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Voltage-activated potassium currents in acutely dissociated hippocampal dentate gyrus neurons from neonatal rats
DEVELOPMENTAL BRAIN RESEARCH
ELSEVIER
Developmental Brain Research 81 (1994) 309-313
Short communication
Voltage-activated potassium currents in acutely dissociated hippocampal dentate gyrus neurons from neonatal rats Scott C. Baraban Departments of n Pharmacology and
b
a,
Eric W. Lothman
b,*
Neurology, University of Virginia, Charlottesville, VA 22908, USA Accepted 7 June 1994
Abstract We have studied outward currents of neurons acutely dissociated from the dentate gyrus region of hippocampus using whole-cell and perforated patch recordings. Depolarizing voltage commands activated sustained outward currents at all ages tested (P5-P30). Outward currents were blocked by tetraethylammonium 00 mM) but not 4-aminopyridine (25 mM). Comparison of sustained potassium current during postnatal development showed a significant increase in current amplitUde with age reaching a peak between P20 and P30. These results suggest an overall increase in the number of voltage-dependent ionic channels during development, specifically those underlying TEA-sensitive potassium currents.
Key words: Acute cell dissociation; Amphotericin B; Hippocampus; Nystatin; Potassium current; Tetraethylammonium
The rat dentate gyrus (DG) is unique in that the majority of DG neurons (at least 85%) become postmitotic during the postnatal period [1,18]. DG cells arise from the hilus and migrate to the granule cell layer during the first two postnatal weeks [16,18]. Although some newly born DG cells are glia, recent studies indieate that most cells born in the DG differentiate into neurons [3,10]. Neurons in the DG may be critieal in the development of abnormal hippocampal function [19,22]. Therefore, electrophysiological characterization of DG cells is important for understanding both hippocampal function and neurogenesis. In the present study, experiments were performed to examine the postnatal development of potassium currents in acutely dissociated DG neurons. Neurons were acutely dissociated as described previously [4,6,11]. Briefly, the dentate gyrus was dissociated from immature postnatal (P) day 5 to P30 day rats anesthetized with halothane. The brain was rapidly removed and 400-450 j,Lm vibratome sections were cut in ice cold artificial cerebrospinal fluid (ACSF) solution containing (in mM) sucrose 5, glucose 10, NaHC0 3
* Corresponding author. University of Virginia Medical Center, Box 394, Charlottesville, VA 22908, USA. Fax: (1) (804) 982-1726. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-3806(94 )001 06-A
26, NaH 2 P04 1.25, KCI 3, MgC1 2 2, CaCl 2 2. The slices were then incubated in PIPES medium containing (in mM) NaCI 120, PIPES 20, glucose 25, KCl 5, MgCl 2 1, CaC1 2 1, gassed with 95% O 2 /5% CO 2 for 1 h. The hippocampal regions were microdissected and incubated in oxygenated PIPES medium with 30 mg/rol Sigma protease XXIII for 33 min. After enzyme treatment the slices were washed and incubated in PIPES medium gassed with 95% O 2 /5% CO 2 for 30-45 min. The DG region was subdissected and mechanically dissociated by trituration. Acutely dissociated neurons were plated onto LUX 5521 culture plates in HEPES extracellular recording solution containing (in mM) NaCl 150, KCI 5, MgCl 2 1, CaCl 2 2, HEPES 10, sucrose 10 (320-325 mOsm measured on an osmomoter; Wescor). These cells were stored in a well oxygenated, humidified chamber until recording. Electrophysiologieal recordings were performed within 3.5 h of cell dissociation. Two types of voltageclamp techniques were used to record outward currents 0) the conventional whole-cell configuration [7] and (ii) the perforated patch configuration [8,15J. In whole-cell configuration intracellular pipette solutions contained (in mM) KMeS0 4 160, HEPES 10, EGTA, 11, CaCl 2 1, MgCl 2 2 (310-320 mOsm). In perforated patch configuration intracellular pipette solutions Contained (in mM) KCI 55, K 2 S04 75, HEPES 10, MgCl 2
310
S.c. Baraban, E. w: Lothman / Developmental Brain Research 81 (J994) 309-313
2 (300-310 mOsm); nystatin or amphotericin B was added to the recording pipette to a final concentration of 150 ,ug/ml. Tetraethylammonium chloride (TEA) and 4-aminopyridine C4-AP) (Sigma; St. Louis, MO) were applied to the bath solution from multi-barrel pipettes positioned within 500 ,urn from the cell surface. The resistance of fire-polished patch-pipettes filled with intracellular recording solution was 2-7 Mn. Voltage steps were delivered via a multi-channel stimulator (Winston Electronics Co.). The current and voltage were recorded with a Axoclamp 2 amplifier (single-electrode voltage-clamp mode) and monitored on a storage oscilloscope (Nicolet). Voltage-clamp data was sampled at 2.8-3.5 kHz, filtered at 3 kHz and collected with an IBM PC 486 computer using pClamp software. Leak and capacitive currents which were comparable at all ages tested were subtracted. All experiments were carried out at room temperature (23°-25°C). Data were analyzed on a Macintosh using AXOGRAPH software (Axon Instruments). Data values are given as mean ± S.E.M. After a one-way analysis of variance, a Kruskal-Wallis H-test was used to
A1. control
81. control
Jr----
...J.::===========r A2. TEA
l
82.4-AP
JI--:~---------------r'
A3. wash
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Fig. 1. Effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) application on voltage-activated currents in P15-P20 neurons. A1: currents evoked in a DG neuron with the use of a standard protocol (bottom right). A2: currents evoked during 10 mM TEA application. A3: currents evoked after washout of 10 mM TEA. B1: Currents evoked in a different DG neuron under the same conditions. B2: currents evoked during 25 mM 4-AP application. Note the inhibition of voltage-activated current during TEA application and the lack of effect of 4-AP.
analyze the effect of age on peak current amplitude. Significance level was taken as P < 0.05. Acute dissociation of the DG region of the hippocampus yielded a cell population of phase-bright neurons. These cells excluded the dye Trypan blue, a standard test for evaluating cell viability [13,17]. Cells were typically characterized as three-dimensional with well differentiated dendrites (examples in Figs. 2A2 and 2B2). Voltage-clamp recordings demonstrated electrophysiological viability of these neurons for up to 3.5 h after dissociation. After establishing a gigaohm seal in current-clamp mode, neurons were voltage-clamped at a holding potential of - 50 to - 60 m V. Voltage-clamp recordings were obtained using whole-cell and perforated-patch recording techniques. Outward currents were elicited by depolarizing voltage commands from the holding potential. Sustained outward currents were elicited at potentials more positive than - 30 mY. Voltageactivated outward currents were comparable in amplitude and longevity in whole-cell and perforated-patch configurations and this data was pooled. The outward currents in DG cells were not stable for recording periods > 15 min and amplitude data was compared from voltage protocols generated within the first 5 min of voltage-clamp recording. Drug applications were performed at all ages. However, stable and prolonged recordings were optimal during the P15 to P20 period. Detailed and systematic pharmacological analysis of outward currents was limited to DG cells from this age period (P15-P20), although comparable results were obtained at other ages (P5 and PlO). Sustained voltage-activated outward currents elicited in DG cells were significantly reduced during 10 mM TEA application (inhibition: 71.6 ± 2.4%; n = 6; Fig. 1A1-1A3). Application of 1 mM TEA did not reduce outward currents (n = 3). Application of 25 mM 4-AP had no significant effect on voltage-activated outward currents (n = 5; Fig. 1B11B2). In the first 3 weeks of postnatal development, there was a progressive increase in the amplitude of outward potassium current (I K) evoked with depolarizing commands. Voltage-step protocols from the holding potential (- 50 to - 60 mV) to more positive potentials (+ 10 to + 20 mV) revealed larger sustained outward currents at later stages of development (compare Figs. 2Al and 2B1). This was especially evident for current evoked with depolarizations positive to - 10 mY. All DG voltage-clamp recordings were made on comparably sized cells as shown (Figs. 2A2 and 2B2). Therefore, the increase in I K amplitude with age could not be explained by an increase in cell size. The amplitude of sustained potassium current obtained in P5-P30 neurons was compared for a depolarization command to + 10 mV (Fig. 2C). Measurements were obtained at
S.c. Baraball, E. W Lothman / Developmental Brain Research 81 (1994) 309-313
the 200-ms mark of a 250-ms depolarizing command, at a potential where potassium currents reached a steady state. The mean amplitude of IK was 1.8 ± 0.3 nA (n = 6) at P20 and 2.4 ± 0.4 nA (n = 7) at P30; both values were significantly greater than IK measured at P5 (0.9 ± 0.1 nA; n = 8). The results of the present study demonstrate that DG neurons in the rat hippocampus undergo changes in their excitable membrane properties during postnatal development. Voltage-activated potassium channels playa central role in the excitability of neurons. These channels contribute to the maintenance of the resting membrane potential, the shaping of the action potential and the control of the frequency of action potential discharge. During the first month of postnatal development we observed a progressive age-related increase in
A1.
I
311
the amplitude of potassium current generated in DG cells (Fig. 2C). The I K increase occurred in comparably sized DG neurons (Figs. 2A2 and 2B2) and therefore is not due to an age-related cell size difference. Previous studies of immature hippocampal neurons in slices using conventional intracellular recording electrodes reported a longer action potential width in young (PIP7) animals [20,21]. The longer duration action potentials observed in these animals would correspond with the smaller I K present at this stage of development. In the rat, the great majority of DG cells are generated postnatally with the peak of cell proliferation occurring toward the end of the first postnatal week [1,16]. Postnatal DG neurogenesis is unique in that the principal cells in the hippocampus migrate and develop during the embryonic period. Thus, the DG of the rat
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Fig. 2. Changes in the voltage-activated outward currents recorded during postnatal development. Ai: currents evoked in a P5 DO neuron with the use of a standard protocol (inset). A2: example of a typical P5 DO neuron. B1: currents evoked in a P20 DO neuron under the same conditions. B2: example of a typical P20 DO neuron. Calibration bar (in A2) = 10 JLm for A2 and B2. Note the size of isolated DG neurons is comparable at these 2 ages but the voltage-activated current is larger at P20. C: peak current amplitUde measured during a voltage step to + 10 mY as a function of age. The data represent the mean ± S.E.M. Number of cells recorded is indicated in parentheses. Current evoked at P20 and P30 was significantly greater than current evoked at PS (significance taken as P < 0.05).
312
S.C. Baraban, E. W. Lothman / Developmental Brain Research 81 (J994) 309-313
offers the opportunity to study neurobiological aspects of the maturation of nerve cells over the period from postmitotic birth to full postnatal maturity. Previous biochemical studies have utilized autoradiography and tritiated-thymidine tracing to demonstrate the unique development of DG cells (1,3,16]' In this study, we provide electrophysiological evidence in isolated DG cells of a postnatal developmental change in the function of potassium ion channels. Voltage-activated potassium currents appear during early postnatal development (24]. The appearance of potassium current coincides with the developmental expression of potassium channels. Specifically, several potassium channel isoforms (Kvl, Kv2, Kv2.1, Kv3.1a, RCKI and RCKS) demonstrate increased levels of expression between PI and P30 (2,14,25,26]. Luneau et al. have shown that TEA-sensitive potassium currents can be elicited in cells expressing K + channel mRNA [12]. In this study, we identified TEA-sensitive potassium currents in isolated DG cells (Fig. lA) from developing rats. Further, [K evoked in isolated DG cells progressively increased between P5 and P30 (Fig. 2). This finding provides additional evidence for a developmental increase in K + channel expression and is in agreement with previous studies where IK has been shown to increase during postnatal development [5,9,23,27]. An age-dependent increase in ion channel expression is one hypothesis for the developmental change in potassium current observed. A second possibility is an increase in the conductance or mean open time of individual potassium ion channels. Clearly, additional electrophysiological approaches, in combination with molecular biological studies, will be required to fully understand the development of potassium ion channels.
This work was supported by grant support from the NINDS.
[1] Bayer, S.A., Development of the hippocampal region in the rat. I. Neufogenesis examined with 3H-thymidine autoradiography, 1 Compo Neurol., 190 (1980) 87-114. [2] Beckh, S. and Pongs, 0., Members of the RCK potassium channel family are differentially expressed in the rat nervous system, EMBO 1,9 (1990) 777-782. [3] Cameron H.A., Woolley C.S., McEwen B.S. and Gould, E., Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat, Neuroscience, 56 (1993) 337-344. [4] Coulter, D.A., Huguenard, J.R and Prince, D.A., Differential effects of petit mal anticonvulsants and convulsants on thalamic neurones: GABA current blockade, Br. 1. Pharmacol., 100 (1990) 807-813. [5] Chad, J.E., Stanford, I., Wheal, H.Y., Williamson, Rand Woodhall, G., Dissociated neurons from adult rat hippocampus. In In J. Chad and H. Wheal (Eds.), Cellular Neurobiology: A Practical Approach, Oxford University Press, New York, 1991.
[7] Chen, F., Wetzel, G.T., Friedman, W.F. and Klitner, T.S., Single-channel recording of inwardly rectifying potassium currents in developing myocardium, f. Mol. Cell. Cardiol., 23 (1991) 259-267. [8J Hamill, D.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, Pjliigers Arch., 391 (1981) 85-100. [9] Horn, R. and Marty, A., Muscarinic activation of ionic currents measured by a new whole-cell recording method, J. Gen. Physiol., 92 (1988) 145-159. [10] Josephson, I.R and Sperelakis, N., Developmental increases in the inwardly rectifying potassium current of embryonic chick ventricular myocytes, Biochim, Biophys. Acta, 1052 (1990) 123127. [11] Kaplan M.S. and Hinds J.w., Neurogenesis in the adult rat: electron microscopic analysis of light radio autographs, Science, 197 (1977) 1092-1094. [12] Kay, A.R. and Wong, KS., Isolation of neurons suitable for patch-clamp recording from adult mammalian central nervous system, f. Nel/rosci. Meth., 16 (1986) 227-238. [13] Luneau, C.J., Williams, J.B., Marshall, J., Levitan, E.S., Oliva, C., Smith, J.S., Antanavage,J., Folander, K, Stein, R.B., Swanson, R, Kaczmarek, L.K and Buhrow, S.A., Alternative splicing contributes to the generation of K + channel diversity in the mammalian central nervous system, Proc. Natl. Acad. Sci. USA, 88 (1991) 3932-3936. [14] Michel, P.P., Yyas, S. and Agid, Y., Toxic effects of iron for cultured mesencephalic dopaminergic neurons derived from rat embryonic brains, f. Neurochem., 59 (1992) 118-127. [15] Perney, T.M., Marshall, J., Martin, KA., Rockfield, S. and Kaczmarek, L.K, Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain, f. Neurophys., 68 (1992) 756-766. [16] Rae, J., Cooper, K, Gates, P. and Watsky, M., Low access resistance perforated patch recordings using amphotericin B, 1. Neurosci. Meth., 37 (1991) 15-26. [17] Rickmann, M., Amaral, D.G. and Cowan, W.M., Organization of radial glial and mature astrocytes studied with a monoclonal antibody to vimentin, 1. Comp. Neurol., 264 (1987) 449-479. [18] Samples, S.D. and Dubinsky, J.M., Aurintricarboxylic acid protects hippocampal neurons from glutamate excitotoxicity in vitro, f. Neurochem., 61 (1993) 382-385. [19] Schlessinger, A.R, Cowan, W.M. and Gottlieb, D.J., An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat, 1. Compo Neurol., 159 (1975) 149-176. [20] Scharfman, H.E. and Schwartzkroin, P.A., Responses of cells of the rat fascia dentata to prolonged stimulation of the perforant path: sensitivity of hilar cells and changes in granule cell excitability, Neuroscience, 35 (1990) 491-504. [21] Schwartzkroin, P.A. and Altschuler, R.I., Development of kitten hippocampal neurons, Brain Res., 134 (1977) 429-444. [22] Schwartzkroin, P.A., Development of rabbit hippocampus: physiology, Del). Brain Res., 2 (1982) 469-486. [23] Sloviter, RS., Decreased hippocampal inhibition and a selective loss of interneurons in experimental epilepsy, Science, 235 (1987) 73-76. [24] Spigelman, 1., Zhang, L. and Carlen, P.L., Patch-clamp study of postnatal development of CAl neurons in rat hippocampal slices: membrane excitability and K+ currents, 1. Neurophys., 68 (1992) 55-69. [25] Spitzer, N.C., A developmental handshake: neuronal control of ionic currents and their control of neuronal differentiation, 1. Neurobiol., 22 (1991) 659-673. [26] Swanson, R., Marshall, J., Smith, J.S., Williams, J.B., Boyle, M.B., Folander, K., Luneau, C.J., Antanavage, J., Oliva, C.,
S.c. Barahan, E. W. Lothman I Developmental Brain Research 81 (1994) 309-313 Buhrow, S.A., Vennett, c., Stein, R.B. and Kaczmarek, L.K., Cloning and expression of eDNA and genomic clones encoding three delayed rectifier potassium channels in rat brain, Neuron, 4 (1990) 929-939. [27] Trimmer, J.S., Expression of Kv2.1 delayed rectifier K+ chan-
313
nel isoforms in the developing rat brain, FEBS Lett., 324 (1993) 205-210. [28] Wahler, G.M., Developmental increases in the inwardly rectifying potassium current of rat ventricular myocytes, Am. J. Physiol., 262 (1992) C1266-C1272.
ELSEVIER
Developmental Brain Research 81 (1994) 309-313
Short communication
Voltage-activated potassium currents in acutely dissociated hippocampal dentate gyrus neurons from neonatal rats Scott C. Baraban Departments of n Pharmacology and
b
a,
Eric W. Lothman
b,*
Neurology, University of Virginia, Charlottesville, VA 22908, USA Accepted 7 June 1994
Abstract We have studied outward currents of neurons acutely dissociated from the dentate gyrus region of hippocampus using whole-cell and perforated patch recordings. Depolarizing voltage commands activated sustained outward currents at all ages tested (P5-P30). Outward currents were blocked by tetraethylammonium 00 mM) but not 4-aminopyridine (25 mM). Comparison of sustained potassium current during postnatal development showed a significant increase in current amplitUde with age reaching a peak between P20 and P30. These results suggest an overall increase in the number of voltage-dependent ionic channels during development, specifically those underlying TEA-sensitive potassium currents.
Key words: Acute cell dissociation; Amphotericin B; Hippocampus; Nystatin; Potassium current; Tetraethylammonium
The rat dentate gyrus (DG) is unique in that the majority of DG neurons (at least 85%) become postmitotic during the postnatal period [1,18]. DG cells arise from the hilus and migrate to the granule cell layer during the first two postnatal weeks [16,18]. Although some newly born DG cells are glia, recent studies indieate that most cells born in the DG differentiate into neurons [3,10]. Neurons in the DG may be critieal in the development of abnormal hippocampal function [19,22]. Therefore, electrophysiological characterization of DG cells is important for understanding both hippocampal function and neurogenesis. In the present study, experiments were performed to examine the postnatal development of potassium currents in acutely dissociated DG neurons. Neurons were acutely dissociated as described previously [4,6,11]. Briefly, the dentate gyrus was dissociated from immature postnatal (P) day 5 to P30 day rats anesthetized with halothane. The brain was rapidly removed and 400-450 j,Lm vibratome sections were cut in ice cold artificial cerebrospinal fluid (ACSF) solution containing (in mM) sucrose 5, glucose 10, NaHC0 3
* Corresponding author. University of Virginia Medical Center, Box 394, Charlottesville, VA 22908, USA. Fax: (1) (804) 982-1726. 0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-3806(94 )001 06-A
26, NaH 2 P04 1.25, KCI 3, MgC1 2 2, CaCl 2 2. The slices were then incubated in PIPES medium containing (in mM) NaCI 120, PIPES 20, glucose 25, KCl 5, MgCl 2 1, CaC1 2 1, gassed with 95% O 2 /5% CO 2 for 1 h. The hippocampal regions were microdissected and incubated in oxygenated PIPES medium with 30 mg/rol Sigma protease XXIII for 33 min. After enzyme treatment the slices were washed and incubated in PIPES medium gassed with 95% O 2 /5% CO 2 for 30-45 min. The DG region was subdissected and mechanically dissociated by trituration. Acutely dissociated neurons were plated onto LUX 5521 culture plates in HEPES extracellular recording solution containing (in mM) NaCl 150, KCI 5, MgCl 2 1, CaCl 2 2, HEPES 10, sucrose 10 (320-325 mOsm measured on an osmomoter; Wescor). These cells were stored in a well oxygenated, humidified chamber until recording. Electrophysiologieal recordings were performed within 3.5 h of cell dissociation. Two types of voltageclamp techniques were used to record outward currents 0) the conventional whole-cell configuration [7] and (ii) the perforated patch configuration [8,15J. In whole-cell configuration intracellular pipette solutions contained (in mM) KMeS0 4 160, HEPES 10, EGTA, 11, CaCl 2 1, MgCl 2 2 (310-320 mOsm). In perforated patch configuration intracellular pipette solutions Contained (in mM) KCI 55, K 2 S04 75, HEPES 10, MgCl 2
310
S.c. Baraban, E. w: Lothman / Developmental Brain Research 81 (J994) 309-313
2 (300-310 mOsm); nystatin or amphotericin B was added to the recording pipette to a final concentration of 150 ,ug/ml. Tetraethylammonium chloride (TEA) and 4-aminopyridine C4-AP) (Sigma; St. Louis, MO) were applied to the bath solution from multi-barrel pipettes positioned within 500 ,urn from the cell surface. The resistance of fire-polished patch-pipettes filled with intracellular recording solution was 2-7 Mn. Voltage steps were delivered via a multi-channel stimulator (Winston Electronics Co.). The current and voltage were recorded with a Axoclamp 2 amplifier (single-electrode voltage-clamp mode) and monitored on a storage oscilloscope (Nicolet). Voltage-clamp data was sampled at 2.8-3.5 kHz, filtered at 3 kHz and collected with an IBM PC 486 computer using pClamp software. Leak and capacitive currents which were comparable at all ages tested were subtracted. All experiments were carried out at room temperature (23°-25°C). Data were analyzed on a Macintosh using AXOGRAPH software (Axon Instruments). Data values are given as mean ± S.E.M. After a one-way analysis of variance, a Kruskal-Wallis H-test was used to
A1. control
81. control
Jr----
...J.::===========r A2. TEA
l
82.4-AP
JI--:~---------------r'
A3. wash
J 500 pA 10ms
Fig. 1. Effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) application on voltage-activated currents in P15-P20 neurons. A1: currents evoked in a DG neuron with the use of a standard protocol (bottom right). A2: currents evoked during 10 mM TEA application. A3: currents evoked after washout of 10 mM TEA. B1: Currents evoked in a different DG neuron under the same conditions. B2: currents evoked during 25 mM 4-AP application. Note the inhibition of voltage-activated current during TEA application and the lack of effect of 4-AP.
analyze the effect of age on peak current amplitude. Significance level was taken as P < 0.05. Acute dissociation of the DG region of the hippocampus yielded a cell population of phase-bright neurons. These cells excluded the dye Trypan blue, a standard test for evaluating cell viability [13,17]. Cells were typically characterized as three-dimensional with well differentiated dendrites (examples in Figs. 2A2 and 2B2). Voltage-clamp recordings demonstrated electrophysiological viability of these neurons for up to 3.5 h after dissociation. After establishing a gigaohm seal in current-clamp mode, neurons were voltage-clamped at a holding potential of - 50 to - 60 m V. Voltage-clamp recordings were obtained using whole-cell and perforated-patch recording techniques. Outward currents were elicited by depolarizing voltage commands from the holding potential. Sustained outward currents were elicited at potentials more positive than - 30 mY. Voltageactivated outward currents were comparable in amplitude and longevity in whole-cell and perforated-patch configurations and this data was pooled. The outward currents in DG cells were not stable for recording periods > 15 min and amplitude data was compared from voltage protocols generated within the first 5 min of voltage-clamp recording. Drug applications were performed at all ages. However, stable and prolonged recordings were optimal during the P15 to P20 period. Detailed and systematic pharmacological analysis of outward currents was limited to DG cells from this age period (P15-P20), although comparable results were obtained at other ages (P5 and PlO). Sustained voltage-activated outward currents elicited in DG cells were significantly reduced during 10 mM TEA application (inhibition: 71.6 ± 2.4%; n = 6; Fig. 1A1-1A3). Application of 1 mM TEA did not reduce outward currents (n = 3). Application of 25 mM 4-AP had no significant effect on voltage-activated outward currents (n = 5; Fig. 1B11B2). In the first 3 weeks of postnatal development, there was a progressive increase in the amplitude of outward potassium current (I K) evoked with depolarizing commands. Voltage-step protocols from the holding potential (- 50 to - 60 mV) to more positive potentials (+ 10 to + 20 mV) revealed larger sustained outward currents at later stages of development (compare Figs. 2Al and 2B1). This was especially evident for current evoked with depolarizations positive to - 10 mY. All DG voltage-clamp recordings were made on comparably sized cells as shown (Figs. 2A2 and 2B2). Therefore, the increase in I K amplitude with age could not be explained by an increase in cell size. The amplitude of sustained potassium current obtained in P5-P30 neurons was compared for a depolarization command to + 10 mV (Fig. 2C). Measurements were obtained at
S.c. Baraball, E. W Lothman / Developmental Brain Research 81 (1994) 309-313
the 200-ms mark of a 250-ms depolarizing command, at a potential where potassium currents reached a steady state. The mean amplitude of IK was 1.8 ± 0.3 nA (n = 6) at P20 and 2.4 ± 0.4 nA (n = 7) at P30; both values were significantly greater than IK measured at P5 (0.9 ± 0.1 nA; n = 8). The results of the present study demonstrate that DG neurons in the rat hippocampus undergo changes in their excitable membrane properties during postnatal development. Voltage-activated potassium channels playa central role in the excitability of neurons. These channels contribute to the maintenance of the resting membrane potential, the shaping of the action potential and the control of the frequency of action potential discharge. During the first month of postnatal development we observed a progressive age-related increase in
A1.
I
311
the amplitude of potassium current generated in DG cells (Fig. 2C). The I K increase occurred in comparably sized DG neurons (Figs. 2A2 and 2B2) and therefore is not due to an age-related cell size difference. Previous studies of immature hippocampal neurons in slices using conventional intracellular recording electrodes reported a longer action potential width in young (PIP7) animals [20,21]. The longer duration action potentials observed in these animals would correspond with the smaller I K present at this stage of development. In the rat, the great majority of DG cells are generated postnatally with the peak of cell proliferation occurring toward the end of the first postnatal week [1,16]. Postnatal DG neurogenesis is unique in that the principal cells in the hippocampus migrate and develop during the embryonic period. Thus, the DG of the rat
A2.
I
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30
Fig. 2. Changes in the voltage-activated outward currents recorded during postnatal development. Ai: currents evoked in a P5 DO neuron with the use of a standard protocol (inset). A2: example of a typical P5 DO neuron. B1: currents evoked in a P20 DO neuron under the same conditions. B2: example of a typical P20 DO neuron. Calibration bar (in A2) = 10 JLm for A2 and B2. Note the size of isolated DG neurons is comparable at these 2 ages but the voltage-activated current is larger at P20. C: peak current amplitUde measured during a voltage step to + 10 mY as a function of age. The data represent the mean ± S.E.M. Number of cells recorded is indicated in parentheses. Current evoked at P20 and P30 was significantly greater than current evoked at PS (significance taken as P < 0.05).
312
S.C. Baraban, E. W. Lothman / Developmental Brain Research 81 (J994) 309-313
offers the opportunity to study neurobiological aspects of the maturation of nerve cells over the period from postmitotic birth to full postnatal maturity. Previous biochemical studies have utilized autoradiography and tritiated-thymidine tracing to demonstrate the unique development of DG cells (1,3,16]' In this study, we provide electrophysiological evidence in isolated DG cells of a postnatal developmental change in the function of potassium ion channels. Voltage-activated potassium currents appear during early postnatal development (24]. The appearance of potassium current coincides with the developmental expression of potassium channels. Specifically, several potassium channel isoforms (Kvl, Kv2, Kv2.1, Kv3.1a, RCKI and RCKS) demonstrate increased levels of expression between PI and P30 (2,14,25,26]. Luneau et al. have shown that TEA-sensitive potassium currents can be elicited in cells expressing K + channel mRNA [12]. In this study, we identified TEA-sensitive potassium currents in isolated DG cells (Fig. lA) from developing rats. Further, [K evoked in isolated DG cells progressively increased between P5 and P30 (Fig. 2). This finding provides additional evidence for a developmental increase in K + channel expression and is in agreement with previous studies where IK has been shown to increase during postnatal development [5,9,23,27]. An age-dependent increase in ion channel expression is one hypothesis for the developmental change in potassium current observed. A second possibility is an increase in the conductance or mean open time of individual potassium ion channels. Clearly, additional electrophysiological approaches, in combination with molecular biological studies, will be required to fully understand the development of potassium ion channels.
This work was supported by grant support from the NINDS.
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