Electrophysiological properties of in vitro hippocampal pyramidal cells from normal and staggerer mutant mice

Brain Research, 311 (1984) 87-96 Elsevier

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Electrophysiological Properties of in vitro Hippocampal Pyramidal Cells From Normal and Staggerer Mutant Mice EMMANUEL FOURNIER* and FRANCIS CREPEL

Unitd INSERM U 97, 2 Ter rue d'Aldsia, 75014 Paris (France) (Accepted February 28th, 1984)

Key words: hippocampus - - calcium spikes - - staggerer mice - - afterpotentials - - potassium conductances

Electrophysiological properties of intracellularly recorded CA1 pyramidal cells from normal and staggerer mice were compared by using hippocampal slices maintained in vitro. In staggerer mice, the passive membrane properties of these neurons as well as their synaptic potentials elicited by stratum radiatum stimulation were very similar to those observed in normal mice. In control and mutant mice and in standard Krebs solution, CA1 pyramidal cells mainly fired tetrodoxin (TFX)°sensitive fast spikes but could also generate slow spikes. In both groups, replacement of calcium (Ca) by barium (Ba) or introduction of TEA in the bathing medium prolonged the repolarization of the fast spikes and suppressed the brief spike afterhyperpolarization which normally followed them, thus suggesting that both events involve fast potassium conductances. Furthermore, in both groups of animals, TEA and Ba enhanced the slow spikes and induced the appearance of prolonged depolarizations. These slow events were TI'X-resistant and were abolished by the Ca channel blockers cadmium or cobalt, thus suggesting that they are Ca-dependent. On the whole, the present results indicate that the staggerer mutation which yields marked abnormalities in the bioelectrical properties of cerebellar Purkinje cells has no such effect on CA1 pyramidal cells. INTRODUCTION

tO determine to what extent the staggerer mutation may affect Ca-conductances in other classes of neu-

It is now well established that the excitability of in-

rons in the central nervous system, even in the ab-

vertebrate and vertebrate neurons is d e p e n d e n t on several types of ionic conductances 1. In unicellular

sence of any other striking abnormalities. Indeed,

animals and in invertebrates, the genetic control of the development of these ionic channels has already been investigated to some extent by the use of point mutations selectively affecting a given class of ionic channels, as for instance sodium (Na) or potassium (K) conductances in DrosophilaX6,25, 43 and calcium (Ca) conductances in Parameciumlg. In vertebrate neurons, such a genetic dissection of m e m b r a n e conductances had never been studied until recent work~O.1~ established that cerebellar Purkinje cells, which can normally generate Ca spikes 21,22, selectively lack these regenerative responses in homozygous staggerer (sg/sg) mutant mice, probably because of a direct effect of the mutation on these neurons. Therefore, the question arises

previous anatomical observations 34,35 support the view that the impact of this point mutation is restricted to the cerebellum, where, besides the abnormality mentioned before, marked morphological, electrophysiological and biochemical alterations are also apparentg,35,37,3s. Hippocampal pyramidal cells provide a suitable model for such studies since the presence of voltagedependent Ca conductances in these neurons is now well documented in guinea pig 6,7,17.30,39,40,42. We have compared the electrophysiological properties of CA1 pyramidal cells in normal mice and in staggerer mutant mice, with special reference to their passive electrical properties, their synaptic inputs and their ionic conductances. As in the companion paper 12, this was achieved by studying the responses of intra-

* Present address: Laboratoire de Physiologie, Facult6 de M6decine, Piti6 Salp&ri6re, 91 Bd. de l'H6pital, 75623 Paris Cedex 13, France. Correspondence: F. Crepel, Unit6 INSERM U97, 2 Ter rue d'Al6sia, 75014 Paris, France.

cedures were the same as those reported in ref, 12. lntracellular recordings from CA1 pyramidal cells were obtained at a somatic level under direct visualization of the stratum pyramidale, using glass microelectrodes filled with 3 M KCI (80-120 M ~ D C resistance) or with 3 M K citrate (100-250 M ~ D C resistance). The identification of CA1 pyramidal cells was confirmed by their antidromic and orthodromic activation elicited by electrical bipolar stimulation of alveus and of stratum radiatum respectively. Cells selected for study had a resting membrane potential of at least - 6 0 mV and a spike amplitude of more than 65 mV.

cellularly recorded neurons in hippocampal slices maintained in vitro. MATERIALS AND METHODS Experiments were performed on 6 month old normal and staggerer mutant mice. H o m o z y g o u s staggerer mice were obtained by intercrossing heterozygous hybrids of C57BL and D B A 2 J mice bearing the mutation. The clinical expression of staggerer straits was evident as early as in the first postnatal month. The age of 6 months was chosen in order to study the mouse hippocampus after the end of its postnatal development. Transverse slices of hippocampus ( 4 0 0 ~ m thick), prepared as described in ref. 12, were maintained in the recording chamber fed with a standard Krebs solution containing (mM): NaCI (124), KCI (5), KH2PO 4 (1.15), MgSO4"TH20 (1.15), CaC12 (2.5), N a H C O 3 (25), glucose (10) and continuously gassed with Oz (95%) and CO2 (5%). The varied bathing solutions used in this set of experiments, as well as the stimulating and recording pro-

A

RESULTS In normal and in staggerer mice, stable intracellular recordings were obtained from 51 and 40 CA1 pyramidal cells, respectively. Resting potentials averaged -63.2 + 2.1 mV in controls and -61.7 + 2.7 mV in the mutant.

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Fig. 1. Responses of CA1 pyramidal cells from normal mice to hyperpolarizing current pulses. A: transmembrane voltage changes (upper traces) induced by negative current pulses (lower traces). Note that a discharge of the celt was elicited at the end of the hyperpolarizing response. B: plot of the transmembrane voltage drop at the pleateau of polarization (V) against'current values for the same cell as in A. The straight line was obtained by applying least-squares criterion to the first 7 data points. Input resistance was 51.7 MQ. C: semilogarithmic plot of the slope of the voltage transient (dV/dt) against time after the onset of a current step of 0.25 nA for the same cell as in A and B. The straight line was obtained by applying least-squares criterion to the last 8 semilogarithmic data points. Membrane time constant was 18.3 ms.

89 Passive electrical constants

Current-voltage ( I - V ) curves were obtained by measuring the transmembrane voltage drop produced by hyperpolarizing current pulses (more than 100 ms in duration) applied through the recording microelectrode (Fig. 1A). Input resistances were determined from the slope of the linear portion of this curve produced by injecting small hyperpolarizing current pulses (less than 0.5 nA) (Fig. 1B). Mean input resistances were very similar in control and in mutant mice (37.1 + 3.3 Mr2 and 38.6 + 4.1 MQ, respectively). The membrane time constant of CA1 neurons was determined by analyzing the time course of the voltage transient produced by current pulses of less than 0.3 nA, i.e. in the linear portion of the I - V curves. In normal and staggerer mice, the late phase of this time course (more than 5 ms after the onset of the current pulse) was closely fitted to a single exponential (Fig. 1C), the time constant of which represents the membrane time constant according to Rall's model 24. Membrane time constant averaged 15.4 + 1.8 ms in the controls and 16.8 + 2.0 ms in the mutants. Table I summarizes some of the characteristics of these neurons measured in standard Krebs solution. Synaptic activation

In CA1 neurons from staggerer mice, electrical stimulation of the stratum radiatum induced responses similar to those seen in normal mice. In all tested cells and in both groups of animals, low stimulus intensities elicited an excitatory postsynaptic potential (EPSP), the amplitude of which was graded from 2 to 12 mV with the stimulus

strength (Fig. 2A1 and B1). Rise times (from base to peak) and half-decay times (from peak to half-amplitude) of these EPSPs (Table I) were determined from neurons in which such responses were not contaminated by inhibitory postsynaptic potentials (IPSPs) as assessed by further testing (see below). In about 20% of the neurons recorded with KCI filled microelectrodes (7 among 38 cells in normal mice and 7 among 37 cells in staggerer mice), these EPSPs were followed by a hyperpolarizing potential, 1 - 4 m V in amplitude, 8 0 - 1 5 0 m s in duration (Fig. 2A2 and B2), which was converted into a depolarizing potential by chloride injection in the cell. These presumed IPSPs were also encountered when neurons were recorded with K citrate filled microelectrodes. Only 4 among these 16 neurons exhibited clearcut IPSPs. The absence of IPSPs in the other tested cells was assessed by the absence of any membrane conductance change following EPSPs evoked by stratum radiatum stimulation (Fig. 2A3). In normal mice and in staggerer mice, a single spike or a burst of spikes was also routinely triggered by stratum radiatum stimulation. When only one spike was evoked, it was followed by a brief afterhyperpolarization, 5-12 mV in amplitude and less than 5 ms in duration (Fig. 2A3). This brief spike afterhyperpolarization increased in amplitude when the cells were depolarized by steady currents applied through the recording microelectrode, and reversed into a depolarizing afterpotential (DAP) when the cells were hyperpolarized (not illustrated). In both groups of animals, the number of spikes within a burst of spikes evoked by stratum radiatum stimulation varied from 2 to 7, even with a constant

TABLE I Summary of some characteristics of CA1 pyramidal cellsfrom normal and staggerer mice in standard Krebs solution

Values are mean + S.D. Values in parentheses are the number of cells contributing to the mean value.

Resting potential (mV) Input resistance (MQ) Membrane time constant (ms) Spike Amplitude (mV) Duration (ms) EPSP Rise time (ms) Half-decay time (ms)

Normal mice

Staggerer mice

-63.2 _ 2.1 (51) 37.1 + 3.3 (17) 15.4 ± 1.8 (7)

-61.7 + 2.7 (40) 38.6 + 4.1 (13) 16.8 ± 2.0 (7)

69.2 _+3.6 (38) 1.1 __+0.1 (38)

70.3 ± 4.3 (31) 1.2 + 0.1 (31)

7.7 __+1.0 (25) 11.5 + 1.4 (25)

9.2 + 1.3 (18) 13.4 + 1.7 (18)

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Fig. 2. Responses of CA1 pyramidal cells from normal and staggerer mice to electrical stimulation of the stratum radiatum. Intracellular recordings, except lower traces in A1, A2, B1 and B2 which show the field potentials after withdrawal of the microelectrode. Superimposed sweeps in A1, A3, A4, B1 and B4. All records were taken from various neurons. A: in normal mice, the amplitude of EPSPs was graded with the stimulus strength (A1). In another cell recorded with a KCI filled microelectrode; the EPSP was followed by an IPSP (A2). In a cell recorded with a K citrate filled microelectrode, the EPSP triggered a single spike followed by a brief spike afterhyperpolarization (A3). The superimposed successive sweeps in A3 illustrate the absence of any change in the response of this cell to a hyperpolarizing current pulse (not illustrated) injected before, during and after the synaptic response. In another cell, a burst of spikes was elicited by stratum radiatum stimulation (A4). B: as in A in staggerer mice. Stratum radiatum elicited either EPSP (BI), or an EPSP-IPSP sequence (B2), or bursts of spikes (B3 and B4). Note that the burst of spikes in B3 was followed by a prolonged hyperpolarization which might represent either a late IPSP or a long-lasting afterhyperpolarization (AHP).

stimulation strength. In addition, these bursts of spikes could always be dissected into two c o m p o nents (Fig. 2A4, B4), The first one consisted of the E P S P and of a train of 2 - 3 fast spikes (less than 1.5 ms in duration and m o r e than 65 mV in amplitude) riding on the rising phase of the EPSP. The second c o m p o n e n t of the burst arose on the late phase of the EPSP and consisted of slower spikes (3.3 +_ 0.4 ms in duration and 51.3 + 4.5 m V in amplitude) s u p e r i m p o s e d on a p r o l o n g e d depolarization up to 25 m V in amplitude. This second c o m p o n e n t was labile and occurred in an all-or-none fashion. Since in guinea pig, these slow spikes and this prolonged d e p o l a r i z a t i o n have b e e n attributed to the activation of Ca conductances 3°,39 this suggests that the same is true in normal and staggerer mice. Therefore, in staggerer mice, present results indicate that CA1 p y r a m i d a l cells might fire Ca spikes in s t a n d a r d

Krebs solution, in contrast to cerebellar Purkinje cells. Response o f CA1 neurons to direct electrical stimulation In standard Krebs solution and in both groups of animals, depolarizing current pulses passed through the recording m i c r o e l e c t r o d e elicited a m a r k e d depolarization of the cells which in turn g e n e r a t e d a repetitive firing of fast spikes, followed each by a brief spike a f t e r h y p e r p o | a r i z a t i o n (Fig. 3A). In normal and staggerer mice, no slow spikes were e v o k e d in these conditions, even at relatively high intensity of stimulation (up to 1.5 n A ) (Fig. 4 A I and B1). A t t h e end of the current pulse, the m e m b r a n e potential decayed rapidly to its resting level. W h e n the cell was h y p e r p o l a r i z e d by steady currents a p p l i e d through the microelectrode, the brief spike afterhyperpolari-

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Fig. 3. Effects of barium on CA1 cell responses to direct electrical stimulation in staggerer mice. In this figure and in the subsequent ones, upper traces show intracellular records of CA1 pyramidal cell responses to depolarizing current pulses (lower traces) applied through the microelectrode. All records were taken from the same cell. A: response in standard Krebs solution. Note the brief spike afterhyperpolarization following each fast spike and the absence of any evoked slow spike. B: response after substitution of CA by 2.5 mMBa in the bath. Note the replacement of the brief spike afterhyperpolarizations by DAPs, the appearance of burst of spikes (B1), slow spikes and prolonged depolarizations (B2). C: idem as in B after introducing 0.5 ~M TTX in the bath. Note the persistence of slow spikes. D: idem as in C after addition of 3 mM Co C12 in the bath. E: slow spikes were in part recovered in the Ba-containing solution when drugs were washed out. zations were reversed into D A P s . A d d i t i o n of 0.5 ~tM tetrodoxin (TTX) in the bath abolished the fast spikes, suggesting that they are m e d i a t e d by Na currents.

Effect of barium and tetraethylammonium on responses of CA1 pyramidal cells to direct electrical stimulation or to synaptic activation Barium (Ba) is a divalent cation that is r e p o r t e d to block fast K conductances 18~31 and to cross Ca channels with greater facility than Ca itself 13. Tetraethylammonium ( T E A ) is a cation known to block fast K conductances 1~.29,32.In normal and in staggerer mice, effects induced in CA1 neurons by replacing the standard Krebs solution by either a 15 m M T E A containing solution or by a Ca-free bathing m e d i u m containing 2.5 m M B a were very similar.

Spike repolarization and brief spike afterhyperpolarization. In both groups of animals, the initial changes seen after substitution of Ca by Ba or after introduction of T E A in the bath were a b r o a d e n i n g of the fast spikes (Table lI) and a replacement o f the brief spike afterhyperpolarizations by p r o m i n e n t D A P s (see first e v o k e d spike in Fig. 4B2). This effect was observed in the case of spikes e v o k e d by depolarizing current pulses as well as for spikes elicited by stratum radiatum stimulation (Fig. 5A2 and B2). In addition, under Ba or T E A , depolarizing current pulses also triggered bursts of fast and slow spikes (Fig. 3B1).

Slow spikes and prolonged depolarizations. A f t e r complete substitution of Ca by Ba or when T E A exerted its full effect, slow spikes and prolonged depolarizations (20-50 mV in amplitude and up to

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Fig. 4. Comparative effects of TEA on CA 1 cell responses to direct electrical stimulation in normal and staggerer mice. A: successive responses of a CA1 pyramidal cell from a normal mouse in standard Krebs solution (A1), after exposure to a 15 mM TEA containing solution (A2), after perfusing a solution containing 15 mM TEA plus lm M Cd (A3) and finally after introducing TTX in the bath, B: idem as in A in a CA1 pyramidal cell from a staggerer mouse.

500 ms in duration) were also elicited in normal mice as well as in staggerer mice by depolarizing current pulses (Figs. 3B2, 4A2 and B2) or by stratum radiaturn stimulation (Fig. 5A2 and B2). Table I1 shows the absence of any significant difference between the characteristics of the slow spikes recorded in normal mice and in staggerer mice u n d e r these conditions. The prolonged depolarizations were triggered by fast or slow spikes, and generated in turn fast and slow spikes. In some cells, prolonged p l a t e a u x of depolari-

zation of 20 mV in amplitude a n d of up to 2 0 s in duration were elicited by repetitive (1 Hz) depolarizing current pulses. These p l a t e a u x of d e p o l a r i z a t i o n terminated abruptly when depolarizing current pulses were converted into hyperpolarizing ones. It should be noted that n o n e of these responses was ever followed by an A H P . In contrast with fast spikes, the slow spikes and the prolonged depolarizations were left unaltered by introduction of T T X in the bath, in staggerer mice

TABLE II Comparison o f the spike characteristics in standard Krebs solution and in B A or T E A containing solution

Data from Ba and TEA containing solution were pooled, since there was no significant difference in the mean values between them. Values are means _+S.D. n represents the number of cells contributing to the mean value. Standard Krebs

Fast spike amplitude (mV) duration (ms) Slow spike amplitude (mV) duration (ms)

Ba or TEA

Normal (n = 38)

Staggerer (n = 31)

Normal (n = 27)

Staggerer (n = 25)

69.2 _+3.6 1.1 + 0.1

70.3 _+4.3 1.2 _+0.1

67.8 ___4.5 2.5 + 0.3

68.2 __+4.7 2.4 +_0.4

51.3 + 4.5 3.3 + 0.4

52.7 ___4.9 3.8 + 0.5

48.7 +-_6.1 7.4 _+_+1.4

49.6 + (i).2 7.6 _+_1.5

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passive component due to the termination of spike repolarization at a more positive level under Ba or T E A than in standard Krebs solution. Slow spikes and prolonged depolarizations. In both groups of animals, introduction of Co and Cd in the bath abolished the TTX-resistant slow spikes and prolonged depolarizations induced under Ba or T E A (Fig. 3D, 4A3 and B3). These effects were at least in part reversible when drugs were washed out (Fig. 3E). DISCUSSION

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Fig. 5. Effects of TEA on synaptic responses of CA1 pyramidal cells from normal and staggerer mice. A: response of a CA1 pyramidal cell from normal mice to stratum radiatum stimulation in standard Krebs solution (A1) and in TEA-containing solution (A2). B: same experiment as in A in a CA1 pyramidal cell from staggerermice.

(Fig. 3C) as well as in normal mice.

Effects of Ca channel blockers on the responses induced under Ba or TEA. Cobalt (Co) and Cadmium (Cd) are two cations known to block Ca channels 5. After addition of 3 mM Co in the Ba-containing solution or after replacement of the TEA-containing solution by a Cafree solution containing 15 mM T E A plus 1 mM Cd, the results observed in staggerer mice and in normal mice were again very similar. Synaptic responses. In both cases, all the synaptic responses were abolished in the presence of Co or Cd. Fast spikes and DAPs. The TTX-sensitive fast spikes elicited by either depolarizing current pulses or by antidromic activation of the cells were left unaltered by the presence of Cd. They were still followed by the TEA-induced DAPs (Fig. 4A3 and B3), suggesting that these DAPs are probably not entbely mediated by Ca conductances. The time course of these DAPs closely fitted to an exponential with a time constant similar to the membrane time constant (comparison established on 5 neurons). This indicates that the Ba- or TEA-induced DAPs include a

The experiments presented here extend previous findings on the bioelectrical properties of in vitro CA1 pyramidal cells and suggest that they are left unaffected by the staggerer mutation, in contrast to the abnormalities previously reported in cerebellar Purkinje cells.

Passive membrane properties In normal mice, the passive membrane properties of CA1 pyramidal cells are similar to those previously described in guinea pig 8.26,27. Since, in staggerer mice, input resistances and membrane time constants of these cells did not significantly differ from those measured in normal mice, it is likely that the specific membrane resistivity of CA1 pyramidal cells is left unaltered by the mutation. Synaptic inputs In staggerer mice, the synaptic responses of CA1 pyramidal cells to stratum radiatum stimulation were very similar to those seen in normal mice, both in terms of amplitude and time course of the synaptic potentials (EPSPs as well as IPSPs). Furthermore, stimulation of the stratum radiatum elicited clearcut IPSPs in the same proportion of tested cells in both groups. These results provide an electrophysiological confirmation to previous anatomical studies 34,35 which have not reported any morphological abnormality of the neuronal network outside the cerebellum. It should be noted that the proportion of CA1 pyramidal cells which exhibited clearcut IPSPs in our slices was smaller than that previously reported from other species2,3,4,26, 28. In these species (rat, guinea pig, rabbit . . . . ), the slicing procedure includes first

94 a dissection of the hippocampus and then cuts along a plane perpendicular to its longitudinal axis 36. Because of the very small size of the mouse hippocampus (about 1 mm in width), it was not dissected away from surrounding structures, and hippocampal slices were directly taken from sections of cerebral hemisphera. Although efforts were made to keep the plane of sections perpendicular to the longitudinal axis of the hippocampus, we cannot preclude that some slices might have been cut with an inappropriate orientation for preserving inhibitory pathways.

Fast spikes and brief spike afterhyperpolarizations In staggerer and in normal mice, CA1 neurons recorded at a somatic level mainly fired TTX-sensitive fast spikes in standard Krebs solution. As previously reported in guinea pig29, the repolarization of these presumed Na-mediated fast spikes was prolonged in the presence of Ba or T E A known to block the fast K conductances~8,31, 32, suggesting that this spike repolarization involves fast K conductances. Our results show that fast spikes in CA1 pyramidal cells are followed by a brief spike afterhyperpolarization when they are elicited by either synaptic activation or by direct stimulation of the cells, i.e. when they are superimposed on an induced depolarization of the cells. Since these spike afterhyperpolarizations were replaced by well-developed DAPs after exposure to Ba or T E A even in the presence of Ca channel blockers, it is likely that they are mediated by fast K conductances in CA1 pyramidal cells from normal and staggerer mice as in many other species of neu-

ronslS,31,32.

Slow spikes and prolonged depolarizations As in guinea pig 14'15.29,30, Ba or T E A enhanced the slow spikes and, in addition, induced prolonged depolarizations in CA1 pyramidal cells from normal and staggerer mice. Since all these events were TTXresistant and were abolished by the Ca channel blockers Co or Cd, this strongly suggests that they are mediated by Ca conductances. The present comparative study indicates that these Ca conductances might be very similar in CA1 pyramidal cells from both groups of animals. In particular, slow spikes were observed in standard Krebs so-

lution within the late phase of the bursts of spikes elicited by synaptic activation of CA1 pyramidal cells from staggerer mice. Since in guinea pig, these slow spikes have been considered as Ca-dependent responses 3°,3~,41, it is likely that ( ' A t pyramidal cells from staggerer mice can fire Ca spikes even in control conditions. These results are in contrast with the lack of Ca spikes in cerebellar Purkin}e cells from staggerer mice ~()

Impact of the staggerer mutation On the whole, the present report provides evidence for normal cable properties and synaptic responses, as well as for the presence of well-developed Ca conductances in CA1 pyramidal cells from staggerer mice. Furthermore, preliminary observations show that granule cells of dentate gyrus in the mutant also possess Ca channels and normal synaptic responses to perforant path stimulation (Fournier and Crepel, unpublished results). These results are in marked contrast with previous observations on cerebellar Purkinje cells from staggerer mice, which exhibit a selective absence of Ca spikes t°, in addition to marked abnormalities in their morphology 2°,33,35y. in their wiring 9,23 and in their surface antigenic determinants 3s. The differential effect of the mutation on Ca channels borne by Purkinje cells and hippocampal cells might result either from a different genetic control of Ca channels in these neurons, or, perhaps more likely, from an impact of the mutation limited to the former class of cells. Furthermore, in Purkinje cells, we cannot decide at the moment if the effect of the staggerer mutation on Ca spikes is direct or if it is due to other alterations of these neurons, which secondarily lead to abnormal activation properties of Ca conductances.

ACKNOWLEDGEMENTS We wish to thank M. W. Debono and J. R. Teilhac for helpful technical assistance, and Christine Cah6rec for valuable secretarial assistance. This work was supported by the Minist6re de la Recherche et de l'lndustrie (Grant 'Action Dynamique du Neurone').

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