114
\'eurusctencc Resc~uch, ,~ (199()) i 1,4 ',i Elsevier Scientific i~ublishcrs I r c l ~ d i t~l
N E U R E S 00341
Excitatory amino acid response in isolated nucleus tractus solitarii neurons of the rat T a k a s h i N a k a g a w a , T e t s u y a Shirasaki, M i n o r u W a k a m o r i , A t s u o F u k u d a * and N o r i o A k a i k e Department of Neuroph.vsiology, Tohoku Unioersity School of Medicine, Sendal (Japan) (Received 13 December 1989; Revised version received 25 January 1990; Accepted 10 February 1990)
Key words." Rat; Nucleus tractus solitarii; Dissociated neurons; Internal perfusion; Voltage clamp; Excitatory amino acid
SUMMARY The excitatory amino-acid-induced currents in nucleus tractus solitarii neurons freshly isolated from rats were investigated in a whole-cell recording mode using a conventional patch-clamp technique. At a holding potential of - 7 0 mV, L-glutamate (Glu), N-methyl-D-aspartate ( N M D A ) with 10 - 9 M glycine, kainate (KA), quisqualate (QA) and L-aspartate (Asp) evoked inward currents. The currents increased in a sigmoidal fashion with increasing agonists concentration. The half-maximum concentration (ECs0) values were 5 × 10 - s M for Glu, 10 6 M for QA, 1 0 - 4 M for KA, 6 × 10-5 M for N M D A and 5 × 10-5 M for Asp. The Hill coefficients of the Glu-, QA-, KA-, N M D A - and Asp-induced respones were 1.0, 1.3, 1.1, 1.3 and 1.1, respectively. The Glu-. QA-, N M D A - and Asp-induced currents consisted of a transient initial peak and a successive steady-state component showing no desensitization. These currents had the same reversal potential near + 5 mV. In the c u r r e n t - v o l t a g e (l-V) relationships for the (31u-, N M D A - and Asp-induced currents, slight outward rectifications were observed in Mg2+-free external solution at membrane potentials negative to 0 inV. In the presence of extracelhilar M g 2+, the currents induced by Glu, N M D A and Asp were suppressed at negative membrane potentials, but the suppression was less for the Glu response. The I-V relationships for QA- and KA-induced responses were almost linear at a m e m b r a n e potential between - 9 0 and + 50 mV with or without the presence of Mg 2+.
INTRODUCTION
The nucleus tractus solitarii (NTS) is a first relay center of the autonomic nervous system and plays an important role in the integration of autonomic functions such as cardiovascular control 26'27"29. Primary afferent fibers arising from arterial baroreceptors, chemoreceptors, cardiopulmonary receptors and other visceral receptors terminate upon neurons within the N T S 26'29"3°. L-Glutamate (Glu) is probably one of the excitatory transmitters of the arterial baroreceptor afferentfl4'24"25 and may participate in cardiovascular reflex control in the N T S 14'2L31"32. However, the mechanisms of glutaminergic action on the NTS have not yet been studied because of difficulties in making stable intracellular
* Present address: Department of Neurology, Stanford University School of Medicine, CA 94305, U.S.A. Correspondence: N. Akaike, Ph.D., Department of Neurophysiology, Tohoku University School of Medicine. Sendai 980, Japan. 0168-0102/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
115 recordings from relatively small NTS neurons by a conventional microelectrode technique. Glu receptor is thought to consist of 3 subtypes: N-methyl-D-aspartate ( N M D A ) , quisqualate (QA) and kainate (KA) receptors 3s. They could be involved in mediation of glutaminergic synaptic transmission in the NTS. Therefore, these excitatory amino acid (EAA) receptors may play an important role in the regulation of cardiovascular reflexes 6'9. Miller and Felder 19 have suggested that these 3 subtypes of EAA receptors mediate the afferent input to the NTS in extracellular recordings of neuronal activities using brain preparations in vitro. Direct evidence for these 3 subtypes in the NTS neuron still remains to be demonstrated. Recently, we developed a technique for acute isolation of neurons in the mammalian central nervous system 11. We were also able to isolate successfully NTS neurons of the rat. In the present study, the physiological and pharmacological properties of EAA receptors were therefore investigated in isolated NTS neurons under voltage-clamp conditions. M ETHODS
Isolation of N T S neurons NTS neurons were freshly isolated from 1-2-week-old Wistar rats. Briefly, several transverse slices of the brainstem were made with the thickness of 350 ttm, and were incubated in a Krebs solution containing 1000 IUflml Dispase (Godo Shusei) at 31°C for 1 h. The NTS regions were micropunched out from the slices and dissociated mechanically by gentle pipetting. The isolated cells adhered to the bottom of the culture dish (35 ram, Falcon ~3801) within 1 h. Figure 1 shows a NTS neuron dissociated from a 10-day-old rat. All the dissociated cells used in the present experiments maintained well their original morphological features, such as dendritic processes. Solutions The standard external solution contained (in mM) NaC1 150, KC1 5, CaCI2 2, MgCI 2 1, HEPES 10, and glucose 10. The p H was adjusted to 7.4 with Tris-OH. For Mg2+-free external solution, MgCI 2 was subtracted from the standard external solution. The internal solution containing K + (in mM) consisted of K-gluconate 120, KCI 20, NaC1 10, MgCI 2 1, Mg-ATP 2, CaCI 2 0.5, E G T A 5, and HEPES 10. The pH of the internal solution was adjusted to 7.2 with Tris-OH. In the internal solution containing Cs +, K + was completely replaced with equimolar Cs +. Eh'ctrical recordings Electrical measurements were performed with a conventional patch-clamp technique in a whole-cell voltage-clamp mode 8. The resistance between the recording electrode filled with internal solution and the reference electrode in the bath was 4 to 8 M~2. The current was measured using a patch-clamp amplifier (List Medical EPC-5 or EPC-7) and was simultaneously recorded on a pen-recorder (Recti-Horiz-8K) after low-pass filtering at 1 kHz, and a video cassette recorder (Mitsubishi HV-F32) after the signals were changed to a digital mode by the digital audio processor (Sony PCM-501ES). Drugs The drugs used in the present experiments were L-glutamate (Glu) (Tokyo Kasei), L-aspartate (Asp) (Ishizu Pharmaceutical Co.), kainate (KA), quisqualate (QA) and N-methyl-D-aspartate ( N M D A ) (Sigma). The N M D A response is suppressed in the presence of extracellular Mg 2+ 2,3.18. Thus, all drugs were dissolved in Mg2+-free external
116 solution just before use. Since glycine (Gly) potentiates N M D A responses ~, and Gly is thought to be necessary for activating the N M D A receptors I3, N M D A was al,~ays dissolved in test solution containing 10- 9 M Gly throughout the present experiments. At this concentration, Gly effectively potentiated the N M D A response 2~. Drugs were applied by means of a rapid application method termed the ' Y - t u b e ' method 1"2° (Fig. 2). With this technique, the solution surrounding an isolated N T S neuron could be exchanged within 20 ms.
Fig. 1. An example of nucleus tractus solitarii (NTS) neuron freshly dissociated from a 10-day-old rat. Taken by phase-contrast optics. The dissociated cells maintained the original morphological features such as dendritic processes. Scale bar: 20 ,ttm.
117
est Solution S UC
E~ele( '
1
-
!
' Inlet
ValV%l---- Suction Outlet
Fig. 2. A schematic illustration of the 'Y-tube' method. The solution was ejected from the tip of the Y-tube. The solution in the tube was changed rapidly by suction, which was controlled by an electromagnetic valve. NS = normal solution; Rec. Elec. = recording electrode; Ref. Elec. = reference electrode. Data are presented as mean _+ SE in the text and the SE is indicated by a vertical bar in the figures. A vertical bar is not drawn for the cases in which the value of the SE is smaller than the symbol for the data point. RESULTS
Excitatory amino-acid-induced responses The isolated NTS neurons were constantly perfused with Mg2+-free external solution at a holding potential (VH) of - 7 0 mV. The application of excitatory amino acids (EAAs) such as L-glutamate (Glu), L-aspartate (Asp), kainate (KA), quisqualate (QA) and N-methyl-D-aspartate ( N M D A ) evoked the inward currents which increased in a concentration-dependent manner (Fig. 3). Regardless of the different cell configurations and the regions of the NTS dissociated, all tested cells responded not only to Glu and Asp but also 3 kinds (QA, KA and N M D A ) of Glu agonists. The inward currents induced by Glu, Asp, QA and N M D A exhibited an initial transient peak component followed by a steady-state component at a higher concentration, while the KA-induced current exhibited only a steady-state component. Recently, it was reported that n o n - N M D A receptors desensitize rapidly in the order of 5-10 ms 34. Complete exchange of the external solution by our method takes more time (20 ms). The desensitization might therefore be partially contaminated in the peak component observed in the present experiments, and the amplitude of the peak component might be underestimated.
Concentration-response relationships for EAA responses The concentration-response relationships for these EAA-induced currents are summarized in Figure 4. The concentration-response relations can be represented by the following expression: 1 = 1.... / ( 1 +
(C/Ka) n)
118 VH
Glu
=:
-70mY
10-5M ~'-~-. i j
3x10-5 7
10-7M
3x10 -7
[Mg2* ]o-free 10-4 3x10 -4 m
1200 pA 10-6
3x10 -6
i
10-5M
3x10-5
]
10 .4
3x 10"4
12oo
_S
KA
L
10-5M
3x10-5
10-4
3x10 -4
NMDA
20
10-s M Asp
3x 10, 5
10 -4
3X10 -4
T)ir 20 sec
Fig. 3. Representative currents induced by the excitatory amino acids (EAAs), such as L-glutamate (Glu), quisqualate (QA), kainate (KA), N-methyl-D-aspartate (NMDA), and L-aspartate (Asp). The holding potential (VH) was -70 mV. Horizontal bar above each current indicates the period of drug application. The currents induced by Glu, QA, and KA, and those induced by NMDA and Asp were recorded from two different cells. Note the initial peak component of Glu-, QA-, NMDA- and Asp-induced currents followed by the steady-state component. where I is the observed current, 1,,,x is the m a x i m u m value of the current, C the drug concentration, K a the dissociation constant, and n the Hill coefficient. T h e half-maxim u m concentrations (ECs0) were 5 x 10 -5 M for Glu, 10 -6 M for QA, 10 -4 M for KA, 6 × 10 -5 M for N M D A , and 5 x 10 -5 M for Asp. The Hill coefficients of Glu-, QA-, KA-, N M D A - and A s p - i n d u c e d responses were 1.0, 1.3, 1.1, 1.3 and 1.1, respectively. Q A evoked the response at a concentration lower than that of other agonists.
Current-voltage relationships of excitatory amino-acid-induced currents The c u r r e n t - v o l t a g e (l-V) relationships for the EAA-activated responses were investigated using the internal solution containing Cs ÷ which blocks the K ÷ channel. Figure 5 shows the I-V relationships for the E A A - i n d u c e d responses with or without the external Mg 2+. The reversal potentials of all the E A A - i n d u c e d currents were near + 5 mV regardless of the presence of M g 2÷. The measured reversal potential was close to the theoretical value ( + 1 mV) calculated from the N e r n s t equation for non-selectively permeable intra- and extracellular cations. The results suggested that E A A s activated a large cation channel in N T S neurons. Similarly, E A A s activated large cation channels in the h i p p o c a m p a l pyramidal cells of rats and guinea-pigs 7, and the cultured spinal cord
119
Glu (Peak) • Glu (Steady)
8 6
© QA (Peak) • QA (Steady)
4
~
L ~_l
~ : l
~/
KA
Z;Jm~
0
~ / /
II1[11
I
Q
I IIIIIH
I
I IIIII1[
I
I I[lllll
I
I
.m~
IPeakl (Steady) v NMDA (Peak) ] _v NMOA (
2
1
Asp
•
Asp
10-7
10-6
o~J"l ~,y~...f.~
\'eurusctencc Resc~uch, ,~ (199()) i 1,4 ',i Elsevier Scientific i~ublishcrs I r c l ~ d i t~l
N E U R E S 00341
Excitatory amino acid response in isolated nucleus tractus solitarii neurons of the rat T a k a s h i N a k a g a w a , T e t s u y a Shirasaki, M i n o r u W a k a m o r i , A t s u o F u k u d a * and N o r i o A k a i k e Department of Neuroph.vsiology, Tohoku Unioersity School of Medicine, Sendal (Japan) (Received 13 December 1989; Revised version received 25 January 1990; Accepted 10 February 1990)
Key words." Rat; Nucleus tractus solitarii; Dissociated neurons; Internal perfusion; Voltage clamp; Excitatory amino acid
SUMMARY The excitatory amino-acid-induced currents in nucleus tractus solitarii neurons freshly isolated from rats were investigated in a whole-cell recording mode using a conventional patch-clamp technique. At a holding potential of - 7 0 mV, L-glutamate (Glu), N-methyl-D-aspartate ( N M D A ) with 10 - 9 M glycine, kainate (KA), quisqualate (QA) and L-aspartate (Asp) evoked inward currents. The currents increased in a sigmoidal fashion with increasing agonists concentration. The half-maximum concentration (ECs0) values were 5 × 10 - s M for Glu, 10 6 M for QA, 1 0 - 4 M for KA, 6 × 10-5 M for N M D A and 5 × 10-5 M for Asp. The Hill coefficients of the Glu-, QA-, KA-, N M D A - and Asp-induced respones were 1.0, 1.3, 1.1, 1.3 and 1.1, respectively. The Glu-. QA-, N M D A - and Asp-induced currents consisted of a transient initial peak and a successive steady-state component showing no desensitization. These currents had the same reversal potential near + 5 mV. In the c u r r e n t - v o l t a g e (l-V) relationships for the (31u-, N M D A - and Asp-induced currents, slight outward rectifications were observed in Mg2+-free external solution at membrane potentials negative to 0 inV. In the presence of extracelhilar M g 2+, the currents induced by Glu, N M D A and Asp were suppressed at negative membrane potentials, but the suppression was less for the Glu response. The I-V relationships for QA- and KA-induced responses were almost linear at a m e m b r a n e potential between - 9 0 and + 50 mV with or without the presence of Mg 2+.
INTRODUCTION
The nucleus tractus solitarii (NTS) is a first relay center of the autonomic nervous system and plays an important role in the integration of autonomic functions such as cardiovascular control 26'27"29. Primary afferent fibers arising from arterial baroreceptors, chemoreceptors, cardiopulmonary receptors and other visceral receptors terminate upon neurons within the N T S 26'29"3°. L-Glutamate (Glu) is probably one of the excitatory transmitters of the arterial baroreceptor afferentfl4'24"25 and may participate in cardiovascular reflex control in the N T S 14'2L31"32. However, the mechanisms of glutaminergic action on the NTS have not yet been studied because of difficulties in making stable intracellular
* Present address: Department of Neurology, Stanford University School of Medicine, CA 94305, U.S.A. Correspondence: N. Akaike, Ph.D., Department of Neurophysiology, Tohoku University School of Medicine. Sendai 980, Japan. 0168-0102/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
115 recordings from relatively small NTS neurons by a conventional microelectrode technique. Glu receptor is thought to consist of 3 subtypes: N-methyl-D-aspartate ( N M D A ) , quisqualate (QA) and kainate (KA) receptors 3s. They could be involved in mediation of glutaminergic synaptic transmission in the NTS. Therefore, these excitatory amino acid (EAA) receptors may play an important role in the regulation of cardiovascular reflexes 6'9. Miller and Felder 19 have suggested that these 3 subtypes of EAA receptors mediate the afferent input to the NTS in extracellular recordings of neuronal activities using brain preparations in vitro. Direct evidence for these 3 subtypes in the NTS neuron still remains to be demonstrated. Recently, we developed a technique for acute isolation of neurons in the mammalian central nervous system 11. We were also able to isolate successfully NTS neurons of the rat. In the present study, the physiological and pharmacological properties of EAA receptors were therefore investigated in isolated NTS neurons under voltage-clamp conditions. M ETHODS
Isolation of N T S neurons NTS neurons were freshly isolated from 1-2-week-old Wistar rats. Briefly, several transverse slices of the brainstem were made with the thickness of 350 ttm, and were incubated in a Krebs solution containing 1000 IUflml Dispase (Godo Shusei) at 31°C for 1 h. The NTS regions were micropunched out from the slices and dissociated mechanically by gentle pipetting. The isolated cells adhered to the bottom of the culture dish (35 ram, Falcon ~3801) within 1 h. Figure 1 shows a NTS neuron dissociated from a 10-day-old rat. All the dissociated cells used in the present experiments maintained well their original morphological features, such as dendritic processes. Solutions The standard external solution contained (in mM) NaC1 150, KC1 5, CaCI2 2, MgCI 2 1, HEPES 10, and glucose 10. The p H was adjusted to 7.4 with Tris-OH. For Mg2+-free external solution, MgCI 2 was subtracted from the standard external solution. The internal solution containing K + (in mM) consisted of K-gluconate 120, KCI 20, NaC1 10, MgCI 2 1, Mg-ATP 2, CaCI 2 0.5, E G T A 5, and HEPES 10. The pH of the internal solution was adjusted to 7.2 with Tris-OH. In the internal solution containing Cs +, K + was completely replaced with equimolar Cs +. Eh'ctrical recordings Electrical measurements were performed with a conventional patch-clamp technique in a whole-cell voltage-clamp mode 8. The resistance between the recording electrode filled with internal solution and the reference electrode in the bath was 4 to 8 M~2. The current was measured using a patch-clamp amplifier (List Medical EPC-5 or EPC-7) and was simultaneously recorded on a pen-recorder (Recti-Horiz-8K) after low-pass filtering at 1 kHz, and a video cassette recorder (Mitsubishi HV-F32) after the signals were changed to a digital mode by the digital audio processor (Sony PCM-501ES). Drugs The drugs used in the present experiments were L-glutamate (Glu) (Tokyo Kasei), L-aspartate (Asp) (Ishizu Pharmaceutical Co.), kainate (KA), quisqualate (QA) and N-methyl-D-aspartate ( N M D A ) (Sigma). The N M D A response is suppressed in the presence of extracellular Mg 2+ 2,3.18. Thus, all drugs were dissolved in Mg2+-free external
116 solution just before use. Since glycine (Gly) potentiates N M D A responses ~, and Gly is thought to be necessary for activating the N M D A receptors I3, N M D A was al,~ays dissolved in test solution containing 10- 9 M Gly throughout the present experiments. At this concentration, Gly effectively potentiated the N M D A response 2~. Drugs were applied by means of a rapid application method termed the ' Y - t u b e ' method 1"2° (Fig. 2). With this technique, the solution surrounding an isolated N T S neuron could be exchanged within 20 ms.
Fig. 1. An example of nucleus tractus solitarii (NTS) neuron freshly dissociated from a 10-day-old rat. Taken by phase-contrast optics. The dissociated cells maintained the original morphological features such as dendritic processes. Scale bar: 20 ,ttm.
117
est Solution S UC
E~ele( '
1
-
!
' Inlet
ValV%l---- Suction Outlet
Fig. 2. A schematic illustration of the 'Y-tube' method. The solution was ejected from the tip of the Y-tube. The solution in the tube was changed rapidly by suction, which was controlled by an electromagnetic valve. NS = normal solution; Rec. Elec. = recording electrode; Ref. Elec. = reference electrode. Data are presented as mean _+ SE in the text and the SE is indicated by a vertical bar in the figures. A vertical bar is not drawn for the cases in which the value of the SE is smaller than the symbol for the data point. RESULTS
Excitatory amino-acid-induced responses The isolated NTS neurons were constantly perfused with Mg2+-free external solution at a holding potential (VH) of - 7 0 mV. The application of excitatory amino acids (EAAs) such as L-glutamate (Glu), L-aspartate (Asp), kainate (KA), quisqualate (QA) and N-methyl-D-aspartate ( N M D A ) evoked the inward currents which increased in a concentration-dependent manner (Fig. 3). Regardless of the different cell configurations and the regions of the NTS dissociated, all tested cells responded not only to Glu and Asp but also 3 kinds (QA, KA and N M D A ) of Glu agonists. The inward currents induced by Glu, Asp, QA and N M D A exhibited an initial transient peak component followed by a steady-state component at a higher concentration, while the KA-induced current exhibited only a steady-state component. Recently, it was reported that n o n - N M D A receptors desensitize rapidly in the order of 5-10 ms 34. Complete exchange of the external solution by our method takes more time (20 ms). The desensitization might therefore be partially contaminated in the peak component observed in the present experiments, and the amplitude of the peak component might be underestimated.
Concentration-response relationships for EAA responses The concentration-response relationships for these EAA-induced currents are summarized in Figure 4. The concentration-response relations can be represented by the following expression: 1 = 1.... / ( 1 +
(C/Ka) n)
118 VH
Glu
=:
-70mY
10-5M ~'-~-. i j
3x10-5 7
10-7M
3x10 -7
[Mg2* ]o-free 10-4 3x10 -4 m
1200 pA 10-6
3x10 -6
i
10-5M
3x10-5
]
10 .4
3x 10"4
12oo
_S
KA
L
10-5M
3x10-5
10-4
3x10 -4
NMDA
20
10-s M Asp
3x 10, 5
10 -4
3X10 -4
T)ir 20 sec
Fig. 3. Representative currents induced by the excitatory amino acids (EAAs), such as L-glutamate (Glu), quisqualate (QA), kainate (KA), N-methyl-D-aspartate (NMDA), and L-aspartate (Asp). The holding potential (VH) was -70 mV. Horizontal bar above each current indicates the period of drug application. The currents induced by Glu, QA, and KA, and those induced by NMDA and Asp were recorded from two different cells. Note the initial peak component of Glu-, QA-, NMDA- and Asp-induced currents followed by the steady-state component. where I is the observed current, 1,,,x is the m a x i m u m value of the current, C the drug concentration, K a the dissociation constant, and n the Hill coefficient. T h e half-maxim u m concentrations (ECs0) were 5 x 10 -5 M for Glu, 10 -6 M for QA, 10 -4 M for KA, 6 × 10 -5 M for N M D A , and 5 x 10 -5 M for Asp. The Hill coefficients of Glu-, QA-, KA-, N M D A - and A s p - i n d u c e d responses were 1.0, 1.3, 1.1, 1.3 and 1.1, respectively. Q A evoked the response at a concentration lower than that of other agonists.
Current-voltage relationships of excitatory amino-acid-induced currents The c u r r e n t - v o l t a g e (l-V) relationships for the EAA-activated responses were investigated using the internal solution containing Cs ÷ which blocks the K ÷ channel. Figure 5 shows the I-V relationships for the E A A - i n d u c e d responses with or without the external Mg 2+. The reversal potentials of all the E A A - i n d u c e d currents were near + 5 mV regardless of the presence of M g 2÷. The measured reversal potential was close to the theoretical value ( + 1 mV) calculated from the N e r n s t equation for non-selectively permeable intra- and extracellular cations. The results suggested that E A A s activated a large cation channel in N T S neurons. Similarly, E A A s activated large cation channels in the h i p p o c a m p a l pyramidal cells of rats and guinea-pigs 7, and the cultured spinal cord
119
Glu (Peak) • Glu (Steady)
8 6
© QA (Peak) • QA (Steady)
4
~
L ~_l
~ : l
~/
KA
Z;Jm~
0
~ / /
II1[11
I
Q
I IIIIIH
I
I IIIII1[
I
I I[lllll
I
I
.m~
IPeakl (Steady) v NMDA (Peak) ] _v NMOA (
2
1
Asp
•
Asp
10-7
10-6
o~J"l ~,y~...f.~
