Electrical activity in cerebellar cultures determines Purkinje cell dendritic growth patterns

Neuron, Vol. 7,891~902, December, 1991, Copyright 0 1991 by Cell Press

Electrical Activity in Cerebellar Cultures Determines Purkinje Cell Dendritic G rowth Patterns

Karl Schilling, Michael H. Dickinson,* John A. Connor, and James I. Morgan Department of Neurosciences Roche Institute of Molecular Biology Roche Research Center Nutley, New Jersey 07110

Summary

In primary dissociated cultures of mouse cerebellum a number of Purkinje cell-specific marker proteins and characteristic ionic currents appear at the appropriate developmental time. During the first week after plating, Purkinje cell dendrites elongate, but as electrical activity emerges the dendrites stop growing and branch. If endogenous electrical activity is inhibited by chronic tetrodotoxin or high magnesium treatment, dendrites continue to elongate, as if they were still immature. At the time that branching begins, intracellular calcium levels become sensitive to tetrodotoxin, suggesting that this cation may be involved in dendrite growth. Even apparently mature Purkinje cells alter their dendritic growth in response to changes in activity, suggesting long-term plasticity.

Introduction

During the development of the vertebrate nervous system there is generally a close temporal correlation between the morphological maturation of neurons and the emergence of their mature electrical properties. This raises several fundamental questions. First, do the two processes proceed independently of one another or are they coordinately regulated? Second, what are the mechanisms that orchestrate the ontog- eny of these properties of the neuronal phenotype?

Several studies have suggested that neurotransmitters may behave as cell-extrinsic signals during development (for reviews, see Black et al., 1984; Mattson, 1988; Kater and Mills, 1991). Therefore, while synapses are regarded as the unique characteristic of neurons that provides the means for information storage and transfer, they may function also in another context as a locus for the transduction of developmental signals. Indeed, the synaptotropic hypothesis posits that dendritic growth and branching are initiated and maintained by synapses formed on dendritic growth cones (Vaughn, 1989). This raises the question of the mechanism by which synapses regulate dendritic growth and also implies that electrical activity might be capa- ble of regulating some aspects of neurodevelopment.

For several technical reasons the rodent cerebellum is a particularly useful model for investigating the pro-

* Present address: Department of Organismal Biology and Anat- omy, University of Chicago, Chicago, Illinois 60637.

cesses that regulate neurodevelopment (Slemmon et al., 1984). Furthermore, two indirect lines of evidence support the idea that electrical activity can regulate neurodevelopment in this structure. First, the expression of several genes in Purkinje neurons is dependent upon synaptic input (Slemmon et al., 1988; Bala- ban et al., 1989). Second, mutations and treatments that affect afferent input to Purkinje cells result in dramatic alterations in the morphology of these neurons, particularly with respect to their dendritic branching (e.g., Berry and Bradley, 1976a; Privat and Drian, 1976; Mariani et al., 1977; for review, see ho, 1984). Indeed, alterations in Purkinje cell morphology have provided some of the experimental basis for the synaptotropic hypothesis of dendritic growth (re- viewed in Vaughn, 1989).

To pursue the question of the relationship between electrical activity and Purkinje cell morphology, a primary dissociated culture system is described and characterized. The emergence of electrical activity in cultured Purkinje neurons and the development of dendritic morphology are described both under normal growth conditions and following chronic synaptic/electrical blockade. In cultured Purkinjecells,dendritic morphology appears to be dictated by the consequences of the development of excitability.

Results

Murine cerebellar Purkinje neurons in primary dissociated culture have been characterized with regard to their developmental expression of specific marker molecules, electrophysiological properties, and morphology.

Developmental Expression of Purkinje Cell Markers Developing Purkinjecells in culture have been identified immunohistochemically by their expression of three marker proteins, calbindin 28K (Jande et al., 1981) , PEP-19 (Ziai et al., 1986, 1988; Mugnaini et al., 1987; Sangameswaran et al., 1989), and L7 (Oberdick et al., 1988), aswell as by expression of a fusion transgene comprising the L7 promoter and B-galactosidase (L7BGal)(Oberdicketal.,1990).At16days invitro(DIV) all four genes were expressed in cells having dense, relatively short (I-3 cell body diameters) perisomatic dendrites and what appears to be a single axon (Fig- ures IA-ID). Cell counts revealed that essentially the same number of cells were labeled by the three markers in cultures older than 8 DIV (Figure 2). At earlier times of culture both the number of cells expressing the markers and their morphology changed in a ste- reotypic manner. Within 24 hr of plating (embryonic day 16) Purkinje cells expressed calbindin 28K and PEP-19 but not L7 or L7BGal (Figure 2; Figure 3A). Pur- kinje cells at this stage were typically round or spindle shaped with a scant cytoplasm (Figure 3A). Within the

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Regulation of Purkinje Cell Dentritogenesis a93

Table 1. Dendrite Length of Developing Purkinje Cells Cultured in the Presence or Absence of TTX

Mean Dendrite Length bun) i 1 SD

DIV Control TTX Treated

1 24.4 f 14.6 3 51.2 f 15.0 4 46.4 f 17.1 5 58.4 * 37.3" 7 28.2 f a.9 9 20.7 f 7.8" 13 16.1 f 4.2 17 33.3 * 10.0 24 48.4 + a.2 26 51.4 f 13.5

ND ND ND

51.4 * 10.7a 25.4 + 6.4 16.4 * 5.4" 30.4 * 6.6 67.1 f 17.8"

142.9 f 33.7b 178.6 f 47.5b

Values were compared using the Tukey-Kramer multiple comparison procedure (Sokal and Rohlf, 1981). ND, not determined. a Statistically significant decrease in dendritic length in cells between 5 and 9 DIV, both in thecontrol and thelTX-treated group. b Statistically significant (p < 0.05) differences between controls and lTX-treated cells. While this conservative test suggests that the dendritic length of control (13 DIV) and TTX-treated (13 DIV) cells is not different, comparison of these conditions in isolation by Student’s t test shows TTX-treated dendrites to be significantly longer (p < 0.001).

1200 -

1000 -

800-

- PEP-19 - L7 - Calbindin

600-

400-

200-

0--, ’ I ’ I I t 0 4 8 12 16 20

Days in vitro Figure 2. Numbers of Cells Expressing PEP-19, Calbindin 2gK, or L7 after Various Times of Cultivation

Neurons immunoreactive for PEP-19 and calbindin 28K could be seen at 1 DIV, whereas L7-immunoreactive neurons could only be detected after 3 DIV. Numbers of L7-expressing neurons increased to reach levels of PEP-19-and calbindin ZBK-immunore active cells during the second week in vitro.

next5DIVthenumberofcellsexpressing bothcalbin- din 28Kand PEP-19 peaked and then declined by about 60% to a stable value by 8 DIV (Figure 2). The decline in calbindin- and PEP-19-positive cells is attributable to neuronal cell death that is not limited to Purkinje cells (Figure 2; data not shown). L7 was not expressed until 5 DIV, and subsequently the number of labeled cells rose to the same value observed with the other markers (Figure 2).

Between 1 and 5 DIV the Purkinje cells grew several smooth, tapering processes (Figures 3B-3D). One of the processes grew to a considerable length while remaining essentially unbranched except for terminal arborization. Based upon its morphology (Figures IA-IC; Figure 3D arrowheads) this long process is an axon. Morphometric analysis of the processes showed that they elongated rapidly between plating and 3 DIV (Table 1). Between 3 and 5 DIV elongation slowed. Between 5 and 13 DIV the neurites, except for

the single axon, became markedly shorter, thereby giving the Purkinje cell a polarity. These processes subsequently assumed the morphology of dendrites, By 7 DIV, the time at which neurites began to shorten, they began to branch, eventually to form a halo of dendrites whose density increased with time (Table 1; Figures 3F, 3H, 3J, 3L, and 3N). Linear growth of dendrites subsequent to 13 DIV was very slow when compared with the early period of neurite extension (Table 1). Finally, the volume of the soma increased substantially during the first 2 weeks in culture (Fig- ures 3A-3D, 3F, 3H, 3],3L, and 3N). While the developmental sequence described above applied to all cultured Purkinje neurons, the actual timing varied by *I day for individual cells. Such asynchronous development has been observed both for Purkinje cell development in vivoand in microexplantcultures(Heck- roth et al., 1990; Gruol and Franklin, 1987).

There are obvious differences between mature Pur-

Figure 1. lmmunocytochemical Characterization of Cerebellar Cultures (A-D) Purkinje cells grown for 16 DIV and immunostained for PEP-19 (A), calbindin 28K (B), and L7 (C). The Purkinje cell in (D) was obtained from a transgenic animal carrying a L7-/acZfusion gene and was identified histochemically by its expression of B-galactosidase. The presumed axon (see text) is marked by arrowheads in (A), (B), and (C). (E-G) 16 DIV cerebellar cultures immunostained for glial fibrillary acidic protein (E), neuron-specific enolase (F), and microtubule- associated protein 2 (C). Cultures contained a variety of morphologically distinct neurons. Arrows in (F) and (C) point out Purkinje neurons. (H-K) Control (H and I) and TTX-treated (J and K) Purkinje cells grown for 16 DIV and stained for PEP-19 (H and J) and the secretory vesicle marker protein ~38 (I and K). In (I), the cell body of the Purkinje cell is essentially unstained, while the dendrites are covered by immunostained presynaptic elements (see text). In TTX-treated cells (K), synapses were concentrated on the terminal branchings of the dendrites. There was also increased cytoplasmic immunostaining for p3B. (Land M) Calcium level in the soma and proximal dendrites of a Purkinje neuron (15 DIV) under control conditions (L) and after addition of 1 uM TTX (M). Cells were loaded with fura- (see Experimental Procedures), and 340/3BO nm ratio images were obtained in Krebs saline at room temperature. Addition of lTX resulted in an acute drop of cytoplasmic free calcium from about 140 nM to about 80 t-&i. Bar, 50 urn in (A)-(K); 20 urn in (L) and (M).

Figure 3. Morphological Development of Cultured Purkinje Cells and its Modulation by Chronic Treatment with TTX Cultures were maintained for 1 (A), 3 (B), 4 (C), 5 (D and E), 7 (F and G), 9 (H and I), 13 (J and K), 17 LL and M), or 26 (N-Q) days, and Purkinje neurons were visualized by staining for calbindin 28K. The cells shown in (E), (G), (I), (K), (M), and (Q) were treated with 1 uM TTX every other day, starting on the 2nd DIV. The cell shown in (0) was grown for 26 DIV and treated with TTX during the last 3 days of cultivation. Note that the dendrites are smoother and less branched than in controls (N), resulting in a much less dense dendritic tree. The arrow points to the axon that originates from a dendrite (arrowheads). This type of morphology is rather typical for Purkinje cells that have been exposed to TTX late during cultivation. (P) Cells grown in TTX from 2-17 DIV and then switched back to normal medium and grown for another 9 days (17-26 DIV). Note the comparatively thick dendrites covered with numerous protrusions in (P). Arrowheads in (D) indicate presumptive axon. Bars fin A and L), 50 pm.

Regulation of Purkinje Cell Dentritogenesis 895

L r

Nl?UVXl 8%

A B

C

D

I3

F

G + Ci iQX

+ I’TX -c--. -. ---.-

Figure 4. Basic Electrophysiological Properties of Cultured Mouse Purkinje Cells

(A) Sample records of free-running membrane voltage recorded in Purkinje neurons at three different developmental ages. Around day 7 the cells began exhibiting spontaneous activity in the form of action potentials and postsynaptic potentials. (B) Membrane currents recorded at the same three ages with soma voltage clamped at -80 mV. This blocked the generation of action potentials in the cell but left spontaneous postsynaptic currents that became prominent around day 7. (C) Membrane voltage records from Purkinje cell after 14 days in culture. The cells displayed spontaneous sodium spikes and longer plateau potentials, as well as an abundance of synaptic input. Typical resting potentials ranged from -50 to -65 mV. (D)Responseof Purkinjecelltoa50msiontophoreticapplication of glutamate or CABA. Glutamate induced a rapid transient increase in the firing rate, riding on a large depolarization. GABA inhibited the spontaneous firing rate of the Purkinje cells and caused a transient hyperpolarization. (E-GJBlockageof postsynapticcurrentsinculturedPurkinjecells (14 DIV) by the quisqualatelkainate glutamate receptor antagonist CNQX and the GABAA antagonist PTX. (E) Spontaneous synaptic currents in normal saline wrth cell soma clamped at -80 mV. (F) Postsynaptic currents 5 min after theaddition of 10 PM CNQX to the recording solution. Most of the spontaneous synaptic input was blocked. (C) Current records 5 min after the addition of 100 FM PTX and 10 PM CNQX. The residual spontaneous input in the presence of CNQX was blocked by PTX. The effects of both blockers were completely reversible with short periods of washing.

kinje cell morphology in vivo and that observed in older cultures. For example, cultured Purkinje cells typically possessed several main dendrites that were shorter than their in vivo counterparts. In addition,

the axon ot cultured Purkinje neurons occasionally originated from a dendrite (see arrow, Figure 30). The morphology of cultured Purkinjecells is most reminis- cent of normal Purkinje cells transplanted into adult pcd cerebellum (Sotelo and Alvarado-Mallart, 1987) and those Purkinje cells in the reeler mutant that develop ectopically (Mariani et al., 1977; Caviness and Rakic, 1976). In addition, the Purkinje neurons of ro- dents that are deficient in granule cells have atypical dendriticgeometry(e.g., Berry and Bradley, 1976a; Pri- vat and Drian, 1976). It is believed that the shape of the Purkinjecell dendriticarbor is partially determined by the anatomical arrangement of parallel and climbing fibers. Thus the loss, or disorientation, of these inputs is thought to be responsible for the aberrant Purkinje cell morphology (for review, see Ito, 1984). It is likely that a similar explanation accounts for the morphology of Purkinje cells in culture, which lack extracere- bellar afferents and normal lamination.

In addition to Purkinje cells, these cultures contained both glia, as revealed by glial fibrillary acidic- protein immunostaining (Figure IE), and other neurons,asshown bystainingfor neuron-specificenolase (Figure IF) and microtubule-associated protein 2 (Fig- ure IG). Besides Purkinje cells, which were only a minor population in these cultures, several classes ot neurons could be distinguished by their morphology after immunostaining for both neuron-specific enolase and microtubule-associated protein 2 (Figures IF and IG). We assume most of the abundant small neurons to be cerebellar granule cells. The neuron3 formed a dense neuritic network, and staining for the secretory marker protein p38 (Navone et al., 1986; Schilling and Gratzl, 1988) revealed the presence of numerous presynaptic elements (Figure II) (Schilling et al., 1989). Many of these presynaptic elements were concentrated on the dendritic field of the Purkinje cells (Figure II). This suggests that active neuronal networks were being established in culture.

Electrophysiological Characterization of Developing Purkinje Neurons The electrical activity and synaptic input of developing Purkinje cells in culture were characterized using perforated patch-clamp techniques (Horn and Marty, 1988). At 5 DIV Purkinje cells exhibited neither spontaneous activity nor synaptic currents (Figures 4A and 48). However, Purkinje cells were responsive to iontophoretically applied glutamate and y-aminobu- tyric acid (GABA), showing the presence of postsynaptic receptors, but lacked either presynaptic structures or ongoing electrical activity in presynaptic neurons (data not shown). In 7-day-old cultures, Purkinje cells showed occasional spiking and synaptic currents, with the frequency of both these events increasing over the next few days. Examples from g-day-old cultures are given in Figures 4A and 48. Again these cells were responsive to glutamate and GABA (data not shown). By 14 DIV, the Purkinje neurons were sponta- neously active, firing tetrodotoxin (-X)-sensitive ac-

Regulation of Purkinje Cell Dentritogenesis a97

A 10 mV

10 set

control c

control D

T-l-X --e-.., 25pA

--I 5sec

Mg2+

I- ,--

Figure 5. Effects of TTX and 12 mM Magnesium on the Spontane- ous Activity and Postsynaptic Currents of Purkinje Cells Grown for 14 DIV

(A) Application of 0.5 PM TTX completely abolished the spontaneous activity of Purkinje cells and resulted in a 5 mV hyperpolarization. (B) Saline containing 12 mM magnesium also eliminated the spontaneous firing rate of Purkinje cells and, after a lag of ap proximately 15 s, caused a minor tonic depolarization. This small depolarization was a typical feature of the response to elevated magnesium. (C) Blockage of spontaneous synaptic currents (left-hand record) by 0.5 PM TTX (right-hand record). Soma voltage clamped to -a0 mV. (D) Blockage of synaptic input by 12 mM magnesium saline.

tion potentials from a resting potential of typically-55 mV, often riding on longer plateaus (Figure 4C). In 14 DIV cultures, 50 ms pulses of glutamate, applied iontophoretically (see Experimental Procedures), typically caused l-2 s depolarizations accompanied by a transient increase in spike rate(Figure4D). In contrast, application of GABAcaused a transient hyperpolarization and decrease in spike rate (Figure 4D). Voltage- clamp recordings from 14 DIV Purkinje cells reveal a high level of spontaneous synaptic input (Figure 4E). The spontaneous synaptic currents were blocked by a cocktail of 10 uM 6-cyano-7-nitroquinoxaline-2,3- dione (CNQX) and 100 uM picrotoxin (PTX), specific antagonists of quisqualate and GABAA receptors, re- spectively (Figure 4G). The CNQX-sensitive, PTX-in- sensitive synaptic currents showed a reversal potential of approximately0 mV. The PTX-sensitive currents reversed at approximately -50 mV, similar to the reversal potential for exogenously applied GABA. The

proportion of CNQX-sensit ive currents, in terms of absolute rates of synaptic events, was typically higher than the PTX-sensitive component (Figure 4F). These results indicate that Purkinje cells become integrated into a functional neural network and after some 7-9 DIV receive both excitatory and inhibitory synaptic input.

The electrical characteristics of the cultured Pur- kinje cells reported here are qualitatively similar to those described for Purkinje cells in dissociated cultures from rat (Hockberger et al., 1989) and explant cultures (Cruel and Franklin, 1987).

Relationship between Electrical Activity and Morphological Development of Purkinje Cells The data demonstrate a temporal coincidence between the emergence of electrical activity in Purkinje neurons (7-9 DIV; Figures 4A and 4B) and the begin- ning of dendritic branching (7-9 DIV; Figures 3F and 3H). To determine whether there is a causal relationship between branching and electrical activity, sodium currents were blocked with TTX, and synaptic communication was impaired by supplementing the medium with high levels of magnesium.

Electrophysiological recording revealed that both 1 PM TTX and the addition of 12 m M magnesium had a similar effect of blocking spontaneous activity and/ or synaptic input in Purkinje neurons (Figures 5A-5D). Prior to 9 DIV there were no differences in either the length or morphology of dendrites of control and chronically T-TX-treated Purkinje cells (Figures 3D-31; Table 1). However, at and subsequent to 13 DIV, Pur- kinje cells grown in TTX showed a dramatically altered morphology (Figures 3)-3Q). At 13 DIV, T-TX-treated Purkinje cells typically had two to three primary dendrites with branches confined to their distal ends (Figure 3K), which can be contrasted with the halo of short, branched dendrites seen in the untreated culture (Figure 3)). In addition, although not quite significant by the Tukey-Kramer test, the dendrites had begun to elongate as judged by Student’s t test (Table 1). Subsequently the linear growth of dendrites in TTX became pronounced, and by 26 DIV the dendrites were 3-4 times longer than in control Purkinje cells (Table 1; Figures 3N and 3Q). In contrast to the dense halo of branched dendrites in control cells, TTX- treated Purkinje cells exhibited little dendritic branching except occasionally at their tips (Figures 3M and 34; Table 1). As judged at the light microscopic level, dendrites in T-TX-treated cultures were smooth, while those in control cultures possessed numerous protuberances that may represent spines. The appearance of these protuberances can best be seen when com- paring long-term T-TX-treated Purkinje cells with those that have had the agent removed for 9 days (Figures 3P and 34). The presumptive axon was not detectably affected by TTX treatment. Similar morphological changes occurred after culturing Purkinje cells with elevated magnesium (data not shown).

Since spontaneous activity is only observed in Pur-

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a a a

0 4 8 12 16 20 24 28

days in vitro

Figure 6. Summary of 11-10 Effects of Dlttvrc’nt mX Treatmvnt Schedules on Purkinje Cell Morphology

Purkinje neurons were grown for 5-26 DI\’ and treated with 1 PM TTX for the time periods indicated (shaded areas). Thry were then immunoztained for calbindin 28K or PEP-19, and thrlr morphologywas compared with that of controls grown for Iden- tical t ime periods. The resulting morphology is indicated at the end of the row (n, normal morphology, I.?.. not different tram controls; a, altered morphology).

kinje cells after 7 DIV (Figures 4A and 4B), TTX should not be able to affect cell morphology up to this time. Furthermore, dendrite development under normal growth conditions appeared to continue (Figures 3A- 3D, 3F, 3H, 31, 3L, and 3N), suggesting that the morphology of apparently differentiated Purkinje cells may be TTX sensitive and that the TTX effect might be reversible. These possibilities were assessed by ex- posing cerebellar cultures to 1 W M TTX for various periods of time (schematically summarized in Figure 6). As shown in Figures 3D-31, chronic treatment of cultures with TTX starting on the second DIV has no detectableeffect upon Purkinjecell morphology prior to 13 DIV. Furthermore, transient exposure to TTX duringearlytimes in culturedid not result in morphological changes at 16 DIV (Figure 6). However, a 3 day treatment of 23 DIV cultures with TTX was sutficient to induce morphological changes (Figure 30; Figure 6). While the dendritic tree of cells treated for only 3 days was not larger than that of controls, individual dendrites were less branched and had longer, smooth segments. Consequently, the dendritic tree ot TTX- treated cells was much less dense than that ot controls. Together, the above results suggest that there is a time during development at which Purkinje cell morphology becomes sensitive to TTX.

To test whether the morphological changes are per- manent, cultures were exposed to TTX up to 17 DIV and then either maintained in TTX or returned to control medium for a further 9 days (26 DIV). The presence of TTX produced a marked abnormality in dendrite morphology at 17 DIV (Figure 3M) and 26 DIV (Figure 3Q). The withdrawal of TTX for 9 days, between 17 and 26 DIV, resulted In a partial reversal of dendrite

morphology (Figure 3P). While the TTX-withdrawn cells still had longer dendrites than controls (Figure 3N), these were markedly shorter and thicker than those maintained in TTX. Moreover the dendrites of TTX-withdrawn cells were covered with numerous small protrusions (Figure 3P), while TTX-treated dendrites were smooth (Figure 3Q). Therefore, TTX withdrawal resulted in a cessation of linear growth of main dendrites and a resumption ot branching. Since branching (an resume in TTX-treated Purkinje cells b\ simply removing the agent, it also demonstrates that selective cell killing is not the mechanism by which TTX exerts it\ action on dendrite growth.

Influence of Electrical Activity on Purkinje Cell Biochemistry One issue raised by the data is whether the blockadtb of electrical activity alters the development of the Pur, kinje c-ell in terms of synapse formation and the maturation of its electrophysiological properties. Chronic exposure to TTX had no obvious qualitative effect on the expression of any of the marker proteins (data not shown). Furthermore, p38 immunoreactivity was still present over Purkinje cells, suggesting that synapse\ remained (Figure IK). This conclusion is supported b\, measurements of the electrophysiological properties of TTX-treated Purkinje cells. After the acute washout of TTX, both spontaneous firing and synaptic currents were qualitatively the same as in controls (data not shown).

One ot the primary consequences ot the tiring oi action potentials is the gating of calcium ions, whit-h is known to represent an important intracellular signaling event. Therefore, we investigated theontogen\, of cytosolic (al<-ium levels in Purkinje cells grown in normal medium or in the presence ot TTX or high magnesium These measurements were tarried out under two bet\ of conditions, as dictated by technical considerations. Stable electrical recordings required temperatures below 30°C with the pH of Krebs saline stabilized bv an organic buffer. Growth condition< (see Experimental Procedures) employed bicarbonate buffer in basal medium Eagle at 37OC and 10% CO The effects of TTX or high magnesium on talcium levels were qualitatively the same under both sets 01 conditions, but the absolute levels differed-the values measured under electrical recording condition5 being lower than those under growth conditions. In addition, variability between cells was much greater in growth medium. The physiological basis and signit- icance ot these differences in absolute values between the two recording conditions are unknown, but cannot be attributed to differences in the inorganic ion complement, since it is the same under both conditions. It is likely that the Krebs saline condition result< in the loss ot some aspects of calcium homeostasi\, although our data do not permit us to identity the actual mechanism.

Figure 7 illustrates the intracellular calcium data ob tained in kreb\ saline. At 5 DIV, Purkinje cells tested


300

I

300 Figure 7. Levels of Cytoplasmic Free Cal- A p

3w 300 C

T I

D cium as Measured with Fura- in Cultured

I Purkinje Cells

Cultures grown for 5 (A) or 14 (B-D) DIV were loaded with fura-2, and calcium levels were recorded either under resting conditions (PRE) or after addition of TTX (1 PM) or magnesium (Mg, 12 mM). The data in (C] were obtained from cells grown in the

0 i

presence of TTX. Values represent means 0 0 0 f 1 SEM (n = 16). Note that calcium levels

PRE TTX PRE TTX WP.SH TTX WASH TTX PRE f.q WASH in 5-day-old cultures do not change upon treatment with TTX. In 14 DlVcultures grown in normal medium, both TTX (B) and high magnesium (D) resulted in a decrease of cytosolic free calcium by at least 50 nM. These changes were reversible upon washout of the agents. Cultures grown chronically in TTX (C) had a resting cytosolic calcium level comparable to that seen in controls after acute treatment with TTX (compare leftmost column in [C] to middle column in LB]). Upon TTX washout, their cvtosolic calcium levels increased by some 120 nM; it fell again after reapplication of TTX.

in Krebs saline had a resting level of 85 f 3 nM (mean + SEM; n = 16) free calcium. This level was unaltered if the cells were acutely treated with TTX (Figure 7A). In growth medium, calcium levels were always greater than 250 nM with a very wide range observed in the population of neurons, but again lTX had no effect (data not shown). In 14 DIV cultures measured in Krebs saline, the mean calcium level was 142 + 11 nM (n = 16) (Figure IL; Figure 7B). Measured in growth medium the mean level was 393 & 40 nM (n = 14). Thus, under the recording conditions, there was an increase in free calcium from 85 nM at 5 DIV to 142 nM at 14 DIV, while in growth medium there was no clear increment in free calcium. However, under both measurement paradigms, TTX caused a reduction in freecalcium in 14 DIV Purkinjecells. Thus, acute treatment of 14 DIV cultures with TTX significantly reduced free calcium in a reversible manner, from 142 + 11 to 88 + 7 nM (n = 16) in Krebs saline (Figure IM; Figure 78) and from 393 k 40 to 256 k 28 nM (n = 14) in growth medium. To ensure that chronic exposure to TTX had the same effect as acute treatment, cells were maintained from 2 DIV to 14 DIV in TTX. These cells had basal calcium levels of 98 * 8 nM (n = 16) in Krebs saline, which rose to 219 + 32 nM (n = 16) following washout of the TTX (Figure 7C). Readdition of TTX reduced calcium levels back to prewashout values (Figure 7C). Acute treatment of 14 DIV cultures with 12 m M magnesium resulted in similar changes in free calcium to those observed following TTX (Figure 7D). Thus, mean free calcium in Purkinje cells is regulated by electrical activity in later development. Further- more, since calcium levels are the same in both 5 DIV and 14 DIV Purkinje cells when measured in growth medium and since calcium in 5 DIV neurons is unre- sponsive to TTX under both measuring paradigms, we conclude that determinants other than electrical activity contribute to calcium homeostasis in younger Purkinje cells. We do not understand these mechanisms at present.

Discussion

The electrophysiological, biochemical, and morpho-

logical properties of Purkinje cells have been characterized in primary dissociated cerebellar cultures. The development of specific marker proteins in Purkinje cells in these cultures closely mirrors the order and time course of their expression in vivo (Thomasset et al., 1982; Ziai et al., 1986; Sangameswaran et al., 1989; Smeyne et al., 1991), suggesting that the cells in culture execute a normal developmental program. Fur- thermore, electrophysiological analyses indicate that cultured Purkinje cells, as in vivo (Woodward et al., 1971), express GABA and glutamate receptors before they become integrated into a functional neuronal network. These properties suggest that cultured Pur- kinje neurons are comparable, in many respects, to their in vivo counterparts despite the lack of extracer- ebellar input and three-dimensional organization.

As Purkinjecells mature in culture, increasing spontaneous electrical activity appears between 7-9 DIV. During this transition period, Purkinje cell dendrites cease linear growth, retract, and begin to branch pro- fusely. This raises the possibility that electrical activity might control dendrite growth pattern. This notion is supported by the observation that blockade of electrical activity in older cultures by either TTX or high magnesium results in the continued elongation of Purkinje cell dendrites and a failure to branch. Coinci- dent with the time at which alterations of electrical and morphological properties occur, there are also changes in the characteristics of intracellular calcium regulation. Prior to 7 DIV, there is negligible electrical activity, and TTX is ineffective in lowering intracellular calcium levels. Subsequent to 7-9 DIV, the blockade of electrical activity by TTX (or magnesium) causes a decrease (between 40% and 70%) in intracellular calcium levels. From this we infer that electrical activity is responsible for a portion of ambient calcium levels in older Purkinje cells. This is not surprising given the large influxes of calcium that are associated with action potential firing and presynaptic input (e.g., Hockberger et al., 1989). One possible interpretation of these data is that dendrite elongation is the default growth pattern in Purkinje neurons and that this is overridden by activity-dependent alterations in calcium homeostasis. Such an interpretation isconsis-

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tent with the results of Holliday and Spitzer (1990) obtained in amphibian neurons. Further experimen- tation is required to determine the magnitude and cellular distribution of the calcium changes that contribute to dendrite growth.

The evidence supporting the synaptotropic hypothesis in cerebellum is based upon a correlation between dendritic branching and afferent input both during normal development (Berry and Bradley, 1976b) and where granule cells or climbing fiber inputs have been ablated (e.g., Berry and Bradley, 1976a; Privat and Drian, 1976). The in vitro Purkinje cell model described here provides a means of addressing the molecular and biochemical details of the relation- ships among synapse formation, activity, and branching. Our data indicate that for branching to occur, not only are synaptic contacts necessary but they must be functional, both in terms of neurotransmission and the generation of postsynaptic activity, as postulated by Bradley and Berry (1978). In addition, we propose that in the absenceof synaptic activity, main dendrites will elongate until they encounter active presynaptic elements. This would result in the cessation of elongation and initiation of branching. These conclusions are all consistent with the synaptotropic hypothesis and with the suggestion that Purkinje cell dendrites can grow in the absence of synaptic contacts (Privat and Drian, 1976).

A final issue raised by these results is the nature of the signals and molecules that maintain dendrite growth or branching. Branching might require the same molecules that are involved in linear dendrite growth except that they could be posttranslationally modified. In this context, calcium transients could be a local signal shaping growth pattern (see Mattson et al., 1988; Kater et al., 1988; Ho!liday and Spitzer, 1990; Kater and Mills, 1991) by calcium-dependent proteoly- sis or phosphorylation of critical proteins. Another possibility is that activity triggers the induction of genes encoding proteins involved specifically in branching of Purkinje cell dendrites. The latter possibility has been raised previously based upon the observation of calcium conductances in developing Pur- kinje cell dendrites (Llinas and Sugimori, 1979). It is now known that neuronal excitation and elevated intracellular calcium can elicit the induction of immediate early genes (Sheng and Greenberg, 1990; Curran et al., 1990; Morgan and Curran, 1991). Since many of these genes encode transcription factors, they could provide a mechanistic link between activity and gen- eral transcription. The culture system described here can be used to investigate the consequences of synaptic activity (or its absence) upon gene expression.

While activity dictates dendritogenesis, the emergence of several membrane conductances, as well as the expression of glutamate and GABA receptors, in Purkinjecells is independent of both activity and dendrite morphology. However, this does not preclude the possibilitythat activity might regulate more subtle aspects of development that pertain to Purkinje cell

physiology. Recently it has been shown that the culture system described here can be used to study long- term depression in Purkinje cells (Linden et al., 1991). Since long-term depression demands not only the presence of particular receptors and channels but also coupling to intracellular signaling systems, its as- sessment provides a much more powerful assay to delineate developmental effects of activity.

Experimental Procedures

Cell Culture Primary dissociated cultures were prepared trom the cerebellar anlageof l&day-old embryonic mice (day of insemination equals day 0; Harlan Sprague-Dawley, B6C3Fl). Cerebellar tissue was incubated at room temperature in 0.1% trypsin (Gibco) in PBS for 15 min. Two volumes of 10% fetal calf serum in PBS were added, and the tissue was triturated with a plastic pipette. The solution was then filtered through a 200 pm mesh and centrt- fuged (500 x g, 10 min, at room temperature). Cells were resus- pended in growth medium supplemented with 5% horse serum. They were grown at a density of 200,000 cells per cm2 in poly+ lysine-coated dishes. After 24 hr in vitro, cultures were switched to basal medium Eagle supplemented with 2 mM sodium pyr- uvate and hormones as described by Fischer (1982). Low calcium/ high magnesium medium was prepared by mixing 9 parts of basal medium Eagle containing 0.3 mM CaCI, with a 120 mM MgCI, solution. This resulted in isotonic medium containing 0.27 mM calcium and 12.7 mM magnesium.

lmmunostaining and Histochemistry for fi-Galactosidase Thefollowingantiserawereused(suppllerorreferenceanddllu- tion in parentheses): anti-calbindin 28K (Sigma, 1400); anti-glial fibrillary acidic protein (ICN, 1:lOOO); anti-L7 (Oberdick et al., 1988) (1:1000); anti-PEP-19 (Ziai et al., 1988) (1:2000); anti-p38 (Navone et al., 1986; Schilling et al., 1989) (1:2000); anti-neuron-specific enolase (NSE; Dako, 1:2000); and anti-microtubule-associated protein 2 (MAPZ; Boehringer Mannheim, 1:lOO). Antibody binding was visualized with the avidin-biotin-peroxidase method (Hsu et al., 1981) with reagents from Vector. 8-Ca- lactosidase activity was developed as described (Oberdick et al., 1990). Cells staining for calbindin 28K and PEP-19 were consrd- ered to be Purkinje cells. In cultures older than 8 days, expression of L7 was used as a further criterion to identify Purkinje neurons. Cells were viewed and photographed using a Leitz in- verted microscope fitted with 10x eyepieces and 4-25x objec- tives.

Morphometric and Statistical Analyses Measurements of dendrite length were made from photomicro- graphs (final magnification, 280x). These determinations were made on a minimum of ten neurons per well and at least three independent cultures per condition. Datawere compared using the Tukey-Kramer multiple comparison method. The choice of this conservative method was dictated by the fact that multiple groups had to be compared (Sokal and Rohlf, 1981).

Electrophysiology To limit washout of calcium conductances and endogenous biochemical pathways, current and voltage recordings of cultured Purkinje cells were obtained with the perforated patch-clamp technique (Horn and Marty, 1988). Extracellular solution, unless otherwise noted, contained 150 mM NaCI, 5 mM KCI, 1 mM CaCb, 0.8 mM MgCb, 10 mM HEPES, 10 mM glucose (pH 7.35). Pipette solutions contained 95 mM KzS04, 15 mM KCI, 0.8 mM MgCb, 10 mM HEPES (pH 7.35). A stock solution of 50 mg/mI nystatinldimethyl sulfoxide was added to the internal saline at a ratio of I:200 and sonicated for 30 s to yield a working pipette solution. Pipette tips were filled with normal internal saline and backfilled with the internal solution containing nystatin. Perma-


nent copies of both voltage and current records were obtained directlyon IineusingaCouldoscillographic recorder.Allexperi- ments were conducted at room temperature (2OOC).

The perforated patch recordings were made from cells 5-14 days in culture. In bright-field illumination, the Purkinje cells could be distinguished from the more numerous small granule cells by their size and from the large glial cells by a smoother appearance and higher profile. Phenotype was also confirmed by immunostaining. lontophoretic pipettes contained 10 mM solutions of either glutamate or CABA, delivered by negative pulses applied through a positive backing current.

Measurement of Intracellular Calcium Measurements of intracellular calcium were made by standard (340/380 nm) ratio imaging using the indicator dye fura- (Con- nor, 1986; Hockbergeretal., 1989). Thecerebellum cultures were loaded for 20 min at 37OC with the acetoxymethylester form of fura-2. A stock solution of 1 mM fura- acetoxymethylester in dimethyl sulfoxidewas sonicated into the tissueculture medium for a final concentration of 1 PM. Experiments were conducted either at room temperature, using the physiological salines described above, or in complete culture medium equilibrated with CO, at 37OC. Calibration of intracellular calcium concentration was calculated by previously described methods (Connor, 1986; Hockberger et al., 1989).

We wish to thank Dr. R. Jahn (Munich, Federal Republic of Cer- many) for the antibody to ~38 and Dr. J. Oberdick for the antibody to L7. We are also grateful to M. Smeyne for her expert help with cell cultures and calcium imaging. K. S. is supported by a research fellowship from the Deutsche Forschungsgemein- schaft (Schi 271/2-2).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemen? in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received June 6, 1991; revised August 1, 1991.

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Electrical activity in cerebellar cultures determines Purkinje cell dendritic growth patterns