Neuroscience Letters, 135 (1992) 231-234

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© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00 NSL 08378

Young CA1 pyramidal cells of rats, but not dentate gyrus granule cells, express a delayed inward rectifying current with properites of IQ J a s m i n e Stabel, E c k h a r d F i c k e r a n d U w e H e i n e m a n n Institut fiir Neurophysiologie, Zentrurn fiir Physiologic und Pathophysiologie, Universitgitzu K6ln, K61n ( E R. G.)

(Received 9 October 1991; Revised version received 29 October 1991; Accepted 4 November 1991) Key words: CA1 pyramidal cell; Dentate gyrus granule cell; Inward rectifier; Io; Ontogenesis; Rat

In hippocampal CA I pyramidal cells (CA1PC) and dentate gyrus granule cells (DGGC) we compared the expression of currents which could cause differences in discharge behaviour. Negative current injections cause a uniform hyperpolarization in DGGC whereas in CA1PC the initial hyperpolarization is followed by a repolarization towards resting membrane potential. The underlying inward current can be classifiedas IQ. It is sensitive to CsC1, activated at -80 mV, and it has a mean amplitude of-109.8 pA and a mean activation time constant of 187 ms with voltagejumps from -40 to -120 inV. We conclude that some of the differences in response properties of DGGC and CA 1PC upon repetitive stimulation can be attributed to differences in the expression of Io.

Both dentate gyrus granule cells and hippocampal pyramidal cells display slow inhibitory postsynaptic potentials (IPSPs) upon afferent stimulation [2, 10, 15]. In dentate gyrus slow IPSPs largely account for frequency habituation [11], i.e. a decrease in stimulus response upon repetitive stimulation. In hippocampal pyramidal cells repetitive stimulation regularly leads to frequency potentiation in spite of the generation of slow IPSPs [1, 6]. This may point to differences in expression of current components activated near resting membrane potential. We therefore compared cell properties of visually identified dentate gyrus granule cells and CA1 pyramidal cells in a thin slice preparation [5] using current clamp and voltage clamp recording techniques. The studies were performed on transverse hippocampal slices obtained from 10-20 day old Wistar rats. The method described by Edwards et al. [5] was followed. The animals were decapitated under deep ether anaesthesia, the brain was removed and submerged in oxygenated ice-cold artificial cerebrospinal fluid (ACSF). The bisected brain was fixed with cyanoacrylate glue to a Campden vibroslicer and horizontal slices of a thickness of 150/lm were made. After cutting out the hippocampus the hippocampal slices were stored on a net in oxygenated ACSF containing (in mM): NaC1 124, NaH2PO 4 1.25,

Correspondence: J. Stabel, Institut for Neurophysiologie, Zentrum ftir Physiology und Pathophysiologie, Universit~.t zu K6ln, Robert Koch Stral3e 39, D-W-5000 K61n 41, F.R.G.

MgSO4 1.8, CaC12 1.6, KC1 3, glucose 10 and NaHCO3 26. Temperature was held at 34°C and p H at 7.4. One slice at a time was transferred into a measuring chamber in an upright Zeiss microscope and superfused with oxygenated ACSF (2 ml/min) at r o o m temperature. The ACSF, otherwise identical to the ACSF used for storage, contained 5 m M KC1 and 1 0 / I M bicuculline; MgSO4 was replaced by MgC12 and NaH2PO 4 was omitted. For studies on effects of Cs + and Ba 2+ those agents were added as C1- salts to the ACSF in a concentration of 1 mM. In order to block Na + spikes, tetrodotoxin (TTX) was added in a concentration of 1/~M. In the measuring chamber the slice was held down with parallel nylon threads stretched 1 m m apart over an u-shaped silver frame. Cells were visually identified and their surface was cleaned by a stream of ACSF blown out and sucked in through a 5 - 1 0 / l m cleaning pipette [14]. Recordings were performed with patch pipettes (5-8 M~2) made from borosilicate glass in the whole cell mode using a discontinuous single electrode amplifier (SEC1L npi electronic) both in the current and voltage clamp mode. The switching frequency was usually about 25 kHz with 25% duty cycle. Pipette solution contained (in mM): KC1 120, E G T A 11, MgC12 2, CaC12 1 and H E P E S 10. Osmolarity was adjusted to 300 m O s m with glucose and p H was 7.1. The data were obtained from 31 dentate gyrus granule cells ( D G G C ) and 29 CA1 pyramidal cells (CA1PC). The cells were always first recorded in the current clamp mode without TTX, in order to judge the quality of the

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recorded cells. Cells were accepted for further study when the resting membrane potential was below -50 mV, the input resistance above 250 MD and when the amplitude of action potentials (AP) was greater than 70 mV. The average resting membrane potential of D G G C was -73.6 + 10.3 mV (n=31), of C A I P C 65.7 _+ 9.4 mV (n=29). The mean input resistance was 541.1 + 144.8 M ~ (n=31) for D G G C and 420.3 + 172.7 M,Q (n--29) for CA 1PC. Fig. 1 shows typical current clamp recordings of D G G C and CA IPC at a membrane potential o f - 7 0 mV. Fig. 1A illustrates the differences in discharge characteristics of both cell types. In D G G C the sharp APs are followed by afterhyperpolarizations (in 30 out of 31 cells) whereas in CA1PC the broader APs only very rarely (3 out of 29 cells) are followed by afterhyperpolarizations. In D G G C hyperpolarizing current injection with -10 to - 1 6 0 pA causes an electrotonical hyperpolarization with little rectification (Fig. 1B). In CA1PC similar current injections cause increasing hyperpolarizations with a subsequent repolarization towards resting membrane potential. This results in the inward rectification indicated by the I/Vplot in Fig. 1C. After termination of the current injection a rebound depolarization develops (Fig. I B). Such rebound depolarizations are often ascribed to activation of a low voltage activated (LVA) (Ttype) Ca 2+ current [8, 9, 13]. This is unlikely in this case

tbr several reasons: the cells were held at - 7 0 mV which is below the threshold for activation of these Ca 2+ currents [3]. Secondly the T- channel antagonist ethosuximide [4] (1 or 5 raM) did not block the rebound depolarizations in 10 experiments (not shown) and thirdly neither CoCL (1.5 mM, 2 cells) nor CoCI 2 (2.3 mM) and reduced Ca 2` (0.1 mM, 2 cells) nor NiCI 2 (1 mM, 1 cell) were able to block the rebound depolarizations (not shown). Fig. 2A control panel shows voltage clamp recordings during hyperpolarizing voltage commands from a holding potential of -70 mV. The membrane potential was changed for 1 s to more negative levels in steps of 10 mV between - 8 0 and -120 mV. Hyperpolarization to -80 mV elicits an inward current which steadily increases with stronger hyperpolarizations. Upon return to holding potential an inward tail is apparent, which is, however, sometimes obscured by a transient outward current component (e.g. Fig. 3A). The amplitude of the inward current varied between -31 and -202 pA and was on average 109.8 _+ 55.0 pA (n=12), when determined at -120 inV. The inward current could show some run down behaviour over time. The mean time constant r of activation was 187 +_ 95 ms (n--16, A + B exp(-t/r)) for hyperpolarizing steps from holding potentials of - 4 0 to -120 mV at room temperature. No voltage dependency of the time constant could be statistically secured. Control

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Fig. I. Comparison of current clamp recordings of dentate gyrus granule cells and CAI pyramidal cells. A: membrane potential adjusted at - 7 0 mV. Depolarizing and hyperpolarizing current pulses of +95 pA and -45 pA to a 19 day old granule cell and pulses o f + 6 5 pA and -60 pA to a 15 day old pyramidal cell. B: hyperpolarizing current pulses to a l 4 day old granule cell (0 pA to 160 pA) and to a l 5 day old pyramidal cell ( - 3 0 pA to -210 pA). Filled circles and squares mark times at which values for I / V plots in C were taken. C: 1IV curves tbr pulse protocols shown in B. Values were taken at times marked in B.

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Fig. 2+ Effect of I m M CsC1 on the inward rectifying current. A: voltage clamp recording of a 15 day old CA1 pyramidal cell. Holding potential --70 mV, hyperpolarizing steps between - 8 0 mV and -120 mV. B: current clamp recording of a 15 day old pyramidal cell. Membrane potential adjusted at 70 inV. C: l, V curve of voltage clamp recording. Holding potential 40 inV. The same cell as in B. Open circles, control: filled circles, 1 mM CsCI: squares, wash. D: l / V c u r v e of current clamp recording of the same cell as in B and C. Open circles, control; filled circles, I mM CsCI: squares, wash.

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Cesium, applied with 1 mM in the perfusing solution reversibly reduced the current, as shown in Fig. 2A. The effect of Cs + on the inward rectifying current is also illustrated in the I / V plot in Fig. 2C. This plot, where the cell was held at - 4 0 mV, shows activation of the inward current at about - 8 0 mV. Half maximal activation could not be determined because the current showed no saturation behaviour in the investigated voltage range. The figure also shows that 1 mM Cs + did not completely remove the inward rectifying current. Cs + often had a small additional suppressing effect on the leak current. Fig. 2B shows the effects of Cs ÷ in current clamp recordings. In addition to a small increase in input resistance, 1 mM Cs + removed almost all inward rectification as also shown in the I/V relationship (Fig. 2D). Interestingly, a very delayed rebound depolarization was still present in 1 mM Cs ÷. The rebound depolarization was usually sufficient to trigger action potentials when recorded in the absence of TTX (Fig. 2B). We also tested the effect of Ba 2+ on the inward rectifying current. A typical experiment is shown in Fig. 3 where the cell was held at - 4 0 inV. Ba 2+ suppressed a leak conductance but had almost no effect on the inward rectifying current. This becomes particularly obvious in the I/V plot of current recording in the presence and absence of Ba -~+ (Fig. 3B). These findings show that granule cells and CA1 cells differ with respect to inward rectifying properties. Upon hyperpolarization CA1 cells activate an inward current which counteracts a hyperpolarization, as for example induced by activation of y-aminobutyric acid~ (GABAB) receptors. Indeed it is a general experience that already the resting membrane potential of D G G C is somewhat more negative than that of CA1PC. The inward current activated upon hyperpolarization has typical properties Control

1 mM BaCl=

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Fig. 3. Effect of BaCI2 on a 15 day old CA1 pyramidal cell. A: voltage clamp recording. Holding potential -40 mV, hyperpolarizing steps between -50 and -120 m V. B: 1/V curve of voltage clamp recording of the same cell as in A. Open circles, control; filled circles, 1 mM BaC12; squares, wash.

of IQ, the mixed K+/Na + conductance known from measurements with microelectrodes on CA1PC [7] and from cultured hippocampal neurons [12]. It is activated at about - 8 0 mV, sensitive to Cs + and insensitive to Ba 2+. The current seems to deactivate slowly. This could account at least for part of the rebound depolarization following a hyperpolarizing current injection observed under current clamp conditions. However, this rebound depolarization was only delayed but not blocked in the presence of Cs ÷. It therefore seems that these depolarizations have more than one component even when observed at - 7 0 mV. Cs + had also both a small effect on leak currents as well as on input resistance. Ba 2+ had a much stronger effect on input resistance and leak conductance possibly related to block of K + and C1- channels. These findings suggest that some of the differences of synaptic responses during repetitive stimulation may be accounted for by differences in the expression of Q currents in D G G C and CA1PC. This research was supported by D F G Grant He 1128/ 6-1 and the SFB 194 B3. We are indebted to Ms. M. Bullmann, A. Specht and G. Heske for excellent technical assistance in preparing the experiments and the manuscript. 1 Alger, B.E., Characteristics of slow hyperpolarizing synaptic potential in rat hippocampal pyramidal cells in vitro, J. Neurophysiol., 52 (1984) 892-910. 2 Alger, B.E. and Nicoll, R.A., Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studied in vitro, J. Physiol., 328 (1982) 105-123. 3 Carbone, E. and Lux, H.D., A low voltage-activated, fully inactivating Ca channel in vertebrate sensory neurones, Nature, 310 (1984) 501-502. 4 Coulter, D.A., Huguenard, J.R. and Prince, D.A., Specific petit mal anticonvulsants reduce calcium currents in thalamic neurons, Neurosci. Lett., 98 (1989) 74-78. 5 Edwards, F.A., Konnerth, A., Sakmann, B. and Takahashi, T., A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system, Pfl(igers Arch., 414 (1989) 600-612. 6 Hablitz, J.J. and Thalmann, R.H., Conductance changes underlying a late synaptic hyperpolarization in hippocampal CA3 neurons, J. Neurophysiol., 58 (1987) 160-179. 7 Halliwell, J.V. and Adams, P.R., Voltage-clamp analysis of muscarinic excitation in hippocampal neurons, Brain Res., 250 (1982) 71 92. 8 Llin~s, R.R. and Yarom, Y., Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro, J. Physiol., 315 (1981) 569 584. 9 Llin~is, R.R. and Yarom, Y., Electrophysiology of mammalian inferior olivary neurons in vitro: different types of voltage-dependent ionic conductances, J. Physiol., 315 (1981) 549-567. 10 Nicoll, R.A. and Alger, B.E., Synaptic excitation may activate a calcium dependent potassium conductance in hippocampal pyramidal cells, Science, 212 (1981) 957 959. 11 Rausche, G., Sarvey, J.M. and Heinemann, U., Slow synaptic inhi-

234 bition in relation to frequency habituation in dentate granule cells of rat hippocampal slices, Exp. Brain Res., in press. 12 Segal, M. and Barker, J.L., Rat hippocampal neurons in culture: potassium conductances, J. Neurophysiol., 51 (1984) 1409-1433. 13 Suzuki, S. and Rogawski, M.A., T-type calcium channels mediate the transition between tonic and phasic firing in thalamic neurons, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 7228-7232.

14 Takahashi, T., Intracellular recordings from visually identified motoneurones in rat spinal cord slices, Proc. R. Soc. Lond., Ser. B, 202 (1978) 417 421. 15 Tbalmann, R.H. and Ayala, G.F., A late increase in potassium conductance follows synaptic stimulation of granule neurons of the dentate gyrus, Neurosci. Lett., 29 (1982) 243-284.