Restrained Dendritic Growth of Adult-Born Granule Cells Innervated by Transplanted Fetal GABAergic Interneurons in Mice with Temporal Lobe Epilepsy

Experimental timeline for RV labeling of adult-born GCs in naïve and SE mice. A, Different groups of mice studied and the experimental timelines for each group including: timing of retroviral injections (RV), day of transplantation of E13.5 MGE progenitors (TX), day of optogenetic and patch-clamp electrophysiological experiments, and 3D neuronal reconstructions and Sholl analyses. B, RV expression (mCherry, red) in a dentate GC showing labeling of dendritic arbor and axon. C, Patterns of RV-labeled GCs in an adult naïve mouse. D–G, Patterns of RV labeling in adult-born GCs in TLE mice following retroviral injections at one, two, six, or 12 weeks post-SE. Scale bars: 20 µm (B) and 200 µm (C–G).

Functional inhibition from transplanted ChR2-expressing MGE-derived GABAergic interneurons onto a mature adult-born GC labeled with RV one week post-SE. Transplanted interneurons were optogenetically stimulated while performing patch-clamp recordings from adult-born, RV-labeled GCs in hippocampal slices. A, Low-magnification image of the DG in an SE mouse that received injections of RV at one week post-SE and a transplant at two weeks post-SE. Boxed area shows RV-labeled GCs (red), and white arrow indicates the RV-labeled GC that was further characterized by patch-clamp electrophysiology, optogenetic stimulation, and biocytin staining (blue). Various interneuron subtypes comprising the transplant are shown in Extended Data Figure 2-1. B, Higher magnification view of the boxed region in A; the electrophysiologically-characterized and biocytin-filled GC is indicated by a white arrow in the group of adult-born GCs (red). C, Magnified view of the same group of RV-labeled adult-born GCs (red) showing the presence of dense plexus of MGE-derived ChR2-EYFP-expressing GABAergic axons (green). Note that due to photobleaching, the density of axons appears to be reduced around the cell bodies. D, Same biocytin-filled GC (white) surrounded by transplanted MGE-derived ChR2-EYFP-expressing GABAergic interneurons and their axonal arbors (green). E, High-magnification single optical slice showing dendritic segment of the biocytin-filled GC (white) and transplant-derived putative synaptic boutons (green). Arrows indicate sites of putative synaptic contacts by the transplanted interneurons onto this GC. F, A partial reconstruction of the same biocytin-filled GC (red). The yellow dots indicate the locations of putative inhibitory synaptic contacts from the transplants. G, Electrophysiological recording of this GC showing optogenetically-induced IPSCs, following blue-light stimulation of the ChR2-expressing transplanted interneurons (vertical blue bars indicate blue light pulses of 5-ms duration and 200-ms interpulse interval). Scale bars: 200 µm (A), 50 µm (B, C), 20 µm (D), and 2 µm (E).

Amplitudes of optogenetically-activated IPSCs in GCs strongly correlate with number of synaptic inputs from transplanted GABAergic interneurons. A–C, Optogenetically-induced synaptic currents were measured in adult-born GCs generated two, six, or 12 weeks post-SE, by whole-cell patch clamping and subsequently filled with biocytin (white). A’–C’, Merged images of filled GCs showing mCherry expression (red), biocytin-fill (blue), and transplant arborization (green) in these slices. A’’–C’’, Synaptic innervation of adult-born GCs shown in A–C demonstrated by optogenetic activation of transplanted interneurons. D, E, GCs located outside of the region innervated by the transplanted GABAergic interneurons did not show optogenetically-induced currents. F, Summary graph from all recorded GCs (both RV-labeled and non-labeled) showing strong correlation between the density of transplant-derived boutons and the peak amplitude of light-induced inhibitory currents. Adult-born GCs with confirmed RV labeling are indicated by colored triangle-shaped symbols. The blue triangles are GCs labeled one week post-SE; the green triangles are GCs labeled two weeks post-SE; the red triangles are GCs labeled 12 weeks post-SE. The gray circles are GCs with unknown birthdates.

Transplanted GABAergic interneurons innervate adult-born GCs in naïve mice. A, Low-magnification image showing RV-labeled adult-born GCs (red) surrounded by a transplant of ChR2-eYFP-expressing interneurons (green) in the DG of a naïve mouse. Boxed region in A, biocytin-filled (blue) and RV-labeled (red) adult-born GC that was characterized by whole-cell patch clamp electrophysiology. B, Magnified view of the boxed region in A, the same biocytin-filled GC is shown to co-express mCherry. C, Biocytin-filled GC (pseudo-colored white for enhanced visibility) surrounded by neuropil from transplanted MGE-derived GABAergic interneurons (green). D, Electrophysiological recording from this GC showing postsynaptic IPSCs induced by exciting the ChR2-expressing interneurons in this transplant. Vertical blue bars indicated blue light pulses delivered to the slice. E, Sholl analyses of the dendritic arbor of this GC provide quantification of dendritic crossings every 10-µm interval from the cell’s soma. F, IMARIS 3D reconstruction of this GC showing distribution of putative transplant-derived synapses (yellow dots). Scale bars: 100 µm (A), 50 µm (B), and 20 µm (C).

Transplanted GABAergic interneurons innervate adult-born GCs born six weeks post-SE. A, Low-magnification image showing adult-born GCs (red) surrounded by ChR2-eYFP expressing transplants (green) in a mouse that had SE. Boxed region shows the biocytin-filled (blue) adult-born GC that was recorded in this slice using whole-cell patch clamping. B, Magnified view of the boxed region, showing co-labeling with biocytin and mCherry. C, View of the biocytin-filled GC (white) and surrounding axons from transplanted MGE-derived GABAergic interneurons (green). D, Electrophysiological recording from this GC showed a moderate response to optogenetic activation of the ChR2-expressing transplanted interneurons and a distinct IPSC can be seen in response to the first light-pulse. E, Sholl analysis graph of the number of dendritic intersections. F, Complete neuronal reconstruction of this GC and the sites of putative transplant-derived synapses (yellow dots). Scale bars: 100 µm (A), 50 µm (B), and 20 µm (C).