Fig 2: A direct excitatory projection from EC-6b to hippocampal CA3.a Representative horizontal section stained for CPLX3 and DAPI (left) alongside an expanded view of the cortex (right). b Representative STED images from LEC-1, stained for CPLX3 alongside either VGluT1 (top left) or VGAT (bottom left). Expanded inserts are shown to the right of each image above their corresponding mask, demonstrating the calculation of colocalization for each marker. c Analysis of synaptic density, using STED images acquired from the fields denoted by green dashed squares in (a). Synaptic density was calculated here as the ratio between the signal area and total image area. d Relative eCPLX3 and iCPLX3 signal density of the total VGluT1 or VGAT signal area. e Unilateral injection of retAAV into the CA1 enables genetic targeting of the contralateral CA3 for retrograde labeling, exploiting the abundance of commissural axons of CA3 pyramidal neurons (top schematics). Representative horizontal sections from the mid-dorsal hippocampus of the ipsilateral (bottom left) and contralateral (bottom center) hemispheres for RetAAV-cre injection. Double-labeled neurons in the enlarged inset (bottom right) are putative starter cells. f, g Representative horizontal section from the ventral hippocampus following CA3-specific retrograde labeling reveals a population of neurons in the deepest layer of the EC (f), which immunolabels positive for the subplate-specific marker CPLX3 (72/78 cells counted from 5 animals, g). h Representative horizontal sections of the layer 6b following retrograde labeling with CVS-N2c(deltaG)-tdTomato, from CA3 neurons of a GAD1-EGFP mouse (left). Numbers above the bars in the right-hand panel indicate the total number of double-labeled tdTomato+/EGFP+ somata out of the total tdTomato+ somata per each of the regions examined (P = 1.2 × 10-9; two-sided Fisher’s exact test). i Two biocytin-labeled CA3-projecting pSPNs (magenta) from an acute hippocampal section (left) and an enlargement of a dendritic segment (right). Experiment has been reproduced for 12 cells with identical results. j Representative traces of spontaneous EPSCs (top trace) and membrane potential following current injections of -50 pA (blue), 100 pA (red), and 200 pA (black; bottom traces). Summary plot below shows the firing frequency as a function of current amplitude (N = 31 cells). Scale bars for magnified representative EPSCs (top trace) and first AP at rheobase current (bottom trace) indicate 25 ms/20 pA and 2 ms/20 mV, respectively. N in c, d represents number of individual sections from 6 different animals. Data in c, d, j is shown as mean and SEM.

Fig 3: ?2 is essential for GABAAR synaptic localization in the brain.(A, B) Localization of GABAAR subunits in the cerebellar granule cell (GC)-?2 knockout (KO) mice and age matched controls without Cre expression (Control). Inhibitory presynaptic VGAT and excitatory postsynaptic GluN1 were co-stained. (A) Loss of ?2 was observed specifically in the granular layer in ?2 GC-KO mice, whereas a1 and ß2 remained. The images are representative of four independent experiments. (B and C) High-magnification representative images showed protein distribution on each glomerulus. Inhibitory inputs project to outer edges of the glomerulus, whereas excitatory inputs project to inner edges of the glomerulus. In the ?2 GC-KO, the fraction of a1 colocalized with VGAT was reduced, whereas the fraction of GluN1 colocalized with a1 was increased (n = 30 areas/2 animal each). (D–F) Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded from granule cells in acute cerebellar slices, and representative traces are shown (D). In ?2 GC-KO mice, sIPSC frequency (E) and amplitude (F) were dramatically reduced, but not completely eliminated (n = 4 bins (E), n = 69–1740 events (F), see Materials and methods). The asterisk indicates a sIPSC recorded from a ?2 GC-KO mouse. Picrotoxin (100 µM) blocked all sIPSCs. (G and H) Representative images show localization of gephyrin in ?2 GC-KO and control mice. Gephyrin colocalized with VGAT at the glomerular periphery in controls. In the ?2 GC-KO, the fraction of gephyrin colocalized with VGAT was reduced, and at the same time, the fraction of GluN1 colocalized with gephyrin was reduced (n = 30 areas/2 animal each). Scale bars: 60 µm (A), 5 µm (B, G). Data are given as mean ± s.e.m.; p values were determined using student’s t test.

Fig 4: SIM and dSTORM reveal NL2 is organized into nanoscale structures at inhibitory synapses. (A) 3D-SIM maximum projection of hippocampal dendrite labeled with antibodies to NL2, gephyrin and VGAT. Scale bar = 1 µm. (B) Magnifications of synapses from 3D-SIM images, with line-scans showing nanoscale subsynaptic domains (SSDs) of NL2 and gephyrin. White lines show path of line scans on the right. Arrows indicate NL2 and gephyrin SSDs. Scale bar = 500 nm. (C) 3D reconstruction of inhibitory synapse in panel (B), upper panel, labeled for NL2, gephyrin and VGAT. XY scale bar = 320 nm, Z scale bar = 600 nm. Also see Supplementary Movie 1. (D) dSTORM rendered images of VGAT-positive inhibitory synapses labeled with antibodies to NL2 and gephyrin. Arrows indicate high-density regions. Scale bar = 250 nm. (E) Localization plots of dSTORM data from panel (D).

Fig 5: PC collaterals preferentially inhibit mGluR1+ UBCs via fast GABAAR-mediated feedback.(A) Schematic showing a mossy fiber (MF) and a PC collateral innervating a UBC. A whole-cell electrode was used to voltage clamp the UBC, and responses were measure for either a glutamate puff applied with a nearby electrode or to optical stimulation of PC collaterals. (B) Whole-cell recordings of glutamate-evoked currents (left) and to optically evoked PC inhibition (right) are shown for cell #1 of (D). (C) As in (B), but for cell #41 of (D). (D) Summary of cells in which responses to glutamate puffs were measured, with red corresponding to an excitatory inward current and blue corresponding to an inhibitory outward current. Cells with light-evoked inhibitory synaptic current are indicated with a black dot (n=41). (E) Light-evoked synaptic currents were recorded in baseline (black) and after the addition of the GABAA receptor antagonist gabazine (gray). (F) Normalized cumulative distributions of charge evoked by a glutamate puff for cells that either had light-evoked responses (black) or that did not (gray). (G) Average currents evoked by a glutamate puff for cells that had PC feedback (black) and that did not have PC feedback (gray). (H) Schematic as in (A), but for application of the mGluR1 agonist DHPG. DHPG-evoked increases in firing were measured with a cell-attached electrode, and then optically evoked synaptic currents were measured with a whole-cell voltage-clamp recording. (I) DHPG-evoked increases in firing (left) and light-evoked synaptic currents (right) are shown for cell #35 in (K). (J) Same as (I) but for cell #45 in (K). (K) Summary of cells in which firing evoked by DHPG puffs was quantified. Cells with light-evoked inhibitory synaptic current are indicated with a black dot (n=60). (L) Normalized cumulative distributions of charge evoked by a DHPG puff for cells that either had light-evoked responses (black) or that did not (gray). (M) Average currents evoked by a DHPG puff for cells that had PC feedback (black) and that did not have PC feedback (gray). (N) Scatter plot of IPSC amplitudes as a function of the peak firing rates for the cells in (K). DHPG, (S)-3,5-Dihydroxyphenylglycine; IPSC, inhibitory post-synaptic current; mGluR1, metabotropic glutamate receptor type 1; PC, Purkinje cell; VGAT, vesicular GABA transporter.