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Electrical properties of acutely isolated, identified rat spinal dorsal horn projection neurons
Neuroscience Letters, 82 (1987) 267-272
267
Elsevier Scientific Publishers Ireland Ltd. NSL 04973
Electrical properties of acutely isolated, identified rat spinal dorsal horn projection neurons Li-Yen Mac Huang Marine Biomedical Institute and Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.)
(Received 15 July 1987; Revised version received 4 August 1987; Accepted 10 August 1987) Key words: Spinothalamic neuron; Trigeminothalamic neuron; Acutely isolated spinal cord cell; Na
channel; Ca channel; K channel; Patch-clamp We used a retrograde marker, rhodamine-labeled fluorescent latex microspheres and the enzyme papain to isolate a group of identified, dorsal horn projection neurons from rat spinal cord. This method has allowed us to study the electrical properties of these acutely dissociated Cells with patch-clamp techniques. It will also provide us the opportunity to understand the transmitter responses of these neurons that had not undergone culturing manipulation. The isolated projection cells have a short-lasting action potential and fire repetitively under a sustained depolarization. The ionic conductance mechanisms underlying the action potentials were studied under voltage-clamp conditions. Voltage-dependent inward sodium, inward calcium, outward potassium and calcium-activated potassium currents were observed in these cells.
D o r s a l horn p r o j e c t i o n cells, which include s p i n o t h a l a m i c a n d t r i g e m i n o t h a l a m i c neurons, have been shown to have i m p o r t a n t roles in relaying i n f o r m a t i o n related to n o x i o u s stimuli [2, 20]. T h e c o n d u c t a n c e m e c h a n i s m s o f the p r o j e c t i o n n e u r o n s a n d their responses to v a r i o u s a m i n o acids a n d p e p t i d e s are central to an u n d e r s t a n d ing o f h o w p a i n is m e d i a t e d a n d c o n t r o l l e d by the n e r v o u s system. O n e o f the impedim e n t s in the studies o f spinal n e u r o n s has been difficulties with i n t r a c e l l u l a r r e c o r d ings. M o s t studies o f channel p r o p e r t i e s in these cells were d o n e in p r i m a r y cultures o f fetal a n d n e o n a t a l tissues [7]. U n d e r such e x p e r i m e n t a l c o n d i t i o n s , the identification o f the cells were lost a n d the p r o p e r t i e s o f the cells might be altered due to the c u l t u r i n g technique. In o r d e r to c i r c u m v e n t these p r o b l e m s , we have d e v e l o p e d p r o cedures to identify a n d isolate d o r s a l h o r n p r o j e c t i o n n e u r o n s so that they are suitable for p a t c h - c l a m p recordings w i t h o u t u n d e r g o i n g c u l t u r i n g m a n i p u l a t i o n s . Prelim i n a r y results o f these studies have a p p e a r e d in an a b s t r a c t [8]. R h o d a m i n e - l a b e l e d fluorescence latex m i c r o s p h e r e s [12] were chosen to label the cells following r e t r o g r a d e t r a n s p o r t . M i c r o s p h e r e s have several characteristics that Correspondence: L.-Y.M. Huang, Marine Biomedical Institute and Department of Physiology and Bio-
physics, University of Texas Medical Branch, Galveston, TX 77550, U.S.A. 0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
268
are important for our application. First, there is little spread of the label at the injection site. Second, once taken up by the neurons, the microspheres remain inside those cells even after an enzymic dissociation procedure. Third, incorporation of the label does not change the viability of the cells. Long--Evans rats, 12-17 days old, were anesthetized with metofane and placed in a stereotaxic apparatus. A total of 1.6 ktl of fluorescent latex microspheres solution was injected into the lateral and/or medial part of the thalamus on both sides of the brain with a Hamilton microsyringe. After a 2-7 day survival time, the rats were sacrificed; the brain and the upper cervical regions of the spinal cord were removed from
O
Fig. 1. Retrograde labeling of projection cells in rats (a) Light and (b) fluorescent photomicrographs of rat brain showing the location of an injection site. (c) A labeled trigeminothalamic neuron viewed under an epifluorescent microscope. B a r = 12 /tm. (d) The distribution of labeled cells at different levels of medulla and spinal cord. Within the medulla, the caudal nucleus of the trigeminal spinal tract, gracile and cuneate nuclei were heavily labeled. Fewer labeled neurons were found in the cord; a majority of them were in the neck of the dorsal horn,
269 the animal. To confirm the injection sites, or to study the distribution of the labeled cells, the removed tissue was immediately placed on dry-ice. The tissue was then sectioned at 16/tm on a cryostat and mounted on presubbed microscope slides. To obtain isolated projection neurons, we removed the lateral portion o f the caudal medulla and the cervical spinal cord from an injected rat, cut into small pieces and incubated in a buffer solution plus 30 U/ml papain, 2 m M cysteine at 36.5°C. The buffer solution contained (mM): NaCl 120, KCI 5, CaCl2 l, MgCI2 l, glucose 30, HEPES 20. After l h, the tissue pieces were taken out of the enzyme solution and washed with buffer solution containing l mg/ml bovine serum albumin and 2 mg/ml trypsin inhibitor ovomucoid. A single cell suspension was obtained by triturating the tissue. Cells were then resuspended in the recording solution and allowed to settle to the b o t t o m of the experimental chamber. This cell dissociation procedure was similar to that described for hippocampal neurons with modifications [1 l, 13]. Electrophysiological experiments were performed at r o o m temperature unless otherwise noted. The ionic currents were studied using the patch electrode voltage-clamp method [6]. Fig. I a,b gives an example of an injection site in the ventral basal complex thalamus; distribution o f the labeled cells in the lower medulla and spinal cord is given in Fig. I d. Many cells were found in the dorsal column nuclei as well as in the nucleus caudalis of the trigeminal complex. A few cells were also observed in the neck of the dorsal horn and in the intermediate gray zone of the spinal cord. These results are in agreement with previous anatomical studies of dorsal horn projection neurons [4, 5, 9]. A labeled neuron in unfixed frozen tissue is shown in Fig. l c. These neurons normally displayed a granular fluorescence in the cytoplasm of their somata and proximal dendrites. When those cells were enzymatically isolated, the healthy ones gave a highly refractile appearance under a phase microscope (see Fig. 2a). The projection cells had bright granular fluorescence present in their cytoplasm similar to that observed in unfixed tissue slices (Fig. 2b). Less than 10% of the isolated cells were labeled with fluorescence. The electrical properties of the isolated labeled cells were studied using the whole cell recording configuration of the patch-clamp technique. Current-clamp recordings were used to establish the passive membrane and action potential properties. The average resting potential of these cells was in the range of - 65 to - 75 mV (n = 6). The
Fig. 2. Example of an isolated, identified projection cell. (a) Phase photomicrograph of a freshly dissociated cell isolated from the lower medulla. This healthy cell had a highly refractile appearance. The proximal dendritic structure was preserved; the distal dendritic processeswere usually lost with enzymatictreatment. (b) The same cell as in (a) viewed under a fluorescent microscope. Distinct granular microspheres could be observed in the cytoplasm of this cell. Bar = 15/tm.
270 input resistance was about 0.5+0.2 GQ. Action potentials were elicited when the membrane pc:ential was depolarized beyond - 5 0 mV. The action potential had an amplitude of 70-90 mV, and the rate of rise was around 100 V/s. The spike had a duration of 5-7 ms, and a hyperpolarizing afterpotential. The cell fired action potentials repetitively with reduced amplitude when a maintained depolarizing pulse was applied (Fig. 3a). Under voltage-clamp conditions, depolarizing potentials elicited both inward and outward currents. A fast inactivating inward current component was observed in those cells that had been treated with tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Cs instead of K was used as an intracellular cation in these experiments. A series of superimposed current records obtained from these cells is shown in the inset of Fig. 3b. The currents, similar to INa from other central neurons [10, 15, 18] could be blocked completely by 2/tM tetrodotoxin (TTX) and were greatly reduced upon decreasing the extracellular Na concentration. The peak INa vs voltage relationship (Fig. 3b) shows that the current activated at potentials more depolarized than - 60 mV, peaked at - 15 mV and reversed around + 37 mV. A slow activating inward current was also present in these cells. This current component was isolated when INa was blocked by 2/tM TTX, and the outward K current was suppressed by replacing intracellular K with Cs, Ba was used as the current carrier. The predominant Ba current started to activate at - 2 5 mV and did not inactivate substantially at the end of a 100-ms depolarizing pulse (Fig. 3c, inset). A second component of Ba current, which activated at more hyperpolarized potentials and inactivated relatively rapidly, was also observed. COC12 was able to block both types of slow inward currents. The current-voltage curves are illustrated in Fig. 3c. These currents have many of the characteristics of the inward long lasting and transient Ca current observed in other preparations [3, 14, 16, 17]. Outward currents were studied with K as the major intracellular cation, with the addition of 2 ~tM TTX to block 1Na. There were at least two types of K channels present in the projection cells - voltage-dependent and Ca-dependent K channels. In some cells, the IK remained constant for the duration of the pulse. In other cells the outward current rose slowly during the depolarizing pulse. Some IK current exhibited a slow decaying phase. The inactivating portion of the outward current (not shown), which decreased with extracellular 4-AP and was insensitive to the Ca channel blocker COC12, was identified as going through voltage-dependent potassium channels. The steady-state IK in some cells could be blocked substantially by 30 mM TEA and had an N-shaped current-voltage curve (Fig. 3d) that disappeared with superfusion of COC12, showing the characteristics of Ca-dependent K channels [1, 19]. This work is the first to record from acutely isolated, identified projection dorsal horn neurons from spinal cord of postnatal (14-24 days) rats. The procedure discussed here has taken advantage of the availability of a new retrograde marker and recent technical advances in electrophysiology. By combining these techniques, we have studied the membrane properties of identified projection neurons. This approach will not only enable us to characterize the effects of transmitters important in mediating nociceptive information in projection cells in the future, but will also be useful for many kinds of studies of other centrally projecting spinal neurons.
271
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-2.C Fig. 3. Electrical properties of projection cells. The basic compositions o f the solutions used were (mM): external solution NaC1 130, KCI 5, CaCI2 1, MgC12 1, HEPES 10; internal solution - K F 130, HEPES 10, Solution pH was 7.2 7.5. The osmolarity o f the solutions was adjusted with o-glucose. Changes made in the composition of individual solutions are indicated below. (a) Action potential elicited by depolarizing current pulses from a holding potential - 6 5 mV, a duration of 5 ms, and a hyperpolarizing afterpotential. The cell fired repetitively in response to a long depolarizing pulse. (b) Current-voltage curve of the peak IN, of a projection cell. Inset: lNa records o f the same cell. In order to keep IN, controlled under voltageclamp conditions, it was measured in low external Na (40 m M ) solution and at 14--15°C. Intracellular K was replaced with Cs and 30 m M T E A was added in the external solution. Calibration: 4 ms, 0.5 nA. (c) The voltage dependence of steady-state 1~ ( e ) (measured at t = 100 ms), and the ( t r a n s i e n t - s t e a d y state) 1,, ((3). Inset: IBa records. The external solution contained 2/~M TTX, 10 m M BaC12, 30 m M TEA and the internal solution had 60 m M CsF, 10 m M EGTA. The slowly inactivated c o m p o n e n t of Ba currents activated around - 25 mV and maintained at a similar level for the duration of the pulse. The transient Ba current activated and peaked at more hyperpolarizing potentials than the slowly inactivated component. No leakage and capacity current subtraction was made in this case. Calibration: 10 ms, 0.3 nA. (d) Current-voltage relationship of the Ca-dependent K current. T w o / I M TTX was added to the bath. The steady-state K currents measured at the end o f 200 ms pulses decreased when applied potentials were between + 6 0 and ÷ 100 mV. The current-voltage curve is N-shaped, a characteristic of the Ca-dependent K current. Calibration: 37 ms, 2.2 nA.
272 T h e a u t h o r t h a n k s D r . W . D . Willis f o r his i n t e r e s t a n d c o m m e n t s ; D r s . A. K a y , A. R i t c h i e , K . W e s t t u n d - H i g h , a n d R . S . W o n g f o r d i s c u s s i o n s ; K . E . F o r n i f o r t e c h n i cal a s s i s t a n c e a n d L. S i m m o n s , M . W a t s o n f o r p r e p a r i n g t h e m a n u s c r i p t . T h i s w o r k is s u p p o r t e d b y N I H N S 23061 a n d R C D A N S 0 1 0 5 0 . t Dubinsky, J.M. and Oxford, G.S., Ionic currents in two strains of rat anterior pituitary tumor cells, J. Gen. Physiol., 83 (1984) 309-339. 2 Dubner, R. and Bennet, G.J., Spinal and trigeminal mechanisms of nociception, Annu. Rev. Neurosci., 6 (1983) 381 408. 3 Fishman, M.C. and Spector, I., Potassium current suppression by quinidine reveals additional calcium currents in neuroblastoma cells, Proc. Natl. Acad. Sci. USA, 78 (1981) 5254 5249. 4 Giesler, G.J., Spiel, H.R. and Willis, W.D., Organization of spinothalamic tract axons within the rat spinal cord, J. Comp. Neurol., 195 (1981) 243-252. 5 Granum, S.L., The spinothalamic system of the rat. 1. Location of the cells of origin, J. Comp. Neurol., 247(1986) 159 180. 6 Hamill, O.P., 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, Ptliigers Arch. ges. Physiol., 391 (1981) 85 100. 7 Huang, L.-Y.M., What we have learned from patch clamp recordings of cultured cells. In J. Bottenstein and D. Sato (Eds.), Cell Culture in the Neuroscience, Plenum, New York, 1985, pp. 339 372. 8 Huang, L.-Y.M., Electrical properties of isolated spinothalamic tract cells, Soc. Neurosci. Abstr., 12 (I986) 1147~ 9 Huang, L.-Y.M., Carlton, S.M. and Willis, W.D., Identification of spinothalamic tract cells in fresh, unfixed rat spinal cord, J. Neurosci. Meth., 14 (1985) 91 96. 10 Huang, L.-Y.M., Moran, N. and Ehrenstein, G., Batrachotoxin modified the gating kinetics of sodium channels in internally perfused neuroblastoma cells, Proc. Natl. Acad. Sci. USA, 79 (1982) 2082 2085. 11 Huettner, J.E. and Baughman, R.W., Primary culture of identified neurons from the visual cortex of postnatal rats, J. Neurosci., 6 (1986) 3044~3060. 12 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 (London), 310 (1984) 498 500. 13 Kay, A.R. and Wong, R.S., Isolation of neurons suitable for patch clamping from adult mammalian central nervous systems, J. Neurosci. Meth., 16 (1986) 227 238. 14 Matteson, D.R. and Armstrong, C.M., Properties of two types of calcium channels in clonal pituitary cells, J. Gcn. Physiol., 87 (1986) 161-182. t 5 Moonlenaar, W.H. and Spector, I., Ionic currents in cultured mouse neuroblastoma cells under voltage clamp conditions, J. Physiol. (London), 278 (1978) 265-286. 16 Narahashi, T., Tsunoo, A. and Yoshii, M., Characterization of two types of calcium channels in mouse neuroblastoma cells, J. Physiol. (London), 383 (1987) 231-249. 17 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Three types of neuronal calcium channels with different calcium agonist sensitivity, Nature (London), 316 (1985)44~443. 18 Quandt, F.N. and Narahashi, T., Isolation and kinetic analysis of inward currents in neuroblastoma cells, Neuroscience, 13 (1984)24~262. 19 Ritchie, A.K., Two distinct calcium-activated potassium currents in a rat anterior pituitary cell line, J. Physiol. (London), 385 (1987) 591--609. 20 Willis, W.D., Control of nociceptive transmission in the spinal cord. In W.D. Willis (Ed.), Progress in Sensory Physiology, Springer, Berlin, 1982, 159 pp.
267
Elsevier Scientific Publishers Ireland Ltd. NSL 04973
Electrical properties of acutely isolated, identified rat spinal dorsal horn projection neurons Li-Yen Mac Huang Marine Biomedical Institute and Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.)
(Received 15 July 1987; Revised version received 4 August 1987; Accepted 10 August 1987) Key words: Spinothalamic neuron; Trigeminothalamic neuron; Acutely isolated spinal cord cell; Na
channel; Ca channel; K channel; Patch-clamp We used a retrograde marker, rhodamine-labeled fluorescent latex microspheres and the enzyme papain to isolate a group of identified, dorsal horn projection neurons from rat spinal cord. This method has allowed us to study the electrical properties of these acutely dissociated Cells with patch-clamp techniques. It will also provide us the opportunity to understand the transmitter responses of these neurons that had not undergone culturing manipulation. The isolated projection cells have a short-lasting action potential and fire repetitively under a sustained depolarization. The ionic conductance mechanisms underlying the action potentials were studied under voltage-clamp conditions. Voltage-dependent inward sodium, inward calcium, outward potassium and calcium-activated potassium currents were observed in these cells.
D o r s a l horn p r o j e c t i o n cells, which include s p i n o t h a l a m i c a n d t r i g e m i n o t h a l a m i c neurons, have been shown to have i m p o r t a n t roles in relaying i n f o r m a t i o n related to n o x i o u s stimuli [2, 20]. T h e c o n d u c t a n c e m e c h a n i s m s o f the p r o j e c t i o n n e u r o n s a n d their responses to v a r i o u s a m i n o acids a n d p e p t i d e s are central to an u n d e r s t a n d ing o f h o w p a i n is m e d i a t e d a n d c o n t r o l l e d by the n e r v o u s system. O n e o f the impedim e n t s in the studies o f spinal n e u r o n s has been difficulties with i n t r a c e l l u l a r r e c o r d ings. M o s t studies o f channel p r o p e r t i e s in these cells were d o n e in p r i m a r y cultures o f fetal a n d n e o n a t a l tissues [7]. U n d e r such e x p e r i m e n t a l c o n d i t i o n s , the identification o f the cells were lost a n d the p r o p e r t i e s o f the cells might be altered due to the c u l t u r i n g technique. In o r d e r to c i r c u m v e n t these p r o b l e m s , we have d e v e l o p e d p r o cedures to identify a n d isolate d o r s a l h o r n p r o j e c t i o n n e u r o n s so that they are suitable for p a t c h - c l a m p recordings w i t h o u t u n d e r g o i n g c u l t u r i n g m a n i p u l a t i o n s . Prelim i n a r y results o f these studies have a p p e a r e d in an a b s t r a c t [8]. R h o d a m i n e - l a b e l e d fluorescence latex m i c r o s p h e r e s [12] were chosen to label the cells following r e t r o g r a d e t r a n s p o r t . M i c r o s p h e r e s have several characteristics that Correspondence: L.-Y.M. Huang, Marine Biomedical Institute and Department of Physiology and Bio-
physics, University of Texas Medical Branch, Galveston, TX 77550, U.S.A. 0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
268
are important for our application. First, there is little spread of the label at the injection site. Second, once taken up by the neurons, the microspheres remain inside those cells even after an enzymic dissociation procedure. Third, incorporation of the label does not change the viability of the cells. Long--Evans rats, 12-17 days old, were anesthetized with metofane and placed in a stereotaxic apparatus. A total of 1.6 ktl of fluorescent latex microspheres solution was injected into the lateral and/or medial part of the thalamus on both sides of the brain with a Hamilton microsyringe. After a 2-7 day survival time, the rats were sacrificed; the brain and the upper cervical regions of the spinal cord were removed from
O
Fig. 1. Retrograde labeling of projection cells in rats (a) Light and (b) fluorescent photomicrographs of rat brain showing the location of an injection site. (c) A labeled trigeminothalamic neuron viewed under an epifluorescent microscope. B a r = 12 /tm. (d) The distribution of labeled cells at different levels of medulla and spinal cord. Within the medulla, the caudal nucleus of the trigeminal spinal tract, gracile and cuneate nuclei were heavily labeled. Fewer labeled neurons were found in the cord; a majority of them were in the neck of the dorsal horn,
269 the animal. To confirm the injection sites, or to study the distribution of the labeled cells, the removed tissue was immediately placed on dry-ice. The tissue was then sectioned at 16/tm on a cryostat and mounted on presubbed microscope slides. To obtain isolated projection neurons, we removed the lateral portion o f the caudal medulla and the cervical spinal cord from an injected rat, cut into small pieces and incubated in a buffer solution plus 30 U/ml papain, 2 m M cysteine at 36.5°C. The buffer solution contained (mM): NaCl 120, KCI 5, CaCl2 l, MgCI2 l, glucose 30, HEPES 20. After l h, the tissue pieces were taken out of the enzyme solution and washed with buffer solution containing l mg/ml bovine serum albumin and 2 mg/ml trypsin inhibitor ovomucoid. A single cell suspension was obtained by triturating the tissue. Cells were then resuspended in the recording solution and allowed to settle to the b o t t o m of the experimental chamber. This cell dissociation procedure was similar to that described for hippocampal neurons with modifications [1 l, 13]. Electrophysiological experiments were performed at r o o m temperature unless otherwise noted. The ionic currents were studied using the patch electrode voltage-clamp method [6]. Fig. I a,b gives an example of an injection site in the ventral basal complex thalamus; distribution o f the labeled cells in the lower medulla and spinal cord is given in Fig. I d. Many cells were found in the dorsal column nuclei as well as in the nucleus caudalis of the trigeminal complex. A few cells were also observed in the neck of the dorsal horn and in the intermediate gray zone of the spinal cord. These results are in agreement with previous anatomical studies of dorsal horn projection neurons [4, 5, 9]. A labeled neuron in unfixed frozen tissue is shown in Fig. l c. These neurons normally displayed a granular fluorescence in the cytoplasm of their somata and proximal dendrites. When those cells were enzymatically isolated, the healthy ones gave a highly refractile appearance under a phase microscope (see Fig. 2a). The projection cells had bright granular fluorescence present in their cytoplasm similar to that observed in unfixed tissue slices (Fig. 2b). Less than 10% of the isolated cells were labeled with fluorescence. The electrical properties of the isolated labeled cells were studied using the whole cell recording configuration of the patch-clamp technique. Current-clamp recordings were used to establish the passive membrane and action potential properties. The average resting potential of these cells was in the range of - 65 to - 75 mV (n = 6). The
Fig. 2. Example of an isolated, identified projection cell. (a) Phase photomicrograph of a freshly dissociated cell isolated from the lower medulla. This healthy cell had a highly refractile appearance. The proximal dendritic structure was preserved; the distal dendritic processeswere usually lost with enzymatictreatment. (b) The same cell as in (a) viewed under a fluorescent microscope. Distinct granular microspheres could be observed in the cytoplasm of this cell. Bar = 15/tm.
270 input resistance was about 0.5+0.2 GQ. Action potentials were elicited when the membrane pc:ential was depolarized beyond - 5 0 mV. The action potential had an amplitude of 70-90 mV, and the rate of rise was around 100 V/s. The spike had a duration of 5-7 ms, and a hyperpolarizing afterpotential. The cell fired action potentials repetitively with reduced amplitude when a maintained depolarizing pulse was applied (Fig. 3a). Under voltage-clamp conditions, depolarizing potentials elicited both inward and outward currents. A fast inactivating inward current component was observed in those cells that had been treated with tetraethylammonium (TEA) and 4-aminopyridine (4-AP). Cs instead of K was used as an intracellular cation in these experiments. A series of superimposed current records obtained from these cells is shown in the inset of Fig. 3b. The currents, similar to INa from other central neurons [10, 15, 18] could be blocked completely by 2/tM tetrodotoxin (TTX) and were greatly reduced upon decreasing the extracellular Na concentration. The peak INa vs voltage relationship (Fig. 3b) shows that the current activated at potentials more depolarized than - 60 mV, peaked at - 15 mV and reversed around + 37 mV. A slow activating inward current was also present in these cells. This current component was isolated when INa was blocked by 2/tM TTX, and the outward K current was suppressed by replacing intracellular K with Cs, Ba was used as the current carrier. The predominant Ba current started to activate at - 2 5 mV and did not inactivate substantially at the end of a 100-ms depolarizing pulse (Fig. 3c, inset). A second component of Ba current, which activated at more hyperpolarized potentials and inactivated relatively rapidly, was also observed. COC12 was able to block both types of slow inward currents. The current-voltage curves are illustrated in Fig. 3c. These currents have many of the characteristics of the inward long lasting and transient Ca current observed in other preparations [3, 14, 16, 17]. Outward currents were studied with K as the major intracellular cation, with the addition of 2 ~tM TTX to block 1Na. There were at least two types of K channels present in the projection cells - voltage-dependent and Ca-dependent K channels. In some cells, the IK remained constant for the duration of the pulse. In other cells the outward current rose slowly during the depolarizing pulse. Some IK current exhibited a slow decaying phase. The inactivating portion of the outward current (not shown), which decreased with extracellular 4-AP and was insensitive to the Ca channel blocker COC12, was identified as going through voltage-dependent potassium channels. The steady-state IK in some cells could be blocked substantially by 30 mM TEA and had an N-shaped current-voltage curve (Fig. 3d) that disappeared with superfusion of COC12, showing the characteristics of Ca-dependent K channels [1, 19]. This work is the first to record from acutely isolated, identified projection dorsal horn neurons from spinal cord of postnatal (14-24 days) rats. The procedure discussed here has taken advantage of the availability of a new retrograde marker and recent technical advances in electrophysiology. By combining these techniques, we have studied the membrane properties of identified projection neurons. This approach will not only enable us to characterize the effects of transmitters important in mediating nociceptive information in projection cells in the future, but will also be useful for many kinds of studies of other centrally projecting spinal neurons.
271
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i
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.!
-~0
~O
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30/
50
,I b
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I (hA) 8,0,
4,0,
-~o
2.0
30
-30
-gO
OJ
90
~LSO v (mv)
-2.C Fig. 3. Electrical properties of projection cells. The basic compositions o f the solutions used were (mM): external solution NaC1 130, KCI 5, CaCI2 1, MgC12 1, HEPES 10; internal solution - K F 130, HEPES 10, Solution pH was 7.2 7.5. The osmolarity o f the solutions was adjusted with o-glucose. Changes made in the composition of individual solutions are indicated below. (a) Action potential elicited by depolarizing current pulses from a holding potential - 6 5 mV, a duration of 5 ms, and a hyperpolarizing afterpotential. The cell fired repetitively in response to a long depolarizing pulse. (b) Current-voltage curve of the peak IN, of a projection cell. Inset: lNa records o f the same cell. In order to keep IN, controlled under voltageclamp conditions, it was measured in low external Na (40 m M ) solution and at 14--15°C. Intracellular K was replaced with Cs and 30 m M T E A was added in the external solution. Calibration: 4 ms, 0.5 nA. (c) The voltage dependence of steady-state 1~ ( e ) (measured at t = 100 ms), and the ( t r a n s i e n t - s t e a d y state) 1,, ((3). Inset: IBa records. The external solution contained 2/~M TTX, 10 m M BaC12, 30 m M TEA and the internal solution had 60 m M CsF, 10 m M EGTA. The slowly inactivated c o m p o n e n t of Ba currents activated around - 25 mV and maintained at a similar level for the duration of the pulse. The transient Ba current activated and peaked at more hyperpolarizing potentials than the slowly inactivated component. No leakage and capacity current subtraction was made in this case. Calibration: 10 ms, 0.3 nA. (d) Current-voltage relationship of the Ca-dependent K current. T w o / I M TTX was added to the bath. The steady-state K currents measured at the end o f 200 ms pulses decreased when applied potentials were between + 6 0 and ÷ 100 mV. The current-voltage curve is N-shaped, a characteristic of the Ca-dependent K current. Calibration: 37 ms, 2.2 nA.
272 T h e a u t h o r t h a n k s D r . W . D . Willis f o r his i n t e r e s t a n d c o m m e n t s ; D r s . A. K a y , A. R i t c h i e , K . W e s t t u n d - H i g h , a n d R . S . W o n g f o r d i s c u s s i o n s ; K . E . F o r n i f o r t e c h n i cal a s s i s t a n c e a n d L. S i m m o n s , M . W a t s o n f o r p r e p a r i n g t h e m a n u s c r i p t . T h i s w o r k is s u p p o r t e d b y N I H N S 23061 a n d R C D A N S 0 1 0 5 0 . t Dubinsky, J.M. and Oxford, G.S., Ionic currents in two strains of rat anterior pituitary tumor cells, J. Gen. Physiol., 83 (1984) 309-339. 2 Dubner, R. and Bennet, G.J., Spinal and trigeminal mechanisms of nociception, Annu. Rev. Neurosci., 6 (1983) 381 408. 3 Fishman, M.C. and Spector, I., Potassium current suppression by quinidine reveals additional calcium currents in neuroblastoma cells, Proc. Natl. Acad. Sci. USA, 78 (1981) 5254 5249. 4 Giesler, G.J., Spiel, H.R. and Willis, W.D., Organization of spinothalamic tract axons within the rat spinal cord, J. Comp. Neurol., 195 (1981) 243-252. 5 Granum, S.L., The spinothalamic system of the rat. 1. Location of the cells of origin, J. Comp. Neurol., 247(1986) 159 180. 6 Hamill, O.P., 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, Ptliigers Arch. ges. Physiol., 391 (1981) 85 100. 7 Huang, L.-Y.M., What we have learned from patch clamp recordings of cultured cells. In J. Bottenstein and D. Sato (Eds.), Cell Culture in the Neuroscience, Plenum, New York, 1985, pp. 339 372. 8 Huang, L.-Y.M., Electrical properties of isolated spinothalamic tract cells, Soc. Neurosci. Abstr., 12 (I986) 1147~ 9 Huang, L.-Y.M., Carlton, S.M. and Willis, W.D., Identification of spinothalamic tract cells in fresh, unfixed rat spinal cord, J. Neurosci. Meth., 14 (1985) 91 96. 10 Huang, L.-Y.M., Moran, N. and Ehrenstein, G., Batrachotoxin modified the gating kinetics of sodium channels in internally perfused neuroblastoma cells, Proc. Natl. Acad. Sci. USA, 79 (1982) 2082 2085. 11 Huettner, J.E. and Baughman, R.W., Primary culture of identified neurons from the visual cortex of postnatal rats, J. Neurosci., 6 (1986) 3044~3060. 12 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 (London), 310 (1984) 498 500. 13 Kay, A.R. and Wong, R.S., Isolation of neurons suitable for patch clamping from adult mammalian central nervous systems, J. Neurosci. Meth., 16 (1986) 227 238. 14 Matteson, D.R. and Armstrong, C.M., Properties of two types of calcium channels in clonal pituitary cells, J. Gcn. Physiol., 87 (1986) 161-182. t 5 Moonlenaar, W.H. and Spector, I., Ionic currents in cultured mouse neuroblastoma cells under voltage clamp conditions, J. Physiol. (London), 278 (1978) 265-286. 16 Narahashi, T., Tsunoo, A. and Yoshii, M., Characterization of two types of calcium channels in mouse neuroblastoma cells, J. Physiol. (London), 383 (1987) 231-249. 17 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Three types of neuronal calcium channels with different calcium agonist sensitivity, Nature (London), 316 (1985)44~443. 18 Quandt, F.N. and Narahashi, T., Isolation and kinetic analysis of inward currents in neuroblastoma cells, Neuroscience, 13 (1984)24~262. 19 Ritchie, A.K., Two distinct calcium-activated potassium currents in a rat anterior pituitary cell line, J. Physiol. (London), 385 (1987) 591--609. 20 Willis, W.D., Control of nociceptive transmission in the spinal cord. In W.D. Willis (Ed.), Progress in Sensory Physiology, Springer, Berlin, 1982, 159 pp.