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. 2006 Jul 6;575(Pt 3):789–806. doi: 10.1113/jphysiol.2006.114272

Analysis of the effects of cannabinoids on identified synaptic connections in the caudate-putamen by paired recordings in transgenic mice

Ilka Freiman 1, Alexandra Anton 1, Hannah Monyer 2, Michal J Urbanski 1, Bela Szabo 1
PMCID: PMC1995699  PMID: 16825300

Abstract

CB1 cannabinoid receptors are expressed in many neurons in the caudate-putamen. However, it is not known how the activation of these receptors influences synaptic transmission between different neuron classes. The aim was to establish a method for studying identified synaptic connections in the caudate-putamen, and to determine the effects of cannabinoids on these connections. Brain slices were prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) in parvalbumin-positive fast spiking interneurons (PV-FSNs). PV-FSNs were identified based on their fluorescence. Non-fluorescent medium-sized neurons were considered to be medium spiny neurons (MSNs). Synaptic transmission was studied by simultaneous patch-clamp recording from identified neuron pairs. In the case of PV-FSN → MSN neurotransmission, the synthetic cannabinoid receptor agonist WIN55212-2 lowered the success rate of transmission and the amplitude of successful postsynaptic events. Analysis of miniature inhibitory postsynaptic currents indicated that WIN55212-2 inhibited synaptic transmission presynaptically. WIN55212-2 did not elicit somatodendritic effects in PV-FSNs: membrane potential, membrane current and evoked firing were not changed. WIN55212-2 also depressed the MSN → MSN neurotransmission. The inhibitory synaptic input to MSNs was only weakly suppressed by endocannabinoids released by depolarized postsynaptic MSNs. The results show that the combined use of transgenic animals and paired-recording techniques allows the study of synaptic connections between rare neurons. Using these techniques, we showed that activation of CB1 receptors on axon terminals of (i) PV-FSNs and (ii) MSNs leads to presynaptic inhibition of GABAergic synaptic transmission between these axons and their postsynaptic targets, the MSNs. The cannabinoids acted preferentially on axon terminals without effects on the somatodendritic region of the neurons.

The Gαi/o protein-coupled CB1 cannabinoid receptor is the primary neuronal target of the phytocannabinoid Δ9-tetrahydrocannabinol and of the endogenous cannabinoids (endocannabinoids) anandamide and 2-arachidonoylglycerol (Howlett et al. 2002; Abood, 2005; Pertwee, 2005). The CB1 receptor is widely distributed in the nervous system (Herkenham et al. 1991b; Tsou et al. 1998; Egertova et al. 2003). Activation of CB1 receptors leads to presynaptic inhibition of synaptic transmission in several regions of the central and peripheral nervous system (Freund et al. 2003; Szabo & Schlicker, 2005).

The present work focuses on the function of CB1 receptors in the caudate-putamen where the receptor density is high and many of the CB1 receptors are localized on presynaptic axon terminals (Herkenham et al. 1991b; Tsou et al. 1998; Rodriguez et al. 2001; Köfalvi et al. 2005). A portion of the CB1 receptors is on terminals of glutamatergic afferent axons, and activation of these receptors leads to presynaptic inhibition of glutamatergic synaptic transmission (Gerdeman & Lovinger, 2001; Huang et al. 2001).

The aim of the present work was to determine, how activation of CB1 receptors on GABAergic axon terminals modulates synaptic transmission between identified neurons in the caudate-putamen. In a previous study, activation of CB1 receptors led to inhibition of GABAergic neurotransmission in the caudate-putamen (Szabo et al. 1998). However, since all inputs of the recorded neurons were non-selectively stimulated in this latter study, the involved synapses could not be identified.

The principal neurons of the caudate-putamen are the GABAergic medium spiny neurons (MSNs). They constitute 97% of the neuronal population and project to the substantia nigra pars reticulata and globus pallidus, and send recurrent axon collaterals to neighbouring MSNs (see Fig. 1A) (Plenz, 2003; Gerfen, 2004; Tepper & Bolam, 2004; Tepper et al. 2004). The parvalbumin-positive fast spiking neurons (PV-FSNs) are interneurons; although they constitute only 0.7% of the neuronal population, they are the most important source of GABAergic input to MSNs (Kawaguchi et al. 1995; Gerfen, 2004; Tepper & Bolam, 2004; Tepper et al. 2004). The neuropeptide Y/NOS/somatostatin-positive interneurons comprise 0.8% of the striatal neurons and give inhibitory input to MSNs (Koos & Tepper, 1999; Tepper & Bolam, 2004). The calretinin-positive striatal interneurons are also GABAergic (Tepper & Bolam, 2004). Neurons of the caudate-putamen receive GABAergic input also from other nuclei, for example, from the globus pallidus (Gerfen, 2004).

Figure 1. Recording of synaptic transmission (i) between parvalbumin-positive fast spiking neurons (PV-FSNs) and medium spiny neurons (MSNs), and (ii) between recurrent axon collaterals of MSNs and MSNs.

Figure 1

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A, the GABAergic PV-FSNs innervate MSNs. The GABAergic MSNs project to the substantia nigra pars reticulata and the globus pallidus and send recurrent axon collaterals to neighbouring MSNs. CB1 cannabinoid receptor mRNA is synthesized in PV-FSNs and MSNs, and the receptor protein is probably localized in the axon terminals of these neurons. Patch-clamp pipettes were positioned in presynaptic PV-FSNs and MSNs to elicit action potentials (APs), and the resulting postsynaptic events were recorded in MSNs. B, a PV-FSN displaying fluorescence due to enhanced green fluorescent protein (EGFP) – colour-coded fluorescence picture. C, paired patch-clamp recording from a PV-FSN (the same neuron as in B) and a neighbouring MSN – near-infrared video microscopical picture. D, synaptic connection between the neurons shown in C. Shown are 20 presynaptic APs and the corresponding postsynaptic events.

In situ hybridization anatomical studies suggest that PV-FSNs possess CB1 receptors (Fig. 1A) (Marsicano & Lutz, 1999; Hohmann & Herkenham, 2000). Accordingly, the main hypothesis of the present study was that cannabinoids presynaptically inhibit synaptic transmission between PV-FSNs and MSNs. In order to test the hypothesis, it was necessary to establish a technique that allowed analysis of synaptic transmission between identified neurons. In order to facilitate identification of the rare PV-FSNs, we used transgenic mice which express enhanced green fluorescent protein (EGFP) in parvalbumin-positive interneurons (Meyer et al. 2002). Non-fluorescent medium-sized neurons were considered to be MSNs. Synaptic transmission was analysed by simultaneous patch-clamp recording from identified PV-FSN–MSN pairs (see Fig. 1). For a more complete analysis of the effects of cannabinoids on GABAergic synaptic transmission in the caudate-putamen, we also studied cannabinoid effects on transmission between recurrent collaterals of MSNs and MSNs (Fig. 1A). MSNs also possess CB1 receptors (Matsuda et al. 1993; Marsicano & Lutz, 1999; Hohmann & Herkenham, 2000).

Methods

The experiments conformed to the European Community law regulating the use of animals in biomedical research. All efforts were made to minimize both the suffering and the number of animals used. The methods were similar to those previously described (Than & Szabo, 2002; Szabo et al. 2004).

Brain slices

For easier identification of parvalbumin-positive fast spiking neurons (PV-FSNs), transgenic mice were used. An EGFP expression cassette was inserted into the parvalbumin gene contained on a bacterial artificial chromosome, and transgenic mice containing multiple copies of this transgene were used (see Meyer et al. 2002, for details of the generation and characterization of these mice). Ninety-six per cent of neurons showing green fluorescence in the caudate-putamen were immunpositive for parvalbumin (Meyer et al. 2002). EGFP expression in the brain begins on postnatal days 10–11; accordingly, 12–18-day-old-mice were used in the present study. Similar transgenic mice (with one copy of the EGFP/parvalbumin transgene) have already been successfully used for identifying parvalbumin-positive hippocampal neurons for patch clamping (Bartos et al. 2002).

Mice were anaesthetized with isoflurane (>3%) and decapitated. The brains were rapidly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) of the following composition (mm): NaCl 126, NaH2PO4 1.2, KCl 3, MgCl2 5, CaCl2 1, NaHCO3 26, glucose 20, Na-lactate 4, pH 7.3–7.4 (after the solution was gassed with 95% O2/5% CO2). Sagittal slices of the caudate-putamen that were 300 μm thick were cut. The slices were protected from bright light and stored in a Gibb chamber containing ACSF of the following composition (mm): NaCl 126, NaH2PO4 1.2, KCl 3, MgCl2 1, CaCl2 2.5, NaHCO3 26, glucose 10, Na-lactate 4, pH 7.3–7.4. In order to support regeneration processes in neurons, the temperature was raised to 35°C for 45 min. Thereafter, the slices were stored at room temperature until patch clamping started up to 6 h later.

Brain slices were fixed at the glass bottom of a superfusion chamber with a nylon grid on a platinum frame, and superfused with ACSF at room temperature at a flow rate of 1.5 ml min−1. The superfusion ACSF was of the following composition (mm): NaCl 126, NaH2PO4 1.2, KCl 3, MgCl2 1, CaCl2 2.5, NaHCO3 26, glucose 10, pH 7.3–7.4. The slices were observed by a fixed-stage Zeiss Axioskop microscope (Zeiss, Göttingen, Germany). PV-FSNs were identified using the following fluorescence filter set: excitation filter, 475AF45; dichroic mirror, 505DRLP; emission filter, 510ALP (Omega Optical, Brattleboro, VT, USA). Illumination of the slices was kept short to minimize fluorescence bleaching and phototoxicity. After fluorescence identification, PV-FSNs were observed with near-infrared video microscopy for patch clamping (see Fig. 1). Medium-sized neurons without fluorescence were regarded to be MSNs.

Patch-clamp recording techniques

Pipettes were pulled from borosilicate glass and had resistances of 2–5 MΩ when filled with intracellular solution. Patch-clamp recordings were obtained with an EPC-9-double amplifier, and data were evaluated with the TIDA for Windows software (HEKA Elektronik, Lambrecht, Germany). Series resistance compensation of 50% was usually applied. Data were filtered at 1–2.9 kHz and stored with sampling rates at least twice the filtering frequency. Series resistance was measured before and after recordings, and experiments with major changes in series resistance (>20%) were discarded. The basic electrical properties of neurons were determined with pipettes containing (mm): K gluconate 145, CaCl2 0.1, MgCl2 2, Hepes 5, EGTA 1.1, ATP-Mg 5, GTP-Tris 0.3, pH 7.4.

Recording of synaptic transmission between neuron pairs

The superfusion ACSF contained AP-5 (2.5 × 10−5m) and DNQX (10−5m) in order to suppress fast glutamatergic neurotransmission. For studying PV-FSN → MSN neurotransmission, presynaptic PV-FSNs were patched with pipettes containing (mm): K gluconate 145, CaCl2 0.1, MgCl2 2, Hepes 5, EGTA 1.1, ATP-Mg 5, GTP-Tris 0.3, pH 7.4. Postsynaptic MSNs were patched with pipettes containing (mm): CsCl 142, MgCl2 1, Hepes 10, EGTA 1, ATP-Na2 4, N-ethyl-lidocaine Cl (QX-314) 2, pH 7.4. In a subset of experiments, in which depolarization-induced suppression of inhibition (DSI) was studied, postsynaptic MSNs were patched with pipettes containing (mm): CsCl 147, MgCl2 1, Hepes 10, EGTA 1, ATP-Na2 4, GTP-Na2 0.4, N-ethyl-lidocaine Cl 2, pH 7.4. For studying MSN → MSN neurotransmission, a pipette solution suitable for presynaptic as well as for postsynaptic neurons was used (mm): K gluconate 93, KCl 36, MgCl2 2, Hepes 10, EGTA 0.02, ATP-Na 3, GTP-Tris 0.3, pH 7.4. This latter solution allowed testing of synaptic connections between neuron pairs in both directions; this was necessary, because the coupling ratio between MSNs was low (see Table 1). Single action potentials in presynaptic neurons were elicited in current-clamp mode, by depolarizing current injections (500–800 pA for 3–5 ms). Postsynaptic events were recorded at a holding potential of −60 mV (PV-FSN → MSN transmission) or −70 mV (MSN → MSN transmission). Synaptic transmission was evaluated by a program written by us in SigmaPlot (for details see Supplemental material and Supplemental Figs 1 and 2). Usually, 40 (PV-FSN → MSN transmission) and 100 synaptic events (MSN → MSN transmission) were analysed. The program searched at first for peaks within a preset period after presynaptic actions potentials. Peaks surpassing a preset threshold were termed synaptic ‘successes’, otherwise they were termed as ‘failures’. Finally, the program determined success rate, mean amplitude of successes, mean amplitude of all events following a presynaptic action potential (including successes and failures), coefficient of variation (s.d./mean) and mean2/s.d.2. A change in mean amplitude of all events reflects changes in both success rate and amplitude of successes; it corresponds to changes in postsynaptic summation currents determined in most electrophysiological experiments in which many afferent inputs of a neuron are simultaneously stimulated. A decrease in the parameter mean2/s.d.2 indicates decreased transmitter release probability from axon terminals (compare with Clements, 1990; Koos & Tepper, 2002; Galante & Marty, 2003).

Table 1.

Parameters of synaptic transmission between PV-FSNs and MSNs, and between recurrent axon collaterals of MSNs and MSNs

PV-FSN → MSN MSN → MSN
Coupling ratio%a 20 (35/173) 12 (29/251)
Number of experiments n = 15 n = 7
Stimulation rate (Hz) 0.067 0.33
Latency (ms)b 1.8 ± 0.1 2.6 ± 0.2*
Success rate (%)c 86 ± 4 18 ± 9*
Amplitude of successes (pA) 66 ± 17 (13 ± 4)
39 ± 12d
Conductance of successes (pS)e 922 ± 24 (412 ± 139)
1236 ± 417d
Amplitude of all events (pA)f 63 ± 18 (2 ± 1)
6 ± 3d*
s.d./mean (coefficient of variation)g 0.660 ± 0.066 3.44 ± 0.70*
Mean2/s.d.2g 4.0 ± 1.0 0.35 ± 0.25*
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Means ±s.e.m. of values determined during the initial reference period (PRE; before administration of solvent or drugs). Significant difference between PV-FSN → MSN and MSN → MSN transmission: P < 0.05.

a

Percentage of synaptically connected neuron pairs (for example, 35 PV-FSN–MSN pairs were connected out of 173 tested pairs). Only 15 of 35 (PV-FSN–MSN) and 7 of 29 (MSN–MSN) connected pairs were included in the analysis, because some pairs were stimulated at different frequencies and because some connections were lost during the initial equilibration period.

b

Interval between the onset of the presynaptic action potential and the onset of the postsynaptic event.

c

Number of synchronous successful postsynaptic events per 100 presynaptic action potentials.

d

Cesium was not included in postsynaptic pipettes for studying MSN → MSN transmission. This leads to an underestimation of currents and conductances by a factor of ∼3 (see Koos et al. 2004). For allowing better comparison with PV-FSN → MSN transmission, measured values (13 ± 4 pA, 412 ± 139 pS, 2 ± 1 pA) were multiplied by 3.

e

For better comparison, conductances were calculated (since chloride concentration and holding potential in postsynaptic neurons were different in the groups; see Methods).

f

Amplitude of all synchronous events, including successes and failures. Please note that the following equation is valid for the individual experiments but not for the means included in the table: amplitude of all events = amplitude of successes × success rate/100.

g

s.d./mean and mean2/s.d.2 were calculated from the amplitude of all synaptic events.

Figure 2. Characterization of PV-FSNs and MSNs.

Figure 2

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A, responses of a PV-FSN. B, responses of an MSN. Membrane potential was recorded in the whole-cell and current-clamp mode with pipettes containing a K gluconate-based solution. Without current injection (0 pA), both types of neurons were silent with stable resting membrane potential (RMP). Current injections of 300 ms (150 pA, 300 pA, 450 pA and 600 pA) depolarized the neurons and elicited firing. The action potentials marked by * and # are shown with an enhanced time scale in the lowermost traces. C, statistical evaluation of firing rates. Firing rates were calculated from the intervals between the first two action potentials in the salvos. Means ±s.e.m. of 19 (PV-FSN) and 15 (MSN) neurons (s.e.m. is sometimes smaller than the symbol). Significant difference from MSN: *P < 0.05.

Recording of miniature inhibitory postsynaptic currents (mIPSCs) in MSNs

The superfusion ACSF contained AP-5 (2.5 × 10−5m), DNQX (10−5m) and tetrodotoxin (3 × 10−7m). The patch pipettes contained (mm): CsCl 142, MgCl2 1, Hepes 10, EGTA 1, ATP-Na2 4, N-ethyl-lidocaine Cl (QX-314) 2, pH 7.4. mIPSCs were recorded at a holding potential of −60 mV in 120-s periods, and detected and analysed with the MiniAnalysis software (version 6.0.1; Synaptosoft, Decatur, GA, USA). The detection parameters in MiniAnalysis (for example, minimum peak amplitude) were determined for each neuron for allowing detection of most mIPSCs without detection of noise currents. Several recordings from one neuron (during PRE and WIN55212-2 superfusion) were always evaluated using identical detection parameters.

Recording of membrane potential, holding current and evoked neuronal firing in the perforated patch-clamp mode

The superfusion ACSF contained AP-5 (2.5 × 10−5m), DNQX (10−5m) and bicuculline (2 × 10−5m) in order to suppress fast glutamatergic and GABAergic inputs. The perforated patch-clamp mode was used in these experiments for preserving the intracellular milieu of the neurons. The pipettes contained (mm): K gluconate 145, CaCl2 0.1, MgCl2 2, Hepes 5, EGTA 1.1, ATP-Mg 5, GTP-Tris 0.3, pH 7.4. Amphotericin B 300 μg ml−1 was added for establishing perforated patches. Neuronal firing was evoked every minute by depolarizing current injections (30–150 pA for 500 ms).

Protocols and statistics

Recordings started 15–30 min after establishment of the whole-cell or perforated-patch configuration (the period is long, because two neurons had to be patched and equilibrated with intracellular solution). Zero time in the figures is the time when recording began. Solvent and drug superfusion is indicated in the figures. Values of parameters during superfusion with solvent or drugs were expressed as percentages of the initial reference values (PRE; the PRE periods are indicated in the figures).

Means ± s.e.m. are given throughout. The non-parametric two-tailed Mann–Whitney and Wilcoxon signed rank tests were used to identify significant differences between groups (drug versus solvent) and within groups (drug versus PRE), respectively. P < 0.05 was taken as the limit of statistical significance, and only this level is indicated, even if P was < 0.01 or < 0.001. In a few cases, outlying observations were identified using Grubbs' test (Grubbs & Beck, 1972).

Drugs

Drugs were obtained from the following sources. Alamone Labs (Jerusalem, Israel): N-ethyl-lidocaine Cl (QX-314); Sanofi (Montpellier, France): rimonabant (previously called SR141716A); Sigma (Deisenhofen, Germany): amphotericin B, baclofen; Tocris Cookson (Bristol, England): R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (WIN55212-2) (RS)-3,5-dihydroxyphenylglycine (DHPG).

The cannabinoid ligands WIN55212-2 and rimonabant were dissolved in dimethylsulphoxide (DMSO). Stock solutions were stored at −32°C. Further dilutions were made with superfusion buffer; the final concentration of DMSO in the superfusion fluid was 1 ml l−1. Control solutions always contained the appropriate concentration of DMSO. Since cannabinoid ligands cannot be washed out from brain slices, only one concentration per drug was tested in a brain slice.

Results

Characterization of PV-FSNs and MSNs

PV-FSNs were identified in the brain slice based on their green fluorescence (Fig. 1B). Medium-sized neurons lacking fluorescence were considered to be MSNs. In an initial series of experiments, we determined the electrical properties of 19 PV-FSNs and 15 MSNs (see Fig. 2). In current-clamp mode and at 0 pA holding current, both kinds of neurons were silent. The resting membrane potential of PV-FSNs was less negative than that of MSNs (PV-FSN: −63 ± 1 mV; MSN: − 71 ± 2 mV; P < 0.05). Depolarizing current injections elicited action potential salvos (Fig. 2). The action potential threshold was identical in the two populations (PV-FSN: −39 ± 1 mV; MSN: −39 ± 2 mV; P = 0.577). The amplitude of action potentials was determined relative to the threshold potential. It was very similar in the two kinds of neurons (PV-FSN: 96 ± 1 mV; MSN: 98 ± 2 mV; P = 0.521). The afterhyperpolarization following the action potentials was determined relative to the threshold potential. It was more prominent in PV-FSNs than in MSNs (PV-FSN: − 15 ± 1 mV; MSN: −9 ± 2 mV; P < 0.05) (see also Fig. 2A and B). The width of action potentials was determined at the half amplitude of the action potentials. PV-FSNs had shorter action potentials than MSNs (PV-FSN: 0.70 ± 0.04 ms; MSN: 1.12 ± 0.11 ms; P < 0.05) (see also Fig. 2A and B, lowermost traces). Increasing depolarizing currents (150–600 pA) led to increasing firing rates in both neurons, and PV-FSNs were able to fire at higher rates than MSNs (Fig. 2C). Increasing the depolarizing current above the values shown in Fig. 2C led to firing rates above 200 Hz in many PV-FSNs (not shown). In contrast, the effects of stronger depolarizing currents were frequently limited by sodium channel inactivation in MSNs (not shown). Intermittent firing has not been observed in PV-FSNs. Finally, the input resistance of the two kinds of neurons was similar (PV-FSN: 273 ± 29 MΩ; MSN: 272 ± 42 MΩ; P = 0.535).

Thus, the two neuronal populations significantly differed in four parameters: resting membrane potential, afterhyperpolarization, action potential width and firing rate. We also analysed the distributions of individual values of these parameters. There were considerable overlaps between the distribution plots of the two neuronal populations in the case of all four parameters (not shown). This indicates that a reliable identification of neurons solely on the basis of their electrophysiological properties is not possible in our slice preparation.

Synaptic transmission between PV-FSNs and MSNs

Characterization of synaptic transmission

The main aim of the study was to determine the effects of cannabinoids on PV-FSN → MSN neurotransmission. Pairs of neurons with green fluorescence (PV-FSN) and without fluorescence (MSN) were patch clamped (Fig. 1B and C). Table 1 shows the parameters of synaptic transmission between PV-FSN–MSN pairs during the initial reference period (PRE). The coupling ratio between PV-FSN–MSN pairs was 20%. In the case of synaptically coupled pairs, depolarization of presynaptic PV-FSNs every 15 s (0.067 Hz) elicited postsynaptic events in MSNs with a latency of 1.8 ms. The success rate of synaptic transmission was 86%. The mean amplitude of successful action potential-coupled postsynaptic events was 66 pA. The mean amplitude of all action potential-coupled postsynaptic events (including successes and failures) was 63 pA (somewhat lower than the amplitude of successes, because success rate was less than 100%). Synaptic transmission between PV-FSNs and MSNs showed the typical properties of action potential-elicited GABAergic neurotransmission. Thus, synaptic transmission was abolished if action potential development in PV-FSNs was inhibited by tetrodotoxin. Ten minutes after the beginning of tetrodotoxin (3 × 10−7m) superfusion, the amplitude of postsynaptic events decreased to 3 ± 2% of the initial reference value (n = 5; P < 0.05). Cadmium, by blocking voltage-dependent calcium channels in presynaptic axon terminals and consequently transmitter release, also inhibited neurotransmission. Ten minutes after the beginning of cadmium (5 × 10−5m) superfusion, the amplitude of postsynaptic events decreased to 1 ± 1% of the initial reference value (n = 4; P = 0.07). Blockade of postsynaptic GABAA receptors by bicuculline abolished postsynaptic events as well. Ten minutes after the beginning of bicuculline (2 × 10−5m) superfusion, the amplitude of postsynaptic events decreased to 7 ± 2% of the initial reference value (n = 9; P < 0.05). Finally, we determined the reversal potential of the postsynaptic current by varying the holding potential of the postsynaptic neuron between −60 mV and +30 mV. The determined reversal potential, +3 mV (n = 13), was nearly identical with the calculated equilibrium potential of chloride, +2 mV, suggesting that chloride was the charge carrier of the postsynaptic currents.

For evaluation of cannabinoid-evoked changes, all further values of synaptic parameters were expressed as percentages of PRE (the PRE period is shown in Figs 3A and 4). In control experiments, in which solvent (SOL) was superfused, the parameters remained fairly constant (Fig. 4).

Figure 3. Effects of the synthetic CB1/CB2 cannabinoid receptor agonist WIN55212-2 (WIN) and the CB1 receptor antagonist rimonabant (RIM) on synaptic transmission between PV-FSNs and MSNs: original tracings.

Figure 3

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Action potentials in PV-FSNs were elicited every 15 s by depolarizing current injections and the following postsynaptic events were recorded in MSNs. A, • represent the amplitudes of postsynaptic events. B, 40 events recorded during the initial reference period (PRE), during superfusion of WIN55212-2 and during combined superfusion of WIN + RIM (the 10-min recording periods are indicated in A). The success rate was 60%, 20% and 48% during PRE, WIN and WIN + RIM, respectively. C, averages of successes and all events (including successes and failures) from the 40 events shown in B.

Figure 4. Effects of the synthetic CB1/CB2 cannabinoid receptor agonist WIN55212-2 (WIN), the CB1 receptor antagonist rimonabant (RIM) and solvent (SOL) on synaptic transmission between PV-FSNs and MSNs: statistical evaluation.

Figure 4

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Action potentials in PV-FSNs were elicited every 15 s by depolarizing current injections and the following postsynaptic events were recorded in MSNs. Parameters of synaptic transmission were calculated for 40 events (recorded during 10-min periods) and expressed as percentages of the initial reference value PRE. Slices of the control group were superfused with SOL during the whole experiment. The other group received WIN and RIM as indicated. Means ±s.e.m. of 6 (WIN + RIM) and 6 (SOL) experiments. Significant difference from SOL: *P < 0.05.

Cannabinoids inhibit synaptic transmission

Effects of the synthetic CB1/CB2 cannabinoid receptor agonist WIN55212-2 and of the CB1 receptor antagonist rimonabant on synaptic transmission are shown in Fig. 3 (original tracings) and Fig. 4 (statistical analysis). WIN55212-2 (5 × 10−6m) lowered the success rate of neurotransmission (Figs 3A and B and 4A) and the amplitude of successes (Figs 3A and C and 4B). Consequently, the averaged amplitude of all events (including successes and failures) – which reflects changes both in success rate and amplitude of successes – decreased strongly (Figs 3C and 4C). The parameter mean2/s.d.2 also decreased (Fig. 4D). Importantly, WIN55212-2 did not affect action potentials recorded in the presynaptic PV-FSNs (not shown). Additional superfusion of the antagonist rimonabant (10−6m) attenuated all effects of WIN55212-2 (Figs 3A–C and 4A–D).

In three additional experiments, rimonabant (10−6m) was superfused at first, and then WIN55212-2 (5 × 10−6m) was superfused in the continued presence of rimonabant. In the presence of rimonabant alone, success rate (99 ± 2% of PRE, n = 3), amplitude of successes (93 ± 5% of PRE, n = 3), amplitude of all synaptic events (92 ± 6% of PRE, n = 3) and mean2/s.d.2 (113 ± 16% of PRE, n = 3) were not changed. Lack of effect of the antagonist alone indicates that synaptic transmission was not tonically inhibited by endocannabinoids. In the presence of rimonabant, WIN55212-2 did not significantly change the success rate (98 ± 9% of the value preceding superfusion of WIN55212-2; n = 3), amplitude of successes (78 ± 18% of the value preceding superfusion of WIN55212-2; n = 3), amplitude of all synaptic events (80 ± 24% of the value preceding superfusion of WIN55212-2; n = 3) and mean2/s.d.2 (116 ± 42% of the value preceding superfusion of WIN55212-2; n = 3). Antagonism of the effects of WIN55212-2 under this protocol and under the protocol shown in Figs 3 and 4 strongly suggests that CB1 receptors were involved in the inhibition of neurotransmission by WIN55212-2.

Cannabinoids inhibit synaptic transmission presynaptically

The decreases in the success rate of neurotransmission and in the parameter mean2/s.d.2 point to a presynaptic mode of inhibition. As a next step, we carried out a conventional analysis of mIPSCs in order to verify the presynaptic action.

mIPSCs were recorded in the presence of tetrodotoxin (3 × 10−7m). During the initial reference period (PRE), the amplitude and frequency of mIPSCs were 28 ± 3 pA and 1.2 ± 0.3 Hz (n = 17), respectively. In solvent-treated control slices, the amplitude of mIPSCs decreased slightly (Fig. 5E), but the frequency of mIPSCs remained stable (Fig. 5F). In absolute numbers: the amplitude and frequency were 32 ± 4 pA and 1.0 ± 0.5 Hz, respectively, at PRE and 29 ± 4 pA and 1.1 ± 0.5 Hz at t = 20 min in the presence of solvent (n = 4). In the presence of WIN55212-2 (5 × 10−6m), the amplitude of mIPSCs was not significantly changed (Fig. 5B, C and E); in contrast, the frequency was lowered (Fig. 5A, D and F). In absolute numbers: the amplitude and frequency were 26 ± 4 pA and 1.3 ± 0.4 Hz, respectively, at PRE and 25 ± 3 pA and 0.9 ± 0.3 Hz at t = 20 min in the presence of WIN55212-2 (n = 13). Lack of effect of WIN55212-2 on the amplitude of mIPSCs indicates that WIN55212-2 does not interfere with the effect of synaptically released GABA on postsynaptic MSNs. Exclusion of the postsynaptic effect of WIN55212-2 is indirect proof of the presynaptic inhibition of neurotransmission. The decrease in mIPSC frequency shows that WIN55212-2 interferes with the vesicular release machinery in GABAergic axon terminals innervating MSNs.

Figure 5. Effects of WIN55212-2 (WIN) and solvent (SOL) on miniature IPSCs (mIPSCs) recorded in MSNs in the presence of tetrodotoxin (3 × 10−7m).

Figure 5

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mIPSCs were recorded during the initial reference period (PRE) and during superfusion with WIN (5 × 10−6m) or solvent (SOL). A, original tracings from an experiment with WIN. B, averaged mIPSCs from an experiment with WIN (same experiment as in A). C and D, cumulative probability distribution plots of amplitudes and interevent intervals of mIPSCs from an experiment with WIN (same experiment as in A). Significant difference from PRE (Kolmogorov–Smirnov test):+P < 0.05. E and F, mIPSC amplitudes and frequencies were expressed as percentages of the initial reference value PRE. Means ±s.e.m. of 13 (WIN) and 4 (SOL) experiments. Significant difference from SOL: *P < 0.05.

Lack of somatodendritic effects of cannabinoids on PV-FSNs

PV-FSNs synthesize CB1 receptors. The results described above show that cannabinoids inhibit GABA release by acting on CB1 receptors on axon terminals of PV-FSNs. The aim of the next series of experiments was to determine the consequences of activation of CB1 receptors in the somatodendritic region of PV-FSNs. Recordings were carried out in the perforated-patch mode, in order to preserve the physiological internal milieu of PV-FSNs.

In the first experiments, membrane potential was recorded in current-clamp mode and 500-ms depolarizing current injections were applied to elicit action potential salvos (Fig. 6). During the initial reference period (PRE), resting membrane potential was −60 ± 4 mV (n = 8), and the depolarization-elicited action potentials occurred at a frequency of 25 ± 6 Hz (n = 8). In solvent-treated slices, the resting membrane potential and the frequency of salvos remained constant (Fig. 6A and B). WIN55212-2 (5 × 10−6m) also did not change the resting membrane potential and the frequency of salvos (Fig. 6A–C). Thus, the cannabinoid had no obvious effect on neuronal excitability.

Figure 6. Effects of WIN55212-2 (WIN) and solvent (SOL) on resting membrane potential and evoked firing of PV-FSNs.

Figure 6

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Membrane potential was recorded in the perforated-patch mode. Firing was elicited every min by 500-ms depolarizing current injections. AP-5 (2.5 × 10−5m), DNQX (10−5m) and bicuculline (2 × 10−5m) were present in the superfusion buffer in order to block fast glutamatergic and GABAergic inputs to PV-FSNs. Means ±s.e.m. of five (WIN) and three (SOL) experiments. A, resting membrane potential. Note that the potentials were slightly different in the WIN and SOL groups already during the PRE period, probably due to statistical variability. B, firing rates during depolarizing current injections were expressed as percentages of the initial reference value PRE. C, rectangular depolarizing current and firing recorded at time points 1 and 2 (shown in B) in a typical experiment with WIN.

In the next experiments, the holding current was measured in voltage-clamp mode at a holding potential of Vh =–60 mV. During superfusion of solvent for 15 min, the holding current increased very slightly (+ 2 ± 1 pA; n = 4). The change during 15-min WIN55212-2 (5 × 10−6m) superfusion (+ 4 ± 1 pA; n = 6) was not significantly different from the change observed in the solvent group. In contrast, the GABAB receptor agonist baclofen (10−5m) caused an outward current of 14 ± 4 pA (n = 8; P < 0.05).

Effect of depolarization of the postsynaptic neuron on synaptic transmission

At many synapses, depolarization of the postsynaptic neuron leads to inhibition of transmitter release from the presynaptic axon terminal. This form of retrograde signalling is termed ‘depolarization-induced suppression of inhibition’ (DSI) in the case of GABAergic synapses and ‘depolarization-induced suppression of excitation’ (DSE) in the case of glutamatergic synapses. DSI and DSE are frequently mediated by endocannabinoids which are synthesized and released by depolarized postsynaptic neurons (for review see Wilson & Nicoll, 2002; Freund et al. 2003; Diana & Marty, 2004). In order to find a physiological role for CB1 receptors and endocannabinoids at PV-FSN → MSN synapses, we searched for DSI at these synapses.

In the first series of experiments, PV-FSN → MSN transmission was elicited every second, and the postsynaptic MSNs were depolarized (from −60 mV to 0 mV for 100 ms) by a series of nine pulses applied at 1 Hz. This depolarization did not influence the following synaptic events (Fig. 7A–C). In contrast, in cerebellar Purkinje cells the same depolarization protocol caused a robust and long-lasting (>60 s) decrease in the cumulative amplitude of spontaneous IPSCs (to 47 ± 6% of the initial reference value; n = 6; P < 0.05). This latter result verifies that the experimental conditions were suitable for observing DSI and that there was no fundamental flaw in our methods.

Figure 7. Effect of depolarization of MSNs on synaptic transmission between PV-FSNs and MSNs.

Figure 7

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Action potentials in PV-FSNs were elicited every 1 s by depolarizing current injections and the following postsynaptic events were recorded in MSNs. After 60 s (60 events), MSNs were depolarized (from −60 mV to 0 mV for 100 ms) by a series of nine pulses applied at 1 Hz. A, amplitudes of postsynaptic events were expressed as percentages of the initial reference value PRE and, in addition, moving averages including five events were calculated. Means ±s.e.m. of 13 trials in 7 neurons. B, ten events recorded immediately before the depolarizing pulses (during the initial reference period, PRE) and immediately after the depolarizing pulses. C, averages of the events shown in B.

We also used a more ‘aggressive’ protocol for eliciting DSI (see Trettel & Levine, 2003). PV-FSN → MSN neurotransmission was elicited every 3 s for 11 min. During the sixth minute, MSNs were depolarized before every synaptic event (from −60 mV to 0 mV; the depolarization started 650 ms before a synaptic event and lasted for 150 ms). However, even under this ‘aggressive’ protocol, no meaningful DSI occurred (17 trials in 8 neurons; data not shown).

Finally, we attempted to elicit endocannabinoid-mediated retrograde signalling by combining depolarization with activation of Gαq/11 protein-coupled receptors in the postsynaptic cells (see Kreitzer & Malenka, 2005; Maejima et al. 2005; Edwards et al. 2006). The model was simplified in these experiments: MSNs were patch clamped and their GABAergic input was activated by a stimulating electrode positioned in the vicinity of the recorded MSNs (i.e. all GABAergic inputs were activated; compare with Fig. 1). Neurons were depolarized before and during superfusion of the group I metabotropic glutamate receptor agonist DHPG (see Fig. 8). Nine depolarizing pulses (from −70 mV to 0 mV for 100 ms) applied at 1 Hz caused a small DSI (14% suppression) before DHPG superfusion (Fig. 8Aa). DHPG (5 × 10−5m) significantly lowered the amplitude of IPSCs (Fig. 8Ab). DSI during DHPG superfusion was stronger (21% suppression) and lasted longer than before DHPG superfusion (compare Fig. 8Ac with Fig. 8Aa). The difference between DSI in the absence and presence of DHPG was statistically significant (P < 0.05) at 10 time points. When the entire experiment was carried out in the presence of the CB1 receptor antagonist rimonabant, the effect of DHPG on IPSCs was attenuated (Fig. 8Bb; see also Edwards et al. 2006; for less than full blockade of the effects of DHPG on IPSCs by a CB1 receptor antagonist). Rimonabant fully blocked DSI before (Fig. 8Ba) and during (Fig. 8Bc) DHPG superfusion, suggesting that endocannabinoids and CB1 receptors were involved in DSI.

Figure 8. Effects of depolarization of MSNs and the metabotropic glutamate receptor agonist DHPG on IPSCs recorded in MSNs.

Figure 8

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MSNs were patched-clamped and IPSCs were elicited with an electrode positioned in their vicinity. The experiments had three phases. Aa, during the first phase, IPSCs were elicited every 1 s. After 60 s, MSNs were depolarized (from −70 mV to 0 mV for 100 ms) by a series of nine pulses applied at 1 Hz. IPSC amplitudes were expressed as percentages of the initial reference value PRE (average of IPSCs during the first 60 s). Means ±s.e.m. of 24 trials in 12 neurons. Significant difference from PRE: +P < 0.05. The inset shows IPSCs recorded before (continuous line) and after (broken line) depolarization. Ab, during the second phase, IPSCs were elicited every 6 s and DHPG was superfused after 10 min. IPSCs were averaged every 2 min and expressed as percentages of the initial reference value PRE (average of IPSCs during the first 10 min). Means ±s.e.m. of 12 experiments. Significant difference from PRE: +P < 0.05. Ac, during the third phase, DHPG was superfused further. IPSCs were elicited every 1 s, and MSNs were depolarized after 60 s as during the first phase. Means ±s.e.m. of 22 trials in 12 neurons. Ba–Bc, the protocol was identical with that shown in Aa–Ac, but all experiments were carried out in the presence of the CB1 receptor antagonist rimonabant (10−6m). Ba, means ±s.e.m. of 12 trials in 6 neurons. Bb, means ±s.e.m. of 6 experiments. Significant difference from PRE: +P < 0.05. Bc, means ±s.e.m. of 9 trials in 6 neurons.

Synaptic transmission between recurrent axon collaterals of MSNs and MSNs

Characterization of synaptic transmission

The secondary aim of the present study was to determine the effects of cannabinoids on neurotransmission between MSNs. MSN–MSN pairs were patched, and synaptic connections between the pairs were tested (in most cases in both directions). Table 1 shows the parameters of synaptic transmission during the initial reference period (PRE). The coupling ratio between MSN–MSN pairs was 12%. In the case of synaptically coupled pairs, depolarization of presynaptic MSNs every 3 s (0.33 Hz) elicited postsynaptic events in MSNs with a latency of 2.6 ms. The success rate of synaptic transmission was 18%. The mean amplitude of successful action potential-coupled postsynaptic events was 13 pA. The mean amplitude of all action potential-coupled postsynaptic events (including successes and failures) was 2 pA (much lower than the amplitude of successes, due to low success rate). The data in Table 1 indicate that the MSN → MSN synaptic connection is weaker than the PV-FSN → MSN connection, with marked differences in coupling ratios, success rates and amplitudes of all events. Abolition of postsynaptic events by bicuculline verifies that synaptic transmission between MSNs was mediated by GABAA receptors (Fig. 11A and B; see below). For evaluation of cannabinoid-evoked changes, all further values of synaptic parameters were expressed as percentages of PRE.

Figure 11. Effects of WIN55212-2 (WIN), rimonabant (RIM) and bicuculline on synaptic transmission between recurrent axon collaterals of MSNs and MSNs: original tracings.

Figure 11

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In this particular experiment, action potentials in presynaptic MSNs were elicited every 0.5 s by depolarizing current injections and the following synaptic events were recorded in postsynaptic MSNs. A, 200 events recorded during the initial reference period (PRE), during superfusion of WIN55212-2, during combined superfusion of WIN + RIM and during superfusion of bicuculline. B, averages of successes and all events (including successes and failures) from the 200 events shown in A.

Cannabinoids inhibit synaptic transmission

Effects of WIN55212-2 on synaptic transmission are shown in Fig. 9 (original tracings) and Fig. 10 (statistical analysis). WIN55212-2 (5 × 10−6m) lowered the success rate of neurotransmission (Figs 9A and B and 10A). The amplitude of successes was only slightly lowered (Figs 9C and 10B). The amplitude of all events (including successes and failures) strongly decreased (Figs 9C and 10C), reflecting the strong decrease in success rate. The parameter mean2/s.d.2 also decreased (Fig. 10D). Importantly, WIN55212-2 did not affect action potentials recorded in the presynaptic MSNs (not shown).

Figure 9. Effects of WIN55212-2 (WIN) on synaptic transmission between recurrent axon collaterals of MSNs and MSNs: original tracingsM.

Figure 9

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Action potentials in presynaptic MSNs were elicited every 3 s by depolarizing current injections and the following synaptic events were recorded in postsynaptic MSNs. A, • represent the amplitudes of postsynaptic events. B, 100 events recorded during two initial periods (PRE-1 and PRE-2) and during superfusion of WIN55212-2 (the 5-min recording periods are indicated in A). The success rate was 69%, 69% and 31% during PRE-1, PRE-2 and WIN, respectively. C, averages of successes and all events (including successes and failures) from the 100 events shown in B. Synaptic transmission remained stable during the two initial periods PRE-1 and PRE-2; WIN55212-2 caused marked inhibition.

Figure 10. Effects of WIN55212-2 (WIN) on synaptic transmission between recurrent axon collaterals of MSNs and MSNs: statistical evaluation.

Figure 10

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Action potentials in presynaptic MSNs were elicited every 3 s by depolarizing current injections and the following synaptic events were recorded in postsynaptic MSNs. Parameters of synaptic transmission were calculated for 100 events (recorded during 5-min periods) and expressed as percentages of the initial reference value PRE. Means ±s.e.m. of six experiments. Significant difference from PRE: +P < 0.05.

In one experiment, WIN55212-2 (10−6m) was superfused at first and then the antagonist rimonabant (10−6m) was given in addition (Fig. 11). WIN55212-2 eliminated the successes but successes reappeared in the presence of rimonabant (Fig. 11A and B). Antagonism by rimonabant points to the involvement of CB1 receptors in synaptic inhibition also at the MSN → MSN synapse.

The changes observed in the presence of WIN55212-2 were most probably not due to run-down. First, because all four determined parameters remained stable (or even increased slightly) during the initial reference period PRE (see the two time points during PRE in Fig. 10 and the PRE-1 and PRE-2 periods in Fig. 9). Second, because the changes developed with a time pattern matching very well WIN55212-2 superfusion (see Fig. 9). Third, the reversal of the effects of WIN55212-2 by rimonabant (Fig. 11) also argues against run-down. Unfortunately, no more experiments could be performed with the protocol of Fig. 11, because the MSN → MSN transmission experiments were extremely time consuming (note that 251 MSN → MSN synaptic connections have been tested; Table 1).

Lack of somatodendritic effects of cannabinoids on MSNs

As in the case of PV-FSNs, we searched for somatodendritic actions of cannabinoids on MSNs. Again, the recordings were carried out in the perforated-patch mode. Membrane potential was recorded in current-clamp mode and 500 ms depolarizing current injections were applied to elicit action potential salvos. During the initial reference period (PRE), resting membrane potential was −57 ± 3 mV (n = 5), and the depolarization-elicited action potentials occurred at a frequency of 21 ± 1 Hz (n = 5). WIN55212-2 (5 × 10−6m) did not change the resting membrane potential or the frequency of salvos (n = 5; not shown).

Discussion

PV-FSNs in the caudate-putamen were unambiguously identified by use of transgenic mice. Simultaneous electrophysiological recording from identified neuron pairs showed that GABAergic synaptic transmission between PV-FSNs and MSNs and between axon collaterals of MSNs and MSNs operates in the mouse, and that activation of CB1 cannabinoid receptors leads to presynaptic inhibition of transmission at both synapses.

Properties of neurons in the caudate-putamen

PV-FSNs constitute only 0.7% of the neuronal population in the caudate-putamen (Tepper & Bolam, 2004). The expression of EGFP in the caudate-putamen is high specific for parvalbumin-containing neurons in the transgenic mouse line used by us (Meyer et al. 2002). Use of these mice and fluorescence optical techniques allowed the unambiguous identification of PV-FSNs based on a neurochemical marker before electrophysiological recording. PV-FSNs do not greatly differ morphologically from the majority of neurons in the caudate-putamen when observed with near-infrared video microscopy during patch-clamping. Although the electrophysiological properties of PV-FSNs differ from the properties of MSNs, the differences are not sufficiently robust for the unambiguous identification of PV-FSNs (at least in the slice preparation used by us). Thus, the fluorescent marker is an important advantage for identifying these rare neurons, and the identification method may significantly facilitate electrophysiological studies on PV-FSNs in the future.

Since MSNs constitute 97% of the neuron population in the caudate-putamen (Tepper & Bolam, 2004), the probability is very high (>98%) that medium-sized neurons without fluorescence were MSNs (our selection criteria exclude large cholinergic interneurons and PV-FSNs).

Several properties of the PV-FSNs in our study (high firing rates, short action potentials, marked afterhyperpolarization) are similar to those seen in previous studies (Kawaguchi, 1993; Plenz & Kitai, 1998; Koos & Tepper, 1999; Bracci et al. 2002; Centonze et al. 2003). However, in contrast to these previous studies, no intermittent firing was observed in our study. The resting membrane potential in our study was similar to that determined by Plenz & Kitai (1998), but higher than the values determined by Kawaguchi (1993) and Bracci et al. (2002). Three major differences in experimental conditions may explain the differences between our observations and the previous observations. (i) Mice were used in the present study, whereas rats were used in most of the previous studies. (ii) Twelve- to 18-day-old-animals were used at present, whereas young adult or adult animals were used in most of the previous studies. (iii) Recordings were carried out at room temperature in the present study, whereas higher temperatures (>31°C) were used in the previous studies. The properties of MSNs have also been determined in previous studies (for example, Kawaguchi et al. 1989; Nisenbaum et al. 1994; Plenz & Kitai, 1998; Tepper et al. 1998; Koos & Tepper, 1999; Czubayko & Plenz, 2002). The resting membrane potential of MSNs in our study was somewhat higher than in the previous studies. Moreover, slow depolarizing ramps were not apparent in the present study, but were observed in the previous studies. The three above-mentioned differences in experimental conditions also exist in the case of MSNs, and may explain the differing observations on the properties of MSNs. We believe, however, that the different age of the animals is the most important reason for the differences, because the properties of MSNs change strongly during the first three postnatal weeks (Tepper et al. 1998). According to this latter study, the membrane potential is higher, and inward rectification is only weakly expressed in animals having the age of the animals used in our study.

Synaptic connections between identified neurons in the caudate-putamen

Synaptic connection between FSNs and MSNs has been recently studied in slices prepared from brains of adults rats (Koos & Tepper, 1999, 2002; Koos et al. 2004). In these studies FSNs were identified based on their morphology, electrophysiological properties and the presence of parvalbumin (detected by immunohistochemistry in one study). It was only recently that the operation of synaptic connections between axon collaterals of MSNs and MSNs has been unequivocally demonstrated in rat brain by paired recording (Tunstall et al. 2002; Czubayko & Plenz, 2002; Koos et al. 2004). A synopsis of these studies indicates that the MSN → MSN synaptic connection is weaker than the PV-FSN → MSN connection, as reflected by lower coupling ratios, success rates of synaptic transmission and numbers of involved release sites (for review see Plenz, 2003; Tepper et al. 2004).

Our results extend these previous observations by showing that synaptic connections between PV-FSNs and MSNs, and between recurrent axon collaterals of MSNs and MSNs, also operate in the mouse. Like in the rat, MSN → MSN synaptic connections were weaker than PV-FSN → MSN connections. Some differences between our observations (for example, the success rate of the MSN → MSN transmission was somewhat low in our study) and the above-mentioned studies may be due to species differences and differences in experimental conditions. Thus, 12–18-day-old-mice were used in the present study, and recordings were carried out at room temperature. In contrast, in most of the above-mentioned studies, adult rats were used and recordings were carried out at 32–36°C.

Activation of CB1 receptors inhibits synaptic transmission

In our previous study (Szabo et al. 1998) and in a more recent study (Centonze et al. 2005), all GABAergic inputs to MSNs were non-selectively stimulated; accordingly, the synapses inhibited by the cannabinoids could not be identified. The present study clearly shows that activation of CB1 receptors leads to inhibition of both PV-FSN → MSN and MSN → MSN synaptic transmission. This is an important advancement of knowledge, because the two synaptic connections play different roles in the function of the basal ganglia.

Although WIN55212-2 is a synthetic mixed CB1/CB2 cannabinoid receptor agonist (Howlett et al. 2002; Pertwee, 2005), it is very likely that it inhibited neurotransmission by acting on CB1 receptors. First, because CB2 receptors are only very weakly expressed in the rodent brain (Munro et al. 1993; Mackie, 2005), second, because effects of WIN55212-2 were abolished by the CB1-selective antagonist rimonabant. Importantly, WIN55212-2 has no affinity for a wide range of receptors and ion channels (Kuster et al. 1993).

The CB1 receptor antagonist rimonabant, superfused alone, did not enhance the PV-FSN → MSN synaptic transmission. This observation indicates that endocannabinoids do not tonically inhibit synaptic transmission between the two types of neurons.

Two kinds of depolarizing protocols did not elicit DSI at the PV-FSN → MSN synapse. When IPSCs due to stimulation of all GABAergic inputs to MSNs were studied, a small DSI (14% suppression) was observed. It is likely that this DSI is due to endocannabinoid-mediated retrograde inhibition of the MSN → MSN synaptic transmission. This DSI is clearly weaker than DSI and DSE observed under similar experimental conditions in other brain regions (for example, Wilson & Nicoll, 2001; Wallmichrath & Szabo, 2002; Brenowitz & Regehr, 2003; Trettel & Levine, 2003; Szabo et al. 2004; for review see Wilson & Nicoll, 2002; Freund et al. 2003; Diana & Marty, 2004). In agreement with the weak DSI in our experiments, no DSE was observed in MSNs in a recent study (Kreitzer & Malenka, 2005). Taken together, depolarization alone elicits either no or only weak endocannabinoid-mediated retrograde signalling between MSNs and their afferent axons. DSI was enhanced in the presence of the group I metabotropic glutamate receptor agonist DHPG. It has been repeatedly observed that endo- cannabinoid release is enhanced when depolarization is combined with activation of Gαq/11 protein-coupled metabotropic glutamate receptors in the postsynaptic cells (Maejima et al. 2005; Edwards et al. 2006), specifically also in MSNs (Kreitzer & Malenka, 2005). Depolarization combined with high-frequency electrical stimulation of afferent axons can also lead to endocannabinoid release and long-term depression of the glutamatergic input to MSNs (see Gerdeman et al. 2002; Ronesi et al. 2004).

Synaptic transmission is inhibited presynaptically

At both studied synapses, a presynaptic mode of inhibition is supported by results which exclude a postsynaptic action, and by results which directly point to a decreased transmitter release probability from presynaptic axon terminals.

Three observations indicate that the cannabinoid WIN55212-2 did not interfere with the activation of GABAA receptors in postsynaptic MSNs. First, WIN55212-2 did not change the amplitude of mIPSCs. Second, in our previous study, WIN55212-2 also did not change currents evoked by the GABAA receptor agonist muscimol in postsynaptic MSNs (Szabo et al. 1998). Third, in the case of the MSN → MSN synapse, the amplitude of successful synaptic events remained constant in the presence of a strong decrease in success rate (obvious at the 17.5 and 22.5-min evaluation points in Fig. 10).

Three observations point to depressed transmitter release probability from axon terminals of PV-FSNs and recurrent axon terminals of MSNs. (i) The success rate of neurotransmission was decreased. (ii) The decrease in the parameter mean2/s.d.2 is also thought to reflect depressed transmitter release (see Clements, 1990; Koos & Tepper, 2002; Galante & Marty, 2003). (iii) Finally, the reduction of mIPSC frequency is an additional proof of the inhibition of the release of synaptic vesicles (unfortunately, since MSNs receive GABAergic input from several sources, it is not known, from which of the afferent axons release was inhibited). The lowering of mIPSC frequency indicates that WIN55212-2 inhibits transmitter release also when release is not triggered by calcium influx through voltage-dependent calcium channels – probably by directly interfering with the synaptic vesicle release mechanism. Such a mechanism has been repeatedly (for example, Szabo et al. 2004), but not always (for example, Hoffman & Lupica, 2000), observed at other synapses depressed by cannabinoids.

Selective presynaptic versus somatodendritic action

Stimulation of many Gαi/o protein-coupled receptors elicits outward potassium currents and thus leads to hyperpolarization and inhibition of neuronal firing. For example, stimulation of dopamine D2 receptors, α2-adrenoceptors and GABAB receptors lowers the firing rate of neurons (Lacey et al. 1987; Szabo et al. 1996; Than & Szabo, 2002). WIN55212-2 did not change the membrane potential, evoked firing and holding current of PV-FSNs and MSNs. In contrast, the GABAB receptor agonist baclofen elicited the expected outward potassium current in PV-FSNs. The lack of effect of WIN55212-2 is surprising, because PV-FSNs and MSNs express CB1 receptors, and activation of these receptors in the axon terminals of these neurons causes marked presynaptic inhibition. We observed a similar lack of somatodendritic effects in the presence of marked axon terminal effects in the case of cerebellar cortical interneurons (Szabo et al. 2004) and subthalamo-pallidal projection neurons (Freiman & Szabo, 2005). Although cannabinoids sometimes elicit somatodendritic effects (Huang et al. 2001; Kreitzer et al. 2002), a preferential presynaptic action probably is the rule. The reason for the preferential axon terminal effect of cannabinoids is probably the preferential localization of CB1 receptors on axon terminals (Irving et al. 2000). A recent study suggests that this polarity is due to continuous endocytotic removal of CB1 receptors from the somatodendritic cell membrane but not the axonal cell membrane (Leterrier et al. 2006).

Role of CB1 receptors in the basal ganglia

Using the results obtained in the present study, a rather complete picture can be constructed on the localization and function of CB1 receptors in the caudate-putamen. CB1 receptors are present on axon terminals of PV-FSNs and recurrent axon collaterals of MSNs, and the receptor activation leads to presynaptic inhibition of transmission between these neurons and their postsynaptic targets, the MSNs. Other neurons in the caudate-putamen – neuropeptide Y/NOS/somatostatin-positive interneurons, calretinin-positive interneurons and cholinergic interneurons – do not synthesize CB1 receptors (Marsicano & Lutz, 1999; Hohmann & Herkenham, 2000). As mentioned in the Introduction, glutamatergic afferent axons from the cortex possess CB1 receptors, the activation of which leads to inhibition of excitatory neurotransmission. Finally, dopaminergic afferent axons from the substantia nigra and GABAergic afferent axons from the globus pallidus do not possess CB1 receptors (Herkenham et al. 1991a; Matsuda et al. 1993; Szabo et al. 1999; Köfalvi et al. 2005).

A typical motor effect of cannabinoids is catalepsy (Sanudo-Pena et al. 1999). Since cannabinoids modulate GABAergic and glutamatergic neurotransmission in the basal ganglia at least at 11 sites (see Fig. 6 in Szabo & Schlicker, 2005), it is difficult to give a simple and unequivocal explanation for the catalepsy elicited by systemically administered cannabinoids.

Conclusion

We established a technique which permits analysis of synaptic transmission involving rare neurons in the caudate-putamen. Employing transgenic mice allowed identification of parvalbumin-positive neurons based on their green fluorescence. Transmission between identified neurons was studied by paired recording. Activation of CB1 cannabinoid receptors by exogenous agonists led to presynaptic inhibition of PV-FSN → MSN neurotransmission. Exogenous agonists also inhibited MSN → MSN neurotransmission. The GABAergic input to MSNs was only weakly inhibited by endocannabinoids released by depolarized postsynaptic MSNs. Cannabinoids acted with remarkable preference on axon terminals: GABA release from axon terminals was strongly inhibited, but no somatodentritic effects were observed.

Acknowledgments

This study was supported by the Deutsche Forschungs- gemeinschaft (Sz 72/5-1) and the Hermann und Lilly Schilling-Stiftung. We thank Klaus Starke for his comments on the manuscript.

Supplemental material

The online version of this paper can be accessed at:DOI: 10.1113/jphysiol.2006.114272 http://jp.physoc.org/cgi/content/full/jphysiol.2006.114272/DC1 and contains supplemental material on the determination of the parameters of synaptic transmission.

This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com

tjp0575-0789-s1.pdf (160.4KB, pdf)
Supplemental data
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