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The Journal of Neuroscience, March 1, 2000, 20(5):1837-1848
Serotonin-Driven Long-Range Inhibitory Connections in the Cerebellar Cortex
Stéphane Dieudonné1 and Andréa Dumoulin2 1 Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8544, and 2 Laboratoire de Biologie Cellulaire de la Synapse, Institut National de la Santé et de la Recherche Médicale U497, Ecole Normale Supérieure, 75005 Paris, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Disturbances of the serotoninergic neuromodulation in the cerebellar cortex have been involved in several types of ataxia, but the physiological action of serotonin in this structure remains poorly understood. We report that in slices of the rat cerebellar vermis, serotonin triggers the firing of an inhibitory interneuron presynaptic to Golgi cells. The Lugaro cell, a neglected interneuronal type, satisfies the expected criteria for this input, whereas basket cells, stellate cells, or Golgi cells do not. Lugaro cells are selectively excited by serotonin, and their firing behavior (sustained steady frequency in the 5-15 Hz range) resembles the pattern of occurrence of serotonin-evoked IPSCs in Golgi cells. Immunohistochemical stainings and single cell reconstructions show that Lugaro cell axons form a parasagittal plexus but also extend long transverse branches that run parallel to the parallel fibers and are partly myelinated. Electrophysiological data suggest that these transverse axons participate in synaptic contacts of the Lugaro cells with Golgi cells, and we calculated that in the intact cerebellum a given Lugaro cell contacts >100 Golgi cells. Serotonin modulation of Lugaro cells may constitute an intracortical switch involved in information patterning at the level of Golgi cells and granule cells populations, and particularly in synchronizations recorded along the transverse axis in vivo.
Key words: serotonin; cerebellar cortex; inhibition; interneurons; Golgi cell; Lugaro cell; myelinated axons
INTRODUCTION
Reciprocal interactions between the motor system and the serotoninergic modulatory system are well documented (Jacobs and Fornal, 1997
). The cerebellar cortex, like all other motor structures, receives serotoninergic innervation in the form of a plexus of fine varicose fibers that do not face any differentiated postsynaptic element (Ungerstedt, 1971
It has been reported that application of serotonin potentiates the inhibition of Purkinje cells by exogenous GABA (Strahlendorf et al., 1989
In this work we have combined electrophysiological recordings in thin slices of the rat cerebellar cortex with cell reconstruction and immunohistochemical methods to identify a new site of action of serotonin. Recordings from Golgi cells revealed that serotonin (1 µM) evokes numerous IPSCs, whose pattern of occurrence reveals the regular spiking of a small number of inhibitory interneurons presynaptic to Golgi cells. We show that the Lugaro cells, whose connectivity and function have remained poorly known since their initial description (Lugaro, 1894
MATERIALS AND METHODS
Slice preparation.
Cerebellar thin slices were prepared from male Wistar rats aged from 11 to 21 d, following the method originally described by Llinás and Sugimori (1980)
Slices were visualized using a 40× water-immersion objective (0.75 NA, Axioskop, Carl Zeiss) and near-infrared optics (illumination filter 750 ± 30 nm, and a Sony CCD camera from which the infrared blocking filter had been removed). Golgi cells were selected for recording both on visual criteria, as previously explained (Dieudonné, 1995
Patch-clamp recording. All experiments were performed at room temperature (20-25°C). The recording chamber was continuously perfused at a rate of 1.5 ml/min with a saline solution, pH 7.4, containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 25 glucose, bubbled with a mix of 95% O2 and 5% CO2. The same solution was used during dissection and slicing. TTX (Sigma, St. Louis, MO), APV (Tocris), CNQX (Tocris), and gabazine (RBI) were bath-applied. Serotonin (Sigma) was also bath-applied, usually for 3 min, and washed for at least 15 min between applications to avoid desensitization.
Golgi cells were recorded in cell-attached and whole-cell configurations. In both cases the voltage-clamp mode was used with the pipette potential held at
70 mV. Pipettes had a resistance of 2-4 M
and were filled with the following internal solution (in mM): 142 CsCl, 10 HEPES, 1 EGTA, 5 MgCl2, 0.1 CaCl2, 4 Na-ATP, 0.4 Na-GTP; pH adjusted to 7.3 with N-methyl-D-glucamine. Purkinje cells were recorded in the whole-cell configuration with the solution described above and held at
Data analysis. PClamp software (Axon Instruments) was used for the acquisition of all recordings. Data were filtered at 2 kHz and digitized at 10 kHz. Spontaneous synaptic currents were detected automatically using the ACS software kindly provided by P. Vincent (Institut des Neurosciences, Paris). All detected events were subjected to visual inspection, and EPSCs were discarded on the basis of their fast decay time course (Dieudonné, 1998
Morphological reconstruction. For Lugaro cell recordings, biocytin (2-5 mg/ml) was added to the intracellular solution just before the experiment. At the end of whole-cell recordings, the patch pipette was slowly moved away from the cell to obtain an outside-out patch. The slice was then fixed at 4°C in PBS (150 mM) containing 4% w/w paraformaldehyde (PFA) and sometimes 0.05% v/v glutaraldehyde. After 12 hr to 2 weeks of fixation, biocytin was revealed using the ABC elite kit (Vector Laboratories, Burlingame, CA). Slices were preincubated for 10 min in 10% methanol, 89% PBS, and 1% H2O2 to remove endogenous peroxidase activity. They were then permeabilized for 30 min with 0.4% Triton X-100 in PBS and incubated for 2 hr with the avidin derivatives. Peroxidase activity was revealed by incubation in a Tris solution containing 0.015% diaminobenzidine, 0.15% nickel ammonium sulfate, and 0.4 µl/ml of a 1% H2O2 solution, after a preincubation for 20 min in a similar solution without H2O2. Alternatively, biocytin was revealed with FITC-coupled streptavidin (1:200, Transduction Laboratories) for 2 hr. In this case, fixation was performed at 4°C with 4% w/w PFA in PBS for 3-12 hr. The reconstructed image of a Lugaro cell presented in this paper was drawn from serial images taken every 1 µm with a Hamamatsu CCD camera (C5985) mounted on a standard epifluorescence microscope (Leica DMRD).
Fluorescence immunohistochemistry. Female Sprague Dawley rats (180-200 gm, Janvier) were deeply anesthetized with pentobarbital (60 mg/kg body weight, i.p.) and perfused intracardially with 4% w/w PFA in PBS. The cerebellum was removed, post-fixed for 12-15 hr in the same fixative, and cut into 30-µm-thick sections with a vibratome in sagittal or transverse planes. Sections were immersed for 20 min in 50 mM ammonium chloride in PBS and rinsed. After 30 min of preincubation in 0.1% gelatin, 0.1% Triton X-100 in PBS, an overnight incubation at 4°C was performed in the same solution either with a polyclonal anti-calretinin antibody alone (1:5000, SWant) or combined with (1) a monoclonal anti-GAD65 antibody (1:500, Boehringer Mannheim, Indianapolis, IN) (Gottlieb et al., 1986
RESULTS
Inhibition of Golgi cells is under serotoninergic control
Figure 1A,B illustrates the effect of serotonin (1 µM) on the synaptic activity recorded from Golgi cells. Before application of serotonin, synaptic currents occurred at low frequency and at random intervals, as illustrated in Figure 1B. After bath application of serotonin, the synaptic activity dramatically increased. In Figure 1B the fast synaptic currents are EPSCs, which can be blocked by AMPA receptor antagonists (Dieudonné, 1998
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Figure 1. Serotonin increases the inhibitory synaptic activity recorded from cerebellar Golgi cells. The spontaneous synaptic activity was recorded from a Golgi cell held at
For the cell of Figure 1, the amplitude histograms of the IPSCs in control conditions and during serotonin application are displayed in C. In this cell control IPSCs had a mean amplitude of 32 pA (average amplitude: 33 ± 10 pA; n = 8 Golgi cells), and the amplitude of the serotonin-induced IPSCs was 10.7 times that of the spontaneous IPSCs recorded in control conditions. Therefore Golgi cells receive two classes of IPSCs that can be separated by their frequency of occurrence before and during serotonin application and by their amplitude (Fig. 1C). On average, the ratio of the amplitude of serotonin-evoked IPSCs over that of control IPSCs was 4.0 ± 1 (mean ± SEM; n = 13; min = 0.4; max = 12). In some Golgi cells the amplitude of the serotonin-evoked IPSCs decreased markedly during the application of serotonin (Fig. 2A). This rundown might have to do with the state of preservation of the presynaptic cell, which in the case of Figure 2A seemed to display a low level of spontaneous activity before serotonin application (as assessed from the presence of a population of large amplitude IPSCs in control conditions).
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Figure 2. Analysis of the rhythm of the inhibitory IPSCs evoked by serotonin. A, Dot plot of the effect of serotonin on the inhibitory activity recorded from a Golgi cell. Each dot indicates the amplitude and time of occurrence of an IPSC. Note that events with an amplitude similar to the serotonin-induced events are recorded before serotonin application, indicating that in that case the serotonin-sensitive presynaptic interneuron was firing at low frequency in control conditions. A large decrease in the amplitude of the serotonin-induced IPSCs is observed during the effect. B,a, IPSCs recorded during the control period a illustrated in A were aligned on the first point of their rising phase and then averaged. The same operation was performed on groups of 200 consecutive IPSCs taken during the onset (b), the peak (c), and the steady-state (d) of the effect of serotonin. The regularly spaced wavelets in the three averages indicate that during the action of serotonin there is a strong level of autocorrelation in the responses. The number 0 indicates the peak corresponding to the averaged event;
The time course of the effect of serotonin is better illustrated by the frequency of occurrence of the serotonin-evoked IPSCs (separated from control IPSCs by their amplitudes) shown in Figure 1D. The first IPSCs appeared after a delay of ~30 sec after the arrival of serotonin in the bath. This value is calculated by substraction of the bath perfusion delay, which has been assessed from the time course of IPSC inhibition when applying the GABAA antagonist gabazine. This delay for the appearance of serotonin-evoked IPSCs varied largely between cells, from a few seconds to >3 min. The effect of serotonin was entirely reversible within 5-10 min (Fig. 1A,D). The serotonin-induced increase in IPSC frequency could be repeated many times on the same cell, although in several cells a decrease in the peak frequency that was reached could be noted between the first and subsequent applications.
In all of the Golgi cells recorded before P12, serotonin decreased the amplitude and frequency of the spontaneous IPSC. This inhibitory effect was better seen in cells in which serotonin did not evoke a new class of IPSCs. Both effects could also superimpose, in which case the inhibitory effect, having a negligible latency, would precede the appearance of the serotonin-evoked IPSCs. The inhibitory effect was never recorded in cells after P15 and will not be discussed further.
Serotonin induces spike firing of inhibitory interneurons presynaptic to the Golgi cell
The large-amplitude IPSCs induced by serotonin may result from an increase in the release probability at previously active synapses or from the excitation of previously silent interneurons. The fact that serotonin induces a gradual increase in the frequency of the IPSCs but a sharp increase in their amplitude (Figs. 1-3) argues for the second hypothesis. Bath-applied TTX (0.2-1 µM) blocked completely and reversibly the increase in IPSC frequency induced by serotonin (n = 3; data not shown), which indicates that the release probability for miniature events is not affected. Moreover, the effect of serotonin was unaffected by coapplication of CNQX (5 µM) and DL-APV (50 µM) (n = 3; data not shown), indicating that it does not depend on the activity of glutamatergic synapses. Taken together, these data suggest that serotonin directly induces spike firing in a population of presynaptic interneurons that are silent in control conditions but does not increase the release probability at previously active synapses.
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Figure 3. Several serotonin-sensitive interneurons can be presynaptic to the same Golgi cell. A, A 280 sec whole-cell recording shows the beginning of the effect of serotonin (1 µM). The bar chart represents the mean IPSC frequency during 2.5 sec intervals for the same period of recording. The brackets on top of the trace indicate the periods used for the analysis in B and C. B, Amplitude histogram of the IPSCs before serotonin application (control), at the beginning of the effect (a), and after the onset of large-amplitude events (b). The inset in b shows the repartition of the large amplitude IPSCs at a different vertical scale. The bin value is 10 pA, and the detection threshold was
Serotonin transforms silent interneurons into regularly firing ones
The pattern of activity of the serotonin-driven inhibitory afferents to Golgi cells was found to be highly rhythmic. The regular occurrence of serotonin-induced IPSCs can be visually appreciated (Fig. 1B), but to better characterize this rhythm, IPSCs were aligned on the first point of their rising phase and averaged both in control conditions and during serotonin applications. The dot plot in Figure 2A, which indicates the time of occurrence and amplitude of all IPSCs, illustrates a representative experiment in which, after a delay of ~30 sec, the IPSC frequency rose during ~90 sec (period indicated by b) and then settled to a steady-state (periods indicated by c and d). The frequency returned close to control values within 8 min of washout of serotonin. In control conditions the average IPSC rises rapidly from a flat baseline and decays monotonically (Fig. 2B,a). In contrast, the averaged trace obtained during serotonin application (Fig. 2B,b,c,d) was composed of a series of wavelets with equidistant peaks on both sides of the averaged IPSC (marked 0 on Fig. 2B,c). The shape of this crude autocorrelogram means that in the presence of serotonin, IPSCs occur with an extreme regularity. The main frequency can be calculated as the inverse of the interval between peaks. As shown in Figure 2C, the rhythm appeared at the onset of the effect. The firing frequency of the presynaptic interneuron rose gradually (from 4.1 to 7.9 Hz) during the first minute of serotonin application to reach a steady-state rate that did not decrease with time. The rhythmic activity also persisted during most of the offset, decreasing gradually to half of the maximum frequency (4.1 Hz), until rare random events were left. The stability and regularity of the periodic occurrence of IPSCs indicate that serotonin-sensitive interneurons behave, in the presence of serotonin, as oscillators with a preferred frequency. It also suggests that either a single interneuron or synchronized interneurons are responsible for the effect of serotonin in the cell of Figure 2.
Several presynaptic interneurons can be recruited by serotonin with different latencies
In some Golgi cells the onset of the effect of serotonin was clearly multiphasic. As illustrated in Figure 3A, multiple phases can be distinguished relative to both the amplitude and frequency of the serotonin-evoked IPSCs. As illustrated in the left plot of Figure 3B, control IPSCs recorded in this experiment had an amplitude <70 pA. During the first phase of the effect, the serotonin-induced IPSCs had a larger amplitude than control IPSCs but remained <200 pA (Fig. 3B, middle plot). While the frequency of this intermediate class of events still rose, IPSCs >200 pA began to occur with increasing frequency. The successive classes of events did not replace the smaller ones (Fig. 3B, right plot), as would have been the case with an increase in the strength of a single presynaptic unit.
Multiple peaks were observed in the average IPSC calculated (as in Fig. 2) for the initial period a, indicating a preferred firing frequency of 6.4 Hz for the presynaptic units (data not shown). In contrast, multiple peaks did not appear if the average was performed later (Fig. 3, period b). To further analyze period b, IPSCs were separated into two groups according to their amplitudes: 70-200 pA, corresponding to the amplitude of the initial IPSCs induced by serotonin, and over >pA, corresponding to the amplitude of the IPSCs induced later. The distributions of the intervals between successive events, calculated for each population individually, are plotted in Figure 3C. Intervals were distributed in equidistant peaks. The preferred intervals were multiples of 145 msec (6.9 Hz, small IPSCs) and 170 msec (5.9 Hz, large IPSCs), respectively. In contrast, when the IPSCs belonging to the two classes of amplitude were considered together (amplitude >70 pA) (Fig. 3C, right plot) intervals between 0 and 200 msec were found to be equally likely, and longer intervals never occurred. Therefore the two classes of amplitudes correspond to IPSCs coming from two distinct presynaptic units that fire regularly at similar frequencies but independently from each other. In two other cells, in which the IPSCs during the successive phases of the effect of serotonin had very different amplitudes, a similar analysis gave the same results: preferred intervals for each amplitude population alone but short random intervals for the overall population. This confirms that the multiphasic effects of serotonin indicate the sequential excitation of several independent presynaptic interneurons.
Serotonin does not increase the activity of molecular layer interneurons or of Golgi cells
We sought to identify the serotonin-sensitive interneuron presynaptic to Golgi cells. Early studies have shown that Golgi cells are contacted by basket cells and stellate cells (Eccles et al., 1967
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Figure 4. Serotonin decreases the inhibitory synaptic activity recorded from Purkinje cells. Purkinje cells were held at
The effect of serotonin does not involve Purkinje cell to Golgi cell connections. Some authors have proposed that the recurrent collaterals of Purkinje cell axons contact Golgi cells (Ramón y Cajal, 1911
The serotonin effect does not involve Golgi-Golgi interactions. This was made unlikely by the fact that connections between Golgi cells have never been described in morphological studies and by our previous observation (Dieudonné, 1998
In view of the above results, it is unlikely that the serotonin-sensitive inhibitory interneurons presynaptic to Golgi cells are basket cells, stellate cells, Purkinje cells, or Golgi cells.
Serotonin transforms inactive Lugaro cells into regular spiking neurons
The only other possible target of serotonin, given the classification of cerebellar cell types, was the Lugaro cell, a poorly characterized cerebellar interneuron that was first identified by its bipolar shape and location under the Purkinje cell bodies (Lugaro, 1894
Putative Lugaro cells can be identified in the slice by their shape and position. This visual identification remains ambiguous because some Golgi cells can be found in the same location (Sahin and Hockfield, 1990
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Figure 5. Serotonin excites Lugaro cells identified morphologically. A, Reconstruction of a biocytin-filled Lugaro cell in the parasagittal plane. The cell was located at the bottom of a lobule, and the two main dendritic trees marked the curvature of the Purkinje cell layer (PC). A third dendrite, descending in the granular layer, was cut at approximately 50 µm from the cell body. Thin varicose fibers formed an axonal plexus in the parasagittal plane inside the molecular and Purkinje cell layers. This plexus originated (short arrows) from a neurite running among the main dendrites. It was not possible to assess whether this neurite (arrows) was an axon or a dendrite. The indicates a transverse axonal branch emerging from the parasagittal plexus and running toward the surface where it was cut. The
points to a varicose branch of the axon running toward the depth of the slice. The
indicates another transverse neurite that was emitted by a main dendrite. B, Summary of the firing frequency of a Lugaro cell recorded in the cell-attached configuration. C, Part of the cell-attached recording marked in B. D, Whole-cell current-clamp recording of a Lugaro cell. The holding current was set to
In the cell-attached configuration, serotonin (1 µM) evoked the firing of action potentials in three of three Lugaro cells, as illustrated in Figure 5B,C. The firing frequency was sustained in the presence of serotonin, and the spikes occurred at regular intervals (Fig. 5C). On washout of serotonin the firing gradually stopped within 2-3 min. Lugaro cells were also recorded in the whole-cell current-clamp mode. The holding current was fixed as the inverse of the leak current through the seal in the cell-attached configuration just before the membrane was broken (
In both cell-attached and whole-cell modes the latency of the excitatory effect of serotonin on Lugaro cells was not significantly different from the time necessary to perfuse the bath (~60 sec), whereas the effects of serotonin on the inhibitory activity recorded from Golgi cells could take as much as 3 more minutes. When spikes were initiated (Fig. 5D), the delay between the onset of depolarization and the first spike was never longer than 20 sec and could be as short as a few milliseconds (Fig. 6B). Firing was sustained, at 5-15 Hz, during the whole serotonin application, with abrupt changes in firing frequencies at both the onset (Fig. 5E) and offset (Fig. 5F) of the effect. These frequency changes were linked to different levels of depolarizing plateaus, as shown in Figure 5F. During prolonged applications of serotonin, overexcitation of the Lugaro cell eventually led to chaotic firing, with a dramatic reduction in spike amplitude (Fig. 5D,F). This might explain the gradual decrease in the amplitude of serotonin-evoked IPSCs observed in some Golgi cells.
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Figure 6. Mechanisms involved in serotonin excitation of Lugaro cells. A, Moderate depolarizing current pulses evoked Lugaro cell firing with a strong accommodation of the firing frequency. Note that the slow AHP is gradually developing (arrows) at the beginning of the spike train and causes accommodation. B, In the same cell, serotonin induced a moderate depolarization and sustained firing without accommodation. Note the small size of AHPs in the presence of serotonin. C, Effect of a 50 pA hyperpolarizing current pulse before, during, and after application of serotonin in another cell. During serotonin application the holding current was changed to return to the resting potential recorded before serotonin application. Note the depolarizing plateau at the offset of the hyperpolarizing pulse in the presence of serotonin. D, Same as C, for 50 pA depolarizing pulses. Note the increased firing frequency and decreased AHPs during the depolarizing pulse. A depolarizing plateau is also present at the offset of depolarizing pulses in the presence of serotonin.
The changes in active membrane properties of the Lugaro cell, which underlie the effect of serotonin, were further investigated. In current clamp, minor reductions in the hyperpolarizing holding current (from
The effects of hyperpolarizing and depolarizing current pulses in control conditions and in the presence of 1 µM serotonin were compared. The holding current was increased during serotonin application to stop serotonin-induced firing and to bring the cell back to its initial holding potential. In control conditions, a large hyperpolarizing pulse was followed at the repolarization by a small afterdepolarization, which can be attributed to the presence of an Ih current, as evidenced by the sag seen in the membrane potential recordings during hyperpolarization (Fig. 6C). In the presence of serotonin, the amplitudes of the maximal hyperpolarization and of the sag were not markedly affected, but the small afterdepolarization was transformed into a prolonged depolarizing plateau leading to the emission of several spikes (Fig. 6C). Inhibition of potassium conductances active around the resting potential may account for this increased effect of Ih and for the depolarization induced by serotonin. However, surprisingly, an afterdepolarization was also seen after depolarizing current pulses in the presence of serotonin, but not in control conditions (Fig. 6D). This pattern resembles that observed in spinal motoneurons in the presence of serotonin, which has been linked to a nifedipine-sensitive calcium current (Hounsgaard et al., 1988
Organization of the Lugaro cell axonal plexus
The observation of biocytin-filled Lugaro cells confirmed previous data concerning the organization of its axons (Fig. 5A). It is commonly accepted that the Lugaro cell has a parasagittally oriented thin varicose axon (Lugaro, 1894
Some authors have also proposed that Lugaro cells have a transverse axonal plexus in the form of thick axonal branches that extend in the axis of the folia, parallel to the parallel fibers (Lugaro, 1894
To further study the distribution and morphology of Lugaro cells, we took advantage of the fact that calretinin strongly stains unipolar brush cells (UBCs) and Lugaro cells in the cerebellar cortex (Rogers, 1989
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Figure 7. Calretinin immunoreactivity of Lugaro cells in the parasagittal (A, B), coronal (C1, C2), or transverse plane (D, E) of the rat cerebellum. A, Low-magnification reconstruction of a lobule in the sagittal plane. Lugaro cell bodies (arrows) are located beneath the Purkinje cell layer; their dendrites remain in the upper granule cell layer or at the border of the Purkinje cell layer. B, High magnification of a Lugaro cell body, located between two Purkinje cell bodies that are immunonegative for calretinin. Two dendrites (arrows) originate from the opposite poles of the typically fusiform cell body. Immunoreactive spots (arrowheads) corresponding to Lugaro axonal fibers, transversally cut, are observed above the Purkinje cell bodies in the sagittal plane. C1, C2, Lugaro cell dendrites and transverse axon collaterals are orthogonally orientated. Example of a Lugaro cell (asterisk) detected in the coronal plane when focused below the Purkinje cell layer (C1) or above it (C2). C1, The initial segment of a dendrite bifurcates, and dendrites (arrows) coursing amid the Purkinje cell bodies are visible. C2, Same region as in C1, but focused at a level tangential to the low molecular layer. Dendrites are no longer visible, and varicose axons with a transverse course (arrowheads) are detected. Because of the curvature of the lobule, the cell body present in C1 is still detected in C2 (asterisk). D, Low-magnification reconstruction of a lobule in the transverse plane. Three Lugaro cell bodies are present (arrows). Calretinin-positive Lugaro axonal fibers follow a transverse course (arrowheads) in the low molecular layer. Note the calretinin background staining in the upper molecular layer (asterisk). E, Calretinin-positive axonal fibers in the low molecular layer, at high magnification. Most of the fibers display immunoreactive varicosities (arrowheads). The inset shows a recapitulative drawing of a lobule cut in the sagittal plane, where a Lugaro cell has been represented in black. Four transverse axon collaterals are represented in the transverse orientation, which is the orientation of the parallel fibers. m, Molecular layer; P, Purkinje cell body; g, granule cell layer. Scale bars: A, D, 50 µm; B, E, 10 µm; C1, C2, 20 µm.
The Lugaro cells spatial organization was better appreciated in some areas of coronal slices, where the plane of cut was tangential to the bottom of the Purkinje cell layer (Fig. 7C1). Lugaro cell dendrites often deviated from their main parasagittal orientation to form X-shaped lattices centered on the Lugaro cell bodies. The dendrites formed a dense mesh around and under Purkinje cell bodies, and several dendrites coming from different cells were often found in tight apposition. When in the same observation field one focused on the bottom of the molecular layer, calretinin-immunoreactive fibers were seen running perpendicularly to the main orientation of the Lugaro cell dendrites (Fig. 7C2). These fibers differed from dendrites by being straight and varicose in some portions. In transverse sections perpendicular to the plane of the Purkinje cell dendrites, these fibers were located in the low molecular layer, above the Purkinje cell bodies (Fig. 7D,E). These fibers differed from the parallel fibers (Harvey and Napper, 1991
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Figure 8. Lugaro cell axons in the transverse plane. A, Segment of a Lugaro cell axon stained for calretinin that could be followed for >350 µm. B, C, Sections were double-stained with anti-calretinin antibody (green in B1 and C1) and anti-GAD65 (red in B2) or anti-MBP (red in C2). B3 and C3 are the superimposed images B1-B2 and C1-C2. B, Axonal varicosities immunoreactive for calretinin are positive for GAD65 (arrowheads), but the number of GAD65-containing terminals outnumbers the calretinin-positive varicosities (arrows). Some rare spots are calretinin positive and GAD65 negative (double arrow). C, Lugaro cell transverse axons are partially myelinated. Nonvaricose segments of calretinin-positive axons are myelinated (arrowhead), whereas varicose ones are not (arrows). Note that myelination stops (double arrow) in calretinin-positive fibers when the fiber starts to be varicose. Some MBP immunoreactivity is not associated with calretinin-labeled fibers (asterisks). Scale bars: A, 30 µm; B, C, 10 µm.
The varicosities of these calretinin-positive fibers were GAD65-positive (Fig. 8B), confirming that they are not parallel fibers but rather the axon of inhibitory Lugaro cells. GAD65 immunoreactivity was also found in terminals where it was not associated with calretinin. These terminals presumably correspond to the synapses made by basket cell axons and Purkinje cell recurrent collaterals in the low molecular layer. Colocalization of calretinin and MBP in transverse fibers indicated that the transverse plexus of Lugaro cell axons is myelinated in part. Thick unvaricose segments were most frequently myelinated, whereas segments bearing varicosities were not (Fig. 8C); the two types of immunoreactive pattern were often observed along the same axonal fiber. Because almost all of the MBP-positive axons were calretinin-positive, we may assume that at the age analyzed, Lugaro axons constitute the majority of myelinated fibers in the low molecular layer. The remaining myelinated fibers probably correspond to Purkinje cell recurrent collaterals (Hámori and Szentágothai, 1968
DISCUSSION
Serotonin excitation of Lugaro cells accounts for serotonin-driven IPSCs in Golgi cells
We have shown that serotonin increases the frequency of IPSCs in Golgi cells. This serotonin-driven inhibitory activity arises from the excitation of a small number of presynaptic inhibitory interneurons, which differ from the interneurons responsible for background inhibition of Golgi cells by the amplitude of their IPSCs. We further show that Lugaro cells are selectively excited by serotonin in the slice. Although the connection between Lugaro and Golgi cells is not demonstrated directly in this work, the results that are presented strongly suggest that excitation of Lugaro cells by serotonin accounts for the serotonin-driven inhibitory activity recorded from Golgi cells. As discussed in Results, Golgi or Purkinje cells are not likely candidates, primarily because they do not appear to contact Golgi cells.
The case of molecular layer interneurons necessitates further comments. We report that serotonin induces a slight decrease in the amplitude and frequency of the IPSCs recorded from Purkinje cells. Our results appear to contradict those of Mitoma et al. (1994)
The analysis developed in Results provides a number of positive arguments that point to Lugaro cells as the source of serotonin-driven inhibition of Golgi cells. Lugaro cells are usually silent in the slice. They are excited by serotonin at the same concentration that evokes IPSCs in Golgi cells, and the frequency of firing of Lugaro cells is in the same range as the frequency of serotonin-evoked IPSCs in Golgi cells. Serotonin suppresses the accommodation in the firing of Lugaro cells and transforms these cells into regular spiking neurons, in agreement with the rhythmic occurrence of serotonin-driven IPSCs in Golgi cells. Finally, Lugaro cells are not abundant, which would explain why in the slice a given Golgi cell receives the input of only one or two cells.
What are the likely hypotheses about the action of Lugaro cells' inhibitory input onto Golgi cells? The absence of effect of Lugaro cell inhibition on the basal firing frequency of Golgi cells suggests that these IPSPs do not significantly hyperpolarize Golgi cells (Dieudonné, 1998
Organization of the Golgi cell-Lugaro cell connection
The cell reconstructions presented here demonstrate that the Lugaro cell possesses two axonal plexes. One ramifies in the parasagittal plane, as do most other neurites in the cerebellum, and the other consists of transversal fibers and represents, with the parallel fibers, the only pathway for transverse information flow in the cerebellar cortex. Immunohistochemical stainings reveal that these transverse Lugaro cell axons form a dense plexus in the lower third of the molecular layer. The myelination of this plexus and the large diameter of its fibers would allow a fast action potential conduction, suggesting that a precise timing of the Lugaro cell inhibition is important for cerebellar function. In adult animals, the Lugaro cell transverse axons can be very long: 2 mm in the cat (Lugaro, 1894
Electrophysiological data indicate that the transverse plexus of the Lugaro cell axons participates in the synaptic contacts made with Golgi cells. Lugaro cells used for direct recording, and located at the surface of the slice, respond to serotonin with a negligible latency. In contrast, the serotonin-induced increase of IPSC frequency recorded from Golgi cells has a long latency and progressive onset. In addition, when several presynaptic Lugaro cells are preserved in the slice, excitation of these cells by serotonin occurs with different latencies. This most likely indicates that when we recorded from Golgi cells, serotonin had to reach Lugaro cells located in the depth of the slice. Given the fact that Golgi cell dendrites are restricted to a narrow parasagittal volume (Dieudonné, 1998
Our data allow us to give lower estimates of the level of convergence (C) and divergence (D) of the Lugaro cell to Golgi cell connection, if they are made at random along the 2 mm of the Lugaro cell transverse axons. Because there was on average more than one presynaptic Lugaro cell per Golgi cell in parasagittal slices of 200 µm thickness, each Golgi cell may be contacted by >10 Lugaro cells in the intact cerebellum. Because the total number (N) of connected Lugaro-Golgi pairs is given by N = NG × C = NL × D, where NG is the number of Golgi cells in the cerebellum and NL is the number of Lugaro cells. It follows that an underestimate of the divergence (D) is D = C × (NG/NL).
Our estimation of 1 Lugaro cell for 15 Purkinje cells is close to the 1:30 ratio obtained in the cat (Sahin and Hockfield, 1990
Given the peculiar transverse organization of their axons, Lugaro cells might play a role in the synchrony found between the spikes of Golgi cells situated along the same parallel fiber beam (Vos et al., 1999
Modulation of Lugaro cells by serotonin in vivo
The fact that Lugaro cell excitation and subsequent Golgi cell inhibition can be evoked by submicromolar concentrations of serotonin indicates that these effects are of physiological relevance. Indeed, the densest part of the serotoninergic plexus in the vermis corresponds to the location of the cell body and dendrites of the Lugaro cells (Chan-Palay, 1975
FOOTNOTES Received Aug. 18, 1999; revised Dec. 23, 1999; accepted Dec. 23, 1999.
S.D. was supported by a fellowship from the Fondation Lilly, BIOMEDII BMH4CT 972374, the Ecole Normale Supérieure, and the Centre National de la Recherche Scientifique (UMR 8544). A.D. was supported by BIOMEDII BMH4CT 972374 and the Institut National de la Santé et de la Recherche Médicale (U497). We thank P. Ascher, B. Barbour, H. Gerschenfeld, S. Supplisson, and A. Triller for critical reading of this manuscript.
Correspondence should be addressed to Stéphane Dieudonné, Laboratoire de Neurobiologie, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France. E-mail: sdieudon{at}sfu.ca.
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