- Email: [email protected]
Electrophysiological studies on the postnatal development of intracerebellar nuclei neurons in rat cerebellar slices maintained in vitro. II. Membrane conductances
Developmental Brain Research, 20 (1985) 97-106 Elsevier
97
BRD 50210
Electrophysiological Studies on the Postnatal Development of Intracerebellar Nuclei Neurons in Rat Cerebellar Slices Maintained in Vitro. II. Membrane Conductances ROBERT GARDEqq'E, MARC DEBONO, JEAN-LUC DUPONT and FRANCIS CREPEL INSERM U97, 75014 Paris (France)
(Accepted December 18th, 1984) Key words: intracerebellar nuclei - - postnatal development - - membrane conductances
The development of membrane conductances of intracerebellar nuclei neurons was studied in the rat since birth up to the weaning period by the use of thick sagittal cerebellar slices maintained in vitro. Mature nuclear neurons express fast sodium and slowly inactivating sodium conductances, as well as calcium conductances. As early as birth, fast sodium and calcium conductances appear well developed whereas slowly inactivating sodium conductances mature within the first postnatal week.
INTRODUCTION The study of the development of membrane neuronal excitability is of major importance in order to better understand the role played by these intrinsic properties of nerve cells in the functioning of the central nervous system. Indeed, m e m b r a n e neuronal excitability is dependent on several types of ionic conductancesl, u, the ontogenesis of which can vary in function of the species or the nervous structures 48. Thus, some nerve cells seem to be able to express the whole repertory of their ionic conductances at very early developmental stages41, 43 while others show a progressive establishment of their bioelectrical properties, for instance sodium (Na) conductances being expressed before calcium (Ca) conductances 6,21,32, or the inverse 4,5,12,39,49,51. If these active electrical properties are already well known for the cerebellar Purkinje cells either during development 32, or at the adult stage10,33,34, or even in some cerebellar mutantslO, 13, those of the intracerebellar nuclei neurons still remained unexplored in the adult as well as during development. Therefore, the development of bioelectrical membrane properties of rat intracerebellar nuclei neurons
was studied from birth up to the weaning period. One aim of the study was to determine the extent to which these cells resemble Purkinje cells in this respect since they derive both from the same region of the dorsal metencephalic plates of neuroepithelial cells2,3,28 with nearly the same birthdates3,18,38, 52. Experiments were performed on thick cerebellar slices maintained in vitro since this preparation allows stable intracellular recordings of nuclear neurons19, 20 and facilitates the study of the effects of various agents known to act upon ionic channels by exchanging the bathing medium or by direct drug application. A preliminary report of this work has been already published 19. MATERIALS AND METHODS The experimental procedures used in this study were identical to those described in a previous paper 20. Nuclear neurons, evenly distributed from birth (postnatal day 0 (PN0)) to the weaning period (PN23) were intracellularly recorded with glass microelectrodes filled with KCI 3 M or potassium citrate 3 M (DC resistance 50-120 MQ). Cells were stimulated by intracellular currents injected through the
Correspondence: R. Gardette, INSERM U97, 2ter, rue d'Alrsia, 75014 Paris, France.
0165-3806/85/$03.30 (E) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
98 recording electrode via a bridge circuit. When necessary, the control Krebs solution was replaced by a Ca-free superfusing fluid containing either BaC12 (Ba, 2.5 mM) or CdCI 2 (Cd, 1 mM), or by a tetraethylammonium (TEA) -containing medium (TEA, 15 mM). The composition of these various bathing solutions was the same as already described elsewhere (Table I in ref. 15). Finally, variations in the composition of these media included addition of tetrodotoxin (TTX, 5 × 10-6 M) or of cobalt chloride (Co, 2 mM) in the Ba- or the TEA-containing solutions. RESULTS Membrane conductances were studied in 64 nuclear neurons selected among cells recorded in a previous study dealing with synaptic responses 20. Identification of the nuclear cells was based upon the direct visual control of the recording microelectrode location within the intracerebellar nuclei and usually further confirmed by their antidromic responses induced by juxtanuclear stimulation (see Fig. 1 in ref. 20). Resting potentials ranged between - 5 0 and - 7 0 mV with no significant difference between young and older stages as already demonstrated 20. Similarly, spikes elicited by direct stimulation of the cells presented the same evolution between immature and mature stages as those antidromically or synaptically evoked 20 (see also following paragraphs). Responses induced by direct electrical stimulation in mature intracerebellar nuclei neurons Responses in control Krebs solution. On PN23 in control Krebs solution, small depolarizing current pulses (less than 0.5 nA) passing through the recording microelectrode elicited a marked depolarization of the cells (n = 25) which gave rise to one (Fig. 1A1) or several (Figs. 1A2, B1, 2A1, B1 and 3A1) fast spikes, depending on the amplitude of the pulse. These spikes could reach 70 mV in amplitude and their duration was 1.0-1.5 ms, thus resembling fast spikes synaptically evoked in these cells by juxtanuclear stimulation 20. Indeed, addition of 5 x 10-6 M q T X in the bath abolished these fast spikes, indicating that they were Na-dependent 9 (Figs. 1B3, 2B3). When the stimulus intensity was further increased up to 1.0 nA or more, the only visible effect was an inac-
A2
!']
I
i,!~;i
}
20ms
B1
Co~qtrol
Ba
83
Ba+TTX
40r~
Fig. 1. Effect of Ba on responses of intracerebellar nuclei n rons to direct electrical stimulations on PN23. A: responses nuclear neuron (upper traces) to stimulating currents of 3 ferent intensities (lower traces) in control Krebs solution small current pulse (0.1 nA, A1) induced the firing of a sin spike. Further increase of the stimulus intensity led first to firing of a train of spikes (0.3 hA, A2) and then to the inacti tion of the response (1 nA, A3). B: effect of Ba and TTX on sponses of nuclear neurons. The 3 responses (upper trac were elicited in the same neuron by direct electrical stimt tions (lower traces) in control Krebs solution (B1), after placement of Ca by Ba in the bath (B2) and after addition TTX to the bath (B3). Note that replacement of Ca by Ba in bath did not elicit any other responses than fast spikes (B2) that addition of TTX suppressed all the active components the electricallyinduced response (B3). tivation of the Na spikes following the initial o (Fig. 1A3), but slower rising spikes, such as I spikes elicited in cerebellar Purkinje cells 10,13,33 we never obtained in these mature nuclear neurons. Responses induced in Ba- or TEA-containing so~ tions. The presence of Ca currents was further test, in these cells by studying the effects of Ba, a divale cation reported to block voltage-dependent potas um (K) conductances 46, to move more easily than ( through Ca channels 22, and not to activate the Ca-d pendent K conductances 14, and of T E A , a cati~ which also blocks K conductances29,44, 47. When Ba replaced Ca in the bathing medium cells), depolarizing current pulses passing throul the recording microelectrode never gave rise to a~ other responses than the fast spikes described befor even when the amplitude of the pulse was as high 0.8 nA (Fig. 1B1, B2). In contrast, when the control Krebs solution w replaced by a 15 mM TEA-containing superfusiz fluid (17 cells), several changes progressively a peared in the responses of the cells. Indeed, this e change of medium led first to a lengthening of the fa repolarizing phase of the fast spikes (single arrow Figs. 2A2, 3A2) and to the appearance of depolm
99 zing afterpotentials ( a r r o w h e a d in Figs. 2A2, 3A2) which could in turn trigger the firing of multiphasic fast spikes (double arrow in Figs. 2A2, 3A2). The second event that t o o k place in such a superfusing fluid was the occurrence of p r o l o n g e d plateaux of depolarization outlasting the current pulse (Fig. 2A3, B2), 2 0 - 5 0 mV in a m p l i t u d e d e p e n d i n g on cells and up to 500 ms in duration. Finally, the frequency of spontaneously occurring spikes as well as the synaptic b a c k g r o u n d noise were also e n h a n c e d (compare Fig. 3A1, A3). W h e n 5 x 10 -6 M T F X was a d d e d to the T E A - c o n taining solution (6 cells), the fast Na spikes, the multiphasic spikes and the plateaux of depolarization totally disappeared, while slow rising spikes up to 55 mV in amplitude and presenting an even decay which could reach up to 150 ms in duration were u n m a s k e d (Fig. 2B3). T h e r e f o r e , these results suggest that multiphasic spikes are N a - d e p e n d e n t whereas slow rising spikes are C a - d e p e n d e n t (see also below). FurA1
Control
A2
A3
TEA
TEA
Ii,llll
A1
Control
'ill
I
A2
p
',ii ~
'lOOms
TEA+Cd
A3
TEA
l,j[~j~
TEA+Cd
iLIIII, jn|tltiiil ' m
40m$
A5
TEA
400ms
A6
TEA+Cd+TTX
Fig. 3. Effect of TEA and of Cd on responses of intracerebellar nuclei neurons on PN23. All responses were elicited in the same neuron by direct electrical stimulations in control Krebs solution (A1), after substitution of the Krebs solution by a 15 mM TEA-containing medium (A2, A3) and after addition of Cd (A4, A5) and TTX (A6) to the TEA-containing medium. Note the lengthening of the fast spike (single arrow in A2) and the appearance of depolarizing afterpotentials (arrowhead in A2) sustaining firing of multiphasic spikes (double arrow in A2) under TEA. Note also, under the same conditions, the enhancement of the synaptic background noise and the occurrence of prolonged depolarizations outlasting the current pulse (A3). In spite of the addition of Cd to the bath, the current pulse still elicited long-lasting depolarizations on which fast and multiphasic spikes were or were not superimposed (A4, A5): Addition of TI'X totally abolished these responses (A6). thermore, because plateaux of depolarization in-
m
~rrB
B1
Control
B2
TEA
m
1O0ms TEA+TTX
Fig. 2. Effect of TEA on responses of intracerebellar nuclei neurons on PN23. A: responses of a nuclear neuron to direct electrical stimulation in control Krebs solution (A1) and after substitution of the Krebs solution by a 15 mM TEA-containing solution (A2, A3). Note that the first effect elicited in this cell by the TEA-containing solution was a lengthening of the repolarizing phase of the fast spikes (single arrow in A2) and the occurrence of small depolarizing afterpotentials (arrow head in A2) which could trigger firing of multiphasic spikes (double arrow in A2). When the exchange of the medium was fully achieved, the same current pulse then elicited prolonged plateaux of depolarization, following the fast spike and outlasting the end of the current pulse (A3). B: responses of another nuclear neuron to direct electrical stimulation in control Krebs solution (B1), after substitution of the Krebs solution by a 15 mM TEA-containing medium (B2) and after addition of TFX to the bath (B3). Note the appearance of prolonged action potentials following the initial fast spike under TEA (B2). Addition of TTX abolished the fast components whereas slow rising and prolonged action potentials were still elicited (B3). The bridge balance in B2 and B3 was less accurate than in B1 and the tips of the spikes were retouched in B1 in this figure and also in Figs. 5A1, A3 and 8A.
duced under T E A alone were no longer present under T E A plus TTX, this suggested that o t h e r conductances such as slowly inactivating Na conductances, were also activated under T E A alone and contributed to the generation of these plateaux of depolarization.
Effects of Ca-channel blockers on responses induced in TEA-containing solutions. The eventual presence of slow Na-conductances was explored in 9 cells after Ca currents were blocked by the replacem e n t of the T E A - c o n t a i n i n g solution by a Ca-free solution containing the same concentration of T E A plus 1 m M of the Ca-channel blocker Cd 8. U n d e r these conditions, depolarizing current pulses elicited either Cd-resistant long-lasting depolarizations, 2 5 - 3 0 m V in amplitude and up to 300 ms in duration, on which fast and multiphasic spikes were superimposed (Fig. 3A4), or p r o l o n g e d plateaux of depolarization, 2 5 - 3 0 m V in amplitude and up to 1.5 s in duration sustaining no spike firing, at least during their initial phase (Fig. 3A5). A closer inspection of these records suggested that these events might result from a m o r e or less complete fusion of depolari-
100 zing afterpotentials such as those previously described (Figs. 2A2, 3A2). A d d i t i o n of T I ' X to the bathing m e d i u m suppressed all these c o m p o n e n t s (Fig. 3A6) suggesting that they were N a - d e p e n d e n t . Finally, the slow spikes described in Fig. 2B3 were absent when Cd was a d d e d to the superfusing fluid, thus enforcing the hypothesis that they were mediated by Ca conductances. In brief, these results strongly suggest the presence of several ionic conductances in these m a t u r e intracerebellar nuclei neurons sustaining N a and Ca spikes on the one hand and N a - d e p e n d e n t depolarizing afterpotentials and plateaux of depolarization on the other hand.
Postnatal development of responses induced by direct electrical stimulation in intracerebellar nuclei neurons With the d e m o n s t r a t i o n of the presence of several types of ionic conductances in m a t u r e intracerebellar nuclei neurons, the question arose to d e t e r m i n e when these conductances first a p p e a r e d and how they evolved during postnatal d e v e l o p m e n t . Therefore, similar experiments as those described before were p e r f o r m e d on 39 cells at various postnatal stages since birth up to PN14.
Responses in control Krebs solution. Since PN0 (4 cells) and at all the postnatal stages studied, direct electrical stimulation of the cells elicited a m a r k e d depolarization which in turn induced the firing of a single spike (Fig. 6A1) or of a train of spikes d e p e n d ing on the stimulus intensity (Figs. 4A1, 5A1, 7A1, 8C1). F u r t h e r increase of the stimulus intensity led to an inactivation of the spikes following the initial one (Fig. 4A2) as already noticed in m a t u r e nuclear neurons (see above and Fig. 1A3). The amplitude of these spikes could reach 6 0 - 7 0 m V even at very young stages (Figs. 4A1, 6A1) whereas their duration decreased from 3 - 5 ms to 1 - 2 ms during postnatal development, according to previous observations on antidromicaUy or synaptically elicited spikes recorded in these neurons after juxtanuclear stimulation 20. These spikes persisted when 1 m M Cd was added to the bathing m e d i u m (Figs. 5A4, 6A3, 8A, B, C2, C3) but were abolished by 5 x 10 -6 M T F X (Figs. 4A5, 5A6, 6A4, 7A3, 8C4) thus indicating that they were m e d i a t e d by fast N a currents throughout postnatal development. A s in adult animals, no C a - d e p e n d e n t spikes were ever e v o k e d in control
A1
A2
Conlroi
It~A ~
,
20ms
Ba
A3
conl,::l
k
~
20ms
A5
Ba+TTX
40ms
A6
~ + ; Tx+c::~
Fig. 4. Effect of Ba on responses of intracerebellar nuclei neurons to direct electrical stimulations on PN0. All responses were recorded in the same neuron in control Krebs solution (AI, A2), after replacement of Ca by Ba in the bath (A3, A4) and after addition of TTX (A5) and Co (A6) to the Ba-containing medium. Note that in control Krebs solution a two-fold increase of the pulse intensity from 0.4 nA (A1) to 0.8 nA (A2) only led to an inactivation of the fast spikes. When Ba replaced Ca in the bath, prolonged action potentials (A3) and prolonged plateaux of depolarization (A4) were now elicited. Note that in A3 the prolonged action potentials occurred spontaneously. Addition of TTX to the Ba-containing medium suppressed the initial fast spike of the responses but did not abolish the plateaux of depolarization (A5) which disappeared after addition of Co to the bath (A6). Krebs solution at any d e v e l o p m e n t a l stages studied. Responses induced in Ba-containing solution. As early as birth and up to PN14 when Ca was replaced A1
Control
A2
Ba
A3
~i
2see Ba+Cd
A5
8a+Cd+TTX
~
Ba+Cd +TTX
Fig. 5. Effect of Ba on responses of intracerebellar nuclei neurons on PN4. A l l responses were elicited in the same neuron by
direct electrical stimulations in control Krebs solution (A1), after replacement of Ca by Ba in the bath (A2, A3) and after addition of Cd (A4) and T r x (A5, A6) to the Ba-containing medium. Note the synaptic noise in control Krebs solution (A1). During the transition from the control solution to the Ba-containing medium, the current pulse first gave rise to small prolonged postdepotarizations (A2) which turned to become true plateaux of depolarization when the exchange of the medium was achieved (A3). Addition of Cd to the bath abolished these plateaux and suppressed the synaptic background noise but left unaffected the fast initial spike of the responses and unmasked depolarizing afterpotentials, longer in duration than the depolarizing afterpotentials recorded on PN0 (compare A4 with 6A3). Further addition of TI'X first suppressed the depolarizing afterpotentials (A5) and then the Cd-resistant fast spike (A6).
101 by 2.5 m M B a in the s u p e r f u s i n g fluid, the first change o b s e r v e d in the electrophysiological prop-
A1
Control
A2 I
TEA /'
erties of the n u c l e a r n e u r o n s (n = 13) was the a p p e a r ance of a p r o l o n g e d d e p o l a r i z a t i o n following s p o n t a n e o u s l y occurring fast spikes (Fig. 4 A 3 ) or of a postdepolarization outlasting the current pulse
40m$
(Fig. 5A2). W h e n the e x c h a n g e of the m e d i u m was fully achieved, p r o l o n g e d p l a t e a u x of d e p o l a r i z a t i o n (Figs. 4A4, 5 A 3 ) , similar to the B a - d e p e n d e n t action
A3
TEA+TTX
A4
TEA+TTX+Co
potentials previously described in cerebellar Purkinje cellsl0,13, 33, were i n d u c e d in n u c l e a r cells by direct electrical s t i m u l a t i o n . T h e y usually followed an initial fast spike a n d their a m p l i t u d e r a n g e d from o n e to several seconds. A d d i t i o n of o n l y o n e of the c h a n n e l blockers T T X , Cd or Co to the B a - c o n t a i n i n g fluid could o n l y partially abolish the electrically i n d u c e d r e s p o n s e s (Figs. 4A5, 5A4), thus suggesting that m e m b r a n e c o n d u c t a n c e s activated u n d e r Ba i n v o l v e d b o t h N a a n d Ca currents. T h e r e f o r e , the effects of these channel blockers were studied as a f u n c t i o n of the se-
A1
Control
A2
TEA
Fig. 7. Effect of TEA on responses of intracerebellar nuclei neurons on postnatal day 9. All responses were elicited in the same neuron by direct electrical stimulations in control Krebs solution (A1), after substitution of the Krebs solution by a 15 mM TEA-containing medium (A2) and after addition of TTX (A3) and Co (A4) to the TEA-containing solution. Note the lengthening of the fast spikes under TEA, reinforced by the appearance of a shoulder on their repolarizing phase (arrow in A2), the replacement of such responses by slow rising action potentials when TI'X was added to the bath (A3) and the abolition of the responses when Co was further added to the TEA plus TTX-containing solution (A4). q u e n c e of their a d d i t i o n to the s u p e r f u s i n g solution. W h e n 5 × 10 -6 M T T X was first a d d e d to the BaA
TEA+Cd
el
Control
C2
TEA+Cd
04
TEA+Cd+TTX
'
A3
TEA+Cd
A4
TEA+Cd+TTX
B
TEA+Cd
~
TEA+Cd
40ms
Fig. 6. Effect of TEA on responses of intracerebellar nuclei neurons to direct electrical stimulations on PN0. All responses were recorded in the same neuron in control Krebs solution (A1), after substitution of the Krebs solution by a 15 mM TEAcontaining medium (A2) and after addition of Cd (A3) and TI'X (A4) to the TEA-containing solution. The current pulse which only gave rise to a single fast spike in control Krebs solution (A1) also gave rise to slower rising spikes under TEA (arrow in A2). The fast spike persisted after addition of Cd whereas the slower rising spikes were abolished (A3). In this TEA plus Cd-containing solution a small increase of the current intensity elicited a depolarizing afterpotential following the fast spike (arrow in A3). Addition of TTX to the solution abolished altogether the fast spike and the depolarizing afterpotentials (A4).
Fig. 8. Effect of TEA and Cd on responses of intracerebellar nuclei neurons on PNs 6, 7 and 8. A, B: responses of two nuclear neurons on PN6 (A) and PN7 (B) to direct electrical stimulation under a 15 mM TEA plus 1 mM Cd-containing solution. Note that, in spite of the presence of Cd in the bathing medium, the current pulse elicited in these cells not only fast spikes, but also long-lasting depolarizations sustaining the firing of fast and multiphasic spikes. C: responses of another nuclear neuron on PN8 to direct electrical stimulation in control Krebs solution (C1), after substitution of the control solution by a 15 mM TEA plus 1 mM Cd containing-medium (C2, C3) and after addition of TI'X to the bathing medium (C4). Note the appearance of prolonged depolarizations (C2, C3) under TEA plus Cd. Addition of TTX suppressed all these responses (C4).
102 containing medium, the depolarizing current pulse still elicited the same plateaux of depolarization as described before whereas the initial fast spikes were completely abolished (Fig. 4A5). These TTX-resistant long-lasting depolarizations were totally suppressed by the addition of the Ca-channel blocker Co (Fig. 4A6), thus indicating that they were most probably due to a Ba conductance involving Ca channels. On the contrary when Cd was first added to the Bacontaining medium, thus abolishing the Ca components of the electrically induced responses precedingly described, as well as the synaptic background noise (compare Fig. 5A2, A4), the current pulse passing through the recording microelectrode still generated other depolarizing events in the form of depolarizing afterpotentials following the initial fast spike, with an even decay reaching up to 150 ms but which did not sustain any spike firing until PN4 (Fig. 5A4). Addition of TTX to the bathing medium suppressed first these depolarizing afterpotentials (Fig. 5A5) and then the fast Na component (Fig. 5A6). These responses, resistant to Ca-channel blockers but sensitive to Na-channel blockers, were thus considered as mediated by Na conductances. They were further investigated in TEA-containing solution since they were also elicited in this bathing medium (see next section). Responses induced in TEA-containing solution. In 26 experiments when the control Krebs solution was replaced by a 15 mM TEA-containing medium, the repolarizing phase of the fast spike was not obviously lengthened until PN6, at least as far as the current pulse intensity was not increased (compare fast spike in Fig. 6A1, A2, A3). However, as early as PN0 stronger stimulations of the cells elicited slow rising and prolonged action potentials (Fig. 6A2) which were encountered at all the postnatal stages studied, with a wide range of their amplitude (25-55 mV) and their duration (25 ms-l.25 s) depending on cells rather than on postnatal stages and resembling Ca spikes described in mature neurons. During postnatal development after PN6 the lengthening of the fast repolarizing phase of the fast spike under TEA became more evident and could even be enhanced by the appearance of a shoulder on this repolarizing phase (arrow in Fig. 7A2). Addition of Cd to the TEA-containing solution suppressed the slow rising and prolonged action potentials and unmasked depo-
larizing afterpotentials following fast Na spikes (see next section, Fig. 6A3). In 4 experiments when TTX was first added to the same TEA-containing medium, the Na-dependent fast rising spikes were replaced by TI'X-resistant slow rising action potentials (Fig. 7A3) which in turn were abolished by the addition of the Ca-channel blocker Co (Fig. 7A4), thus confirming their Ca-dependent nature. Evolution of slow Na conductances in TEA-containing solution. When Cd was first added to the TEA-containing solution or when the recording chamber was directly fed by a 15 mM TEA- plus 1 mM Cd-containing medium (19 cells), the depolarizing current pulses still elicited Cd-resistant responses, the features of which appear to vary as a function of the postnatal stage under study (Figs. 6A3, 8A, B, C2, C3). Indeed, as early as PN0 and under these conditions, the fast Na spikes were usually followed by a Short depolarizing afterpotential, especially when the stimulation strength was slightly increased. However, these depolarizing afterpotenrials never triggered any spike firing (Fig. 6A3) until PN6, as indicated before for cells recorded in Ba plus Cd-containing solution (see above). Addition of TI'X to the bathing medium completely abolished these depolarizing afterpotentials as well as the fast Na spike (Fig. 6A4). Later on, from PN6 to the weaning period, the direct exchange of the control Krebs solution by a TEA plus Cd-containing superfusing fluid produced either the appearance of prolonged plateaux of depolarization following an initial fast spike (25-30 mV in amplitude and 500 ms to more than 1 s in duration) and which did not sustain any spike firing (Fig. 8C2), or the appearance of longlasting depolarizations, 20-30 mV in amplitude and up to 1.25 s in duration, on which fast and multiphasic spikes were superimposed (Fig. 8A, B, C3). These responses were abolished when TTX was added to the TEA plus Cd-containing solution (Fig. 8C4) and were therefore very similar to those generated under the same conditions at mature stages (see above and Fig. 3A4, A5). Thus most of the ionic conductances demonstrated in mature nuclear neurons appear to be already present as early as birth, although it seems that at least the slow Na conductances are not fully developed at very early postnatal stages.
103 DISCUSSION The present experiments as well as those reported in a previous paper20 show that slices of rat intracerebellar nuclei maintained in vitro provide a suitable preparation to investigate bioelectrical properties of developing nuclear neurons. Particularly, stable intracellular recordings permit to explore the ionic conductances of these cells not only at mature stages, but also at very early postnatal stages.
Na conductances Fast Na-dependent spikes. In control Krebs solution, direct electrical stimulation of the nuclear neurons elicited the firing of a single or of a train of fast spikes, whatever the postnatal stage under study. Addition of TTX to the bathing medium abolished these fast spikes, thus indicating that they were sustained by Na currents and corresponded to the wellknown fast Na conductances commonly encountered in central nerve cells. Their amplitude did not significantly differ throughout postnatal development whereas their duration shortened in agreement with the evolution of such Na-dependent responses during development as already reported for these cells20 or for other nerve cells42,43. The longer duration of the fast spike in immature animals than in the adults could result either from different properties of the Na channels in young neurons as compared to more mature cells, or from an immature state of fast K conductances contributing to spike repolarization 23, or from both phenomena. The former possibility is enforced by the fact that in TEA plus Cd-containing medium, where only Na conductances are involved in the generation of the electrically induced response, the duration of the initial fast spikes at the weaning period was always shorter than that at very young stages (1.5-1.8 ms vs 3.0-5.0 ms), indicating that kinetics of activation and/or inactivation of Na conductances probably evolve during postnatal development. An argument in favor of an immature state of fast K conductances during the first postnatal week is the fact that block of K conductances by exposure to T E A did not so markedly prolonge the repolarizing phase of the fast Na spikes (Fig. 6A1, A2, A3) as it did later on during development (Fig. 3A1, A2, A4). Such an evolution of the K conductances could also explain the differ-
ences in the results obtained in the immature neurons as compared to older stages when Ba- or TEA-containing solutions replaced the superfusing control fluid.
Depolarizing afterpotentia!s and Na-dependent long-lasting depolarizations. When the control Krebs solution was replaced by a Ba or T E A plus Cd-containing medium, thus abolishing most K and Ca conductances, the fast Na spikes elicited by direct electrical stimulation of the cells were followed by depolarizing events at all the postnatal stages studied since PN0 (Figs. 3, 5, 6, 8). These responses were suppressed when TI~X was added to the bath thus indicating that they were mediated by Na currents. They appeared as depolarizing afterpotentials following the fast Na spike as early as birth (Figs. 5A4, 6A3) and also, since PN6, as long-lasting depolarizations which eventually sustained spike firing (Figs. 3A4, A5, 8A, B, C2, C3). Therefore it seems that a slowly inactivating Na conductance responsible for the occurrence of depolarizing afterpotentials comparable to the ones already described in other nerve cells under the same conditions15,16 can be unmasked in nuclear cells as early as birth by the block of K conductances. Later on, a more or less complete fusion of these depolarizing afterpotentials could give rise to long-lasting depolarizations as described before 10. However, one cannot preclude that the long-lasting depolarizations recorded in these neurons in spite of the absence of spike firing since PN6, could also result at least in part from a non-inactivating slow Na conductance similar to that observed in cerebellar Purkinje cells33 or in neocortical neurons 50.
Ca conductances In control Krebs solution and at all the postnatal stages studied, no slow rising spikes or slow Ca-dependent depolarizations were ever evoked by direct electrical stimulation of nuclear neurons, in marked contrast with Purkinje cellslO,13,33, hippocampal pyramidal cells16,45,53 or inferior olivary neurons 36. This indicates that under normal conditions, there is no evidence for a noticeable activation of Ca conductances in these cells. Furthermore, the absence of long-lasting afterhyperpolarizing potentials in these neurons, attributed in other classes of cells to a Ca-activated K conductance 24,26,33,36 is consistent with a weak activation of Ca conductances in control
104 Krebs solution. At birth, after exposure to T E A or to Ba, TTX-resistant slow rising spikes and prolonged plateaux of depolarization were recorded in nuclear neurons (Fig. 4A5). These responses were facilitated when larger depolarizing current pulses were passed through the recording microelectrode and were abolished by the addition of Ca channel blockers to the superfusing fluid, strongly suggesting that they were due to the activation of voltage-dependent conductances not activated under normal conditions. These Ca-dependent responses therefore appear well developed as early as birth and remain almost unchanged throughout postnatal development (Figs. 2B3, 7A3). Such Ca conductances, recorded under normal conditions or unmasked by T E A or Ba, appear to represent a general feature of bioelectrical membrane properties of nerve cells and have been described in many mammalian neurons as different as, for instance, cerebellar Purkinje cells 10,13,32,33,34, hippocampal pyramidal and granule cells 15,16,17,45,54, inferior olivary neurons 35,36, thalamic neurons 25,26,31, substantia nigra cells 30, cortical neurons 12, dorsal root ganglion neurons 27,37, or spinal dorsal horn neurons 40. However, in the case of intracerebellar nuclei neurons, whether these conductances are generated in dendrites such as for cerebellar Purkinje cells10,13,32,33,34, cortical neurons 12 or pars compacta cells 30, or at the somatic level as demonstrated for the low threshold Ca responses of olivary36 or thalamic 31 neurons, still remains to be determined. On the whole, it can be concluded that mature in-
REFERENCES 1 Adams, P., Voltage-dependent conductances of vertebrate neurones, Trends Neurosci., 5 (1982) 116-119. 2 Addison, W. H., The development of the Purkinje cells and of the cortical layers in the cerebellum of the albino rat, J. comp. Neurol., 21 (1911) 459-488. 3 Altman, J. and Bayer, S. A., Prenatal development of the cerebellar system in the rat. I. Cytogenesis and histogenesis of the deep nuclei and of the cortex of the cerebellum, J. comp. Neurol., 179 (1978) 23-48. 4 Baccaglini, P. I., Action potentials of embryonic dorsal root ganglion neurones in Xenopus tadpoles, J. Physiol. (Lond.), 283 (1978) 585-604. 5 Baccaglini, P. I. and Spitzer, N. C., Developmental changes in the inward current of the action potential of Rohon-Beard neurones, J. Physiol. (Lond.), 271 (1977) 93-117. 6 Bader, C. R., Bertrand, D., Dupin, E. and Kato, A. C.,
tracerebellar nuclei neurons express a complex set ionic conductances including fast and slowly inac vating Na conductances as well as Ca conductanc, However, Ca spikes are never observed in norn conditions, in marked contrast with cerebellar P1 kinje cells which do exhibit such responses10, 33. least two of these conductances, i.e. the fast Na a Ca conductances, are already well developed as ea: as birth, whereas the slowly inactivating Na condl tances mature progressively during the first postna week. Furthermore, our data suggest that the fast conductance contributing to spike repolarization not fully operating at birth and develops later on. T presence of both Na and Ca conductances since biJ in nuclear neurons is also in contrast with cerebel] Purkinje cells which express first Na and only later, Ca conductances 32. Consequently, if a similar dev opmental pattern of m e m b r a n e excitability exists intracerebellar nuclei neurons it must occur earlier the embryonic development. Indeed, one canr preclude this hypothesis since, for instance, both l and Ca currents are present at very early dev opmental stages in chick ciliary ganglion ce] whereas a developmental sequence of ionic condl tances, with Na currents appearing prior to Ca cl rents, is present in mesencephalic neural crest ce which are the precursors of ciliary ganglia 6. ACKNOWLEDGEMENTS This work was supported by the Minist~re de 1'] dustrie et de la Recherche (Grant No. 83C0910).
Development of electrical membrane properties in cultut avian neural crest, Nature (Lond.), 305 (1983) 808-810. 7 Bader, C. R., Bertrand, D. and Kato, A. C., Membra currents in a developing parasympathetic ganglion, Dev op. Biol., 98 (1983) 515-519. 8 Baker, P. F., Hodgkin, A. L. and Ridgway, E. B., Depo rization and calcium entry in squid giant axons, J. Physi (Lond.), 218 (1971) 709-755. 9 Blankenship, J. E., Action of tetrodotoxin on spinal mo neurons of the cat, J. Neurophysiol., 31 (1968) 186-194. 10 Crepel, F., Dupont, J. L. and Gardette, R., Selective sence of calcium spikes in Purkinje cells of staggerer mut~ mice in cerebellar slices maintained in vitro, J. Physi (Lond.), 346 (1984) 111-125. 11 Crill, W. E. and Schwindt, P. C., Active currents in ma malian central neurones, Trends Neurosci., 6 (19t 236-240. 12 Dichter, M. A., Lisak, J. and Biales, B., Action potenl mechanism of mammalian cortical neurons in cell cultu
105
Brain Res., 289 (1983) 99-107. 13 Dupont, J. L., Gardette, R. and Crepel, F., Bioelectrical properties of cerebellar Purkinje cells in reeler mutant mice, Brain Res., 274 (1983) 350-353. 14 Eckert, R. and Lux, H. D., A voltage-sensitive persistent calcium conductance in neuronal somata of Helix, J. Physiol. (Lond.), 254 (1976) 129-151. 15 Fournier, E. and Crepel, F., Electrophysiological properties of dentate granule cells in mouse hippocampal slices maintained in vitro, Brain Res., 311 (1984) 75-86. 16 Fournier, E. and Crepel, F., Electrophysiologicat properties of in vitro hippocampal pyramidal cells from normal and staggerer mutant mice, Brain Res., 311 (1984) 87-96. 17 Fricke, R. A. and Prince, D. A., Electrophysiology of dentate gyrus granule cells, J. Neurophysiol., 51 (1984) 195-209. 18 Gardette, R., Courtois, M. and Bisconte, J. C., Prenatal development of mouse central nervous structures: time of neuron origin and gradients of neuronal production. A radioautographic study, J. Hirnforsch., 23 (1982)415-431. 19 Gardette, R., Debono, M., Dupont, J. L. and Crepel, F., Development of synaptic responses and membrane conductances of intracerebellar nuclei neurones in rat cerebellar slices maintained in vitro, C.R. Acad. Sci. (Paris), 297 (1983) 43-46. 20 Gardette, R., Debono, M., Dupont, J. L. and Crepel, F., Electrophysiological studies on the postnatal development of intracerebellar nuclei neurons in rat cerebellar slices maintained in vitro. I. Postsynaptic potentials, Develop. Brain Res., 19 (1985) 47-55. 21 Goodman, C. S. and Spitzer, N. C., The development of electrical properties of identified neurones in grasshopper embryos, J. Physiol. (Lond.), 313 (1981) 385-403. 22 Hagiwara, S., Fukuda, J. and Eaton, D. C., Membrane currents carried by Ca, Sr, and Ba in barnacle muscle fiber during voltage clamp, J. gen. Physiol., 63 (1974) 564-578. 23 Hodgkin, A. L. and Huxley, A. F., A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol. (Lond.), 117 (1952) 500-544. 24 Hotson, J. R. and Prince, D. A., A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurons, J. Neurophysiol., 43 (1980) 409-419. 25 Jahnsen, H. and Llinas, R., Electrophysiological properties of guinea pig thalamic neurones: an in vitro study, J. Physiol. (Lond.), 349 (1984) 205-226. 26 Jahnsen, H. and Llinas, R., Ionic basis for the electroresponsiveness and oscillatory properties of guinea-pig thalamic neurones in vitro, J. Physiol. (Lond.), 349 (1984) 227-247. 27 Kameyama, M., Ionic currents in cultured dorsal root ganglion from adult guinea pigs, J. Membrane Biol., 72 (1983) 195-203. 28 Korneliussen, H. K., On the ontogenetic development of the cerebellum (nuclei, fissures and cortex) of the rat, with special reference to regional variations in corticogenesis, J. Hirnforsch., 10 (1968) 379-413. 29 Krnjevic, K., Pumain, R. and Renaud, L., Effects of Ba 2÷ and tetraethylammonium on cortical neurones, J. Physiol. (Lond.), 215 (1971) 223-245. 30 Llinas, R., Greenfield, S. A. and Jahnsen, H., Electrophysiology of pars compacta cells in the in vitro substantia nigra. A possible mechanism for dendritic release, Brain Res., 294 (1984) 127-132.
31 Llinas, R. and Jahnsen, H., Electrophysiology of mammalian thalamic neurones in vitro, Nature (Lond.), 297 (1982) 406-408. 32 Llinas, R. and Sugimori, M., Calcium conductances in Purkinje cell dendrites: their role in development and integration. In M. Cudnod, G. W. Kreutzberg and F. E. Bloom (Eds.), Development and Chemical Specificity of Neurons, Progress in Brain Research, Vol. 51, Elsevier, Amsterdam, 1979, pp. 323-334. 33 Llinas, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 171-195. 34 Llinas, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 197-213. 35 Llinas, R. and Yarom, Y., Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances, J. Physiol. (Lond.), 315 (1981) 549-567. 36 Llinas, R. and Yarom, Y., Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro, J. Physiol. (Lond.), 315 (1981) 569-584. 37 Matsuda, Y., Yoshida, S. and Yonezawa, T., Tetrodotoxin sensitivity and Ca component of action potentials of mouse dorsal root ganglion cells cultured in vitro, Brain Res., 154 (1978) 69-82. 38 Miale, I. L. and Sidman, R. L., An autoradiographic analysis of histogenesis in the mouse cerebellum, Exp. Neurol., 4 (1961) 277-296. 39 Miyaka, M., The development of action potential mechanism in a mouse neuronal cell line in vitro, Brain Res., 143 (1978) 349-354. 40 Murase, K. and Randic, M., Electrophysiological properties of rat spinal dorsal horn neurones in vitro: calciumdependent action potentials, J. Physiol. (Lond.), 334 (1983) 141-153. 41 Peacock, J. H. and Walker, C. R., Development of calcium action potentials in mouse hippocampal cell cultures, Develop. Brain Res., 8 (1983) 39-52. 42 Purpura, D. P., Shofer, R. J. and Scarff, T., Properties of synaptic activities and spike potentials of neurons in immature neocortex, J. Neurophysiol., 28 (1965) 925-942. 43 Schwartzkroin, P. A., Development of rabbit hippocampus: physiology, Develop. Brain Res., 2 (1982) 469-486. 44 Schwartzkroin, P. A. and Prince, D. A., Effects of TEA on hippocampal neurons, Brain Res., 185 (1980) 169-181. 45 Schwartzkroin, P. A. and Slawsky, M., Probable calcium spikes in hippocampal neurons, Brain Res., 135 (1977) 157-161. 46 Schwindt, P. C. and Crill, W. E., Effects of barium on cat spinal motoneurons studied by voltage-clamp, J. Neurophysiol., 44 (1980) 827-846. 47 Schwindt, P. C. and Crill, W. E., Differential effects of TEA and cations on outward ionic currents of cat motoneurons, J. Neurophysiol., 46 (1981) 1-16. 48 Spitzer, N. C., The development of neuronal membrane properties in vivo and in culture. In Developing and Regenerating Vertebrate Nervous Systems, Liss, New York, 1983, pp. 41-59. 49 Spitzer, N. C. and Lamborghini, J. E., The development of the action potential mechanism of amphibian neurons isolated in culture, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 1641-1645.
106 50 Stafstrom, C. E., Schwindt, P. C. and Crill, W. E., Negative slope conductance due to a persistent subthreshold sodium current in cat neocortical neurons in vitro, Brain Res., 236 (1982) 221-226. 51 Strichartz, G., Small, R., Nicholson, C., Pfenninger, K. H. and Llinas, R., Ionic mechanisms for impulse propagation in growing nonmyelinated axons: saxitoxin binding and electrophysiology, Soc. Neurosci. Abstr., 6 (1980) 660. 52 Taber Pierce, E. and Sweet, E., Histogenesis of deep cere-
bellar nuclei as studied autoradiographically with thymidine 3H in the mouse, Anat. Rec., 157 (1967) 301. 53 Wong, R. K. S. and Prince, D. A., Participation of Ca 2÷ spikes during intrinsic burst firing in hippocampal neurons, Brain Res., 159 (1978) 385-390. 54 Wong, R. K. S., Prince, D. A. and Basbaum, A. t., tntradendritic recordings from hippocampal neurons, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 986-990.
97
BRD 50210
Electrophysiological Studies on the Postnatal Development of Intracerebellar Nuclei Neurons in Rat Cerebellar Slices Maintained in Vitro. II. Membrane Conductances ROBERT GARDEqq'E, MARC DEBONO, JEAN-LUC DUPONT and FRANCIS CREPEL INSERM U97, 75014 Paris (France)
(Accepted December 18th, 1984) Key words: intracerebellar nuclei - - postnatal development - - membrane conductances
The development of membrane conductances of intracerebellar nuclei neurons was studied in the rat since birth up to the weaning period by the use of thick sagittal cerebellar slices maintained in vitro. Mature nuclear neurons express fast sodium and slowly inactivating sodium conductances, as well as calcium conductances. As early as birth, fast sodium and calcium conductances appear well developed whereas slowly inactivating sodium conductances mature within the first postnatal week.
INTRODUCTION The study of the development of membrane neuronal excitability is of major importance in order to better understand the role played by these intrinsic properties of nerve cells in the functioning of the central nervous system. Indeed, m e m b r a n e neuronal excitability is dependent on several types of ionic conductancesl, u, the ontogenesis of which can vary in function of the species or the nervous structures 48. Thus, some nerve cells seem to be able to express the whole repertory of their ionic conductances at very early developmental stages41, 43 while others show a progressive establishment of their bioelectrical properties, for instance sodium (Na) conductances being expressed before calcium (Ca) conductances 6,21,32, or the inverse 4,5,12,39,49,51. If these active electrical properties are already well known for the cerebellar Purkinje cells either during development 32, or at the adult stage10,33,34, or even in some cerebellar mutantslO, 13, those of the intracerebellar nuclei neurons still remained unexplored in the adult as well as during development. Therefore, the development of bioelectrical membrane properties of rat intracerebellar nuclei neurons
was studied from birth up to the weaning period. One aim of the study was to determine the extent to which these cells resemble Purkinje cells in this respect since they derive both from the same region of the dorsal metencephalic plates of neuroepithelial cells2,3,28 with nearly the same birthdates3,18,38, 52. Experiments were performed on thick cerebellar slices maintained in vitro since this preparation allows stable intracellular recordings of nuclear neurons19, 20 and facilitates the study of the effects of various agents known to act upon ionic channels by exchanging the bathing medium or by direct drug application. A preliminary report of this work has been already published 19. MATERIALS AND METHODS The experimental procedures used in this study were identical to those described in a previous paper 20. Nuclear neurons, evenly distributed from birth (postnatal day 0 (PN0)) to the weaning period (PN23) were intracellularly recorded with glass microelectrodes filled with KCI 3 M or potassium citrate 3 M (DC resistance 50-120 MQ). Cells were stimulated by intracellular currents injected through the
Correspondence: R. Gardette, INSERM U97, 2ter, rue d'Alrsia, 75014 Paris, France.
0165-3806/85/$03.30 (E) 1985 Elsevier Science Publishers B.V. (Biomedical Division)
98 recording electrode via a bridge circuit. When necessary, the control Krebs solution was replaced by a Ca-free superfusing fluid containing either BaC12 (Ba, 2.5 mM) or CdCI 2 (Cd, 1 mM), or by a tetraethylammonium (TEA) -containing medium (TEA, 15 mM). The composition of these various bathing solutions was the same as already described elsewhere (Table I in ref. 15). Finally, variations in the composition of these media included addition of tetrodotoxin (TTX, 5 × 10-6 M) or of cobalt chloride (Co, 2 mM) in the Ba- or the TEA-containing solutions. RESULTS Membrane conductances were studied in 64 nuclear neurons selected among cells recorded in a previous study dealing with synaptic responses 20. Identification of the nuclear cells was based upon the direct visual control of the recording microelectrode location within the intracerebellar nuclei and usually further confirmed by their antidromic responses induced by juxtanuclear stimulation (see Fig. 1 in ref. 20). Resting potentials ranged between - 5 0 and - 7 0 mV with no significant difference between young and older stages as already demonstrated 20. Similarly, spikes elicited by direct stimulation of the cells presented the same evolution between immature and mature stages as those antidromically or synaptically evoked 20 (see also following paragraphs). Responses induced by direct electrical stimulation in mature intracerebellar nuclei neurons Responses in control Krebs solution. On PN23 in control Krebs solution, small depolarizing current pulses (less than 0.5 nA) passing through the recording microelectrode elicited a marked depolarization of the cells (n = 25) which gave rise to one (Fig. 1A1) or several (Figs. 1A2, B1, 2A1, B1 and 3A1) fast spikes, depending on the amplitude of the pulse. These spikes could reach 70 mV in amplitude and their duration was 1.0-1.5 ms, thus resembling fast spikes synaptically evoked in these cells by juxtanuclear stimulation 20. Indeed, addition of 5 x 10-6 M q T X in the bath abolished these fast spikes, indicating that they were Na-dependent 9 (Figs. 1B3, 2B3). When the stimulus intensity was further increased up to 1.0 nA or more, the only visible effect was an inac-
A2
!']
I
i,!~;i
}
20ms
B1
Co~qtrol
Ba
83
Ba+TTX
40r~
Fig. 1. Effect of Ba on responses of intracerebellar nuclei n rons to direct electrical stimulations on PN23. A: responses nuclear neuron (upper traces) to stimulating currents of 3 ferent intensities (lower traces) in control Krebs solution small current pulse (0.1 nA, A1) induced the firing of a sin spike. Further increase of the stimulus intensity led first to firing of a train of spikes (0.3 hA, A2) and then to the inacti tion of the response (1 nA, A3). B: effect of Ba and TTX on sponses of nuclear neurons. The 3 responses (upper trac were elicited in the same neuron by direct electrical stimt tions (lower traces) in control Krebs solution (B1), after placement of Ca by Ba in the bath (B2) and after addition TTX to the bath (B3). Note that replacement of Ca by Ba in bath did not elicit any other responses than fast spikes (B2) that addition of TTX suppressed all the active components the electricallyinduced response (B3). tivation of the Na spikes following the initial o (Fig. 1A3), but slower rising spikes, such as I spikes elicited in cerebellar Purkinje cells 10,13,33 we never obtained in these mature nuclear neurons. Responses induced in Ba- or TEA-containing so~ tions. The presence of Ca currents was further test, in these cells by studying the effects of Ba, a divale cation reported to block voltage-dependent potas um (K) conductances 46, to move more easily than ( through Ca channels 22, and not to activate the Ca-d pendent K conductances 14, and of T E A , a cati~ which also blocks K conductances29,44, 47. When Ba replaced Ca in the bathing medium cells), depolarizing current pulses passing throul the recording microelectrode never gave rise to a~ other responses than the fast spikes described befor even when the amplitude of the pulse was as high 0.8 nA (Fig. 1B1, B2). In contrast, when the control Krebs solution w replaced by a 15 mM TEA-containing superfusiz fluid (17 cells), several changes progressively a peared in the responses of the cells. Indeed, this e change of medium led first to a lengthening of the fa repolarizing phase of the fast spikes (single arrow Figs. 2A2, 3A2) and to the appearance of depolm
99 zing afterpotentials ( a r r o w h e a d in Figs. 2A2, 3A2) which could in turn trigger the firing of multiphasic fast spikes (double arrow in Figs. 2A2, 3A2). The second event that t o o k place in such a superfusing fluid was the occurrence of p r o l o n g e d plateaux of depolarization outlasting the current pulse (Fig. 2A3, B2), 2 0 - 5 0 mV in a m p l i t u d e d e p e n d i n g on cells and up to 500 ms in duration. Finally, the frequency of spontaneously occurring spikes as well as the synaptic b a c k g r o u n d noise were also e n h a n c e d (compare Fig. 3A1, A3). W h e n 5 x 10 -6 M T F X was a d d e d to the T E A - c o n taining solution (6 cells), the fast Na spikes, the multiphasic spikes and the plateaux of depolarization totally disappeared, while slow rising spikes up to 55 mV in amplitude and presenting an even decay which could reach up to 150 ms in duration were u n m a s k e d (Fig. 2B3). T h e r e f o r e , these results suggest that multiphasic spikes are N a - d e p e n d e n t whereas slow rising spikes are C a - d e p e n d e n t (see also below). FurA1
Control
A2
A3
TEA
TEA
Ii,llll
A1
Control
'ill
I
A2
p
',ii ~
'lOOms
TEA+Cd
A3
TEA
l,j[~j~
TEA+Cd
iLIIII, jn|tltiiil ' m
40m$
A5
TEA
400ms
A6
TEA+Cd+TTX
Fig. 3. Effect of TEA and of Cd on responses of intracerebellar nuclei neurons on PN23. All responses were elicited in the same neuron by direct electrical stimulations in control Krebs solution (A1), after substitution of the Krebs solution by a 15 mM TEA-containing medium (A2, A3) and after addition of Cd (A4, A5) and TTX (A6) to the TEA-containing medium. Note the lengthening of the fast spike (single arrow in A2) and the appearance of depolarizing afterpotentials (arrowhead in A2) sustaining firing of multiphasic spikes (double arrow in A2) under TEA. Note also, under the same conditions, the enhancement of the synaptic background noise and the occurrence of prolonged depolarizations outlasting the current pulse (A3). In spite of the addition of Cd to the bath, the current pulse still elicited long-lasting depolarizations on which fast and multiphasic spikes were or were not superimposed (A4, A5): Addition of TI'X totally abolished these responses (A6). thermore, because plateaux of depolarization in-
m
~rrB
B1
Control
B2
TEA
m
1O0ms TEA+TTX
Fig. 2. Effect of TEA on responses of intracerebellar nuclei neurons on PN23. A: responses of a nuclear neuron to direct electrical stimulation in control Krebs solution (A1) and after substitution of the Krebs solution by a 15 mM TEA-containing solution (A2, A3). Note that the first effect elicited in this cell by the TEA-containing solution was a lengthening of the repolarizing phase of the fast spikes (single arrow in A2) and the occurrence of small depolarizing afterpotentials (arrow head in A2) which could trigger firing of multiphasic spikes (double arrow in A2). When the exchange of the medium was fully achieved, the same current pulse then elicited prolonged plateaux of depolarization, following the fast spike and outlasting the end of the current pulse (A3). B: responses of another nuclear neuron to direct electrical stimulation in control Krebs solution (B1), after substitution of the Krebs solution by a 15 mM TEA-containing medium (B2) and after addition of TFX to the bath (B3). Note the appearance of prolonged action potentials following the initial fast spike under TEA (B2). Addition of TTX abolished the fast components whereas slow rising and prolonged action potentials were still elicited (B3). The bridge balance in B2 and B3 was less accurate than in B1 and the tips of the spikes were retouched in B1 in this figure and also in Figs. 5A1, A3 and 8A.
duced under T E A alone were no longer present under T E A plus TTX, this suggested that o t h e r conductances such as slowly inactivating Na conductances, were also activated under T E A alone and contributed to the generation of these plateaux of depolarization.
Effects of Ca-channel blockers on responses induced in TEA-containing solutions. The eventual presence of slow Na-conductances was explored in 9 cells after Ca currents were blocked by the replacem e n t of the T E A - c o n t a i n i n g solution by a Ca-free solution containing the same concentration of T E A plus 1 m M of the Ca-channel blocker Cd 8. U n d e r these conditions, depolarizing current pulses elicited either Cd-resistant long-lasting depolarizations, 2 5 - 3 0 m V in amplitude and up to 300 ms in duration, on which fast and multiphasic spikes were superimposed (Fig. 3A4), or p r o l o n g e d plateaux of depolarization, 2 5 - 3 0 m V in amplitude and up to 1.5 s in duration sustaining no spike firing, at least during their initial phase (Fig. 3A5). A closer inspection of these records suggested that these events might result from a m o r e or less complete fusion of depolari-
100 zing afterpotentials such as those previously described (Figs. 2A2, 3A2). A d d i t i o n of T I ' X to the bathing m e d i u m suppressed all these c o m p o n e n t s (Fig. 3A6) suggesting that they were N a - d e p e n d e n t . Finally, the slow spikes described in Fig. 2B3 were absent when Cd was a d d e d to the superfusing fluid, thus enforcing the hypothesis that they were mediated by Ca conductances. In brief, these results strongly suggest the presence of several ionic conductances in these m a t u r e intracerebellar nuclei neurons sustaining N a and Ca spikes on the one hand and N a - d e p e n d e n t depolarizing afterpotentials and plateaux of depolarization on the other hand.
Postnatal development of responses induced by direct electrical stimulation in intracerebellar nuclei neurons With the d e m o n s t r a t i o n of the presence of several types of ionic conductances in m a t u r e intracerebellar nuclei neurons, the question arose to d e t e r m i n e when these conductances first a p p e a r e d and how they evolved during postnatal d e v e l o p m e n t . Therefore, similar experiments as those described before were p e r f o r m e d on 39 cells at various postnatal stages since birth up to PN14.
Responses in control Krebs solution. Since PN0 (4 cells) and at all the postnatal stages studied, direct electrical stimulation of the cells elicited a m a r k e d depolarization which in turn induced the firing of a single spike (Fig. 6A1) or of a train of spikes d e p e n d ing on the stimulus intensity (Figs. 4A1, 5A1, 7A1, 8C1). F u r t h e r increase of the stimulus intensity led to an inactivation of the spikes following the initial one (Fig. 4A2) as already noticed in m a t u r e nuclear neurons (see above and Fig. 1A3). The amplitude of these spikes could reach 6 0 - 7 0 m V even at very young stages (Figs. 4A1, 6A1) whereas their duration decreased from 3 - 5 ms to 1 - 2 ms during postnatal development, according to previous observations on antidromicaUy or synaptically elicited spikes recorded in these neurons after juxtanuclear stimulation 20. These spikes persisted when 1 m M Cd was added to the bathing m e d i u m (Figs. 5A4, 6A3, 8A, B, C2, C3) but were abolished by 5 x 10 -6 M T F X (Figs. 4A5, 5A6, 6A4, 7A3, 8C4) thus indicating that they were m e d i a t e d by fast N a currents throughout postnatal development. A s in adult animals, no C a - d e p e n d e n t spikes were ever e v o k e d in control
A1
A2
Conlroi
It~A ~
,
20ms
Ba
A3
conl,::l
k
~
20ms
A5
Ba+TTX
40ms
A6
~ + ; Tx+c::~
Fig. 4. Effect of Ba on responses of intracerebellar nuclei neurons to direct electrical stimulations on PN0. All responses were recorded in the same neuron in control Krebs solution (AI, A2), after replacement of Ca by Ba in the bath (A3, A4) and after addition of TTX (A5) and Co (A6) to the Ba-containing medium. Note that in control Krebs solution a two-fold increase of the pulse intensity from 0.4 nA (A1) to 0.8 nA (A2) only led to an inactivation of the fast spikes. When Ba replaced Ca in the bath, prolonged action potentials (A3) and prolonged plateaux of depolarization (A4) were now elicited. Note that in A3 the prolonged action potentials occurred spontaneously. Addition of TTX to the Ba-containing medium suppressed the initial fast spike of the responses but did not abolish the plateaux of depolarization (A5) which disappeared after addition of Co to the bath (A6). Krebs solution at any d e v e l o p m e n t a l stages studied. Responses induced in Ba-containing solution. As early as birth and up to PN14 when Ca was replaced A1
Control
A2
Ba
A3
~i
2see Ba+Cd
A5
8a+Cd+TTX
~
Ba+Cd +TTX
Fig. 5. Effect of Ba on responses of intracerebellar nuclei neurons on PN4. A l l responses were elicited in the same neuron by
direct electrical stimulations in control Krebs solution (A1), after replacement of Ca by Ba in the bath (A2, A3) and after addition of Cd (A4) and T r x (A5, A6) to the Ba-containing medium. Note the synaptic noise in control Krebs solution (A1). During the transition from the control solution to the Ba-containing medium, the current pulse first gave rise to small prolonged postdepotarizations (A2) which turned to become true plateaux of depolarization when the exchange of the medium was achieved (A3). Addition of Cd to the bath abolished these plateaux and suppressed the synaptic background noise but left unaffected the fast initial spike of the responses and unmasked depolarizing afterpotentials, longer in duration than the depolarizing afterpotentials recorded on PN0 (compare A4 with 6A3). Further addition of TI'X first suppressed the depolarizing afterpotentials (A5) and then the Cd-resistant fast spike (A6).
101 by 2.5 m M B a in the s u p e r f u s i n g fluid, the first change o b s e r v e d in the electrophysiological prop-
A1
Control
A2 I
TEA /'
erties of the n u c l e a r n e u r o n s (n = 13) was the a p p e a r ance of a p r o l o n g e d d e p o l a r i z a t i o n following s p o n t a n e o u s l y occurring fast spikes (Fig. 4 A 3 ) or of a postdepolarization outlasting the current pulse
40m$
(Fig. 5A2). W h e n the e x c h a n g e of the m e d i u m was fully achieved, p r o l o n g e d p l a t e a u x of d e p o l a r i z a t i o n (Figs. 4A4, 5 A 3 ) , similar to the B a - d e p e n d e n t action
A3
TEA+TTX
A4
TEA+TTX+Co
potentials previously described in cerebellar Purkinje cellsl0,13, 33, were i n d u c e d in n u c l e a r cells by direct electrical s t i m u l a t i o n . T h e y usually followed an initial fast spike a n d their a m p l i t u d e r a n g e d from o n e to several seconds. A d d i t i o n of o n l y o n e of the c h a n n e l blockers T T X , Cd or Co to the B a - c o n t a i n i n g fluid could o n l y partially abolish the electrically i n d u c e d r e s p o n s e s (Figs. 4A5, 5A4), thus suggesting that m e m b r a n e c o n d u c t a n c e s activated u n d e r Ba i n v o l v e d b o t h N a a n d Ca currents. T h e r e f o r e , the effects of these channel blockers were studied as a f u n c t i o n of the se-
A1
Control
A2
TEA
Fig. 7. Effect of TEA on responses of intracerebellar nuclei neurons on postnatal day 9. All responses were elicited in the same neuron by direct electrical stimulations in control Krebs solution (A1), after substitution of the Krebs solution by a 15 mM TEA-containing medium (A2) and after addition of TTX (A3) and Co (A4) to the TEA-containing solution. Note the lengthening of the fast spikes under TEA, reinforced by the appearance of a shoulder on their repolarizing phase (arrow in A2), the replacement of such responses by slow rising action potentials when TI'X was added to the bath (A3) and the abolition of the responses when Co was further added to the TEA plus TTX-containing solution (A4). q u e n c e of their a d d i t i o n to the s u p e r f u s i n g solution. W h e n 5 × 10 -6 M T T X was first a d d e d to the BaA
TEA+Cd
el
Control
C2
TEA+Cd
04
TEA+Cd+TTX
'
A3
TEA+Cd
A4
TEA+Cd+TTX
B
TEA+Cd
~
TEA+Cd
40ms
Fig. 6. Effect of TEA on responses of intracerebellar nuclei neurons to direct electrical stimulations on PN0. All responses were recorded in the same neuron in control Krebs solution (A1), after substitution of the Krebs solution by a 15 mM TEAcontaining medium (A2) and after addition of Cd (A3) and TI'X (A4) to the TEA-containing solution. The current pulse which only gave rise to a single fast spike in control Krebs solution (A1) also gave rise to slower rising spikes under TEA (arrow in A2). The fast spike persisted after addition of Cd whereas the slower rising spikes were abolished (A3). In this TEA plus Cd-containing solution a small increase of the current intensity elicited a depolarizing afterpotential following the fast spike (arrow in A3). Addition of TTX to the solution abolished altogether the fast spike and the depolarizing afterpotentials (A4).
Fig. 8. Effect of TEA and Cd on responses of intracerebellar nuclei neurons on PNs 6, 7 and 8. A, B: responses of two nuclear neurons on PN6 (A) and PN7 (B) to direct electrical stimulation under a 15 mM TEA plus 1 mM Cd-containing solution. Note that, in spite of the presence of Cd in the bathing medium, the current pulse elicited in these cells not only fast spikes, but also long-lasting depolarizations sustaining the firing of fast and multiphasic spikes. C: responses of another nuclear neuron on PN8 to direct electrical stimulation in control Krebs solution (C1), after substitution of the control solution by a 15 mM TEA plus 1 mM Cd containing-medium (C2, C3) and after addition of TI'X to the bathing medium (C4). Note the appearance of prolonged depolarizations (C2, C3) under TEA plus Cd. Addition of TTX suppressed all these responses (C4).
102 containing medium, the depolarizing current pulse still elicited the same plateaux of depolarization as described before whereas the initial fast spikes were completely abolished (Fig. 4A5). These TTX-resistant long-lasting depolarizations were totally suppressed by the addition of the Ca-channel blocker Co (Fig. 4A6), thus indicating that they were most probably due to a Ba conductance involving Ca channels. On the contrary when Cd was first added to the Bacontaining medium, thus abolishing the Ca components of the electrically induced responses precedingly described, as well as the synaptic background noise (compare Fig. 5A2, A4), the current pulse passing through the recording microelectrode still generated other depolarizing events in the form of depolarizing afterpotentials following the initial fast spike, with an even decay reaching up to 150 ms but which did not sustain any spike firing until PN4 (Fig. 5A4). Addition of TTX to the bathing medium suppressed first these depolarizing afterpotentials (Fig. 5A5) and then the fast Na component (Fig. 5A6). These responses, resistant to Ca-channel blockers but sensitive to Na-channel blockers, were thus considered as mediated by Na conductances. They were further investigated in TEA-containing solution since they were also elicited in this bathing medium (see next section). Responses induced in TEA-containing solution. In 26 experiments when the control Krebs solution was replaced by a 15 mM TEA-containing medium, the repolarizing phase of the fast spike was not obviously lengthened until PN6, at least as far as the current pulse intensity was not increased (compare fast spike in Fig. 6A1, A2, A3). However, as early as PN0 stronger stimulations of the cells elicited slow rising and prolonged action potentials (Fig. 6A2) which were encountered at all the postnatal stages studied, with a wide range of their amplitude (25-55 mV) and their duration (25 ms-l.25 s) depending on cells rather than on postnatal stages and resembling Ca spikes described in mature neurons. During postnatal development after PN6 the lengthening of the fast repolarizing phase of the fast spike under TEA became more evident and could even be enhanced by the appearance of a shoulder on this repolarizing phase (arrow in Fig. 7A2). Addition of Cd to the TEA-containing solution suppressed the slow rising and prolonged action potentials and unmasked depo-
larizing afterpotentials following fast Na spikes (see next section, Fig. 6A3). In 4 experiments when TTX was first added to the same TEA-containing medium, the Na-dependent fast rising spikes were replaced by TI'X-resistant slow rising action potentials (Fig. 7A3) which in turn were abolished by the addition of the Ca-channel blocker Co (Fig. 7A4), thus confirming their Ca-dependent nature. Evolution of slow Na conductances in TEA-containing solution. When Cd was first added to the TEA-containing solution or when the recording chamber was directly fed by a 15 mM TEA- plus 1 mM Cd-containing medium (19 cells), the depolarizing current pulses still elicited Cd-resistant responses, the features of which appear to vary as a function of the postnatal stage under study (Figs. 6A3, 8A, B, C2, C3). Indeed, as early as PN0 and under these conditions, the fast Na spikes were usually followed by a Short depolarizing afterpotential, especially when the stimulation strength was slightly increased. However, these depolarizing afterpotenrials never triggered any spike firing (Fig. 6A3) until PN6, as indicated before for cells recorded in Ba plus Cd-containing solution (see above). Addition of TI'X to the bathing medium completely abolished these depolarizing afterpotentials as well as the fast Na spike (Fig. 6A4). Later on, from PN6 to the weaning period, the direct exchange of the control Krebs solution by a TEA plus Cd-containing superfusing fluid produced either the appearance of prolonged plateaux of depolarization following an initial fast spike (25-30 mV in amplitude and 500 ms to more than 1 s in duration) and which did not sustain any spike firing (Fig. 8C2), or the appearance of longlasting depolarizations, 20-30 mV in amplitude and up to 1.25 s in duration, on which fast and multiphasic spikes were superimposed (Fig. 8A, B, C3). These responses were abolished when TTX was added to the TEA plus Cd-containing solution (Fig. 8C4) and were therefore very similar to those generated under the same conditions at mature stages (see above and Fig. 3A4, A5). Thus most of the ionic conductances demonstrated in mature nuclear neurons appear to be already present as early as birth, although it seems that at least the slow Na conductances are not fully developed at very early postnatal stages.
103 DISCUSSION The present experiments as well as those reported in a previous paper20 show that slices of rat intracerebellar nuclei maintained in vitro provide a suitable preparation to investigate bioelectrical properties of developing nuclear neurons. Particularly, stable intracellular recordings permit to explore the ionic conductances of these cells not only at mature stages, but also at very early postnatal stages.
Na conductances Fast Na-dependent spikes. In control Krebs solution, direct electrical stimulation of the nuclear neurons elicited the firing of a single or of a train of fast spikes, whatever the postnatal stage under study. Addition of TTX to the bathing medium abolished these fast spikes, thus indicating that they were sustained by Na currents and corresponded to the wellknown fast Na conductances commonly encountered in central nerve cells. Their amplitude did not significantly differ throughout postnatal development whereas their duration shortened in agreement with the evolution of such Na-dependent responses during development as already reported for these cells20 or for other nerve cells42,43. The longer duration of the fast spike in immature animals than in the adults could result either from different properties of the Na channels in young neurons as compared to more mature cells, or from an immature state of fast K conductances contributing to spike repolarization 23, or from both phenomena. The former possibility is enforced by the fact that in TEA plus Cd-containing medium, where only Na conductances are involved in the generation of the electrically induced response, the duration of the initial fast spikes at the weaning period was always shorter than that at very young stages (1.5-1.8 ms vs 3.0-5.0 ms), indicating that kinetics of activation and/or inactivation of Na conductances probably evolve during postnatal development. An argument in favor of an immature state of fast K conductances during the first postnatal week is the fact that block of K conductances by exposure to T E A did not so markedly prolonge the repolarizing phase of the fast Na spikes (Fig. 6A1, A2, A3) as it did later on during development (Fig. 3A1, A2, A4). Such an evolution of the K conductances could also explain the differ-
ences in the results obtained in the immature neurons as compared to older stages when Ba- or TEA-containing solutions replaced the superfusing control fluid.
Depolarizing afterpotentia!s and Na-dependent long-lasting depolarizations. When the control Krebs solution was replaced by a Ba or T E A plus Cd-containing medium, thus abolishing most K and Ca conductances, the fast Na spikes elicited by direct electrical stimulation of the cells were followed by depolarizing events at all the postnatal stages studied since PN0 (Figs. 3, 5, 6, 8). These responses were suppressed when TI~X was added to the bath thus indicating that they were mediated by Na currents. They appeared as depolarizing afterpotentials following the fast Na spike as early as birth (Figs. 5A4, 6A3) and also, since PN6, as long-lasting depolarizations which eventually sustained spike firing (Figs. 3A4, A5, 8A, B, C2, C3). Therefore it seems that a slowly inactivating Na conductance responsible for the occurrence of depolarizing afterpotentials comparable to the ones already described in other nerve cells under the same conditions15,16 can be unmasked in nuclear cells as early as birth by the block of K conductances. Later on, a more or less complete fusion of these depolarizing afterpotentials could give rise to long-lasting depolarizations as described before 10. However, one cannot preclude that the long-lasting depolarizations recorded in these neurons in spite of the absence of spike firing since PN6, could also result at least in part from a non-inactivating slow Na conductance similar to that observed in cerebellar Purkinje cells33 or in neocortical neurons 50.
Ca conductances In control Krebs solution and at all the postnatal stages studied, no slow rising spikes or slow Ca-dependent depolarizations were ever evoked by direct electrical stimulation of nuclear neurons, in marked contrast with Purkinje cellslO,13,33, hippocampal pyramidal cells16,45,53 or inferior olivary neurons 36. This indicates that under normal conditions, there is no evidence for a noticeable activation of Ca conductances in these cells. Furthermore, the absence of long-lasting afterhyperpolarizing potentials in these neurons, attributed in other classes of cells to a Ca-activated K conductance 24,26,33,36 is consistent with a weak activation of Ca conductances in control
104 Krebs solution. At birth, after exposure to T E A or to Ba, TTX-resistant slow rising spikes and prolonged plateaux of depolarization were recorded in nuclear neurons (Fig. 4A5). These responses were facilitated when larger depolarizing current pulses were passed through the recording microelectrode and were abolished by the addition of Ca channel blockers to the superfusing fluid, strongly suggesting that they were due to the activation of voltage-dependent conductances not activated under normal conditions. These Ca-dependent responses therefore appear well developed as early as birth and remain almost unchanged throughout postnatal development (Figs. 2B3, 7A3). Such Ca conductances, recorded under normal conditions or unmasked by T E A or Ba, appear to represent a general feature of bioelectrical membrane properties of nerve cells and have been described in many mammalian neurons as different as, for instance, cerebellar Purkinje cells 10,13,32,33,34, hippocampal pyramidal and granule cells 15,16,17,45,54, inferior olivary neurons 35,36, thalamic neurons 25,26,31, substantia nigra cells 30, cortical neurons 12, dorsal root ganglion neurons 27,37, or spinal dorsal horn neurons 40. However, in the case of intracerebellar nuclei neurons, whether these conductances are generated in dendrites such as for cerebellar Purkinje cells10,13,32,33,34, cortical neurons 12 or pars compacta cells 30, or at the somatic level as demonstrated for the low threshold Ca responses of olivary36 or thalamic 31 neurons, still remains to be determined. On the whole, it can be concluded that mature in-
REFERENCES 1 Adams, P., Voltage-dependent conductances of vertebrate neurones, Trends Neurosci., 5 (1982) 116-119. 2 Addison, W. H., The development of the Purkinje cells and of the cortical layers in the cerebellum of the albino rat, J. comp. Neurol., 21 (1911) 459-488. 3 Altman, J. and Bayer, S. A., Prenatal development of the cerebellar system in the rat. I. Cytogenesis and histogenesis of the deep nuclei and of the cortex of the cerebellum, J. comp. Neurol., 179 (1978) 23-48. 4 Baccaglini, P. I., Action potentials of embryonic dorsal root ganglion neurones in Xenopus tadpoles, J. Physiol. (Lond.), 283 (1978) 585-604. 5 Baccaglini, P. I. and Spitzer, N. C., Developmental changes in the inward current of the action potential of Rohon-Beard neurones, J. Physiol. (Lond.), 271 (1977) 93-117. 6 Bader, C. R., Bertrand, D., Dupin, E. and Kato, A. C.,
tracerebellar nuclei neurons express a complex set ionic conductances including fast and slowly inac vating Na conductances as well as Ca conductanc, However, Ca spikes are never observed in norn conditions, in marked contrast with cerebellar P1 kinje cells which do exhibit such responses10, 33. least two of these conductances, i.e. the fast Na a Ca conductances, are already well developed as ea: as birth, whereas the slowly inactivating Na condl tances mature progressively during the first postna week. Furthermore, our data suggest that the fast conductance contributing to spike repolarization not fully operating at birth and develops later on. T presence of both Na and Ca conductances since biJ in nuclear neurons is also in contrast with cerebel] Purkinje cells which express first Na and only later, Ca conductances 32. Consequently, if a similar dev opmental pattern of m e m b r a n e excitability exists intracerebellar nuclei neurons it must occur earlier the embryonic development. Indeed, one canr preclude this hypothesis since, for instance, both l and Ca currents are present at very early dev opmental stages in chick ciliary ganglion ce] whereas a developmental sequence of ionic condl tances, with Na currents appearing prior to Ca cl rents, is present in mesencephalic neural crest ce which are the precursors of ciliary ganglia 6. ACKNOWLEDGEMENTS This work was supported by the Minist~re de 1'] dustrie et de la Recherche (Grant No. 83C0910).
Development of electrical membrane properties in cultut avian neural crest, Nature (Lond.), 305 (1983) 808-810. 7 Bader, C. R., Bertrand, D. and Kato, A. C., Membra currents in a developing parasympathetic ganglion, Dev op. Biol., 98 (1983) 515-519. 8 Baker, P. F., Hodgkin, A. L. and Ridgway, E. B., Depo rization and calcium entry in squid giant axons, J. Physi (Lond.), 218 (1971) 709-755. 9 Blankenship, J. E., Action of tetrodotoxin on spinal mo neurons of the cat, J. Neurophysiol., 31 (1968) 186-194. 10 Crepel, F., Dupont, J. L. and Gardette, R., Selective sence of calcium spikes in Purkinje cells of staggerer mut~ mice in cerebellar slices maintained in vitro, J. Physi (Lond.), 346 (1984) 111-125. 11 Crill, W. E. and Schwindt, P. C., Active currents in ma malian central neurones, Trends Neurosci., 6 (19t 236-240. 12 Dichter, M. A., Lisak, J. and Biales, B., Action potenl mechanism of mammalian cortical neurons in cell cultu
105
Brain Res., 289 (1983) 99-107. 13 Dupont, J. L., Gardette, R. and Crepel, F., Bioelectrical properties of cerebellar Purkinje cells in reeler mutant mice, Brain Res., 274 (1983) 350-353. 14 Eckert, R. and Lux, H. D., A voltage-sensitive persistent calcium conductance in neuronal somata of Helix, J. Physiol. (Lond.), 254 (1976) 129-151. 15 Fournier, E. and Crepel, F., Electrophysiological properties of dentate granule cells in mouse hippocampal slices maintained in vitro, Brain Res., 311 (1984) 75-86. 16 Fournier, E. and Crepel, F., Electrophysiologicat properties of in vitro hippocampal pyramidal cells from normal and staggerer mutant mice, Brain Res., 311 (1984) 87-96. 17 Fricke, R. A. and Prince, D. A., Electrophysiology of dentate gyrus granule cells, J. Neurophysiol., 51 (1984) 195-209. 18 Gardette, R., Courtois, M. and Bisconte, J. C., Prenatal development of mouse central nervous structures: time of neuron origin and gradients of neuronal production. A radioautographic study, J. Hirnforsch., 23 (1982)415-431. 19 Gardette, R., Debono, M., Dupont, J. L. and Crepel, F., Development of synaptic responses and membrane conductances of intracerebellar nuclei neurones in rat cerebellar slices maintained in vitro, C.R. Acad. Sci. (Paris), 297 (1983) 43-46. 20 Gardette, R., Debono, M., Dupont, J. L. and Crepel, F., Electrophysiological studies on the postnatal development of intracerebellar nuclei neurons in rat cerebellar slices maintained in vitro. I. Postsynaptic potentials, Develop. Brain Res., 19 (1985) 47-55. 21 Goodman, C. S. and Spitzer, N. C., The development of electrical properties of identified neurones in grasshopper embryos, J. Physiol. (Lond.), 313 (1981) 385-403. 22 Hagiwara, S., Fukuda, J. and Eaton, D. C., Membrane currents carried by Ca, Sr, and Ba in barnacle muscle fiber during voltage clamp, J. gen. Physiol., 63 (1974) 564-578. 23 Hodgkin, A. L. and Huxley, A. F., A quantitative description of membrane current and its application to conduction and excitation in nerve, J. Physiol. (Lond.), 117 (1952) 500-544. 24 Hotson, J. R. and Prince, D. A., A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurons, J. Neurophysiol., 43 (1980) 409-419. 25 Jahnsen, H. and Llinas, R., Electrophysiological properties of guinea pig thalamic neurones: an in vitro study, J. Physiol. (Lond.), 349 (1984) 205-226. 26 Jahnsen, H. and Llinas, R., Ionic basis for the electroresponsiveness and oscillatory properties of guinea-pig thalamic neurones in vitro, J. Physiol. (Lond.), 349 (1984) 227-247. 27 Kameyama, M., Ionic currents in cultured dorsal root ganglion from adult guinea pigs, J. Membrane Biol., 72 (1983) 195-203. 28 Korneliussen, H. K., On the ontogenetic development of the cerebellum (nuclei, fissures and cortex) of the rat, with special reference to regional variations in corticogenesis, J. Hirnforsch., 10 (1968) 379-413. 29 Krnjevic, K., Pumain, R. and Renaud, L., Effects of Ba 2÷ and tetraethylammonium on cortical neurones, J. Physiol. (Lond.), 215 (1971) 223-245. 30 Llinas, R., Greenfield, S. A. and Jahnsen, H., Electrophysiology of pars compacta cells in the in vitro substantia nigra. A possible mechanism for dendritic release, Brain Res., 294 (1984) 127-132.
31 Llinas, R. and Jahnsen, H., Electrophysiology of mammalian thalamic neurones in vitro, Nature (Lond.), 297 (1982) 406-408. 32 Llinas, R. and Sugimori, M., Calcium conductances in Purkinje cell dendrites: their role in development and integration. In M. Cudnod, G. W. Kreutzberg and F. E. Bloom (Eds.), Development and Chemical Specificity of Neurons, Progress in Brain Research, Vol. 51, Elsevier, Amsterdam, 1979, pp. 323-334. 33 Llinas, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 171-195. 34 Llinas, R. and Sugimori, M., Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 197-213. 35 Llinas, R. and Yarom, Y., Electrophysiology of mammalian inferior olivary neurones in vitro. Different types of voltage-dependent ionic conductances, J. Physiol. (Lond.), 315 (1981) 549-567. 36 Llinas, R. and Yarom, Y., Properties and distribution of ionic conductances generating electroresponsiveness of mammalian inferior olivary neurones in vitro, J. Physiol. (Lond.), 315 (1981) 569-584. 37 Matsuda, Y., Yoshida, S. and Yonezawa, T., Tetrodotoxin sensitivity and Ca component of action potentials of mouse dorsal root ganglion cells cultured in vitro, Brain Res., 154 (1978) 69-82. 38 Miale, I. L. and Sidman, R. L., An autoradiographic analysis of histogenesis in the mouse cerebellum, Exp. Neurol., 4 (1961) 277-296. 39 Miyaka, M., The development of action potential mechanism in a mouse neuronal cell line in vitro, Brain Res., 143 (1978) 349-354. 40 Murase, K. and Randic, M., Electrophysiological properties of rat spinal dorsal horn neurones in vitro: calciumdependent action potentials, J. Physiol. (Lond.), 334 (1983) 141-153. 41 Peacock, J. H. and Walker, C. R., Development of calcium action potentials in mouse hippocampal cell cultures, Develop. Brain Res., 8 (1983) 39-52. 42 Purpura, D. P., Shofer, R. J. and Scarff, T., Properties of synaptic activities and spike potentials of neurons in immature neocortex, J. Neurophysiol., 28 (1965) 925-942. 43 Schwartzkroin, P. A., Development of rabbit hippocampus: physiology, Develop. Brain Res., 2 (1982) 469-486. 44 Schwartzkroin, P. A. and Prince, D. A., Effects of TEA on hippocampal neurons, Brain Res., 185 (1980) 169-181. 45 Schwartzkroin, P. A. and Slawsky, M., Probable calcium spikes in hippocampal neurons, Brain Res., 135 (1977) 157-161. 46 Schwindt, P. C. and Crill, W. E., Effects of barium on cat spinal motoneurons studied by voltage-clamp, J. Neurophysiol., 44 (1980) 827-846. 47 Schwindt, P. C. and Crill, W. E., Differential effects of TEA and cations on outward ionic currents of cat motoneurons, J. Neurophysiol., 46 (1981) 1-16. 48 Spitzer, N. C., The development of neuronal membrane properties in vivo and in culture. In Developing and Regenerating Vertebrate Nervous Systems, Liss, New York, 1983, pp. 41-59. 49 Spitzer, N. C. and Lamborghini, J. E., The development of the action potential mechanism of amphibian neurons isolated in culture, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 1641-1645.
106 50 Stafstrom, C. E., Schwindt, P. C. and Crill, W. E., Negative slope conductance due to a persistent subthreshold sodium current in cat neocortical neurons in vitro, Brain Res., 236 (1982) 221-226. 51 Strichartz, G., Small, R., Nicholson, C., Pfenninger, K. H. and Llinas, R., Ionic mechanisms for impulse propagation in growing nonmyelinated axons: saxitoxin binding and electrophysiology, Soc. Neurosci. Abstr., 6 (1980) 660. 52 Taber Pierce, E. and Sweet, E., Histogenesis of deep cere-
bellar nuclei as studied autoradiographically with thymidine 3H in the mouse, Anat. Rec., 157 (1967) 301. 53 Wong, R. K. S. and Prince, D. A., Participation of Ca 2÷ spikes during intrinsic burst firing in hippocampal neurons, Brain Res., 159 (1978) 385-390. 54 Wong, R. K. S., Prince, D. A. and Basbaum, A. t., tntradendritic recordings from hippocampal neurons, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 986-990.