Expression of voltage-dependent calcium channels in the embryonic rat midbrain

Developmental Brain Research 139 (2002) 189–197 www.elsevier.com / locate / devbrainres

Research report

Expression of voltage-dependent calcium channels in the embryonic rat midbrain Kathryn A. Whyte, Susan A. Greenfield* Department of Pharmacology, Mansfield Road, Oxford OX1 3 QT, UK Accepted 2 October 2002

Abstract The diversity of expression of high-voltage activated voltage-dependent calcium channels (VDCC) was investigated with whole-cell voltage-clamp recordings from dissociated embryonic rat ventral mesencephalic cells over a 7-day culture period. Cell phenotype was identified post-recording by fluorescent immunocytochemistry as tyrosine hydroxylase positive (TH1) or glutamic acid decarboxylase positive (GAD1). Both TH1 and GAD1 cells displayed high-threshold calcium (Ca 21 ) currents activated by depolarisations positive to 260 mV. In both cell types, pharmacological dissection using selective VDCC inhibitors, v-agatoxin IVA (Aga IVA), v-conotoxin GVIA (GVIA) and nifedipine demonstrated the existence of P/ Q-, N- and L-type VDCC, respectively. The remaining residual current could be blocked by cadmium. It was found that the contribution to the whole-cell current by the N-type channel was greater in TH1 cells than GAD1 cells at each time point examined, whilst the contribution to the whole-cell current by the L-type channel was greater in GAD1 cells than TH1 cells. However, over the 7-day culture period, the expression of VDCC types in both cell phenotypes changed in a similar fashion, with the contribution to the whole-cell current from the N-type current decreasing, and the contribution from the R-type current increasing. Our data could provide new insights into a range of neurodevelopmental mechanisms related to Ca 21 homeostasis in developing mesencephalic neurons.  2002 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Calcium channel structure, function and expression Keywords: Embryonic; Dopamine; Transplantation; Parkinson’s disease; Voltage-dependent calcium channel; Electrophysiology

1. Introduction Preparations of fetal midbrain neurons are used in transplantation as a treatment for Parkinson’s disease. The success of this procedure depends upon survival of, and innervation of the host striatum by embryonic midbrain dopaminergic neurons within the graft and hence ultimately upon the developmental mechanisms operating within these cells. Depolarisation-evoked Ca 21 entry through VDCC is fundamentally important to both neurite extension and survival of developing cells [7,8,23,28,29,32,38]. VDCC provide the primary route of Ca 21 entry into the *Corresponding author. Tel.: 144-1865-271-852. fax: 144-1865-271853. E-mail addresses: [email protected] (K.A. Whyte), [email protected] (S.A. Greenfield).

cell in response to depolarisation [2,25]. Five types of neuronal high-voltage-activated VDCC have been classified according to their electrophysiological and pharmacological properties; L, N, P, Q and R [14,39,43]. L-type VDCC are sensitive to dihydropyridine antagonists such as nifedipine [2,30]. N-type channels are specifically inhibited by GVIA [30,31]. P-type channels show marked sensitivity to Aga IVA with an IC 50 value of |1–10 nM in cerebellar purkinje neurons [26,27]. The Q-type channel is also inhibited by Aga IVA, but at higher concentrations than are observed for the P-type VDCC currents (IC 50 value of 90 nM in cerebellar granule neurons [30]). The R-type or ‘residual’ channel can be isolated following inhibition of the L-, N-, P-, and Q-type channels by nifedipine, GVIA and Aga IVA, respectively. The whole-cell Ca 21 current can be blocked by inorganic ions such as cadmium (Cd 21 ) or cobalt [12,20].

0165-3806 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 02 )00548-5

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Autoradiographic studies in adult animal brain slices have identified dihydropyridine-binding sites [10] and GVIA-binding sites [37] in the substantia nigra (SN), and immunohistochemical studies have localised N- and L-type channels to dopamine neurons of the SN [36]. Furthermore, two studies in dispersed postnatal d3–d10 rat ventral mesencephalon (VM) cells have used specific VDCC inhibitors to characterise voltage-dependent Ca 21 currents by electrophysiology [5,16]. However, the VDCC composition of embryonic midbrain neurons is not currently known. The VM is composed of several brain structures: the subthalamic nucleus, pedunculopontine nucleus, substantia nigra pars reticulata and substantia nigra pars compacta (SNpc) and the ventral tegmental area which, to varying extents, are incorporated into the preparations used for these studies. Incorporation of several brain regions thus gives rise to a heterogeneous cell population, composed for the large majority of cells that are immunoreactive for glutamic acid decarboxylase (GAD), the enzyme involved in the synthesis of g-aminobutyric acid (GABA). An antibody against GAD will therefore detect both glutamatergic and GABA-ergic cells derived from the subthalamic nucleus and substantia nigra pars reticulata [11,22,35,41]. A smaller percentage of the population will be dopaminergic cells, from the SNpc [6,24,34], and cholinergic cells (from the pedunculopontine nucleus and tegmental nucleus) may also be present. Whilst the presence of other cells types in the cultures is acknowledged, TH1 and GAD1 cells account for the large majority of cells within cultures, and all cells examined in this study stained positively for either TH or GAD. This study, therefore, examines the pattern of VDCC expression in TH1 and GAD1 cells within embryonic VM cultures over a 7-day culture period using selective VDCC antagonists Aga IVA, GVIA, nifedipine and cadmium to pharmacologically dissect voltage-dependent Ca 21 currents using whole-cell voltage-clamp electrophysiology.

2. Materials and methods

media in a 75-cm 2 flask with a vented lid pre-coated with poly-D-lysine (PDL) and incubated at 37 8C in a humidified atmosphere comprising 95% air:5% CO 2 for 7 days. Astrocyte flasks were then passaged. Media was removed, the cell layer was washed with Hank’s balanced salt solution (HBSS; Life Technologies) and digested with trypsin to encourage cell removal. The suspension was poured into 3 ml of media and centrifuged for 6 min at 1000 rpm (1673g). The supernatant was discarded and the pellet dissociated in 1 ml of media using a sterile glass pipette. The suspension was then placed into a fresh PDL-coated flask containing 14 ml of media, returned to the incubator and allowed to reach confluency (6–8 days). Astrocytes flasks were then trypsinised as described above and plated onto PDL-coated 13-mm coverslips in 30-mm diameter petri dishes, flooded with 2 ml of media, and incubated for 6 days to produce a confluent extracellular matrix before addition of the dissociated VM preparation.

2.2. Cell preparation and culture of ventral mesencephalon Embryonic VM cultures were prepared according to the method of Branton et al. [4]. Briefly, time-mated Wistar rats at gestational day 14 were anaesthetised using pentobarbitone (140 mg / kg) and decapitated. Embryos were removed and placed in sterile HBSS. The region of the VM was removed from each embryo and stored in HBSS containing 0.05% DNAse (HBSS / DNAse) on ice. Pooled explants were incubated in trypsin (0.1% trypsin in HBSS / DNAse) for 20 min at 37 8C. Explants were then rinsed with 0.1% soyabean trypsin inhibitor in HBSS / DNAse prior to rinsing three times in HBSS / DNAse. Explants were triturated gently in 1 ml of HBSS / DNAse. Viability and cell density were calculated using the trypan blue exclusion method. Cells were plated onto astrocyte-covered coverslips at a density of 2.5310 5 cells / cm 2 . Every 48 h, 0.75 ml of media was removed from each culture and was replaced with 0.75 ml of fresh media. Cultures were used for electrophysiology experiments from day 1 to day 7 in vitro.

2.1. Astrocyte preparation Type I astrocytes were prepared from the brains of decapitated 1-day-old rat pups of either sex. Cerebella were removed and cerebra were rolled on filter paper to remove meninges and chopped into |1-mm 3 pieces. Tissue was then dissociated in 1 ml of media consisting of Dulbecco’s modified eagle medium (4500 mg / l glucose, Life Technologies, UK) supplemented with 10% fetal calf serum (Life Technologies) and 1% penicillin–streptomycin (Sigma–Aldrich, Poole, UK) using a sterile glass pipette and centrifuged for 6 min at 1673g. The supernatant was discarded and the pellet dissociated again in 1 ml of media. One ml of the suspension was then added to 14 ml of

2.3. Electrophysiology Cultures were placed into the recording chamber and perfused with extracellular solution containing (in mM) TEACl, 143; BaCl 2 , 5; MgCl 2 , 1; Hepes, 10; glucose, 10, adjusted to pH 7.4 with TEAOH. The osmolarity was adjusted to 315 mOsm / kg with TEACl. Standard patchclamp recordings [13] (Axopatch 200A whole-cell patchclamp amplifier, Axon Instruments; CED 1401 Cambridge Electronics; Dell XPS PC using Strathclyde Electrophysiology Whole-Cell Program Software) were made using patch-clamp pipettes fabricated from borosilicate

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glass, 1.0-mm outer diameter, 0.58-mm internal diameter, with an internal capillary pulled on a horizontal puller (Sutter Instruments). Pipettes were filled with (in mM): CsCl, 135; MgCl 2 , 1; Hepes, 10; triphosphocreatinine, 14; MgATP, 3.6 and 50 ml / ml creatinine phosphokinase, adjusted to pH 7.1 with caesium hydroxide (CsOH), and 290 mOsm / kg with CsCl. 0.2% biocytin was then added. Pipettes showed resistances of 2–4 MV. Cells were voltage-clamped at a membrane potential of 260 mV. Series resistance and cell capacitance were determined by analysing instantaneous membrane responses to 5 mV, 30 ms voltage steps. Series resistances were 4–6 MV, and partial compensation (60–85%) was applied. For the analysis of toxin actions on voltage-activated Ca 21 currents, a standard protocol was adopted of applying 100 ms steps to a test potential of 0 mV every 30 s. Firstly, IC 50 values were determined for each antagonist. Secondly, experiments were carried out to investigate the VDCC composition of dissociated embryonic VM cells. VDCC antagonists were applied either consecutively to cumulatively inhibit calcium channel components or individually to inhibit a single calcium channel component. Consecutive application was used in order to inhibit separate current components, so that the remaining current that was blocked by cadmium could be attributed to the R-type (‘residual’) current. Single application of antagonists was used to verify that the data obtained for individual antagonists during consecutive application experiments was valid, and not due to rundown of the current as a result of the delay in applying the antagonists consecutively. Currents were filtered by a four-pole, low-pass Bessel filter and were stored on hard disk for subsequent analysis.

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Laboratories, USA) in PBS was applied for 120 min at RT. Monoclonal mouse-anti-TH antibody and polyclonal rabbit-anti-GAD antibody (Chemicon, UK; 1:1000 in 1% donkey serum in PBS) were then co-applied overnight at 4 8C (note, the antibody used against GAD was selective for neurons and did not label astrocytes). Cultures were washed with PBS for 3330 min and fluorescein (FITC)conjugated donkey-anti-mouse and indodicarbocyanine (Cy5)-conjugated donkey anti-rabbit secondary antibodies (Jackson Immunoresearch Laboratories, 1:100 dilution in 1% donkey serum in PBS) were applied overnight at 4 8C. Cultures were washed for 3330 min in PBS, dipped once in distilled water and inverted into Vectashield (Novocastra Laboratories, UK). After excess fluid had dried, coverslips were sealed with nail varnish and stored in the dark at 4 8C until visualised. Labelled cells were examined using a Zeiss Axioplan 1 microscope (Carl Zeiss, UK), with a Zeiss 02 DAPI filter set (Texas Red, 10; Cy5, 15; FITC, 25; Carl Zeiss). A Kodak Spot 2 CCD Camera and Kodak Image Capture software were used to record images.

2.6. Statistical analysis Statistical significance for each data group at each time point was assessed using the Kruskal–Wallis non-parametric test followed by a Dunnett’s multiple comparison test.

3. Results

3.1. Properties of VDCC currents 2.4. Drug application Drugs were delivered to the bath in extracellular solution via a three-way tap system. Complete exchange of the bath solution occurred in approximately 1.5 min. Drugs were applied until the current had stabilised (defined by three depolarisation steps eliciting currents within 2 pA of each other). Cadmium chloride and nifedipine were obtained from Sigma–Aldrich, Aga IVA was obtained from Calbiochem–Novabiochem (UK), and GVIA from Bachem (UK).

2.5. Post-recording cell identification Post-recording, cultures were fixed by placing into paraformaldehyde for 60 min at room temperature (RT). Cultures were then washed with phosphate-buffered saline (PBS) and incubated with streptavidin–Texas Red (Amersham–Pharmacia Biotech, UK; 1:100 overnight at 4 8C) to label the biocytin-fill. Cultures were washed for 3330 min in PBS and 20% donkey serum (Jackson Immunoresearch

The current–voltage (I–V ) relationship for all cells recorded from was examined on each day of recording (day 1 through to day 7 in vitro). For both cell types peak current increased with depolarisation step size, with maximal peak current (Imax ) reached at 0 mV, and a decrease in peak current observed with further depolarisation (Fig. 1A,B). Test potential required to elicit Imax did not vary with ontogenetic age in either cell type. The relationship between cell capacitance and size of current elicited from cells voltage-clamped at 260 mV and depolarised to 0 mV for 100 ms was examined at days 1, 3 and 7 in vitro. Both TH1 and GAD1 cell types demonstrated a positive linear correlation between cell capacitance and current size: TH1 cells (Fig. 1C) r 2 50.635; F5113.3; P,0.0001 (n567), GAD1 cells (Fig. 1D) r 2 50.234, F5179; P,0.0001 (n5111). The data for both cell types were also distributed according to the age in culture, with cells at day 1 in vitro generally having smaller currents and lower capacitance than those at day 3 in vitro. Similarly, cells of both phenotypes tended to have larger currents and greater capacitance at day 7 in vitro than at day 3 in vitro (Fig. 1C,D).

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Fig. 1. Properties of Ca 21 currents in embryonic VM cells. (A) Peak Ca 21 current from TH1 cells plotted against test potential (Vh 5260 mV) (n567). (B) Peak Ca 21 current from GAD1 cells plotted against test potential (Vh 5260 mV) (n5111). Peak current values have been normalised by maximal peak current (Imax ) for both cell types cell. (C) Development of Ca 21 current amplitude and cell capacitance at days 1, 3 and 7 in vitro in TH1 cells. r 2 50.635; F5113.3; P,0.0001 (n567). (D) Development of Ca 21 current amplitude and cell capacitance at days 1, 3 and 7 in vitro in GAD1 cells r 2 50.234, F5179; P,0.0001 (n5111). Lines shown in (C,D) are goodness of fit for entire data sets 695% confidence interval.

3.2. Pharmacologically defined components of the total Ca 21 current IC 50 values were determined for inhibition of specific VDCC components in embryonic VM cells by relevant inhibitors Aga IVA, GVIA and nifedipine (Fig. 2). Then, to determine the complement of VDCC in embryonic VM cells, we carried out a pharmacological dissection of the total Ca 21 current by consecutive addition of Aga IVA (200 nM), GVIA (1 mM), nifedipine (5 mM) and cadmium (20 mM) of TH1 cells at days 1, 3 and 7 in vitro (Table 1 and Figs. 3 and 4) and GAD1 cells at days 1, 3 and 7 in vitro (Table 2 and Figs. 5 and 6). Results demonstrated that VDCC currents in dissociated embryonic TH1 and GAD1 cells from the VM comprised at least five components, distinguished on the basis of their pharmacological properties, referred to as P/ Q- (Aga IVA-sensitive), N- (GVIA-sensitive), L- (nifedipine-sensitive), and R(‘residual’; blocked by cadmium) type currents. Both cell types possessed all five components by day 1 in vitro. However, the contribution to Imax by the component

current subtypes varied over the 7-day culture period (Tables 1 and 2 and Figs. 3 and 5).

4. Discussion

4.1. Current amplitude and cell capacitance of dissociated embryonic VM cells A linear relationship between cell capacitance and Imax was identified in both TH1 and GAD1 cells (Fig. 1C,D). Within these correlations it was also noticeable that current size and capacitance increased, in general, with time in culture for both phenotypes i.e., cells at day 1 in vitro generally had smaller currents and lower capacitance than those at day 3 in vitro. Similarly, cells tended to have larger currents and greater capacitance at day 7 in vitro than at day 3 in vitro. Capacitance can be used as a measure of the surface area of the cell. From observation of cells recorded from, it was apparent that the size of cultured cells increased over the 7-day culture period, so it

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Fig. 2. Dose–response relationships for inhibition of voltage-dependent calcium currents in dissociated embryonic VM cells by VDCC antagonists Aga IVA (A), GVIA (B) and nifedipine (C). In each case, data points represent mean6S.E.M. normalised to maximum inhibition obtained. n54–6. Continuous line represents the best fit of the data with the Hill equation where x5(11IC 50 / [antagonist] y)21 , where x, IC 50 , and y are normalized current, the concentration of antagonist for half-maximal block, and Hill factor, respectively.

is to be expected that capacitance would increase, as has been found to be the case with other developing cell types [3,17,42]. The linear relationship between Imax and capacitance seen in both TH1 and GAD1 cells is indicative that the increase in total current amplitude is proportional to the increase in cell surface area. As the amplitude of the Ca 21

current is proportional to the conductance of the ion through Ca 21 channels, Imax may serve as a measure of the number of VDCC in the cell being recorded from. Thus, the linear relationship between capacitance and Imax seen in the studies presented here may be indicative that the increase in cell surface area is proportional to the increase in the number of Ca 21 channels being expressed in the cell

Table 1 Percentage of Imax attributable to each VDCC component in TH1 cells at days 1, 3 and 7 in vitro Days in vitro 1 3 7

Calcium channel component (% of Imax 6S.E.M.) P/ Q

N

L

R

11.361.3 (9) 9.061.4 (9) 8.961.9 (9)

39.763.8 (10) 42.864.2 (12) 31.662.6 (11)

38.564.1 (14) 36.262.8 (15) 3763.9 (14)

10.561.5 (9)* 12.061.5 (9) 22.562.5 (9)*

Values expressed as % of Imax 6S.E.M. n Values are shown in parenthesis for each treatment at each time point. *Denotes P,0.05; Dunnett’s multiple comparison test for the R-type contribution at days 1 and 7.

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4.2. Voltage-dependent calcium current composition of dissociated embryonic VM cells

Fig. 3. Percentage of Imax attributable to each VDCC component in TH1 cells at days 1, 3 and 7 in vitro. Values expressed as percent of Imax 6S.E.M. For each n59. *Denotes P,0.05; Dunnett’s multiple comparison test for the R-type contribution at days 1 and 7.

membrane. This linearity indicates that the density of Ca 21 channels (the number of channels per unit area of membrane) remained constant over the culture period. An alternative possibility is that the increase in Imax was due to a change in the gating properties of VDCC, resulting in an increase in the Ca 21 conductance from a static number of channels.

The results presented here illustrate that TH1 and GAD1 cells from the VM of embryonic day 14 rat pups possess a full complement of high-voltage-activated VDCC by day 1 in culture. VDCC of dissociated embryonic VM cells have not been previously characterised by electrophysiology. Autoradiographic studies in adult animal brain slices have identified dihydropyridine-binding sites [10] and GVIA-binding sites [37] in the SN, and immunohistochemical studies have localised N- and L-type channels to dopamine neurons of the SN [36]. Furthermore, two studies in dispersed postnatal d3–d10 rat VM cells have used specific VDCC inhibitors to characterise voltage-dependent Ca 21 currents by electrophysiology. The results of these studies, by Cardozo and Bean [5] and Ishibashi et al. [16] revealed similar percentage contributions of current components to the total Ca 21 current; where P/ Q5|33%, N5|24%, L5|32% and R5|12%. These previous findings vary considerably from the findings of this current study where, most noticeably, the P/ Q-type channel contribution to the total Ca 21 current is much lower, both in TH1 and GAD1 cells. The contrast between the results presented here and the studies of

Fig. 4. Representative time course (i) and electrophysiological traces (ii) for whole-cell calcium current pharmacological characterisation of a single TH1 cell at day 1 in vitro. (iii) Post-recording identification of cell phenotype: (a) Identification of the cell recorded from by streptavidin–Texas Red labelling of biocytin. (b) Co-localisation with FITC anti-TH antibody.

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Table 2 Percentage of Imax attributable to each VDCC component in GAD1 cells at days 1, 3 and 7 in vitro Day in vitro 1 3 7

Calcium channel component (% Imax 6S.E.M.) P/ Q

N

L

R

6.160.8 (13) 4.860.7 (13) 4.960.8 (13)

32.562.4 (14)* 25.261.9 (16) 20.661.7 (15)*

47.463.9 (14) 52.962.0 (17) 50.561.3 (17)

14.061.5 (13)** 17.162.3 (13) 24.061.8 (13)**

Values expressed as % of Imax 6S.E.M. n Values are shown in parenthesis for each treatment at each time point. *Denotes P,0.05; Dunnett’s multiple comparison test for the N-type contribution at days 1 and 7. **Denotes P,0.01; Dunnett’s multiple comparison test for the R-type contribution at days 1 and 7.

Cardozo and Bean [5] and Ishibashi et al. [16] could be attributable to differences in experimental design; for example, variations in dissociation procedure, recording solutions, or stimulation protocol. However, a more fundamental factor may be the difference in ages of the cells being investigated; the studies of Cardozo and Bean [5] and Ishibashi et al. [16] used postnatal VM cells, whereas the studies presented here used embryonic VM cells. The results presented here show that the individual channel contributions vary over time, and although caution must be exercised when extrapolating to the in vivo situation, this is indicative that VDCC expression is developmentally regulated. This study illustrates (Tables 1 and 2 and Figs. 3 and 5) that the contribution by the N-type channel as a percentage of the maximal current decreases over the 7-day period, and the contribution from the R-type channel increases, for both cell types. The current contributed by a channel type is proportional to the conductance through that type of channel in the cell membrane. It is possible, as discussed above, that the number of each channel type remains constant over the 7-day culture period, but the conductance of each channel type increases, with the increases in conductance of the N- and R-type channel being disproportionate to the total increase in channel conductance.

Whilst changes in gating patterns of VDCC have been shown to occur under patch-clamp conditions in response to application of neurotransmitters [18,21], changes in VDCC gating patterns in developing VM cells have not been investigated. Thus, it is not possible to say whether this is the case in this study. Alternatively, the size of the current from a particular VDCC subtype could be considered to be a measure of the number of channels of that type in the cell being recorded from. If this view is adopted, the total density of Ca 21 channels appears to remain constant throughout the culture period. The proportional increases in P/ Q- and L-type channel currents are indicative that the density of these channel types also remains constant, whilst the density of N-type channels per cell appears to decrease over the 7-day period, with the density of R-type channels increasing. Changes in N- and R-type channel density could be accounted for by the formation, or lack of formation, of new channels, the formation of new subunits that alter stoichiometry of channel configuration, or, in the case of the increased density of R-type VDCC, the recruitment of previously silent channels.

4.3. Significance of changes in VDCC expression during development

Fig. 5. Percentage of Imax attributable to each VDCC component in GAD1 cells at days 1, 3 and 7 in vitro. Values expressed as percent of Imax 6S.E.M. For each n513. *Denotes P,0.05; Dunnett’s multiple comparison test for the N-type contribution at days 1 and 7. **Denotes P,0.01; Dunnett’s multiple comparison test for the R-type contribution at days 1 and 7.

Changes in VDCC expression in developing cells have been a subject of some debate, since the correlation between developmental process and VDCC subtype expression varies widely in different cell types [1,9,15,19,33,40]. Developmental milestones such as cell differentiation, migration and synaptogenesis are mediated by the activation of secondary messenger enzymes as a result of Ca 21 entry into the developing cell. As such, initiation of these milestones will require mechanisms for regulated Ca 21 influx, for example via VDCC. It is possible that the variation in the correlation between channel subtype expression and developmental process seen in different cell phenotypes depends upon the conductance of the channel subtype in that particular cell phenotype as oppose to the channel subtype per se. The constituent cells of cultures used in the studies presented here are taken from such anatomically different structures as the STN and the SNpc. This study demonstrates that the contribution to the whole-cell current by

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Fig. 6. Representative time course (i) and electrophysiological traces (ii) for whole-cell calcium current pharmacological characterisation in a single GAD1 cell at day 1 in vitro. (iii) Post-recording identification of cell phenotype: (a) Identification of the cell recorded from by streptavidin–Texas Red labelling of biocytin. (b) Co-localisation with Cy5 anti-GAD antibody; arrowheads indicate other GAD1 cells in culture that were not recorded from.

any VDCC component at any of the days examined is different in TH1 and GAD1 cells. The most notable difference is that the contribution to the whole-cell current by the N-type channel is higher, and the L-type channel lower, in TH1 cells than in GAD1 cells at each day examined. This difference may be a reflection of the different functions of the areas that these cells are derived from within the mesencephalon, with particular functions requiring specific Ca 21 levels during development. However, these data also demonstrate a similarity in the pattern of expression of VDCC subtypes in TH1 and GAD1 cells over the 7-day period, wherein P/ Q- and L-type channel contributions to the whole-cell current are constant, the N-type contribution decreases and the R-type contribution increases. It is interesting that the contribution of individual VDCC subtypes to the total Ca 21 current in GAD1 and TH1 cells from within these cultures varies, whilst the pattern of VDCC expression is so similar. Perhaps midbrain TH1 and GAD1 cells have different Ca 21 requirements for certain developmental processes and therefore individual subtype contribution to the total Ca 21 current may vary. However, N- and R-type channels may be fundamentally involved in these processes in both cell types, hence the pattern of expression of these channels is similar. In conclusion, this study presents data that illustrate that

cultured dissociated embryonic GAD1 and TH1 cells of the VM possess a full complement of VDCC within 24 h of culturing. Furthermore, the pattern of expression of VDCC subtypes varies over the 7-day culture period. These results provide the first insight, to our knowledge, into the expression of VDCC in cultured embryonic VM cells, and hence may provide a basis for developing techniques and treatments crucial to the survival and outgrowth of midbrain dopaminergic cells in preparations used in fetal transplantation models of Parkinson’s disease. Acknowledgements This work was funded by Synaptica Ltd. We would like to thank Dr. S.J. Cragg for her helpful comments throughout this project and Nick White within the William Dunn School of Pathology, Oxford for the use of the fluorescent microscope. References [1] S. Arnhold, C. Andressen, D.N. Angelov, R. Vajna, S.G. Volsen, J. Hescheler, K. Addicks, Embryonic stem-cell derived neurones express a maturation-dependent pattern of voltage-gated calcium channels and calcium binding proteins, Int. J. Dev. Neurosci. 18 (2000) 201–212.

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