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.2016 May 23;3(2):ENEURO.0055-16.2016.
doi: 10.1523/ENEURO.0055-16.2016. eCollection 2016 Mar-Apr.

Only a Minority of the Inhibitory Inputs to Cerebellar Golgi Cells Originates from Local GABAergic Cells

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Only a Minority of the Inhibitory Inputs to Cerebellar Golgi Cells Originates from Local GABAergic Cells

Mark D Eyre et al. eNeuro..

Abstract

Cerebellar Golgi cells (GoCs) efficiently control the spiking activity of granule cells through GABAA receptor-mediated tonic and phasic inhibition. Recent experiments provided compelling evidence for the extensive interconnection of GoCs through electrical synapses, but their chemical inhibitory synaptic inputs are debated. Here, we investigated the GABAergic synaptic inputs of GoCs using in vitro electrophysiology and quantitative light microscopy (LM) and electron microscopy (EM). We characterized GABAA receptor-mediated IPSCs in GoCs and Lugaro cells (LuCs), and found that IPSCs in GoCs have lower frequencies, smaller amplitudes, and much slower decay kinetics. Pharmacological and LM immunolocalization experiments revealed that GoCs express α3, whereas LuCs express α1 subunit-containing GABAA receptors. The selective expression and clustered distribution of the α3 subunit in GoCs allowed the quantitative analysis of GABAergic synapses on their dendrites in the molecular layer (ML). EM and LM experiments in rats, and wild-type and GlyT2-GFP transgenic mice revealed that only one third of axon terminals establishing GABAergic synapses on GoC dendrites contain GlyT2, ruling out LuCs, globular cells, and any noncortical glycinergic inputs as major inhibitory sources. We also show that axon terminals of stellate/basket cells very rarely innervate GlyT2-GFP-expressing GoCs, indicating that only a minority of the inhibitory inputs to GoCs in the ML originates from local interneurons, and the majority of their inhibitory inputs exclusively releases GABA.

Keywords: GABA; cerebellum; immunohistochemistry; inhibition; patch clamp; synapses.

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Figures

Figure 1.
Figure 1.
Morphological and electrophysiological characterization of granule cell layer interneurons in the GlyT2-GFP mouse.A, Maximum intensity projection image of a confocal imageZ-stack of anin vitro recorded and biocytin (red)-labeled GoC showing GlyT2-GFP immunoreactivity (green). Scale bar, 20 μm. The Neurolucida reconstruction of this cell is shown on the right (dendrites in blue; axon in black). Scale bar, 20 μm. Voltage responses of this cell to DC current injections are shown on the far right.B, As inA, but showing a Lugaro cell. Although the GFP signal was not detectable in this cell after the recording, prior to patching this cell was clearly GFP positive, as shown in the epifluorescent grayscale image in the inset (arrow; arrowheads indicate adjacent GlyT2-GFP-expressing neurons). The Neurolucida reconstruction of this cell is shown on the right (dendrites in red; axon in black). Scale bar, 20 μm. Voltage responses of this cell to DC current injections are shown on the far right. Note the frequent occurrence of spontaneous IPSPs in the traces. The IPSPs are depolarizing due to the elevated Cl− concentration of the intracellular solution.C, Summary of some electrophysiological properties of morphologically identified Golgi (blue) and Lugaro (red) cells. Rin, Input resistance; Rheobase, minimum current required to generate at least one action potential. **p < 0.01, ***p < 0.001. Testing membrane potential (RMP) and Sag ratio were not different between cell types (p = 0.34 andp = 0.46, respectively). Average rheobase current was significantly different between cell types (p = 0.0002). Comparisons were made by Mann–WhitneyU test or one-way ANOVA.
Figure 2.
Figure 2.
GoCs and LuCs have distinct mIPSC properties and different GABAAR α subunit content.A, Continuous current recordings from a GoC (blue) and a LuC (red) in the presence of 1 μm tetrodotoxin before and after the application of 20 μm SR95531 (traces below). Note the greater frequency and larger amplitude of mIPSCs in the LuC, and that all currents were eliminated by the GABAAR antagonist.B, Average, peak-scaled traces from example cells before (GoC, blue; LuC, red) and after bath application of 100 nm TP003 (left: GoC, teal; LuC, orange) or 100 nm zolpidem (middle: GoC, gray; LuC, dark red). Note the much faster mIPSC decay and selective prolongation by 100 nm zolpidem in the LuC, compared with the slower decay and selective prolongation by 100 nm TP003 in the GoC. Right, The cumulative probability distributions of τw of all individually fitted mIPSCs in GoCs (blue;n =408 mIPSCs) and LuCs (red;n = 2300 mIPSCs).C, Summary of mIPSC mean frequency, peak amplitude, and τw before (GoCs, blue; LuCs, red) and after bath application of 100 nm TP003 or 100 nm zolpidem. Note the large variance in τw in the population of GoCs, and that GoCs with a slower decay show a greater prolongation upon TP003 application. **p < 0.01 (repeated-measures parametric ANOVA, Tukey’s unequaln HSDpost hoc test).
Figure 3.
Figure 3.
GlyT2-GFP-expressing GoC dendrites in the ML are sparsely innervated by GlyT2-GFP-expressing axons.A,B, Immunofluorescent labeling for the GABAAR α3 subunit (α3, green) in the ML (A) and GCL (B) of WT mice is sparse, but overlaps with neuroligin-2-immunopositive (NL2; red) and β3 subunit-immunopositive (β3; cyan) puncta, indicating their synaptic enrichment. Maximum intensity projections of two (A) or four (B) confocal images at 1 μm separation. Scale bars:A,B, 2 µm.C, Immunofluorescent labeling for the GABAAR α3 subunit (red) in the ML of GlyT2-GFP mice is evident as multiple puncta, many of which are associated with GlyT2-GFP-expressing (green) or neurogranin-immunoreactive (blue) dendrites. Scale bar, 10 µm.D, Maximum intensity projection (five confocal sections at 1 μm separation) of the left-hand boxed region inC at a higher magnification showing α3 subunit-immunoreactive puncta (arrows) associated with a GlyT2-GFP-expressing dendrite, but lacking a presynaptic GlyT2-GFP-expressing bouton. Scale bar, 1 µm.E, Maximum intensity projection (five confocal sections at 1 μm separation) of the right-hand boxed region inC at a higher magnification showing intersections between GlyT2-GFP-expressing axons (arrowheads) and a neurogranin-immunoreactive dendrite. Note the presence of additional GABAAR α3 subunit-immunoreactive puncta not associated with a presynaptic GlyT2-GFP-expressing bouton (arrow). Scale bar, 1 µm.F, Schematic proportional representation of α3 subunit-immunoreactive puncta present on GoCs in lobule 5 (left) and lobule 8 (center), categorized as expressing neurogranin (Ng+, blue cells) or only GlyT2-GFP (GlyT2+, green cells). Each punctum was categorized as either facing a presynaptic, GlyT2-GFP-expressing (green) or GlyT2-GFP negative (hollow) axonal bouton, and total puncta density per millimeter GoC dendrite for each GoC type is indicated below each cell. The graph (right) shows the percentage of GlyT2-GFP-positive (green) and -negative (hollow) boutons contacting α3-positive clusters for each type of GoC in the two lobules.G, Electron micrograph showing an asymmetric synapse made by a parallel fiber bouton (PFb) onto a GlyT2-GFP-DAB-labeled dendrite (GFP+d). Scale bar:G (forG–J), 500 nm. H,I, Electron micrographs showing symmetrical synapses formed by unlabeled boutons (b) onto GlyT2-GFP-DAB-labeled dendrites.J, Electron micrograph showing a symmetrical synapse formed by a GlyT2-GFP-DAB-labeled bouton (GFP+b) onto a GFP+d. Two PFbs forming asymmetric synapses onto the same dendrite are also visible. Bar graph shows the percentage of symmetrical synapses formed onto GFP+d by GFP-positive (GFP+b) and GFP-negative (b) boutons.
Figure 4.
Figure 4.
GlyT2-GFP-expressing axons target MLIs.A, Electron micrograph showing a GlyT2-GFP-DAB-positive axon varicosity (GFP+b) making a symmetrical synapse (arrowhead) with a smooth, thin, nonspiny MLI dendrite (MLId). Scale bar, 500 nm.B, Electron micrograph showing a GFP+b establishing a symmetrical synapse with a MLI soma (MLIs). Scale bar, 2 μm.C, Electron micrograph of the boxed region inB showing the synaptic junctions (arrowheads) at a higher magnification. Scale bar, 500 nm.D, Electron micrograph showing a thin GlyT2-GFP-DAB-immunoreactive interbouton axon segment (black precipitate) adjacent to, but lacking a synapse with, a PC dendrite (PCd). Scale bar, 500 nm. Bar graph shows the percentage of synapses formed by GlyT2-GFP-DAB-immunoreactive axons in MLId, MLIs, GlyT2-GFP-DAB-immunoreactive dendrites (GFP+d), and PCd.
Figure 5.
Figure 5.
Only one third of GABAAR α3 subunit-immunoreactive puncta face varicosities immunolabeled for GlyT2.A, Maximum intensity projection (six confocal sections at 1 μm separation) from a Wistar rat section immunolabeled for GABAAR α3 (red) and GlyT2 (cyan). Scale bar, 20 µm.B, Enlarged view of the boxed region inA showing GABAAR α3-immunoreactive puncta (left, red) either not associated with (arrows) or closely associated with (double-headed arrow) GlyT2-immunoreactive terminals (middle, cyan), as can be seen in the overlay (right). Quantification of GABAAR α3-immunoreactive puncta is shown for three genotypes in the lower part of the overlay panel. All images are maximum intensity projections of four confocal sections at 1 μm separation. Scale bar, 2 µm.
Figure 6.
Figure 6.
GlyT2-GFP-expressing dendrites receive negligible inputs from MLI axons.A, Maximum intensity projection image of a confocal imageZ-stack of anin vitro recorded and biocytin (red)-labeled MLI with axons spreading among GlyT2-GFP-expressing processes (green). Scale bar:A (forA–C), 10 μm.B, Neurolucida reconstruction of the soma and axon of the recorded cell (red; black circles indicate varicosities; green circles indicate varicosities next to GlyT2-GFP-expressing dendrites). The number of reconstructed cells and the percentage occurrence of each varicosity type are indicated.C, Boxed area inA shown as a series of confocal image planes highlighting a single putative contact (green arrow). Note that most varicosities of the biocytin-filled axon have unlabeled targets, despite numerous GlyT2-GFP-expressing structures in the vicinity.D, Neurolucida reconstruction of a different MLI (soma and dendrites in blue, axon in red). Scale bar, 50 μm. Boxed regions correspond to the fluorescent images shown inE (left) andF (right).E, The axon of the MLI inD makes a bouton in apposition (red) to a calbindin-immunoreactive (CB; cyan) Purkinje cell dendrite.F, The same axon also has several boutons in close apposition with a weakly GlyT2-GFP-labeled MLI soma (GFP; green). Scale bar:F (forE,F), 5 μm.
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References

    1. Acsády L, Görcs TJ, Freund TF (1996) Different populations of vasoactive intestinal polypeptide-immunoreactive interneurons are specialized to control pyramidal cells or interneurons in the hippocampus. Neuroscience 73:317–334. [TQ1] - PubMed
    1. Ankri L, Husson Z, Pietrajtis K, Proville R, Léna C, Yarom Y, Dieudonné S, Uusisaari MY (2015) A novel inhibitory nucleo-cortical circuit controls cerebellar Golgi cell activity. Elife 4:e06262. 10.7554/eLife.06262 - DOI - PMC - PubMed
    1. Batini C, Buisseret-Delmas C, Compoint C, Daniel H (1989) The GABAergic neurones of the cerebellar nuclei in the rat: projections to the cerebellar cortex. Neurosci Lett 99:251–256. - PubMed
    1. Bäurle J, Grüsser-Cornehls U (1997) Differential number of glycine- and GABA-immunopositive neurons and terminals in the deep cerebellar nuclei of normal and Purkinje cell degeneration mutant mice. J Comp Neurol 382:443–458. 10.1002/(SICI)1096-9861(19970616)382:4<443::AID-CNE2>3.0.CO;2-2 - DOI - PubMed
    1. Billings G, Piasini E, Lrincz A, Nusser Z, Silver RA (2014) Network structure within the cerebellar input layer enables lossless sparse encoding. Neuron 83:960–974. 10.1016/j.neuron.2014.07.020 - DOI - PMC - PubMed

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