IPSC kinetics at identified GABAergic and mixed GABAergic and glycinergic synapses onto cerebellar Golgi cells

Abstract

In the rat cerebellum, Golgi cells receive serotonin-evoked inputs from Lugaro cells (L-IPSCs), in addition to spontaneous inhibitory inputs (S-IPSCs). In the present study, we analyze the pharmacology of these IPSCs and show that S-IPSCs are purely GABAergic events occurring at basket and stellate cell synapses, whereas L-IPSCs are mediated by GABA and glycine. Corelease of the two transmitters at Lugaro cell synapses is suggested by the fact that both GABA(A) and glycine receptors open during individual L-IPSCs. Double immunocytochemical stainings demonstrate that GABAergic and glycinergic markers are coexpressed in Lugaro cell axonal varicosities, together with the mixed vesicular inhibitory amino acid transporter. Lugaro cell varicosities are found apposed to glycine receptor (GlyR) clusters that are localized on Golgi cell dendrites and participate in postsynaptic complexes containing GABA(A) receptors (GABA(A)Rs) and the anchoring protein gephyrin. GABA(A)R and GlyR/gephyrin appear to form segregated clusters within individual postsynaptic loci. Basket and stellate cell varicosities do not face GlyR clusters. For the first time the characteristics of GABA and glycine cotransmission are compared with those of GABAergic transmission at identified inhibitory synapses converging onto the same postsynaptic neuron. The ratio of the decay times of L-IPSCs and of S-IPSCs is a constant value among Golgi cells. This indicates that, despite a high cell-to-cell variability of the overall IPSC decay kinetics, postsynaptic Golgi cells coregulate the kinetics of their two main inhibitory inputs. The glycinergic component of L-IPSCs is responsible for their slower decay, suggesting that glycinergic transmission plays a role in tuning the IPSC kinetics in neuronal networks.

Fig. 1.

Pharmacological characterization of the S-IPSCs and of the L-IPSCs recorded from Golgi cells.A, Gabazine blocks spontaneous IPSCs but not serotonin-evoked IPSCs. The top trace represents 5 min of recordings during which gabazine (30 μm) and serotonin (1 μm) were bath applied at the time indicated byhorizontal bars. The asterisk and thefilled circle indicate the time at which thebottom traces, displayed at a faster time scale, were taken. CNQX (3 μm) was present during the whole experiment to block all EPSCs. B, Summary of a similar experiment in another cell illustrates that strychnine blocks L-IPSCs induced in the presence of gabazine. The cumulative amplitude represents the sum of the peak amplitudes of the IPSCs.C, GABAergic L-IPSCs can be evoked in the presence of strychnine. The top trace represents 4 min of continuous recording in the presence of strychnine and during application of serotonin (1 μm). Bottom traces display the inhibitory activity, at a faster time scale, at the times indicated by the asterisk and filled circle. In this and the following figures, all of the cells were recorded in the whole-cell configuration of the patch-clamp technique with a CsCl-based internal solution and were held at −70 mV.

Fig. 2.

Characterization of the basket cell IPSCs.A, Four examples of the IPSCs evoked in a Golgi cell by electrical stimulation of the basket cell axons in the molecular layer just at the top of a Purkinje cell body are shown. B, Recruitment curve at the same location shows that the amplitude of the IPSCs continues to increase in discrete steps with the stimulation intensity, even when the probability of failure is null. Multiple fibers were recruited at this location. C, Gabazine blocks basket cell IPSCs. Averaged traces of IPSCs before (top), during (middle), and after (bottom) bath application of gabazine (30 μm) are shown.

Fig. 3.

GABA and glycine are coreleased by single Lugaro cells onto Golgi cells. A, The effect of serotonin (1 μm) on the inhibitory synaptic activity recorded from a Golgi cell is summarized in the top plot, where each dot represents an IPSC. The mean frequency of IPSCs over bins of 5 sec is plotted at the same time scale in the bottom graph. Note the monophasic increase in the amplitude and frequency of the IPSCs after application of serotonin. Gabazine (30 μm) decreased both the amplitude and the frequency of the IPSCs in the presence of serotonin.B, Representative sections (2.5 sec) of recording taken before (top; 4 min time point in A) and after (bottom; 5 min time point in A) gabazine application are shown. In the presence of gabazine, IPSCs under the threshold of detection (10 pA) are indicated by anasterisk. C, Amplitude histograms of the IPSCs during the control period (left) and in the presence of 5-HT (right) are shown. D, Histograms show the distribution of the intervals between two successive events during the control period (left) and for the IPSCs <60 pA in the presence of serotonin (right). E, The same histograms for the IPSCs >60 pA in the presence of serotonin (left) and for all the IPSCs recorded after addition of gabazine (right) are shown.

Fig. 4.

Immunocytochemical characterization of Lugaro cell transverse and sagittal axons in the molecular layer. Transverse (A–D) or sagittal (E) sections were double-stained with anti-calretinin antibody (green in A1, B1,C1) and anti-GAD65 (red inA2, B2) or anti-GlyT2 (redin C2) or with anti-GAD65 antibody (green in D1, E1) and anti-GlyT2 (red in D2,E2). A3, B3,C3, D3, and E3 are the superimposed images of A1 and A2,B1 and B2, C1 andC2, D1 and D2, andE1 and E2, respectively.A, Low magnification of a transverse section immunoreacted with calretinin (CR) and GAD65 is shown. Lugaro cell axons (arrowheads) run in the low molecular layer (low molec) of the cerebellar cortex, above the Purkinje cell bodies (P) that are surrounded by GABAergic terminals (arrows). Note the stronger background for the CR staining (asterisk) in the upper molecular layer. B, Varicosities of the calretinin-labeled transverse axons of the Lugaro cell show GAD65 immunoreactivity (arrowheads). Arrowsindicate GAD65-positive terminals devoid of calretinin.C, Transverse Lugaro cell axon stained for GlyT2 is shown. This is mainly visible in its varicosities (arrowheads). Thin fibers, probably parallel fibers, ascending in the molecular layer show faint calretinin immunoreactivity but are GlyT2 negative (arrows). D, GAD65 is also detected in GlyT2-immunoreactive axonal varicosities (arrowheads). In a few GlyT2-positive spots, GAD65 is not colocalized (double arrowheads). Because of their smaller diameter compared with double-stained varicosities, they most likely represent nonvaricose segments of Lugaro cell parasagittal axons cut transversally. E, Expression of GAD65 within varicosities stained for GlyT2 in the sagittal plane is shown. Note that in both transverse (D1) and sagittal (E1) sections, terminals positive for GAD65 only (arrows) outnumber varicosities coexpressing GABA and glycinergic markers (arrowheads), indicating the presence of numerous purely GABAergic boutons. Scale bars: A, 10 μm; B–E, 10 μm.

Fig. 5.

Glycine receptor immunoreactivity of the cerebellar Golgi cell. A, Low-magnification micrograph of a cerebellar section cut in the sagittal plane and immunoreacted with the mAb 4a. Golgi cell somata (arrows) in the granular layer and dendrites (arrowheads) in both granular and molecular layers appear stained for GlyRα/β, as well as some glomerular structures of the granular layer (crossed arrows). B, High magnification of a Golgi cell (reconstruction) located in the upper granule cell layer, showing diffuse staining of the cell body (arrow) and punctate labeling over the dendritic tree in the molecular layer (arrowheads). Note that only Golgi cell dendrites are stained in the molecular layer. The asterisk indicates a Golgi cell body out of focus. C, Diffuse intracellular staining of the Golgi cell body and initial dendritic portion, in contrast with the intense, punctiform labeling of dendrites (arrowheads). Note that the nucleus (n) is unstained. D, Labeled dendrites of Golgi cells in the molecular layer (arrowheads). E, Double immunodetection of GlyRα/β (E1) and VIAAT (E2) in the molecular layer. The slight shift in the immunoreactivity of the two markers indicates their close apposition (arrowheadsshow the location of VIAAT-positive presynaptic terminals). VIAAT-labeled terminals (arrows) not associated with glycine receptor clusters are thought to be involved in the numerous GABAergic synapses in the molecular layer. g, Granular cell layer; m, molecular layer; P, Purkinje cell layer; wm, white matter. Scale bars:A, 50 μm; B, 25 μm; C, 10 μm; D, 20 μm; E, 5 μm.

Fig. 6.

A, B, Colocalization of GlyR and gephyrin with GABA_AR clusters at Golgi cell dendrites in the low molecular layer is shown. Sagittal sections were double-stained with anti-GABA_ARγ2 antibody (greenin A1; red in B1) and anti-GlyRα/β (red in A2) or anti-gephyrin (green in B2) antibodies. A3 and B3 are the superimposed images of A1 and A2 and ofB1 and B2, respectively.A, Clusters positive for the two receptors are intermingled at single postsynaptic sites (A3,arrowheads). B, Clusters of GABA_ARγ2 are closely associated to, but do not colocalize with, gephyrin aggregates at putative postsynaptic sites on Golgi cell dendrites (arrowheads). In the insets(solid rectangles) of A3 andB3, a 2.5× magnification of a region where the labeling is representative (dashed rectangle) is shown enlarged.C–E, Lugaro to Golgi cell axonal contacts are shown. Transverse (C, D) or sagittal (E) sections were double-stained with anti-calretinin (green in C1) or anti-GlyT2 (green in D1, E1) antibodies and with an anti-GlyRα/β antibody (red inC2, D2, E2).C3, D3, and E3 are the superimposed images of C1 and C2,D1 and D2, and E1 andE2, respectively. C, D, Axonal calretinin-stained (C) and GlyT2-stained (D) varicosities are found apposed to GlyRα/β-immunoreactive clusters (arrowheads). Theinsets in C, taken from another section, show a contact at a higher magnification (5×). Note that a few terminals are not associated with GlyR (arrows).E, A GlyT2-immunoreactive varicose fiber from the Lugaro cell sagittal axonal plexus is shown. Some GlyT2 terminals are apposed to GlyR clusters (arrowheads), whereas others are not (arrows). The latter probably correspond to contacts between the Lugaro cell sagittal axon and molecular layer interneurons.F, Double immunostaining of parvalbumin (F1) and glycine receptor (F2) in the molecular layer is shown. Clusters of glycine receptor (arrows) are not associated with parvalbumin-immunoreactive profiles, belonging either to basket, stellate, or Purkinje cells (double arrowheads).Asterisks indicate the cell bodies of the molecular layer interneurons. Pva, Parvalbumin. Scale bars: A, B, F, 5 μm; C–E (bar inF), 10 μm.

Fig. 7.

Variability and coregulation of S-IPSC and L-IPSC decay kinetics. IPSCs were averaged during the control period and during application of serotonin. L-IPSCs were distinguished by their large amplitude. A, Example of averaged S-IPSCs (left; dotted lines) and L-IPSCs (right; solid lines) recorded from two Golgi cells is shown. B, S-IPSCs, as well as L-IPSCs, of cell 1 and cell 2 were scaled and superimposed. Both were faster in cell 1 than in cell 2. C, Plot of the half decay time of the S-IPSCs recorded from 26 Golgi cells against the age of the animals is shown. D, S-IPSCs and L-IPSCs were scaled in cell 1 and in cell 2. L-IPSCs are slower than are S-IPSCs in the same proportion in both cells. E, The half decay times of L-IPSCs measured in 12 Golgi cells are plotted against the half decay times of S-IPSCs in the same cells. The solid linerepresents the linear regression through the points. Its slope is 1.28. The dashed line indicates a slope of 1. L, L-IPSC; PN, Postnatal;S, S-IPSC.

Fig. 8.

The glycinergic components of the serotonin-evoked IPSCs account for their slower decay kinetics.A, S-IPSCs and the GABAergic component of L-IPSCs have the same decay time course. Averaged S-IPSCs and L-IPSCs_GABA recorded from the same cell in control conditions and after addition of serotonin and strychnine (1 μm) are plotted on the left. Biexponential functions fitting the decay of the IPSCs are displayed (solid lines). The time constants are 14 and 52 msec for the S-IPSCs and 20 and 57 msec for the L-IPSCs_GABA. The same IPSCs are scaled to their peak on the right. B, The time constants of decay of S-IPSCs and L-IPSCs_GABA obtained in the same six cells are plotted against each other. The slopes of the linear regressions (solid lines) are 1.16 and 0.86 for the fast (circles) and slow (triangles) components, respectively.C, The glycinergic component of L-IPSCs has a slower decay than S-IPSCs have. Averaged S-IPSCs and L-IPSCs_Glyrecorded from the same cell in control conditions and after addition of serotonin and gabazine (30 μm) are plotted on theleft. The sum of two exponential functions of time constants of 14 and 40 msec is superimposed on the decay of the S-IPSC, whereas the decay of the L-IPSCs_Gly is fitted by a monoexponential function with a time constant of 45 msec. The same IPSCs are scaled to their peak on the right.D, A plot the same as that inB is shown. In all cells the decay of the mean glycinergic IPSCs was appropriately fitted by a single-exponential function, and the single decay time constant of glycinergic IPSCs is compared with both time constants of control IPSCs. The slope of the linear regressions are 2.3 and 0.68.