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6490 The Journal of Neuroscience, July 13, 2005 25(28):6490 6498

Cellular/Molecular

Target-Dependent Use of Coreleased Inhibitory Transmitters at Central Synapses
Guillaume P. Dugue,1* Andrea Dumoulin,2* Antoine Triller,2 and Stephane Dieudonne1
Laboratoire de Neurobiologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique, Unite Mixte de Recherche 8544, and 2Laboratoire de Biologie Cellulaire de la Synapse, Institut National de la Sante et de la Recherche Medicale U497, Ecole Normale Superieure, 75005 Paris, France
Corelease of GABA and glycine by mixed neurons is a prevalent mode of inhibitory transmission in the vertebrate hindbrain. However, little is known of the functional organization of mixed inhibitory networks. Golgi cells, the main inhibitory interneurons of the cerebellar granular layer, have been shown to contain GABA and glycine. We show here that, in the vestibulocerebellum, Golgi cells contact both granule cells and unipolar brush cells, which are excitatory relay interneurons for vestibular afferences. Whereas IPSCs in granule cells are mediated by GABAA receptors only, Golgi cell inhibition of unipolar brush cells is dominated by glycinergic currents. We further demonstrate that a single Golgi cell can perform pure GABAergic inhibition of granule cells and pure glycinergic inhibition of unipolar brush cells. This specialization results from the differential expression of GABAA and glycine receptors by target cells and not from a segregation of GABA and glycine in presynaptic terminals. Thus, postsynaptic selection of coreleased fast transmitters is used in the CNS to increase the diversity of individual neuronal outputs and achieve target-specific signaling in mixed inhibitory networks. Key words: GABA; glycine; corelease; vestibular system; cerebellum; inhibition

Introduction

GABA and glycine are the fast inhibitory transmitters of the mammalian CNS. Although each transmitter acts on specific ionotropic receptors, both inhibitory systems are tightly linked. Unlike other neurotransmitter systems that define segregated groups of neurons, GABA and glycine are frequently accumulated in the same cells (Ottersen et al., 1988; Todd, 1990; Schneider and Fyffe, 1992), which are thus considered to be mixed inhibitory neurons. GABA and glycine colocalize in the majority of inhibitory terminals in the brainstem (Wentzel et al., 1993; Dumba et al., 1998), spinal cord (Ornung et al., 1994; Taal and Holstege, 1994; Todd et al., 1996), and cerebellar granular layer (Ottersen et al., 1988). Additional evidence for the prevalence of mixed interneurons is the colocalization of the glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, with GlyT2, the neuronal plasma membrane glycine transporter (Tanaka and Ezure, 2004). Furthermore, vesicular loading of both transmitters is operated by a common vesicular transporter VIAAT (or VGAT) (McIntire et al., 1997; Sagne et al., 1997). Thus, a large proportion of inhibitory transmission in the hindbrain is mediated by coreleased GABA and glycine. Functional corelease of GABA and glycine by the same vesicle has been demonstrated in the spinal cord (Jonas et al., 1998) and
Received Jan. 4, 2005; revised May 27, 2005; accepted May 28, 2005. This work was supported by Centre National de la Recherche Scientifique, Institut National de la Sante et de la Recherche Medicale, and Ecole Normale Superieure and an Action Concertee Incitative grant from the French Min istry of Research (S.D.). We thank P. Ascher, A. Feltz, and C. Lena for helpful discussion and comments on this manuscript and Boris Barbour for help with data analysis. This work is dedicated to the late Coco Gerschenfeld. *G.P.D. and A.D. contributed equally to this work. Correspondence should be addressed to S. Dieudonne, Laboratoire de Neurobiologie, Unite Mixte de Recherche 8544, Ecole Normale Superieure, 46, rue dUlm, 75005 Paris, France. E-mail: dieudon@biologie.ens.fr. DOI:10.1523/JNEUROSCI.1500-05.2005 Copyright 2005 Society for Neuroscience 0270-6474/05/256490-09$15.00/0
brainstem (OBrien and Berger, 1999; Russier et al., 2002; Nabekura et al., 2004; Awatramani et al., 2005), in which miniature IPSCs display both GABAA and glycine components. These results support the idea that mixed interneurons mediate GABAergic and glycinergic cotransmission. A challenging possibility would be that individual mixed neurons perform cotransmission at some of their synapses but pure glycinergic or GABAergic inhibition at others. Several sets of data suggest that the phenotype of inhibition can be adjusted at individual synapses by a change in the content of neurotransmitters (Nabekura et al., 2004) and/or by the expression of different ratios of postsynaptic GABAA receptors (GABAARs) and glycine receptors (GlyRs) (OBrien and Berger, 2001), which may be modified by maturation processes (Kotak et al., 1998; Korada and Schwartz, 1999; Geiman et al., 2000; Keller et al., 2001). In all cases, however, the comparison of immunohistochemical and/or functional phenotypes of different synaptic terminals of a given mixed neuron is still lacking. In the cerebellar cortex, Golgi cells have been reported to contain both GABA and glycine (Ottersen et al., 1988), although their identified targets, the granule cells, display GABAergic IPSCs exclusively (Kaneda et al., 1995; Rossi and Hamann, 1998; Farrant and Brickley, 2003). It has been postulated recently that Golgi cells also inhibit unipolar brush cells (UBCs) (Mugnaini and Floris, 1994; Dino et al., 2000), a group of excitatory interneurons of the vestibulocerebellum. Using a combination of paired recordings and immunohistochemical techniques, we examined whether GABA and glycine are used differentially at Golgi cell synapses established on granule cells and on UBCs.

from borosilicate glass capillaries (Hilgenberg, Maisfeld, Germany) with a vertical puller (David Kopf Instruments, Tujunga, CA). For Golgi cell recordings, the pipette was filled with an intracellular solution containing the following (in mM): 150 K-gluconate, 6 NaCl, 10 HEPES, 1 MgCl2, 4 ATP-Mg, and 0.4 GTP-Na, with pH adjusted to 7.35 by KOH. For UBC and granule cell recordings, the pipette was filled with an intracellular solution containing the following (in mM): 145 CsCl, 10 HEPES, 1 EGTA, 5 MgCl2, 0.1 CaCl2, 4 ATP-Na2, and 0.4 GTP-Na, with pH adjusted to 7.35 by N-methyl-D-glucamine. Alexa 488 at 100 M (Molecular Probes, Eugene, OR) was added to these intracellular solutions to allow on-line visualization of recorded cells with a high-resolution digital camera (CoolSNAP HQ; Roper Scientific, Trenton, NJ) and a wavelength switcher illumination system (Lambda DG-4; Sutter Instruments, Novato, CA). In some cases, the dye was replaced by 4 mg/ml Neurobiotin (Vector Laboratories) for off-line confocal observation. Recordings were made with an Axopatch 200B and an Axopatch-1D amplifier (Axon Instruments, Union City, CA). The signal was filtered at 2 kHz and sampled at 520 kHz with a Digidata 1200 interface (Axon Instruments). Golgi cells were recorded in the current-clamp mode. Action potentials were elicited by short depolarizing current steps (500 pA for 1 ms). UBCs and granule cells were voltage clamped at 70 mV. Ionotropic glutamate receptors were blocked with 2 M 1,2,3,4-dihydro-2,3-dioxo-benzoquinoxaline-7-sulfonamide (NBQX) and 50 M D-APV. GlyRs were blocked with nM strychnine. GABAARs were blocked with 5 M SR 95531 [6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid hydrobromide] (SR). All drugs were bath applied. Identification of cell types during electrophysiological recordings. Cells could be easily identified according to their capacitive currents and morphology. Granule cells had a fast monoexponential capacitive current with a capacitance of 4 pF. UBCs had a slower capacitive current and a capacitance between 10 and 20 pF. UBCs and granule cells were visualized during recording (see above) and could be easily distinguished according to their morphology. Granule cells had a classical morphology with a small round soma (m) and several dendrites. UBCs had an ovoid cell body (10 m) and a single thick dendrite terminating in a spray of dendrioles. Golgi cells could be unambiguously differentiated from other cells in the granular layer by the size of their soma (m) and their biexponential capacitive current (Dieudonne, 1995). Combined electrophysiological recording and immunohistochemistry. Neurobiotin (4 mg/ml; Vector Laboratories) was added to the intracellular solution. Golgi cells were filled with the tracer through the patch pipette. After recording, slices were fixed overnight in 4% paraformaldehyde at 4C and processed for immunocytochemistry. Neurobiotin was revealed with FITC-coupled streptavidin. An anti-gephyrin monoclonal antibody (see above) was used and revealed by a Cy3-coupled donkey anti-mouse IgG. Two non-overlapping populations of UBCs can be distinguished by their immunoreactivity against the calcium-binding protein CR and mGluR1 (Nunzi et al., 2002). To label all UBCs independently of the expressed marker, a mixture of rabbit polyclonal anticalretinin and rabbit polyclonal anti-mGluR1 antibodies (see above) was revealed with the same secondary antibody (a Cy5-coupled goat anti-rabbit IgG). Images were acquired on a Leica SP2 confocal microscope as described previously. Electrophysiological data analysis. Data were analyzed off-line with custom-made routines written for the Igor PRO analysis environment (WaveMetrics, Lake Oswego, OR). For Golgi cellUBC and Golgi cell granule cell paired recordings, the response latency was measured as the delay between the action potential peak and the half-rise point of the postsynaptic current. IPSCs whose amplitude was below 20 pA were excluded from latency measurements to limit the effect of experimental noise on the position of the half-rise point. A standard algorithm was used to detect spontaneous IPSCs in UBCs and granule cellUBC paired recordings. It consisted of a threshold detection performed on a discrete time derivative of the recorded trace, as described previously (Vincent and Marty, 1993). Cross-correlograms were generated based on the time of occurrence of IPSCs in each cell, as measured at their half-rise point. The average jitter distribution was generated by pooling all data after offsetting the cross-correlogram for each pair by the value of the mean lag. A predicted jitter distribution for granule cellUBC pairs was com-

6492 J. Neurosci., July 13, 2005 25(28):6490 6498
Figure 1. Golgi cells evoke IPSCs in granule cells and UBCs with different pharmacological profiles. A, B, Short depolarizing current steps (500 pA, 1 ms) triggered single action potentials in Golgi cells. Superposition of 10 consecutive action potentials and the corresponding current responses in a granule cell (A) and in a UBC (B). Vh, Holding potential (70 mV). C, E, Histograms of the response latency in a granule cell and in a UBC, respectively. D, F, Average amplitude histograms of the current response in six granule cells and seven UBCs, respectively (bin, 20 pA). Scaled histograms of the noise amplitude (7.9 4.6 pA in granule cells and 4.9 1.6 pA in UBCs) are superimposed in black (bin, 10 pA). G, Example of the effect of 5 M SR on the mean postsynaptic response in a granule cell. H, Plot of the absolute amplitude of postsynaptic responses in six granule cells in control conditions and after application of 5 M SR (*p 0.04, sign test). I, Effect of 600 nM strychnine on the mean postsynaptic response in a UBC. J, Plot of the absolute amplitude of postsynaptic responses in eight UBCs in control conditions, after the application of 600 nM strychnine (*p 0.01, sign test) and after consecutive application of 5 M SR. ctrl, Control; str, strychnine. puted from Golgi cellUBC and Golgi cell granule cell pairs as follows: first, for each pair, jitter distribution was offset by the value of the mean lag; then jitter distributions were pooled for granule cells and UBCs; finally, the two jitter distributions were convolved to produce the prediction for the granule cellUBC jitter based on independence of release at all the synapses. Statistical analysis. Electrophysiological results are reported as mean SD to account for cell-to-cell variability and skewed non-Gaussian distributions. For the same reason, all statistical tests were nonparametric, to prevent any assumption regarding parameter distribution. The MannWhitney U test and the sign test were used to test for statistical differences between two independent and two related samples, respectively. The bootstrap version of the KolmogorovSmirnov test was used to compare two distributions. Morphological quantifications are reported as mean SEM, and Students t test was used to conclude that the percentages of colocalization of different markers were not significantly different between cerebellar lobules. For all tests, significance was assumed if p 0.05.

was similar (MannWhitney U test) in granule cells (pA; n 6) and in UBCs (pA; n 8) (Fig. 1 D, F ). The latency of the responses was measured in both types of paired recordings (Fig. 1C,E), and Gaussian curves were fitted to latency histograms. Their mean value was taken as an estimate of the transmission delay, and their full-width at half-maximum was taken as a measure of the transmission jitter. The transmission delays measured in granule cells (1.10 0.30 ms; n 6) and in UBCs (1.05 0.10 ms; n 6) were similar (MannWhitney U test) and in agreement with a monosynaptic transmission at nearphysiological temperature. The transmission jitter was also similar (MannWhitney U test) in granule cells (0.35 0.15 ms; n 6) and UBCs (0.35 0.10 ms; n 6). Thus, the two synapses are comparable in terms of speed of transmission and postsynaptic conductances. We characterized the pharmacological profile of postsynaptic responses to determine which neurotransmitter was used at each synapse (Fig. 1G,I ). In granule cells, bath application of 5 M SR, an antagonist of GABAARs, decreased the response amplitude to 2.8 5.2% (n 6) of its control value (Fig. 1 H), in agreement with published data (Rossi and Hamann, 1998; Farrant and Brickley, 2003). In contrast, bath application of 600 nM strychnine, an antagonist of GlyRs, decreased significantly ( p 0.01, sign test) the mean amplitude of the response recorded from UBCs to 35 34% (n 8) of its control value (Fig. 1 J). In some cases (n 3), the block was complete, indicating a purely glycinergic transmission. In other cases, a variable strychnine-resistant component remained and was reduced to 1.9 2.8% (n 4) of its value by application of 5 M SR. Thus, Golgi cells evoke GABAergic IPSCs in granule cells and glycinergic or mixed GABAergic and glycinergic IPSCs in UBCs. The absence of a GABAergic component at some Golgi cell to UBC connections may represent the extremum of a continuous distribution of GABAergic contributions at different Golgi cell to UBC connections. To better estimate this distribution, we examined the effect of 600 nM strychnine on the spontaneous synaptic activity recorded from 47 UBCs in the presence of 2 M NBQX and 50 M D-APV (Fig. 2C). In 38% of the cells, the cumulative activity (CA) (the product of the mean amplitude with the mean frequency of spontaneous IPSCs) was totally suppressed, that is, no event was detected (Fig. 2 A). In the other cells, the CA was partially inhibited to an average of 40 31% (n 29) of its control value ( p 0.01, sign test) (Fig. 2 B). The remaining activity was blocked by application of 10 M SR (n 17) (Fig. 2 B). Thus, as with paired recordings, a large fraction of UBCs received pure glycinergic inhibition. In addition, the frequency of spontaneous IPSCs was significantly ( p 0.01, sign test) decreased to 17 20% (n 16) of its control value after application of 500 nM tetrodotoxin, indicating that the majority of spontaneous IPSCs were action potential dependent (data not shown). A single Golgi cell can mediate GABAergic and glycinergic inhibition The expression pattern of several histochemical markers has led to the hypothesis that the cerebellar granular layer may host several subtypes of inhibitory interneurons (Geurts et al., 2003). We thus wondered whether inhibition of UBCs was mediated by a specific Golgi cell subtype. Individual Golgi cells (n 3) were labeled with Neurobiotin, and the putative synaptic contacts made by their axons in the granular layer were identified by their apposition with gephyrin, a postsynaptic marker of both GABAergic and glycinergic synapses (Moss and Smart, 2001). In addition, the dendritic brushes of UBCs were revealed by immu-

Results

Golgi cells mediate GABAergic inhibition of granule cells and glycinergic inhibition of UBCs Paired recordings were performed in cerebellar slices of lobule X (the nodular lobule of the vestibulocerebellum) to characterize the synapses between Golgi cells and their two target neurons, granule cells and UBCs. Action potentials in Golgi cells triggered current responses in 8 of 21 recorded granule cells (Fig. 1 A) and in 11 of 25 recorded UBCs (Fig. 1 B) (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Longlasting paired recordings of connected Golgi cells and granule cells and of connected Golgi cells and UBCs were obtained in six and eight cases, respectively. The mean amplitude of the response
J. Neurosci., July 13, 2005 25(28):6493
types of postsynaptic partners, spontaneous IPSCs were recorded simultaneously from neighboring granule cells (gIPSCs) and UBCs (uIPSCs) (Fig. 3A). Antagonists of ionotropic glutamate receptors (NBQX and D-APV) were added to the bath to prevent synaptically triggered synchronization of presynaptic Golgi cells. In each recorded pair, the temporal relationship between gIPSCs and uIPSCs was estimated by generating cross-correlation histoFigure 2. The spontaneous inhibitory activity recorded from UBCs has a variable sensitivity to strychnine. A, B, Plots of the peak amplitude of detected spontaneous IPSCs, illustrating the two pharmacological profiles observed: a fully glycinergic profile (A) or grams (see Materials and Methods). In a mixed glycinergic and GABAergic profile (B). C, Histogram showing the remaining cumulative activity (product of the amplitude 75% (n 21 of 28) of paired recordings, and frequency) after the bath application of M strychnine (in percentage of the control cumulative activity). The cross-correlograms had a marked peak at approximately zero (Fig. 3B). On average, detection threshold was set to 15 pA. str, Strychnine. this peak accounted for 21 16% of the events detected in UBCs and for 34 26% of the events detected in granule cells. The mean lag between gIPSCs and uIPSCs (mean value of the Gaussian fit) was 0.02 0.18 ms (n 12) and never exceeded 0.36 ms. The average jitter of the lag between gIPSCs and uIPSCs (full-width at halfmaximum of the Gaussian fit) was 0.50 0.12 ms (n 12). The distribution of the jitter was indistinguishable (bootstrap KolmogorovSmirnov) from that obtained by convolution of the data from Golgi granule cell and GolgiUBC pairs (Fig. 3C) (see Materials and Methods), which predicts the jitter for independent release at synapses of the same axon. Thus, synchronous gIPSCs and uIPSCs are elicited by the same presynaptic Golgi cell. The pharmacological profile of gIPSCs and uIPSCs was investigated by bath application of 600 nM strychnine. gIPSCs were detected during the control period, during drug application, and after washout. As expected for purely GABAergic currents, the amplitude and the frequency of spontaneous gIPSCs were not affected (sign test) by strychnine (and 101 35% of their control value, respectively; n 14). Spontaneous gIPSCs are thus reliable reporters of the activity of presynaptic Golgi cells and can be used as time marks to analyze the effect of strychnine on synchronous uIPSCs arising from the same presynaptic Golgi cell. Average synchronous uIPSCs were significantly ( p 0.01, sign test) and reversibly blocked to 30 32% (n 12) of their control amplitude during strychnine application (Fig. 3 D, E). In two Figure 3. Pharmacology of IPSCs triggered by the same presynaptic Golgi cell in granule cells pairs, synchronous uIPSCs were completely blocked by strychand UBCs. A, Synchronous spontaneous IPSCs (arrowheads) in a granule cell (top) and in a UBC nine. Therefore, a single Golgi cell can mediate simultaneously (bottom). Vh, Holding potential (70 mV). B, Corresponding cross-correlogram (bin, 50 s). C, GABAergic inhibition of granule cells and glycinergic or mixed Convolution of pooled latencies at Golgi cellUBC and Golgi cell granule cell synapses (dark inhibition of UBCs. line) superimposed on the distribution of the jitter between uIPSCs and gIPSCs (gray; bin, 50

s). The two distributions do not differ significantly (bootstrap KolmogorovSmirnov). D, Average IPSC in a granule cell (top) and average synchronous current in the UBC (bottom). ctrl, Control; str, strychnine; wsh, washout. E, Average amplitudes, normalized to their control value, of gIPSCs (dark gray) and synchronous currents in UBCs (light gray) during strychnine application (n 12) and after 20 min washout (n 5). The effect of strychnine is significantly different for gIPSCs and for synchronous currents in UBCs (*p 0.01, MannWhitney U test). The error bars represent the SEM.
nodetection of specific markers (see Materials and Methods). Axonal varicosities of a single Golgi cell were found apposed to both gephyrin clusters located in glomeruli devoid of UBCs and gephyrin clusters located on UBC dendrites (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). A total of 36 3% of the glomeruli contacted by the axon of Golgi cells contained the dendritic brush of a UBC (110 glomeruli; n 3 cells). Thus, a single type of Golgi cell may contact both granule cells and UBCs. To determine whether functional contacts are made with both
Presynaptic markers of GABAergic and glycinergic transmission are not segregated The previous results show that Golgi cells are mixed inhibitory interneurons that perform target-specific GABAergic, glycinergic, and mixed transmission. This phenotypic specialization may be of presynaptic (transmitter release) and/or postsynaptic (receptor expression and clustering) origin. Synapse-specific GABA or glycine release could, in principle, be achieved by segregation of GAD and GlyT2 at different varicosities. Doubleimmunostaining experiments showed that GAD and GlyT2 were not strictly colocalized. Only 81.8 2.8% (n 484) of the GADpositive profiles were also immunopositive for GlyT2. This proportion was determined using wide-field CCD camera images and decreased to 49.5 1.3% (n 1234) when the same samples were quantified using single confocal optical sections (Fig. 4 A). This discrepancy could result from the location of GlyT2 on the plasma membrane, as opposed to the intracellular location of
6494 J. Neurosci., July 13, 2005 25(28):6490 6498
GAD. Indeed, GAD and VIAAT, which are both intracellular molecules, were highly colocalized at immunoreactive puncta (98.9 0.4%; n 533; confocal observations) (Fig. 4 B). Thus, GAD, as well as VIAAT (Dumoulin et al., 1999), are reliable markers of inhibitory varicosities in this system. GlyT2, however, could also be detected in axonal segments or at the periphery of varicosities, as defined by their VIAAT immunoreactivity (data not shown), and was almost always detected adjacent to GAD-immunoreactive puncta (Fig. 4 A3). Thus, GADGlyT2 segregation appears to reflect different subcellular distributions rather than the specialization of two populations of varicosities. To confirm that GlyT2 was not expressed selectively at synapses on UBCs, we compared GlyT2 staining in vestibular (lobule X) and nonvestibular (e.g., lobule VI) cerebellar lobules, which contain high and low numbers of UBCs, respectively. We found that GlyT2 immunoreactivity was comparable in lobules X (Fig. 4C) and VI (Fig. 4 D). Furthermore, similar values (Students t test) were found within lobules X and VI for GAD and GlyT2 colocalization (49.5 Figure 4. Immunohistochemical characterization of Golgi cell axon terminals. A, Partial colocalization of the glycinergic 1.3%, n 1234 vs 49.1 1.9%, n 1321, marker GlyT2 (A1) and the GABAergic marker GAD65 (A2) in the same glomerular structures. Note the characteristic GAD65 respectively) and GAD and VIAAT colo- presynaptic terminal staining pattern, contrasting with the broader GlyT2-IR. A3, Superposition of A1 and A2, indicating that calization (98.9 0.4%, n 533 vs 98.8 GlyT2-IR either colocalizes (arrows) or is apposed (asterisks and inset) to GAD65-IR. B, Massive colocalization (arrows) of GAD65-IR (B1) and VIAAT-IR (B2), as seen on superimposed images on B3. C, D, Immunodetection of GlyT2 in lobules X and VI, respectively. 0.5%, n 579, respectively). In the absence of GAD and GlyT2 seg- Note the comparable staining pattern in the granule cell layer of both lobules. Insets, Higher magnification showing GlyT2-IR profiles likely to be Golgi cell axon terminals (arrows) within a glomerulus. E, F, Glycine and GABA immunodetection in Golgi cell regation, synapse-specific transmitter re- terminal. Both are detected in periglomerular profiles (arrows) of lobules X (E , E , E ) and VI (F , F , F ). Note that different lease could be achieved by selective vesic- expression levels are detected within each varicosity. A, B, Single confocal sections; C, D, wide-field 2 3camera images; E, F, CCD ular accumulation of GABA or glycine. We projection of three confocal sections (0.48 m between each section). Scale bars: A, B, E, F, 5 m; C, D, 100 m; inset in A, 1 m; thus investigated directly the presence of insets in C, D, 10 m. glycine and GABA in presynaptic varicosities by double immunostaining. We (Fig. 5A). Such clusters were seen only rarely in the granular layer found that the two amino acids were colocalized in axon termiof lobule VI (Fig. 5B). To quantify GlyR-positive clusters, glonals (Fig. 4 E, F ), in which immunostaining is markedly enhanced meruli were outlined by VIAAT immunostaining of Golgi cell compared with cell bodies or main axodendritic shafts. The numaxonal varicosities. Double detection of GlyR and VIAAT (Fig. ber of costained varicose profiles did not significantly differ be5C,D) showed that GlyR was present in 33.3 2.1% (n 352) of tween lobules X and VI (75.4 2.8%, n 577 vs 81.6 2.7%, the glomeruli in lobule X. The spatial relationship between GlyR n 928, respectively; Students t test). This is consistent with clusters and the dendritic brushes of the two classes of UBCs electron microscopy studies (Ottersen et al., 1988). Because a (Nunzi et al., 2002) was defined using their immunoreactivity similar percentage of colocalization is seen at the level of Golgi toward CR and mGluR1. GlyR aggregates were associated with cell bodies (Ottersen et al., 1988), it may indicate that 20% of the brush of all CR-positive (Fig. 5E) and all mGluR1-positive Golgi cells contain predominantly GABA or glycine. Altogether, (Fig. 5F ) UBCs. When UBCs were labeled simultaneously for CR these data show that presynaptic markers of glycinergic transmisand mGluR1 (see Materials and Methods), 95.2 1.5% (n sion are not preferentially associated with Golgi cell to UBC 212) of GlyR-immunoreactive glomerular-like structures were synapses. associated with UBC brushes. Thus, synaptic clustering of GlyR at UBCs but not in granule cell dendrites can account for the abExpression of postsynaptic receptors correlates with the sence of a glycinergic component of IPSCs in granule cells. transmission phenotype Because Golgi cells provide the only known inhibitory input In the absence of presynaptic differentiation, the target specificity to granule cells and UBCs, the presence or absence of receptors of the transmission at Golgi cell synapses could be attributable to postsynaptic to Golgi cell synapses can be inferred directly from the selective expression and/or clustering of GABAAR and GlyR the response of the postsynaptic cells to bath application of GABA by postsynaptic neurons. If this were true, glycinergic transmisor glycine agonists (Fig. 6). Application of 100 M glycine to sion observed at GolgiUBC synapses would result from the sepostnatal day 19 (P19) to P22 granule cells of lobule X did not lective expression of GlyRs in UBCs but not in granule cells. evoke any single-channel activity, and the response measured as Many aggregates of GlyR-positive clusters were stained by panGlyR immunohistochemistry in the granule cell layer of lobule X an average current (pA; n 7) was not significantly

J. Neurosci., July 13, 2005 25(28):6495
two distinct presynaptic Golgi cells by electrical synapses may provide an alternative explanation for the existence of synchronous IPSCs. The high synchronicity of gIPSCs and uIPSCs in our paired recordings strongly argues against this hypothesis. Whereas the mean lag between synchronous gIPSCs and uIPSCs never exceeded 360 s, the typical delay between action potentials in electrically coupled neurons ranges from one to several milliseconds (Galarreta and Hestrin, 1999; Gibson et al., 1999; Mann-Metzer and Yarom, 1999; Beierlein et al., 2000; Hu and Bloomfield, 2003; Galarreta et al., 2004; Long et al., 2004). The lags between gIPSCs and uIPSCs have a sharp distribution with an average full-width at halfmaximum of 500 s, whereas the delays between action potentials in electrically coupled neurons have broader distributions, with full-widths at half-maximum comprised between one and several milliFigure 5. Expression of GlyRs by UBCs. A, B, Immunodetection of GlyR in lobules X and VI, respectively. Numerous GlyR-IR seconds. Furthermore, electrical coupling structures (arrows) within lobule X granular layer contrasting with small numbers of GlyR-IR profiles (arrows) in lobule VI. C, F, often produces bimodal cross-correlation Double detection of GlyR and different markers in the granular layer of lobule X. C, Codetection of VIAAT (green) and GlyR (red) histograms reflecting the equal probability indicating that only a subpopulation of glomeruli expresses GlyR-IR (arrows). D, High magnification of a glomerulus with presynfor each cell to fire first and entrain the aptic VIAAT-IR (green) apposed (arrows) to postsynaptic GlyR clusters (red). E, GlyR aggregates (red, arrows) on the dendritic other (Galarreta et al., 2004), which was brush of a CR-positive UBC (green). F, GlyR aggregates (red, arrows) are also detected on mGluR1-IR (green) UBC dendrioles. never observed here. Finally, the rate of AF, Wide-field CCD camera images. Scale bars: A, B, 100 m; C, 20 m; DF, 5 m. synchronization in UBC granule cell pairs corresponds to the rate of Golgi cellUBC and Golgi cell granule cell different from zero (sign test), in agreement with previous reconnection, as expected from random connectivity. ports in other lobules (Wall and Usowicz, 1997). Muscimol (5 The pharmacological analysis of synchronous gIPSCs and M) evoked a significantly greater ( p 0.01, MannWhitney U test) response of pA (n 7). UBCs that received mixed uIPSCs confirmed that only uIPSCs are sensitive to strychnine. synaptic inhibition responded to glycine and muscimol applicaWe performed immunohistochemical experiments to determine tions with currents of similar amplitudes (pA, n 11 whether the segregation of the transmission phenotypes at Golgi and pA, n 15, respectively). In contrast, UBCs cell synapses was of presynaptic or postsynaptic origin. No differreceiving pure glycinergic inhibition had a large glycinergic curence could be established between lobules containing a high denrent (pA; n 9) but a response to muscimol signifisity of UBCs and lobules devoid of UBCs when comparing the cantly smaller ( p 0.01, MannWhitney U test) than that of GlyT2 and the GABA and glycine staining profiles of the granular UBCs receiving mixed inhibition (pA; n 19). Thus, the layer. Furthermore, the GABA and glycine stainings appeared to expression pattern of postsynaptic GlyR and GABAAR correlates be highly colocalized, arguing against a functional segregation at with the phenotype of inhibitory transmission at Golgi cell synthe level of the axonal varicosities. In particular, the majority of apses on UBCs and granule cells. Golgi cell varicosities that impinge on granule cells express GlyT2 and contain glycine. In contrast, immunohistochemical detecDiscussion tion of GlyR revealed that UBCs express GlyRs, whereas granule We have shown that Golgi cells evoke GABAergic IPSCs in grancells do not. Bath applications of GABAARs and GlyRs agonists ule cells but predominantly glycinergic IPSCs in UBCs. Labeling on recorded granule cells and UBCs confirmed this result and experiments performed in lobule X showed that the axon of a revealed that the expression of variable amounts of GABAARs in given Golgi cell invades numerous glomeruli, of which 36 5% UBCs can account for the variability of the GABAAR component are occupied by the dendritic brush of a UBC. This corresponds in those cells. The diverse postsynaptic GABAAR expression in to the overall percentage of glomeruli containing the dendrite of UBCs may reflect a functional specialization, because UBCs form a UBC in lobule X (33 2%) (Fig. 5C), indicating that Golgi cell a variety of subpopulations expressing different markers (Nunzi axons innervate both types of glomeruli indifferently. The hyet al., 2002, 2003). Alternatively, it may arise from a variability in pothesis that individual Golgi cells evoke IPSCs with different the maturation state of UBCs at P17P21. The neurogenesis of pharmacological profiles in granule cells and UBCs was investiUBCs spreads over 1 week, although it is at the end of embrygated by recording spontaneous IPSCs simultaneously from onic life (Sekerkova et al., 2004), and, at the morphological level, gIPSCs and uIPSCs. Synchronous gIPSCs and uIPSCs are most maturation of UBC-containing glomeruli is not completed until likely triggered by action potentials occurring in the same presynP28 (Morin et al., 2001). Because mixed inhibitory synapses alaptic Golgi cell because the distribution of their lag could be ways undergo maturation toward more glycinergic transmission predicted from the jitters of the synaptic latencies measured at the (Kotak et al., 1998; Korada and Schwartz, 1999; Smith et al., 2000; Golgi granule cell and GolgiUBC synapses. Synchronization of

6496 J. Neurosci., July 13, 2005 25(28):6490 6498
affinity (Laube et al., 1995) and slow down glycinergic IPSCs (Suwa et al., 2001). Another type of differential control occurs in the dorsal spinal cord, in which pain perception is decreased by neurosteroids and enhanced by inflammation through opposite modulations of GABAergic (Keller et al., 2004) and glycinergic (Harvey et al., 2004) synaptic currents. In this context, targetspecific use of GABAergic or glycinergic inhibition may enable single mixed interneurons to control differentially multiple circuits. This is likely to be the case for Golgi cells that control, on the one hand, granule cells, which relay high-frequency burst firing of afferent mossy fibers (Chadderton et al., 2004), and, on the other hand, UBCs, involved in the divergent amplification and slow temporal integration of vestibular afferent inputs (Rossi et al., 1995). Organization of circuits using cotransmitters The multiple ways in which two transmitters released by the same neuron can be used differentially have been best studied in invertebrates. Presynaptic segregation has been described in Aplysia, in which bag cells release two types of peptides from separate branches of their axon (Sossin et al., 1990). A similar situation occurs in the decapod crustacean stomatogastric nervous system (STNS) in which the modulatory proctolin neuron releases GABA in the commissural ganglia (CoG) and proctolin in the stomatogastric ganglion (Blitz and Nusbaum, 1999). Studies on the STNS have also provided strong evidence that segregated actions of coreleased substances can be based on the selective expression of receptors by different target cells, as in the present study. This is the case for the gastropyloric cell (Katz and HarrisWarrick, 1989), for the modulatory commissural neuron 1 of the CoG (Swensen et al., 2000; Wood et al., 2000) and recently for an unidentified neuron putatively located in the CoG (Thirumalai and Marder, 2002). An intermediate situation in which diffusion and probably recapture and enzymatic degradation differentially affect the action of cotransmitters is found in the frog sympathetic ganglion. Two populations of cells (the B and C cells) receive inputs from separate preganglionic fibers. The fibers innervating the C cells release acetylcholine (ACh) and a neuropeptide, the luteinizing hormone-releasing hormone (LHRH). Both substances elicit a response in C cells, but only the LHRH reaches the B cells, although they also express ACh receptors (Jan and Jan, 1982). At the subcellular level, segregation of receptors may result in a differential effect of coreleased transmitters, as it has been demonstrated in layer I of the adult dorsal spinal cord (Chery and de Koninck, 1999). In this structure, miniature IPSCs are glycinergic, but a GABAergic component can be unmasked by application of benzodiazepines, indicating that GABAARs may be located extrasynaptically and activated by spillover during intense activity. The present work provides the first demonstration of a celltype-specific action of two fast cotransmitters released by the same neuron in the vertebrate CNS. It may apply to a large number of vertebrate neurons, because cotransmission is a common feature of the CNS (Burnstock, 2004). This principle may be used in pathways of the spinal cord and brainstem, in which GABA and glycine are often colocalized (Shupliakov et al., 1993; Ornung et al., 1994) but in which knowledge about synaptic connectivity is less detailed. It may also be used in circuits of the spinal cord dorsal horn, which use GABA and ATP as cotransmitters (Jo and Schlichter, 1999). In those circuits, virtually all neurons express GABAARs (Malcangio and Bowery, 1996; Coggeshall and Carlton, 1997), but only half of them express purinergic P2X recep-

Figure 6. Granule cell and UBC responses to bath application of GABAAR and GlyR agonists. The effects of both agonists were compared in two separate groups of UBCs. UBCs were sorted into these groups according to the effect of strychnine on their spontaneous inhibitory synaptic activity. UBCs were considered to receive, respectively, purely glycinergic or mixed inhibition when their CA was reduced to less or more than 5% of its control value. AC, Average responses of granule cells (A) and UBCs receiving mixed (B) or purely glycinergic (C) inhibition to bath application of 5 M muscimol (dark gray bars) and 100 M glycine (light gray bars). The response of granule cells to 100 M glycine is significantly smaller than their response to 5 M muscimol (*p 0.01, MannWhitney U test) and not significantly different from zero (see Results). The current produced by 5 M muscimol is significantly smaller in UBCs receiving purely glycinergic inhibition than in UBCs receiving mixed inhibition (*p 0.01, MannWhitney U test). The error bars represent the SEM. msc, Muscimol; gly, glycine. D, Model of the postsynaptic selection of cotransmitters by the different targets of the Golgi cell. A Golgi cell releases both GABA and glycine at its terminals, but different types of transmission are recorded on three of its target cells. Specificity of the inhibition relies on the exclusive expression of GABAARs in granule cells, of GABAARs and GlyRs in some UBCs, and of GlyRs only in other UBCs.
Gao et al., 2001; Nabekura et al., 2004; Awatramani et al., 2005), our observation of a postsynaptic specialization between cell types would thus be even stronger in the adult. Functional significance of a target-specific use of GABA and glycine The preferential use of GABAARs or GlyRs by postsynaptic partners of the same mixed neurons has significant functional consequences. First, in all of the systems, GABAAR and GlyR expressed at the same synapse have different kinetics (Jonas et al., 1998; Chery and de Koninck, 1999; OBrien and Berger, 1999; Dumoulin et al., 2001; Gonzalez-Forero and Alvarez, 2005) that speed up in parallel during development (Awatramani et al., 2005). In the spinal cord, fast glycinergic conductances have been proposed to efficiently hyperpolarize the cell, whereas smaller and slower GABAA components would control shunting and the time course of inhibition (Russier et al., 2002). Alternatively, differential modulations of GABAARs and GlyRs may permit fast and synapse-specific adaptation of inhibition to network states of activity. For instance, zinc ions are found in some inhibitory terminals (Wang et al., 2001) and in a fraction of glutamatergic terminals from which they are released and can spill over on nearby synapses (Li et al., 2003). Zinc ions inhibit some GABAARs (Hosie et al., 2003) and, at the same concentration, increase GlyR

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tors (Li and Perl, 1995; Jo et al., 1998). Glutamate and GABA are coreleased during postnatal development of future glutamatergic synapses (Walker et al., 2001; Gutierrez, 2003) or of future inhibitory synapses (Gillespie et al., 2005), as well as in the adult (Kao et al., 2004; Ottem et al., 2004). At specific stages of development, glutamate may also be coreleased with ACh (Li et al., 2004; Nishimaru et al., 2005) and with various monoamines, as assessed from the pattern of expression of vesicular glutamate transporters and vesicular monoamine transporters (Boulland et al., 2004). Thus, target-specific expression and activation of receptors may play a central role during development in the establishment, specification, and refinement of synaptic contacts. This mechanism would be extremely useful, because cell-to-cell signaling is not yet fully constrained by synaptic wiring and synapse independence. Target-specific neurotransmission may also occur when a neurotransmitter is released with one or several neuropeptides, a feature frequently found in the CNS (Burnstock, 2004) and somewhat analogous to the situation in the invertebrate nervous system. In the cases of paracrine and volume transmission, target-specific receptor expression would indeed be the only way to ensure selective cell-to-cell communication by chemical coding of transmission.

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