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Ionic currents in retrogradely labeled trigeminothalamic neurons in slices of rat medulla
66
Neuroscience Letter,~, 110 (1990) 66 71 Elsevier Scientific Publishers Ireland Ltd.
NSL 06669
Ionic currents in retrogradely labeled trigeminothalamic neurons in slices of rat medulla Li C h e n I and Li-Yen M a e H u a n g 1'2 t Marine Biomedical Institute and -'Department Physiology and Biophysics. Universio' ~/" Texas Medical Branch at Galveston, TX 77550 (U.S.A.) (Received l September 1989; Revised version received 17 October 1989; Accepted 20 October 1989) Key word~:
Trigeminothalamic neuron: Thin brain slice; Fluorescent-labeled microsphere; Patch clamp; Sodium current; Calcium current; Potassium current
We have recorded the ionic currents of identified trigeminothalamic neurons in medulla slices in vitro. Trigeminothalamic cells were first retrogradely labeled by injecting fluorescent latex microspheres in the thalamus of a 7- to 10-day-old rat. Two days later, thin slices (80q00 ~m) were prepared from the lower medulla of the injected rat. Whole cell recordings were performed on the labeled cells located in the spinal trigeminal nucleus caudalis using the patch clamp technique. The voltage dependent inward sodium, inward calcium and outward potassium currents are qualitatively similar to those obtained from the enzymatically dissociated trigeminothalamic neurons. Successful application of this thin slice method opens the opportunity of studying synaptic circuitry in the trigeminothalamic system,
Trigeminal nuclei, especially subnucleus caudalis, are generally regarded as an important center for relaying nociceptive information from the craniofacial area to the upper brain [2, I l]. Electrical properties of identified trigeminothalamic tract (TTT) neurons have been examined by combining retrograde labeling and patch clamp techniques [5, 6]. This approach has allowed us to define ion channels and transmitter responses in TTT cells in detail [5-7]. As with any experimental method, ours also has its drawbacks. Because the patch-clamp technique requires that the electrode lbrms a tight seal with the cell membrane, an enzyme was used to clean the surface of our cells. There is always the worry that enzyme treatment may change the membrane properties of cells [1]. Another drawback, a more severe one, is the complete loss of synaptic organization of the isolated cell preparation. Neurons do not function in isolation. In the case of trigeminothalamic neurons, they receive input from primary afferents, interneurons and descending pathways. Axons of these neurons project to the thalamus and send collaterals to the pontomedullary reticular formation and to the regions of the periaquaductal gray. Trigeminothalamic neurons are Correspondence: L.-Y. Mae Huang, University of Texas Medical Branch, Marine Biomedical Institute, 200 University Boulevard, Suite 706A (H-43), Galveston, TX 77550, U.S.A. 03I)4-3940/90/$ 03.50
1990 Elsevier Scientific Publishers Ireland Ltd.
67 not simply relay neurons, but also integrate and disseminate somatosensory information. Thus synaptic transmission in this system is of functional importance when considering the action of various modulators such as enkephalin and serotonin on firing frequency of trigeminothalamic neurons [9, 10]. Edwards et al. [3] have succeeded in recording single channel and whole cell currents in thin slice sections of various brain and spinal cord tissues. Because local circuitry remained intact in the slice, this technique provided an opportunity to study the synaptic transmission at high resolution. Because the surface of the recording cell was blown clean with buffer to allow patch clamping, this method also avoided the problem of possible alteration of membrane properties of neurons by enzyme treatment. We have now developed the thin slice technique in our studies of identified trigeminothalamic neurons. In this paper various ionic currents observed in retrograde labeled trigeminothalamic cells located in the caudal spinal trigeminal nucleus of thin medulla slices will be described. The results obtained from thin slices will be compared with those obtained from isolated cells. Trigeminothalamic neurons were first labelled with rhodamine-labelled fluorescent latex microspheres according to the procedures previously described [5, 6, 8]. Briefly, a 7- to 10-day-old L o n g - E v a n s rat was anesthetized with Metofane vapor inhalant (1.0 ml) and then positioned in a stereotaxic apparatus. A total of 1.6/~1 of fluorescent latex microspheres solution (0.4/~1 for each injection site) was injected into the
,o,
B
Fig. 1. Examples of neurons in spinal trigeminal nucleus. A: spinal trigeminal neurons (indicated by arrows) on the surface of a slice viewed with Hoffman interference optics. The outlines of the cells are easily discernable. B: one of the cells is labeled with fluorescence microspheres when the same slice is viewed with fluorescenceoptics. The diameter of the cell body is about 15 #m.
68 ventrolateral and ventromedial part of the thalamus on both sides of the brain. The marker was transported retrogradely in the axons of projection neurons. After two days, trigeminothalamic neurons could be conveniently identified by the granular fluorescence in the cytoplasm of their somata and proximal dendrites. Horizontal slices of lower medulla were prepared according to the methods described by Edwards et al. [3]. The brain was quickly removed from an injected rat. The tissue was placed in an ice cold oxygenated buffer solution. The solution was composed of 140 mM NaC1, 10 mM KC1, 1 mM CaC12, 6 mM MgC12, l0 mM glucose, 2.5 mM HEPES, and 1 mM KH:PO4. The medulla was isolated with a razor blade. The tissue block was then glued to the stage of the slicer with the dorsal side of the brain facing up. The assembly was put into a Vibratome slicing chamber filled with cold oxygenated buffer solution. The top portion of the tissue was sliced away until the required level (0.5 mm below the coronal plane) was reached. About 2 3 uniform 80-100/~m slice sections were obtained using a blade that moved forward at very slow speed (2--3 scale unit) and a high vibration frequency (8 9 scale units). The slices were incubated in a large chamber containing oxygenated buffer solution at room temperature. After 45-60 rain, one slice was transferred to the recording chamber and was held in place with nylon mesh. The recording chamber was then placed under an inverted microscope equipped with Hoffman objectives. We used Hoffman optics instead of Normarski optics as originally suggested by Edwards. Our optical arrangement provided a longer working distance, therefore, easier maneuverability. The slice was perfused constantly with oxygenated recording solution. The experiment was performed either at 35'~C or room temperature. The surface of the cells was blown clean by a stream of saline solution delivered from a pipette. The patch pipette was then pressed onto the surface of the cell and a tight seal between the glass and membrane was established with gentle suction. Ionic currents were recorded using the whole-cell patch clamp technique [4] on an EPC-7 List or Dagan 8900 patch amplifier. Patch pipettes were pulled from WPI TWF150 or Corning 7502 glass capillaries and were subsequently coated with Sylgard. The pipette resistance was between 4 and 6 Mg2. Current signals were filtered with a 4-pole Bessel filter. The cut-off frequency was set at 3 or 5 kHz. The currents were sampled on-line by a Masscomp computer at 10-200 #s per point. Leakage current and capacitance were subtracted for the analyses. Fig. 1 gives an example of cells in a thin slice section viewed with a Hoffman objective. The cells located in the trigeminal nucleus could be clearly visualized (Fig. I A). A fluorescent labeled cell was readily identifiable (Fig. 1B). The average input resistance of our cells was 0.5 + 0.2 G ~ , and the zero-current membrane potential was between - 4 0 and - 6 5 mV. An action potential could be triggered when the membrane potential was held at - 7 0 mV and then depolarized beyond - 55 mV. The properties of voltage-dependent inward Na current, inward Ca current and various outward currents were studied under voltage clamp conditions. Due to the extensive dendritic processes present in these neurons, whole cell recording sometimes had a space clamp problem. This problem did not affect our recordings of Ic~, and IK. On the other hand, adequate clamping ofly~ was quite diffi-
69
cult because of its large amplitude and rapid activation kinetics. Fast INa was elicited as the depolarized potential reached - 6 0 mV (Fig. 2A upper). The voltage dependence of Na conductance (Fig. 2A lower) and its inactivation (not shown) were similar to those observed in dissociated cells [5]. The inward Ca current (Fig. 2B upper) was isolated by adding tetrodotoxin (TTX) to block the Na channels and by including tetraethylammonium (TEA) and Cs to block the K channels. Inward Ica could be separated into transient (/Ca,V), slow inactivating (/Ca,N) and non-inactivating (/Ca,L) components. ICa,Tactivated with depolarizing pulses more positive than - 6 0 mV and was inactivated with a time constant in the range of 15-50 ms. Its inactivation voltage curve reached a plateau at - 1 0 0 mV and was zero at - 50 mV [6]. ICa,N and ICa,Lwere characterized by positive activation potentials ( - 50 to - 30 mV) and slow inactivation (r > 500 ms) at room temperature. The voltage-dependent properties of ICa.N and Ica,L also are similar to those recorded in isolated cells. Large outward K currents were observed in our preparation (Fig. 3). They were studied with 70 mM K intracellular solution and TTX-containing external solution. I~: could be further separated into three components: the fast inactivating A current (I(A)), slow activating Ca-dependent K current (IK(ca)) and non-inactivating voltagedependent K current (IK¢v))(unpublished observation). IA could be isolated by its vol-
--7O
A
e
:: :;
2
),,/:
B
_ ~
°
nA
-5o
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--40
mB
I (hA)
o
I
6.0.
hD----IOO ~'-le
--'tOO
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(nA)
~..0
mV
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i -20
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60
- :
mV
.
,
-tOO
•
y (mY) iO0
•
1 Q
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0000
--2.0
Fig. 2. Inward Na and Ca currents and their voltage dependence. A (upper): INa elicited at various test potentials from a holding potential (hp) of - 1 0 0 inV. The test potentials used are indicated by the numbers to the right o f each trace. The solutions used were (mM): external solution - - 140 NaCl, 2 KC1 2 KH2PO4, 10 HEPES, 2.5 CaC12, 1.5 MgC12, 10 glucose, 20 sucrose; pipette solution - - 120 N-methyl-Dglutamine fluoride (NMDG-F), 11 BAPTA, 2 TEA, 30 HEPES, 5 Mg-ATP, 200 #M leupeptin. The current was measusred at 35°C. A (lower): the voltage dependence of the peak INa. B (upper): inward Ca currents were measured at 22.5°C in a bath solution consisting of (in mM) l0 Ca, 120 TEA, 10 HEPES, 25 glucose, l0 CsC1, 5 4AP, 50 sucrose and l #M TTX. B (lower): the 1 - V relation of peak/Ca.
70
80
;
T •
1 4. O m A I 1(5
J. I mm
I (hA) ~4.0
hp---80
mV
6.0
•
o o
oo o o oo
__~a~( --~00
I o o
v (my) 100
--2 ° 0
Fig. 3. Outward K currents and their current-voltage relation. Upper: the currents were recorded at 35°C in an external solution containing (in mM) 4 KC1, 150 NaCI, 10 HEPES, 10 glucose, 1.5 MgCI2, 2/~M TTX. The pipette solution consisted of (mM) 70 KF, 70 N M D G - F , 5 HEPES, 90 sucrose. The test potentials used are indicated by the numbers to the left of each trace. Lower: the t V curves for peak ( O ) and steady state (C>) IK.
tage-dependent inactivation and greater sensitivity to 4-amino-pyridine. IK(ca) was distinguishable from the other two K components by the fact that it increased with external Ca 2+ concentration and was blocked by Co 2+ (unpublished observations). All the K components were blocked by TEA. The same types of voltage-dependent K currents also were observed in enzymatically dissociated cells [5]. This report describes for the first time the electrical properties of retrograde labeled trigeminothalamic neurons in the slice preparation. The currents recorded in this preparation are similar to those observed in acutely dissociated trigeminothalamic cells. The major advantages of the thin slice preparation for our application are knowing the exact location of our recorded cells and opening the possibility of studying the synaptic circuitry in the trigeminothalamic system. The work presented here will provide the necessary background for understanding the mechanisms by which transmitters and modulators act on these cells. We wish to thank Dr. A. Konner for his discussions and suggestions, S.-Y. Wong for her technical assistance and L. Simmons and M. Watson for preparing this manuscript. This work was supported by N I H NS23061 and R C D A NS01050. 1 Akaike, N., Kaneda, M., Hori, N. and Krishtal, O.A., Blockade of N-methyl-o-aspartate response in enzyme-treated rat hippocampal neurons, Neurosci. Lett., 87 (1988) 75 79.
71 2 Dubner, R. and Bennet, G.J., Spinal and trigeminal mechanisms of nociception, Annu. Rev. Neurosci., 6 (1983) 381-408. 3 Edwards, F.A., Konnerth, A., Sakmann, B. and Takahashi, T., A thin slice preparation for patch clamp recordings from neurons of the mammalian central nervous system, Pflugers Arch., 414 (1989) 6 0 0 ~ 12. 4 Hamill, O., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pflugers Arch., 391 (1981) 85-100. 5 Huang, L.-Y.M., Electrical properties of acutely isolated, identified rat spinal dorsal horn projection neurons, Neurosci. Lett., 82 (1987) 267 272. 6 Huang, L.-Y.M., Calcium channels in isolated rat dorsal horn neurones, including labelled spinothalamic and trigeminothalamic cells, J. Physiol. (Lond.), 411 (1989) 161 177. 7 Huang, L.-Y.M., The calcium current and the glutamate responses in isolated, identified spinal dorsal horn projection neurons, Soc. Neurosci. Abstr., 13 (1987) 537. 8 Katz, L.C., Burkhalter, A. and Dreyer, W.J., Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex, Nature (Lond.), 310 (1984) 498-500. 9 Willcockson, W.S., Chung, J.M., Hori, Y., Lee, K.H. and Willis, W.D., Effects of iontophoretically released peptides on primate spinothalamic tract cells, J. Neurosci., 4 (1984a) 741 750. 10 Willcockson, W.S., Chung, J.M., Hori, Y., Lee, K.H. and Willis, W.D., Effects of iontophoretically released amino acids and amines on primate spinothalamic tract cells, J. Neurosci., 4 (1984) 732 740. 11 Willis, W.D., Ascending nociceptive tracts, In W.D. Willis (Ed.), The Pain System, Karger, Basel, 1985, pp. 145 212.
Neuroscience Letter,~, 110 (1990) 66 71 Elsevier Scientific Publishers Ireland Ltd.
NSL 06669
Ionic currents in retrogradely labeled trigeminothalamic neurons in slices of rat medulla Li C h e n I and Li-Yen M a e H u a n g 1'2 t Marine Biomedical Institute and -'Department Physiology and Biophysics. Universio' ~/" Texas Medical Branch at Galveston, TX 77550 (U.S.A.) (Received l September 1989; Revised version received 17 October 1989; Accepted 20 October 1989) Key word~:
Trigeminothalamic neuron: Thin brain slice; Fluorescent-labeled microsphere; Patch clamp; Sodium current; Calcium current; Potassium current
We have recorded the ionic currents of identified trigeminothalamic neurons in medulla slices in vitro. Trigeminothalamic cells were first retrogradely labeled by injecting fluorescent latex microspheres in the thalamus of a 7- to 10-day-old rat. Two days later, thin slices (80q00 ~m) were prepared from the lower medulla of the injected rat. Whole cell recordings were performed on the labeled cells located in the spinal trigeminal nucleus caudalis using the patch clamp technique. The voltage dependent inward sodium, inward calcium and outward potassium currents are qualitatively similar to those obtained from the enzymatically dissociated trigeminothalamic neurons. Successful application of this thin slice method opens the opportunity of studying synaptic circuitry in the trigeminothalamic system,
Trigeminal nuclei, especially subnucleus caudalis, are generally regarded as an important center for relaying nociceptive information from the craniofacial area to the upper brain [2, I l]. Electrical properties of identified trigeminothalamic tract (TTT) neurons have been examined by combining retrograde labeling and patch clamp techniques [5, 6]. This approach has allowed us to define ion channels and transmitter responses in TTT cells in detail [5-7]. As with any experimental method, ours also has its drawbacks. Because the patch-clamp technique requires that the electrode lbrms a tight seal with the cell membrane, an enzyme was used to clean the surface of our cells. There is always the worry that enzyme treatment may change the membrane properties of cells [1]. Another drawback, a more severe one, is the complete loss of synaptic organization of the isolated cell preparation. Neurons do not function in isolation. In the case of trigeminothalamic neurons, they receive input from primary afferents, interneurons and descending pathways. Axons of these neurons project to the thalamus and send collaterals to the pontomedullary reticular formation and to the regions of the periaquaductal gray. Trigeminothalamic neurons are Correspondence: L.-Y. Mae Huang, University of Texas Medical Branch, Marine Biomedical Institute, 200 University Boulevard, Suite 706A (H-43), Galveston, TX 77550, U.S.A. 03I)4-3940/90/$ 03.50
1990 Elsevier Scientific Publishers Ireland Ltd.
67 not simply relay neurons, but also integrate and disseminate somatosensory information. Thus synaptic transmission in this system is of functional importance when considering the action of various modulators such as enkephalin and serotonin on firing frequency of trigeminothalamic neurons [9, 10]. Edwards et al. [3] have succeeded in recording single channel and whole cell currents in thin slice sections of various brain and spinal cord tissues. Because local circuitry remained intact in the slice, this technique provided an opportunity to study the synaptic transmission at high resolution. Because the surface of the recording cell was blown clean with buffer to allow patch clamping, this method also avoided the problem of possible alteration of membrane properties of neurons by enzyme treatment. We have now developed the thin slice technique in our studies of identified trigeminothalamic neurons. In this paper various ionic currents observed in retrograde labeled trigeminothalamic cells located in the caudal spinal trigeminal nucleus of thin medulla slices will be described. The results obtained from thin slices will be compared with those obtained from isolated cells. Trigeminothalamic neurons were first labelled with rhodamine-labelled fluorescent latex microspheres according to the procedures previously described [5, 6, 8]. Briefly, a 7- to 10-day-old L o n g - E v a n s rat was anesthetized with Metofane vapor inhalant (1.0 ml) and then positioned in a stereotaxic apparatus. A total of 1.6/~1 of fluorescent latex microspheres solution (0.4/~1 for each injection site) was injected into the
,o,
B
Fig. 1. Examples of neurons in spinal trigeminal nucleus. A: spinal trigeminal neurons (indicated by arrows) on the surface of a slice viewed with Hoffman interference optics. The outlines of the cells are easily discernable. B: one of the cells is labeled with fluorescence microspheres when the same slice is viewed with fluorescenceoptics. The diameter of the cell body is about 15 #m.
68 ventrolateral and ventromedial part of the thalamus on both sides of the brain. The marker was transported retrogradely in the axons of projection neurons. After two days, trigeminothalamic neurons could be conveniently identified by the granular fluorescence in the cytoplasm of their somata and proximal dendrites. Horizontal slices of lower medulla were prepared according to the methods described by Edwards et al. [3]. The brain was quickly removed from an injected rat. The tissue was placed in an ice cold oxygenated buffer solution. The solution was composed of 140 mM NaC1, 10 mM KC1, 1 mM CaC12, 6 mM MgC12, l0 mM glucose, 2.5 mM HEPES, and 1 mM KH:PO4. The medulla was isolated with a razor blade. The tissue block was then glued to the stage of the slicer with the dorsal side of the brain facing up. The assembly was put into a Vibratome slicing chamber filled with cold oxygenated buffer solution. The top portion of the tissue was sliced away until the required level (0.5 mm below the coronal plane) was reached. About 2 3 uniform 80-100/~m slice sections were obtained using a blade that moved forward at very slow speed (2--3 scale unit) and a high vibration frequency (8 9 scale units). The slices were incubated in a large chamber containing oxygenated buffer solution at room temperature. After 45-60 rain, one slice was transferred to the recording chamber and was held in place with nylon mesh. The recording chamber was then placed under an inverted microscope equipped with Hoffman objectives. We used Hoffman optics instead of Normarski optics as originally suggested by Edwards. Our optical arrangement provided a longer working distance, therefore, easier maneuverability. The slice was perfused constantly with oxygenated recording solution. The experiment was performed either at 35'~C or room temperature. The surface of the cells was blown clean by a stream of saline solution delivered from a pipette. The patch pipette was then pressed onto the surface of the cell and a tight seal between the glass and membrane was established with gentle suction. Ionic currents were recorded using the whole-cell patch clamp technique [4] on an EPC-7 List or Dagan 8900 patch amplifier. Patch pipettes were pulled from WPI TWF150 or Corning 7502 glass capillaries and were subsequently coated with Sylgard. The pipette resistance was between 4 and 6 Mg2. Current signals were filtered with a 4-pole Bessel filter. The cut-off frequency was set at 3 or 5 kHz. The currents were sampled on-line by a Masscomp computer at 10-200 #s per point. Leakage current and capacitance were subtracted for the analyses. Fig. 1 gives an example of cells in a thin slice section viewed with a Hoffman objective. The cells located in the trigeminal nucleus could be clearly visualized (Fig. I A). A fluorescent labeled cell was readily identifiable (Fig. 1B). The average input resistance of our cells was 0.5 + 0.2 G ~ , and the zero-current membrane potential was between - 4 0 and - 6 5 mV. An action potential could be triggered when the membrane potential was held at - 7 0 mV and then depolarized beyond - 55 mV. The properties of voltage-dependent inward Na current, inward Ca current and various outward currents were studied under voltage clamp conditions. Due to the extensive dendritic processes present in these neurons, whole cell recording sometimes had a space clamp problem. This problem did not affect our recordings of Ic~, and IK. On the other hand, adequate clamping ofly~ was quite diffi-
69
cult because of its large amplitude and rapid activation kinetics. Fast INa was elicited as the depolarized potential reached - 6 0 mV (Fig. 2A upper). The voltage dependence of Na conductance (Fig. 2A lower) and its inactivation (not shown) were similar to those observed in dissociated cells [5]. The inward Ca current (Fig. 2B upper) was isolated by adding tetrodotoxin (TTX) to block the Na channels and by including tetraethylammonium (TEA) and Cs to block the K channels. Inward Ica could be separated into transient (/Ca,V), slow inactivating (/Ca,N) and non-inactivating (/Ca,L) components. ICa,Tactivated with depolarizing pulses more positive than - 6 0 mV and was inactivated with a time constant in the range of 15-50 ms. Its inactivation voltage curve reached a plateau at - 1 0 0 mV and was zero at - 50 mV [6]. ICa,N and ICa,Lwere characterized by positive activation potentials ( - 50 to - 30 mV) and slow inactivation (r > 500 ms) at room temperature. The voltage-dependent properties of ICa.N and Ica,L also are similar to those recorded in isolated cells. Large outward K currents were observed in our preparation (Fig. 3). They were studied with 70 mM K intracellular solution and TTX-containing external solution. I~: could be further separated into three components: the fast inactivating A current (I(A)), slow activating Ca-dependent K current (IK(ca)) and non-inactivating voltagedependent K current (IK¢v))(unpublished observation). IA could be isolated by its vol-
--7O
A
e
:: :;
2
),,/:
B
_ ~
°
nA
-5o
w --45 3
--40
mB
I (hA)
o
I
6.0.
hD----IOO ~'-le
--'tOO
~0
(nA)
~..0
mV
hla---- i O O
i -20
omj "4 A
V
i O0 0
(my)
,, o
60
- :
mV
.
,
-tOO
•
y (mY) iO0
•
1 Q
-tO.O
0000
--2.0
Fig. 2. Inward Na and Ca currents and their voltage dependence. A (upper): INa elicited at various test potentials from a holding potential (hp) of - 1 0 0 inV. The test potentials used are indicated by the numbers to the right o f each trace. The solutions used were (mM): external solution - - 140 NaCl, 2 KC1 2 KH2PO4, 10 HEPES, 2.5 CaC12, 1.5 MgC12, 10 glucose, 20 sucrose; pipette solution - - 120 N-methyl-Dglutamine fluoride (NMDG-F), 11 BAPTA, 2 TEA, 30 HEPES, 5 Mg-ATP, 200 #M leupeptin. The current was measusred at 35°C. A (lower): the voltage dependence of the peak INa. B (upper): inward Ca currents were measured at 22.5°C in a bath solution consisting of (in mM) l0 Ca, 120 TEA, 10 HEPES, 25 glucose, l0 CsC1, 5 4AP, 50 sucrose and l #M TTX. B (lower): the 1 - V relation of peak/Ca.
70
80
;
T •
1 4. O m A I 1(5
J. I mm
I (hA) ~4.0
hp---80
mV
6.0
•
o o
oo o o oo
__~a~( --~00
I o o
v (my) 100
--2 ° 0
Fig. 3. Outward K currents and their current-voltage relation. Upper: the currents were recorded at 35°C in an external solution containing (in mM) 4 KC1, 150 NaCI, 10 HEPES, 10 glucose, 1.5 MgCI2, 2/~M TTX. The pipette solution consisted of (mM) 70 KF, 70 N M D G - F , 5 HEPES, 90 sucrose. The test potentials used are indicated by the numbers to the left of each trace. Lower: the t V curves for peak ( O ) and steady state (C>) IK.
tage-dependent inactivation and greater sensitivity to 4-amino-pyridine. IK(ca) was distinguishable from the other two K components by the fact that it increased with external Ca 2+ concentration and was blocked by Co 2+ (unpublished observations). All the K components were blocked by TEA. The same types of voltage-dependent K currents also were observed in enzymatically dissociated cells [5]. This report describes for the first time the electrical properties of retrograde labeled trigeminothalamic neurons in the slice preparation. The currents recorded in this preparation are similar to those observed in acutely dissociated trigeminothalamic cells. The major advantages of the thin slice preparation for our application are knowing the exact location of our recorded cells and opening the possibility of studying the synaptic circuitry in the trigeminothalamic system. The work presented here will provide the necessary background for understanding the mechanisms by which transmitters and modulators act on these cells. We wish to thank Dr. A. Konner for his discussions and suggestions, S.-Y. Wong for her technical assistance and L. Simmons and M. Watson for preparing this manuscript. This work was supported by N I H NS23061 and R C D A NS01050. 1 Akaike, N., Kaneda, M., Hori, N. and Krishtal, O.A., Blockade of N-methyl-o-aspartate response in enzyme-treated rat hippocampal neurons, Neurosci. Lett., 87 (1988) 75 79.
71 2 Dubner, R. and Bennet, G.J., Spinal and trigeminal mechanisms of nociception, Annu. Rev. Neurosci., 6 (1983) 381-408. 3 Edwards, F.A., Konnerth, A., Sakmann, B. and Takahashi, T., A thin slice preparation for patch clamp recordings from neurons of the mammalian central nervous system, Pflugers Arch., 414 (1989) 6 0 0 ~ 12. 4 Hamill, O., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pflugers Arch., 391 (1981) 85-100. 5 Huang, L.-Y.M., Electrical properties of acutely isolated, identified rat spinal dorsal horn projection neurons, Neurosci. Lett., 82 (1987) 267 272. 6 Huang, L.-Y.M., Calcium channels in isolated rat dorsal horn neurones, including labelled spinothalamic and trigeminothalamic cells, J. Physiol. (Lond.), 411 (1989) 161 177. 7 Huang, L.-Y.M., The calcium current and the glutamate responses in isolated, identified spinal dorsal horn projection neurons, Soc. Neurosci. Abstr., 13 (1987) 537. 8 Katz, L.C., Burkhalter, A. and Dreyer, W.J., Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex, Nature (Lond.), 310 (1984) 498-500. 9 Willcockson, W.S., Chung, J.M., Hori, Y., Lee, K.H. and Willis, W.D., Effects of iontophoretically released peptides on primate spinothalamic tract cells, J. Neurosci., 4 (1984a) 741 750. 10 Willcockson, W.S., Chung, J.M., Hori, Y., Lee, K.H. and Willis, W.D., Effects of iontophoretically released amino acids and amines on primate spinothalamic tract cells, J. Neurosci., 4 (1984) 732 740. 11 Willis, W.D., Ascending nociceptive tracts, In W.D. Willis (Ed.), The Pain System, Karger, Basel, 1985, pp. 145 212.