Connexin mRNA expression in single dopaminergic neurons of substantia nigra pars compacta

Neuroscience Research 56 (2006) 419–426 www.elsevier.com/locate/neures

Connexin mRNA expression in single dopaminergic neurons of substantia nigra pars compacta Marie Vandecasteele a, Jacques Glowinski b, Laurent Venance a,* a

Laboratoire de Dynamique et Physiopathologie des Re´seaux Neuronaux, Inserm, U667, Colle`ge de France, Univ Pierre et Marie Curie, 11 place Marcelin Berthelot, 75005 Paris, France b Laboratoire de Neurobiologie Pharmacologique, Inserm U114, Colle`ge de France, Paris, France Received 25 July 2006; accepted 22 August 2006 Available online 2 October 2006

Abstract Dopaminergic neurons of the substantia nigra pars compacta play a major role in goal-directed behavior and reinforcement learning. The study of their local interactions has revealed that they are connected by electrical synapses. Connexins, the molecular substrate of electrical synapses, constitute a multigenic family of 20 proteins in rodents. The permeability and regulation properties of electrical synapses depend on their connexin composition. Therefore, the knowledge of the molecular composition of electrical synapses is fundamental to the understanding of their specific functions. We have investigated the connexin mRNA expression pattern of dopaminergic neurons by single-cell RT-PCR analysis, during two periods in which dopaminergic neurons are electrically coupled in vitro (P7–P10 and P17–P21). Our results show that dopaminergic neurons express mRNAs of various connexins (Cx26, Cx30, Cx31.1, Cx32, Cx36 and Cx43) in a developmentally regulated manner. Furthermore, we have observed that dopaminergic neurons display different connexin expression patterns, and that multiple connexins can be expressed in a single dopaminergic neuron. These observations underline the importance of electrical coupling in the development of dopaminergic neurons and raise the question of the existence of functionally distinct electrically coupled networks in the substantia nigra pars compacta. # 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Dopaminergic neuron; Connexin; Single-cell RT-PCR; Electrical synapse; Substantia nigra pars compacta; Gap junction

1. Introduction The basal ganglia are a highly interconnected network of subcortical nuclei involved in adaptative control of behavior, in which the substantia nigra pars compacta (SNc) constitutes the main modulatory nucleus (Graybiel, 1990; Gerfen, 1992). Dopaminergic (DA) neurons composing SNc mainly project to the dorsal striatum, a major input area of basal ganglia which selects relevant cortical information (Wilson, 1995). Dopamine potently modulates the processing of corticostriatal information (Reynolds and Wickens, 2002; Guzman et al., 2003; Bamford et al., 2004a,b). DA neurons activity conveys the motivational component of goal-directed behavior (Schultz, 2004). The consequence of the degeneration of nigro-striatal DA cells, leading to Parkinson’s disease (Obeso et al., 2000), highlights their crucial importance in cognitivo-motor control.

* Corresponding author. Tel.: +33 1 44 27 12 26; fax: +33 1 44 27 12 60. E-mail address: [email protected] (L. Venance).

An important issue concerning SNc DA neurons has long been their local interactions (chemical and electrical transmissions). Concerning chemical transmission it has been reported that dopamine released from dendrites regulates DA cell activity through DA autoreceptors (Groves et al., 1975; Skirboll et al., 1979; Cheramy et al., 1981). Concerning electrical synapses, a pioneering study showed a dye-coupling between SNc DA neurons in vivo in adult rats (Grace and Bunney, 1983b), supporting the existence of an intercellular communication. We have recently shown that SNc DA neurons are connected by functional electrical synapses (Vandecasteele et al., 2005). These electrical synapses were found to be bidirectional, voltage-independent, and displayed strong lowpass filter properties. Such electrical coupling efficiently modulates the spontaneous spiking activity of DA neurons. Electrical synapses refer to connections between two neurons through gap junction channels. These intercellular channels are formed by the apposition of two hemichannels (connexons) each of them being composed by six proteic subunits (connexins, Cxs). In mammals, Cxs constitute a

0168-0102/$ – see front matter # 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2006.08.013

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multigenic family of 20 members (Sohl et al., 2005; Oyamada et al., 2005). The nature of the Cxs composing the gap junction channels determines their permeability and regulation properties (Bruzzone et al., 1996; Harris, 2001). In the central nervous system, electrical synapses are mainly documented within networks of GABAergic interneurons (Galarreta and Hestrin, 2001; Bennett and Zukin, 2004). These electrically coupled networks have been shown to rely on the expression of Cx36 (Bennett and Zukin, 2004; Connors and Long, 2004; Hormuzdi et al., 2004; Sohl et al., 2005). The knowledge of the molecular composition of these electrical synapses has permitted, using animals in which the Cx36 gene had been invalidated, to unveil the crucial role of electrical synapses in the oscillation and synchronization processes in these neuronal networks (for review see Bennett and Zukin, 2004). Concerning DA neurons, the situation appears more complex. Indeed, recent studies report an absence of Cx36 mRNA, the ‘‘neuronal’’ Cx (Lin et al., 2003) and the expression of Cx26, Cx32, Cx43 and Cx45 in the rat midbrain floor (Leung et al., 2002). Following our demonstration of functional electrical synapses between SNc DA neurons (Vandecasteele et al., 2005), we have here analyzed their patterns of Cx expression by single-cell RT-PCR. Several Cx mRNAs were detected (Cx26, Cx30, Cx31.1, Cx32, Cx36 and Cx43) and found to be express in a developmentally regulated manner. Among DA neurons, different Cx expression patterns were observed, suggesting the existence of functionally distinct electrically coupled networks. 2. Materials and methods 2.1. In vitro slice preparation Coronal brain slices were prepared from Sprague–Dawley rats of both sexes (postnatal days 7–21). Animals were killed by decapitation, in accordance with local Ethical Committee and EU guidelines (directive 86/609/EEC). Brain slices (330 mm thick) were cut using a vibrating microslicer (Leica VT1000S, Nussloch, Germany). Slices were subsequently incubated 45 min at 32 8C, in an extracellular solution containing (mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2 and 1 pyruvic acid (sodium salt) bubbled with 95% O2 and 5% CO2 before electrophysiological recordings.

2.2. Electrophysiological recordings Whole-cell recordings were made using borosilicate glass pipettes of 2– 5 MV resistance containing (mM), for electrical coupling experiments: 105 Kgluconate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 ATP-Mg, 0.3 GTP-tris, 0.3 EGTA, and for single-cell RT-PCR experiments: 140 KCl, 5 HEPES, 5 EGTA, 3 MgCl2 (both adjusted to pH 7.35 with KOH). The composition of the extracellular solution was (mM): 125 NaCl, 25 glucose, 25 NaHCO3, 2.5 KCl, 2 CaCl2, 1.25 NaH2PO4, 1 MgCl2, 10 mM pyruvic acid (sodium salt) bubbled with 95% O2 and 5% CO2. All whole-cell recordings were performed at 34 8C using a temperature control system (Bioptechs DTC3, Butler, PA, USA). Signals were amplified using an EPC9-2 amplifier (HEKA Elektronik, Lambrecht, Germany). Current-clamp recordings were filtered at 2.5 kHz and sampled at 5 kHz using the program Pulse-8.50 (HEKA Elektronik). Series resistance compensation was set to 75–90% in whole-cell configuration.

2.3. Data analysis Off-line analysis was performed using PulseFit-8.50 (HEKA Elektronik) and Igor Pro (Wavemetrics, Lake Oswego, OR, USA). All results were

expressed as mean  standard error of mean. Action potential threshold was measured as follows: DA neurons were first clamped to 60 mV and successive depolarization steps (10 pA steps) were applied until the first AP, on which the threshold was then measured. Input resistance was calculated from voltage responses obtained after injecting a hyperpolarizing current (10 pA; 1 s duration). Sag amplitude was measured from voltage responses obtained after injecting a hyperpolarizing current (90 pA; 1 s duration, cell being previously held at 60 mV), between the potential at sag peak (* in Fig. 1) and the potential at steady state (* in Fig. 1). Spike duration was measured between the onset of the spike and the equipotential point during the repolarization phase. Fast after-hyperpolarization amplitude was taken between this last point and the minimum of the after-hyperpolarization.

2.4. Single-cell RT-PCR Single-cell RT-PCR was performed as previously described (Lambolez et al., 1992; Mackler and Eberwine, 1993; Monyer and Lambolez, 1995; Monyer and Jonas, 1995; Venance et al., 2000, 2004; Venance, 2001). Sterilized patch-clamp pipettes (2–3 MV) used for RT-PCR experiments were filled with autoclaved internal solution. Electrical coupling analysis could not be carried out together with single-cell RT-PCR experiments since ATP, GTP and phosphocreatine could not be used due to the required autoclave treatment. After electrophysiological identification, the cytoplasm was aspirated by gentle suction into the patch pipette under visual and electrophysiological controls. Membrane potential, series resistance and input resistance were monitored to control exactly the perfect quality of the seal, and avoid the harvesting of extracellular contaminants. Any significant variation in these parameters led to an interruption of the harvesting of the cytoplasm and rejection of the experiment. The harvested material was subsequently expelled into an autoclaved tube containing poly(dT) primer (2.5 mM final) (Roche, Meylan, France), desoxyribonucleosides triphosphate (250 mM final each) (Invitrogen, Eragny, France), dithiothreitol (5 mM final) (Tebu-bio, Le Perray, France), ribonuclease inhibitor RNAsin (2 Units) (Promega, Charbonnie`res, France) and a mix of Avian Myeloblastosis Virus (0.2 Units final) (Ozymes, St-Quentin, France) and Moloney Murine Leukemia Virus (50 Units final) (Invitrogen) reverse transcriptases and incubated 1 h at 37 8C. Two rounds of PCR amplification were performed, with 4 ml of the first PCR as a template for the second PCR. PCR conditions were the same for both amplification rounds: a hot start at 94 8C for 5 min followed by 30 cycles (94 8C for 30 s; 53 8C for 30 s; 72 8C for 40 s) and a final elongation step at 72 8C for 10 min. PCR 1 and 2 mixes contained 150 mM of each desoxyribonucleosides triphosphate (Invitrogen), 0.2 mM of each primer (Genset Oligos, Paris, France), 2.5 Unit of Taq polymerase (Invitrogen) and various MgCl2 concentrations (see Table 1). The first PCR was multiplex (i.e. amplifying simultaneously different Cxs) and the second one was specific for each Cx, using one nested primer. The 13 Cxs which expression was analyzed were splitted into two multiplex PCR groups (group I being: Cx26, Cx31.1, Cx32, Cx36, Cx43, Cx47 and group II being: Cx30, Cx31, Cx37, Cx40, Cx45, Cx50, Cx57) (Table 1), according to compatibility between primers inside each group and corresponding PCR conditions (PCR1 MgCl2 concentration and primer annealing temperature were optimized on total rat brain cDNA). Primer pairs (Table 1) were chosen to span over the first intron (except for Cx50 and Cx57), to discriminate and exclude from analysis the amplification of incidentally harvested genomic material. Molecular identity of the PCR amplicons was systematically confirmed by direct sequencing (Genset, Montreuil, France). As previously described, in each experiment, contamination artifacts were excluded for both contamination of the PCR and inadvertent harvesting from surrounding material in the slice preparation (controls and special cares are detailed in Monyer and Jonas, 1995; Venance, 2001). A lack of detection of a Cx does not necessarily mean its absence of expression. Indeed, ‘false negatives’ could be due to the very low expression level of neuronal Cx mRNAs (Bruzzone et al., 1996; Rouach et al., 2002; Bennett and Zukin, 2004), the insufficient amounts of harvested material, and/or the loss of the harvested material during the expelling procedure.

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2.5. Dye-loading and immunochemistry Coronal slices (250 mm) were incubated in HEPES based medium (in mM: 140 NaCl, 25 glucose, 10 HEPES, 5 carbohydrazide, 5.5 KCl, 1.8 CaCl2, and 1 MgCl2, adjusted to pH 7.35 with NaOH) for 10 min. After 5 min treatment in low Ca2+ medium (140 NaCl, 25 glucose, 10 HEPES, 5.5 KCl, 5 carbohydrazide, 10 mM CaCl2, 10 mM MgCl2, pH 7.35), slices were incubated for either 2 or 15 additional minutes in low Ca2+ medium containing Lucifer yellow carbohydrazide lithium salt at 1 mg/ml (Sigma). After washing in normal Ca2+ medium, slices were then fixed by overnight incubation in 4%

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PFA. Carbohydrazide (Sigma) was added to prevent non-specific staining of Lucifer yellow carbohydrazide. All experiments were performed at 34 8C. Tyrosine hydroxylase immunostaining was performed by incubation of the dye-loaded slices in a 1/500 diluted rabbit anti-(tyrosine hydroxylase) polyclonal antibody (Chemicon, Temecula, CA, USA) overnight at 4 8C. Goat anti-rabbit secondary antibody, coupled to Cy3 (Amersham Biosciences, Saclay, France) was incubated at dilution 1/500, 2 h at room temperature. Brain slices were visualized using a confocal laser-scanning microscope (Leica TBCS SP2, Wetzlar, Germany).

Fig. 1. Electrophysiological characterization of DA neurons and electrical coupling between SNc DA neurons. (A) Identification of SNc DA neurons in coronal rat brain slices by positive immunoreactivity to tyrosine hydroxylase. (B) Infra-red microphotography of an electrophysiologically identified DA neuron, scale bar: 10 mm. (C) Responses of a DA neuron to current injections (90, 70, 50, 30, 10, +10, +30 pA, left panel, +80 pA, middle panel, holding potential was 60 mV). Right panel: sag peak (*) and steady state (*) I–V relationship (mean of 10 DA neurons). (D) Example of an electrical coupling by simultaneous patchclamp recordings of a SNc DA neuron pair (V1 and I1 referring to cell 1 and V2 and I2 referring to cell 2). Junctional currents (V2, average of 25 and 28 sweeps) were observed after injection of depolarizing (+100 pA) or hyperpolarizing (200 pA) currents in cell 1. The coupling coefficient in this pair (ratio of the membrane potential variations in the two cells) was 4.4  0.5%. Note the difference of scaling between V1 and V2 and the strong low-pass filtering properties illustrated by the non-transmission of the presynaptic spike.

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Table 1 Connexin primer position and sequences used for single-cell RT-PCR, and MgCl2 concentrations used in PCR amplification, listed accordingly to multiplex PCR groups I and II Cxs

[MgCl2] (mM) PCR1/PCR2

Primer location

Primer sequence

Group I Cx26

1.5/1.5

50 30 30 nested

50 -CGGACCTGCTCCTTACAGG-30 50 -CATGATCAGCTGCAGAGCC-30 50 -CGTAGCACACATTCTTACAGCC-30

Cx31.1

1.5/1.5

50 30 30 nested

50 -TCTGATGCTTGCTGAACCC-30 50 -AAGTCCTTCTGGTCGTCACC-30 50 -GCACACGGAAGACAAAGACC-30

Cx32

1.5/1

50 30 30 nested

50 -CACAGACATGAGACCATAGG-30 50 -AACCAAGATGAGTTGCAGG-30 50 -TAGCAGACGCTGTTACAGCC-30

Cx36

1.5/1.5

50 30 30 nested

50 -AATGGACCATCTTGGAGAGG-30 50 -GATCTGGAAGACCCAGTAACG-30 50 -CTGCTCATCATCGTACACCG-30

Cx43

1.5/1

50 30 30 nested

50 -TCCTTTGACTTCAGCCTCC-30 50 -GACTGTTCATCACCCCAAGC-30 50 -TATGAAGAGCACTGACAGCC-30

Cx47

1.5/1.5

50 30 30 nested

50 -TCCCATGACCAACATGAGC-30 50 -AGCAGACGTTGTCACAACCC-30 50 -GAATAGATGGACTCACCACCG-30

1/1.5

50 30 50 nested

50 -TTCCAGTTCACCTCACACGG-30 50 -ACCACGAGGATCATGACTCG-30 50 -TGACTGCCAGAGGAGTAGAAGG-30

Cx31

1/1

50 30 30 nested

50 -GATGCCTCCTTAATGAGTAGGG-30 50 -TGGAGTACTGGTTCACACCG-30 50 -CCTGAAGCTTCTTCCAATCC-30

Cx37

1/1

50 30 50 nested

50 -TAGGAGGAGCTGAGAAAGGC-30 50 -GAAATCAGACTGCTCGTCGC-30 50 -TGAGAAAGGCATTGTGCCC-30

Cx40

1/1.5

50 30 30 nested

50 -CTGGACAGTTGAACAGCAGC-30 50 -TATCACACCGGAAATCAGCC-30 50 -AATGAACAGGACGGTGAGC-30

Cx45

1/1.5

50 30 30 nested

50 -CTCTAAACCACTGCACCAGC-30 50 -AGATGGACTCTCCTCCTACAGC-30 50 -TCGAATGGTTGTGGATCTCC-30

Cx50

1/1

50 30 50 nested

50 -TCTTGGAAGAGGTGAATGAGC-30 50 -TGGAGACGAAGATGATCTGC-30 50 -CAGTGCTCTTCATCTTCCGC-30

Cx57

1/1.5

50 30 50 nested

50 -CATCCTAGAGGAAGTCCACTCC-30 50 -CAGATATTGTTGCAACCGG-30 50 -GAAGATCTGGCTGACCATCC-30

Group II Cx30

PCR 1 was performed using 50 and 30 primers; in PCR 2, one of the primers was replaced by the corresponding nested primer.

3. Results DA neurons (n = 86) located in the SNc (Fig. 1A and B) were distinguished from neighboring GABAergic output neurons of the substantia nigra pars reticulata by their specific electrophysiological features (Grace and Bunney, 1983a; Kita et al., 1986). Indeed, DA neurons display characteristic passive membrane properties and spiking pattern (Fig. 1C), including a pronounced sag (Ih current activation) (15.7  1.1 mV) during hyperpolarization steps (90 pA injected current)

followed by a depolarizing rebound (8.1  1.0 mV) at the stimulus offset, a depolarized action potential threshold (36.8  0.9 mV), a long spike duration (2.8  0.1 ms), a large fast after-hyperpolarization amplitude (25.3  1.3 mV) and finally a slow spontaneous regular spiking activity (3.8  0.5 Hz) (as measured from 20 DA neurons). As we have previously described (Vandecasteele et al., 2005), SNc DA neurons are connected by electrical synapses (see example of an electrically coupled DA neuron pair in Fig. 1D). Such gap junctional communication appears to be developmentally

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regulated (Vandecasteele et al., 2005). Indeed, a high incidence of intercellular coupling was observed between P5 and P10, whereas 20% coupling incidence was detected in juvenile rats (P15–P25). Surprisingly, an interruption of coupling was observed between P10 and P15. Accordingly, we have investigated the Cx expression pattern in DA neurons separately in P7–P10 and in P17–P21 rats. To determine the molecular composition of electrical synapses specifically in DA neurons, the expression of Cx mRNAs was investigated by single-cell RT-PCR. The presence of each Cx mRNAs (Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx33, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, Cx47, Cx50 and Cx57) was first tested in P17 total rat brain cDNA by specific PCRs (data not shown). Amplicons for 13 Cx mRNAs were detected. They were dispatched accordingly to optimum PCR amplification conditions (MgCl2 concentrations, see Table 1, and annealing temperatures) and compatibility between primers into two groups for multiplex RT-PCR on single DA

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neurons (group I: Cx26, 31.1, 32, 36, 43 and 47; group II: Cx30, 31, 37, 40, 45, 50 and 57) (Table 1). Single-cell RT-PCR analysis of Cx mRNAs was performed for both P7–P10 (groups I and II Cxs: n = 24 and 18 cells, respectively) and P17–P21 (groups I and II Cxs: n = 30 and 14 cells, respectively) rats (Fig. 2). The success rate of amplification of at least one Cx per DA neuron decreased between P7–P10 and P17–P21 rats from 50 to 27% for group I Cxs and from 22 to 0% for group II Cxs. Such decrease in the success rate of amplification is in agreement with the decrease observed in tracer (Lucifer yellow and biocytin) and electrical coupling incidences (Walsh et al., 1991; Vandecasteele et al., 2005). The presence of several Cx mRNAs was detected in DA neurons: Cx26 (13%, n = 24), Cx30 (22%, n = 18), Cx32 (8%, n = 24), Cx36 (8%, n = 24) and Cx43 (42%, n = 24) in P7–P10 rats and Cx31.1 (10%, n = 30), Cx36 (13%, n = 30) and Cx43 (7%, n = 30) in P17–P21 animals (Fig. 2). Interestingly, the expression patterns of Cx mRNAs in DA neurons differed with the maturation level of these cells. Indeed, the Cx expression diversity decreased (5 Cxs versus 3 Cxs); 3 Cxs (Cx26, Cx30 and Cx32) were specific to P7–P10 DA neurons and Cx31.1 was specific to P17–P21 neurons (Cx36 and Cx43 being expressed in both groups) (Fig. 2). Moreover, the major Cx switched from Cx43 in P7–P10 to Cx36 in P17–P21 animals. Co-expression of Cx mRNAs was observed in 21% of the P7–P10 DA neurons and systematically involved Cx43 with Cx26 or Cx32 mRNAs (Table 2 and see example in Fig. 2A). Noticeably, such co-expression occurred mainly in P7–P10 rats, since only one P17–P21 DA neuron displayed a co-expression (Cx31.1 and Cx36). The unexpected finding of Cx43 mRNA, a Cx generally found in astrocytes (Rouach et al., 2002; but also reported in some neuronal populations, see review of Bennett and Zukin, 2004) prompted us to ensure that this finding was not an experimental artifact. Such bias could be ruled out since no expression of Cx43 mRNA was found when electrophysioloTable 2 Overview of Cx expression patterns found in DA neurons (P7–P10—group I (Cx26, Cx31.1, Cx32, Cx36, Cx43, Cx47): n = 24 cells and group II (Cx30, Cx31, Cx37, Cx40, Cx45, Cx50, Cx57): n = 18; P17–P21—group I: n = 30 and group II: n = 14) 26

Fig. 2. Cx mRNA expression analyzed by single-cell RT-PCR in SNc DA neurons. (A) Ethidium bromide gel electrophoresis was carried out for RT-PCR amplicons obtained from separate cytoplasm contents harvested from seven different DA neurons, after two rounds of PCR amplification (group I or group II multiplex PCRs, followed by separate specific PCRs). DA neurons 1–4 were obtained from P7 to P10 animals (left part) and DA neurons 5–7 from P17 to P21 animals (right part). Each amplicon migrated at the theoretical size expected knowing Cx sequences and primer locations (Cx26: 293 bp, Cx30: 230 bp, Cx31.1: 199 bp, Cx32: 275 bp, Cx36: 143 bp, Cx43: 175 bp). Amplicons were systematically sequenced. Note the co-expression of Cx32 and Cx43 in the same cell (DA neuron 4). (B) Expression incidence of each Cx found in DA neurons (P7–P10—group I (Cx26, Cx31.1, Cx32, Cx36, Cx43, Cx47): n = 24 cells and group II (Cx30, Cx31, Cx37, Cx40, Cx45, Cx50, Cx57): n = 18; P17–P21—group I: n = 30 and group II: n = 14). Note that Cx43, major Cx for P7–P10 rats, is the minor Cx for P17–P21 rats; in parallel, Cx36 follows the reverse regulation.

30

31

31.1 32

26 0/0 – – 0/0 30 22/0 0/0 – 31 0/0 – 31.1 0/7 32 36 37 40 43 45 47 50 57

0/0 – – 0/0 0/0

36

37

40

43

45

47

50

57

0/0 – – 0/3 0/0 8/10

– 0/0 0/0 – – – 0/0

– 0/0 0/0 – – – 0/0 0/0

13/0 – – 0/0 8/0 0/0 – – 21/7

– 0/0 0/0 – – – 0/0 0/0 – 0/0

0/0 – – 0/0 0/0 0/0 – – 0/0 – 0/0

– 0/0 0/0 – – – 0/0 0/0 – 0/0 – 0/0

– 0/0 0/0 – – – 0/0 0/0 – 0/0 – 0/0 0/0

Values represent percentage of cells displaying amplicons for two different Cxs and in the diagonal italicised values for only one Cx. The first percentage refers to P7–P10 rats and the second one to P17–P21 rats. (–) The non-determined coexpressions, due to the separate analysis of the two Cx groups.

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Fig. 3. Absence of functional hemichannels in DA neurons assessed by dye-loading experiments. (A) Fluorescence confocal microphotographs of dye-loading assays performed to evaluate the presence of functional hemichannels. Slices were incubated in a medium which favors the opening of hemichannels (see Section 2) together with a fluorescent dye (Lucifer yellow) able to cross hemichannels. DA neurons were identified by positive immunoreactivity to tyrosine hydroxylase (left column), and their Lucifer yellow staining (middle column) was compared in control (upper panels) and low Ca2+ medium (lower panels). Calibration bar: 50 mm. (B) Fluorescence confocal microphotographs of dye-loading assays showing the Lucifer yellow uptake by macroglial cells after Ca2+ removal, as a positive control for hemichannel opening. Calibration bar: 10 mm.

gically identified neighboring output neurons from substantia nigra pars reticulata (n = 14) were similarly analyzed in the same brain slices. Detection of Cx43 mRNA in DA neurons led us to look for the eventual presence of functional hemichannels constituted with this Cx (Bennett et al., 2003; Goodenough and Paul, 2003). To assess the eventual presence of hemichannels, dye-loading experiments (Fig. 3) consisting in monitoring the diffusion of extracellular Lucifer yellow into DA neurons through hemichannels opened in low extracellular Ca2+ (10 mM) conditions. DA cells were identified by positive immunoreactivity to tyrosine hydroxylase. No significant difference of Lucifer yellow staining was observed between control and 7 min (8.1  2.5 loaded neurons per SNc slice, n = 12 versus 9.1  1.5, n = 16) or 20 min (7.8  1.4, n = 4 versus 11  2.5, n = 3) low Ca2+ conditions (Fig. 3A). As a positive control, we verified that astrocytic hemichannels were opened by this treatment (Fig. 3B). Additionally, the number of loaded neurons both in control (7.3  1.1, n = 4) and in low Ca2+ (6.8  0.8, n = 4) conditions was not affected by carbenoxolone (250 mM) (data not shown), known to inhibit Cx43 channels and hemichannels. This finding excludes the presence of functional hemichannels in DA neurons. 4. Discussion Cx mRNA expression was investigated by single-cell RTPCR in P7–10 and P17–21 rats. DA cells express various Cx mRNAs (Cx26, Cx30, Cx31.1, Cx32, Cx36 and Cx43). This is

in contrast with the proposal that electrical synapses are mainly constituted by Cx36, as observed in electrically coupled GABAergic interneurons (Venance et al., 2000; Hormuzdi et al., 2004; Bennett and Zukin, 2004). However, it is in accordance with tissue level RT-PCR and immunohistochemistry studies by Leung et al. (2002) that reported the expression of various Cxs (Cx26, Cx32, Cx43 and Cx45) in the midbrain floor where DA neurons are located. It should be added that Cx26, Cx32, Cx43, Cx45, and Cx47 expressions have been reported in several other neuronal populations (Nadarajah et al., 1997; Alvarez-Maubecin et al., 2000; Bittman et al., 2002; Bennett and Zukin, 2004). Surprisingly, Cx43 mRNA was detected in DA neurons. Cx43 is generally found in astrocytes, although its expression has also been reported in mitral cells, motoneurons and locus coeruleus neurons (Bennett and Zukin, 2004). However, according to our previous study by tracer coupling experiments (Vandecasteele et al., 2005), the presence of Cx43 mRNA did not lead to coupling between DA neurons and macroglia. Dye-loading experiments also excluded the presence of functional hemichannels in these DA cells. Consequently, Cx43 could be involved in the assembly of intercellular channels between DA cells. Interestingly, Cx30 mRNA was detected in DA cells of P7–P10 rats. Apoptotic events occur in SNc between P10 and P15, interpreted as a target-dependent selection of DA neurons (Marti et al., 1997; Burke, 2003), and as reported by Condorelli et al. (2002), Cx30 mRNA is transitorily expressed in apoptotic neurons of the adult brain following kainate induced seizures. Therefore, the

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presence of Cx30 mRNA in DA cells of P7–10 rats could reflect the subsequent apoptotic period occurring in these cells. It remains to determine the Cx expression at a protein level. Indeed, due to the sensitivity of the single-cell RT-PCR technique, we cannot exclude that some of the Cx mRNAs detected here belong to the ‘‘gene noise’’ phenomenon, i.e. differences of gene expression patterns within the same clonal population, observed in eukaryotes (Blake et al., 2003). To address this issue while taking into account the very low expression level and the dendritic localization of Cxs expected in neuronal cells, the adequate technique would be immunohistochemistry combined with electronic microscopy. In P17–21 DA neurons three Cxs have been detected with almost no co-expression within the same cell (only one case of co-expression was found). Considering the expected incompatibility between these Cxs (belonging to three different phylogenic Cx classes) (Sosinsky and Nicholson, 2005), this observation suggests that DA neurons could be organized in different interconnected networks. Such coupled cellular subsets could overlap the observed heterogeneities of DA neurons based on electrophysiological characteristics (Gu et al., 1992; Hajos and Greenfield, 1993; Neuhoff et al., 2002), connectivity (Fallon and Moore, 1978; Grace and Onn, 1989; Gauthier et al., 1999), and receptor and peptide expressions (Seroogy et al., 1988; Chen et al., 2001). In P7–P10 DA neurons, the higher diversity and the co-expression of Cxs further extend these network possibilities. It should be noted that according to Cx compatibility studies performed in cellfree systems (Falk et al., 1997), the co-expression of Cx43/ Cx26 and Cx43/Cx32 would not lead to the formation of heteromeric channels, although this remains to be confirmed in situ. The decrease of Cx mRNA expression observed along postnatal development is in accordance with the reduction of electrical coupling incidence in DA neurons (Vandecasteele et al., 2005). However, re-increasing of gap junctional communication could occur in adult animals. Indeed, as shown in the hypothalamus, Cx expression and gap junctional communication are increased during lactation, and are associated with retraction of dendritic glial sheaths (Theodosis, 2002). DA neuron dendrodendritic appositions have been reported to be also separated by glial processes (Reubi and Sandri, 1979; but see Groves and Linder, 1983), which could be similarly involved in the modulation of electrical coupling. A comparison of the elementary properties show SNc electrical synapses are in the weak range of coupling incidence, coupling coefficient and junctional conductance and they display the strongest low-pass filters in the CNS (Vandecasteele et al., in press). Those particularities would minimize the amplitude of the evoked spikelet (electrically transmitted event corresponding to a presynaptic spike) in the coupled cell. Spikelets have been described as preponderant elements of the precision of spike timing and therefore of the synchronization process in electrically coupled GABAergic interneurons (Galarreta and Hestrin, 2001). Besides, DA neurons display slow depolarizations leading to spike firing (Kita et al., 1986; Grace and Onn, 1989). Such slow kinetic events, expected to be

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efficiently transmitted by electrical synapses, could participate to a synchronization of DA neuron activity. The Cx composition of electrical synapses could be responsible for differences in coupling properties observed between DA neurons and GABAergic interneurons (Vandecasteele et al., in press). Indeed, in cortical and hippocampal GABAergic interneurons and in output neurons of the inferior olive and of the suprachiasmatic nucleus, Cx36 is reported as the only Cx expressed and is responsible of the synchronization of activity observed in these structures, as demonstrated by knock-out animals studies (Bennett and Zukin, 2004; Connors and Long, 2004; Hormuzdi et al., 2004). In SNc output neurons, other Cxs are expressed together with Cx36. This difference in Cx composition of intercellular channels is physiologically relevant since it leads to different permeability properties of electrical synapses, suggesting a different impact of electrical transmission in DA neurons network activities. Acknowledgements This work was supported by an ACI ‘‘Jeune Chercheur’’ grant from the French Ministe`re de la Recherche, Fondation de France grant 20020111943, INSERM and the College de France. References Alvarez-Maubecin, V., Garcia-Hernandez, F., Williams, J.T., Van Bockstaele, E.J., 2000. Functional coupling between neurons and glia. J. Neurosci. 20, 4091–4098. Bamford, N.S., Robinson, S., Palmiter, R.D., Joyce, J.A., Moore, C., Meshul, C.K., 2004a. Dopamine modulates release from corticostriatal terminals. J. Neurosci. 24, 9541–9552. Bamford, N.S., Zhang, H., Schmitz, Y., Wu, N.P., Cepeda, C., Levine, M.S., Schmauss, C., Zakharenko, S.S., Zablow, L., Sulzer, D., 2004b. Heterosynaptic dopamine neurotransmission selects sets of corticostriatal terminals. Neuron 42, 653–663. Bennett, M.V., Contreras, J.E., Bukauskas, F.F., Saez, J.C., 2003. New roles for astrocytes: gap junction hemichannels have something to communicate. Trends Neurosci. 26, 610–617. Bennett, M.V., Zukin, R.S., 2004. Electrical coupling and neuronal synchronization in the mammalian brain. Neuron 41, 495–511. Bittman, K., Becker, D.L., Cicirata, F., Parnavelas, J.G., 2002. Connexin expression in homotypic and heterotypic cell coupling in the developing cerebral cortex. J. Comp. Neurol. 443, 201–212. Blake, W.J., Kaern, M., Cantor, C.R., Collins, J.J., 2003. Noise in eukaryotic gene expression. Nature 422, 633–637. Bruzzone, R., White, T.W., Paul, D.L., 1996. Connections with connexins: the molecular basis of direct intercellular signaling. Eur. J. Biochem. 238, 1–27. Burke, R.E., 2003. Postnatal developmental programmed cell death in dopamine neurons. Ann. N. Y. Acad. Sci. 991, 69–79. Chen, L.W., Wei, L.C., Lang, B., Ju, G., Chan, Y.S., 2001. Differential expression of AMPA receptor subunits in dopamine neurons of the rat brain: a double immunocytochemical study. Neuroscience 106, 149–160. Cheramy, A., Leviel, V., Glowinski, J., 1981. Dendritic release of dopamine in the substantia nigra. Nature 289, 537–542. Condorelli, D.F., Mudo, G., Trovato-Salinaro, A., Mirone, M.B., Amato, G., Belluardo, N., 2002. Connexin-30 mRNA is up-regulated in astrocytes and expressed in apoptotic neuronal cells of rat brain following kainate-induced seizures. Mol. Cell. Neurosci. 21, 94–113. Connors, B.W., Long, M.A., 2004. Electrical synapses in the mammalian brain. Annu. Rev. Neurosci. 27, 393–418.

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