = 40) within 10-15 s, declined slightly during continued application (2-6 min), and reversed during 2-3 min when the application was discontinued (Fig. 1). The frequency of the spontaneous oscillations in membrane current was increased by muscarine, and this effect reversed more slowly than the inward current when I
6-2
K. -Z. SHEN AND R. A. NORTH
474
muscarine was washed out (Fig. 1B). Calcium-free solutions were used to test whether muscarine might be acting by releasing other transmitters. Within 5 min of superfusion with a solution containing no calcium (and 10 mM-magnesium) the membrane oscillations disappeared (see Williams et al. 1984), but the current caused Muscarine
B
A
Muscarine
-60 mV
10mV
-80
-100
< 100 pA
1~in 200 pA
1 min
Fig. 1. Depolarization and inward currents caused by muscarine. A, muscarine depolarizes a neurone at its resting potential (-52 mV) and causes discharge of calcium action potentials. Spikes are truncated. Lower trace shows membrane current induced by second application of muscarine to the same cell, voltage clamped at -60 mV. Thick baseline trace in this and other figures results from unclamped current oscillations (see text). Larger downward deflections are spontaneous synaptic currents. TTX (1 uM) present. B, in this cell, as in most (see text), the inward current caused by muscarine became smaller with membrane hyperpolarization but did not reverse polarity.
by muscarine was unaffected (control: 116+11 pA; calcium-free solution: 133 + 15 pA; n = 14). In calcium-free solutions, the decline of the current during the application of muscarine was more marked than in control solutions, but this was not studied in detail. Cadmium (200 or 300 ftM) also had no effect on the peak current. The effective concentrations of muscarine were similar to those required to excite locus coeruleus neurones, measured with extracellular recording (Egan & North, 1985). Therefore, 30 /,tM-muscarine was used in most experiments. (In one group of five cells, the currents evoked by 3, 10, 30 and 100 /tM-muscarine were 68 + 13-7, 95 + 14-8, 130 + 15 6 and 160 + 19-2 pA.) In 100 nM-pirenzepine, muscarine induced a current that was 35 + 2 8 % of control (n = 5), and in 30 nm the muscarine effect was 54*7 + 7-2 % of control (n = 3) (see also Egan & North, 1985). Tetraethylammonium (TEA) blocked the current induced by muscarine. The inhibition was well fitted by a logistic function of Hill coefficient 0-83 and halfmaximal block at 1-2 mM-TEA (concentrations of 0 1, 0 3, 1, 3 and 10 mm were applied; n = 3-6).
MUSCARINE EXCITATION IN LC A
475
B
Control Muscarine
Control Muscarine Wash
Wash
-60 mV -80
-60 mV -80 -100
-100
-120
-120
C
300 pA
D
Potential (mV) -140 -100
Potential (mV) -140 -100
-60 0
-60 0
1-
CL
G)
7
0.
-750
-750
c
-1500 E
F
200
200 Potential (mV) -140 -40 r . . . w
I-C
C.
(AQ
.)
0
lwww
J-200
a, C.,
CL hc-
3
J -200
Fig. 2. Two types of response to muscarine distinguished by voltage dependence; nonreversing (A, C and E) and reversing (B, D and F) current. A and B show current in response to hyperpolarizing voltage commands (150 ms) to -80, -100 and -120 mV (holding potential, -60 mV). In A, the muscarine current showed no reversal, but in B the current was inward at -80 mV and outward at -100 mV. Potassium concentration 6-5 mm in both cases. C and D show the current-voltage relation for two neurones before, during and after muscarine. The currents were recorded in response to a voltage command from a hyperpolarized potential to -60 mV (at 20 mV/s). In D the * and * show the currents recorded in the same neurone at the end of 150 ms steps (the method illustrated in B). E and F show the average current evoked by muscarine at different potentials for neurones of the non-reversing (E) and reversing (F) types. These currents were measured
4K.-Z. SHEN AND
476
R.
A. NORTH
Ionic mechanism of current induced by muscarine Potassium conductance decrease Hyperpolarization to potentials more negative than -80 mV reduced the amplitude of the muscarine-induced current in some cells and had little effect in others. Therefore. cells were arbitrarily divided into two groups, according to whether the current induced by muscarine reversed polarity at a potential less negative than 140 mV. In the majority of neurones (73 of 99), the current evoked by muscarine showed little or no decrease with membrane hyperpolarization to 140 mV (Figs lB and 2A, C and E). These cells had relatively little inward rectification prior to the application of muscarine (conductance was 741 + 0 3 nS at -70 mV and 11 7 + 0 7 nS at -110 mV, n = 46) (Fig. 2C). The inward current was accompanied by either no change or a small decrease in membrane conductance at 110 mV (0 48 + 0 20 nS, n = 46) (Fig. 2A and C). Increasing the potassium concentration also had little or no effect on the current evoked by muscarine at -60 mV (2 5 mM: 82 + 5 7 pA, n = 6; 6 5 mm: 102 + 11 pA. n = 7: 10 5 mm: 102 + 7-4 pA, n = 7). The changes in potassium concentration did not significantly affect the current-voltage relation for muscarine in the potential range -60 to -120 mV, and neither caesium (3 mM) nor barium (100 AtM) changed the current evoked by muscarine. These experiments indicate that the inward current does not result from a reduction in potassium conductance. In the minority of neurones (26 of 99), the current became smaller with hyperpolarization and reversed polarity at 109 + 4 6 mV (n = 12, from ramp voltage commands) or 107 + 2 7 mV (n = 14, from step protocols) (Fig. 2B, D and F). This reversal potential was always more negative than the potassium equilibrium potential (measured from reversal of outward current caused by opioid agonist DAMGO). These neurones had more prominent inward rectification (conductance was 7-9+0-4 nS at -70 mY and 1841 + 1P6 nS at -110 mV, n = 14) and muscarine decreased membrane conductance by 4 1 +0-8 nS at -110 mV (n = 11) (Fig. 2B and D). The reversal potential changed with the extracellular potassium concentration as expected from the Nernst equation (Fig. 3A and B). Caesium (3 mM) or barium (100 Aum) prevented the inward rectification in control conditions (see Williams et al. 1988) and also prevented the reversal of the muscarine current (Fig. 3C and D); the current-voltage plots before and after muscarine became almost parallel, as for the majority of cells. These experiments suggest that potassium conductance decrease contributes more to the inward current in this group of neurones, and that the main potassium current reduced by muscarine is IK, IR. -
-
-
-
-
Cation conductance increase The amplitude of the current induced by muscarine (30 [m) was markedly reduced in solutions with low sodium concentration (control, 126 + 13 pA; in 20 mM-sodium, 28 + 5 pA, n = 29, at -60 mV) (Fig. 4). A similar reduction was seen in the current evoked by quisqualate (3 /tM) (from 529+85 to 199+30 pA at -60 mV, n = 11). from experiments of the type illustrated in A and B. Data from twenty-eight neurones in E (except 26 at -50 mV and 20 at -40 mV) and from twelve neurones in F (except 10 at -50 mVt and 10 at -40 mV). Potassium concentration, 6-5 mM.
MUSCARINE EXCITATION IN LC
477
In the majority of neurones, which showed no current reversal at hyperpolarized potentials, the inward current at all potentials was strongly reduced by low sodium concentration (Fig. 4A and C). In the minority of neurones, which showed current reversal at hyperpolarized potentials, the low sodium concentration (20 mM) caused
A 600
B
10-5
>
E
300
65
0
625
-70
0. cn
A)
-100 -120 5 Potential (mV)
50
0) 0
-120
30
3 10 K concentration
(mM)
D In barium (1 00 ,uM)
C
200
200
0
0
-200
