Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain - PubMed (original) (raw)
Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain
Manuel Alvarez-Dolado et al. J Neurosci. 2006.
Abstract
Embryonic medial ganglionic eminence (MGE) cells transplanted into the adult brain can disperse, migrate, and differentiate to neurons expressing GABA, the primary inhibitory neurotransmitter. It has been hypothesized that grafted MGE precursors could have important therapeutic applications increasing local inhibition, but there is no evidence that MGE cells can modify neural circuits when grafted into the postnatal brain. Here we demonstrate that MGE cells grafted into one location of the neonatal rodent brain migrate widely into cortex. Grafted MGE-derived cells differentiate into mature cortical interneurons; the majority of these new interneurons express GABA. Based on their morphology and expression of somatostatin, neuropeptide Y, parvalbumin, or calretinin, we infer that graft-derived cells integrate into local circuits and function as GABA-producing inhibitory cells. Whole-cell current-clamp recordings obtained from MGE-derived cells indicate firing properties typical of mature interneurons. Moreover, patch-clamp recordings of IPSCs on pyramidal neurons in the host brain, 30 and 60 d after transplantation, indicated a significant increase in GABA-mediated synaptic inhibition in regions containing transplanted MGE cells. In contrast, synaptic excitation is not altered in the host brain. Grafted MGE cells, therefore, can be used to modify neural circuits and selectively increase local inhibition. These findings could have important implications for reparative cell therapies for brain disorders.
Figures
Figure 1.
Distribution of MGE derived cells 3 d after transplantation into neocortex and striatum. A, MGE-derived cells were detected by immunohistochemistry against GFP. Serial sections were used to determine the position of labeled cells. Notice the wide distribution throughout neocortex, striatum, and hippocampus. B, High magnification of area in **A**showing MGE cells moving away from injection site (*). C, Detail of a typical MGE migrating cell. D, Distribution of grafted cells 3 and 60 DAT; number of cells/distance of serial sections. Scale bars: A, 1 mm; B, 250 μm; C, 25 μm. F, Frontal; D, dorsal; L, lateral.
Figure 2.
Acquisition and distribution of mature interneuron morphology at 60 DAT. A, Camera lucida maps indicating the position of MGE graft-derived cells at three rostrocaudal levels after transplantation into neocortex (Ctx), hippocampus (Hp), and striatum (St). B, Detection of grafted cells by immunohistochemistry against GFP in the ipsilateral somatosensory cortex. Note the wide distribution of grafted cells in multiple cortical layers. Compare the dark background in layers I–II and V of the injected hemisphere (B) versus the contralateral hemisphere (C). E–K, GFP detection by immunohistochemistry provides a Golgi-like staining of grafted cells. MGE-derived cells in cortex differentiated into neurons presenting typical morphology of interneuron subtypes, e.g., bitufted or bipolar cells (E), chandelier cells (F) with synaptic boutons resembling candlesticks (arrowheads), basket cells (H), neurons with small body (I), and multipolar cells (J). In hippocampus, grafted cells accumulated in CA1 (D) and dentate gyrus (G). In striatum, the vast majority of cells differentiated into medium aspiny interneuron (K). Scale bars: B–D, F, H, I, 100 μm; E, G, J, K, 50 μm.
Figure 3.
Molecular characterization of MGE graft-derived cells in somatosensory (A–F, J–O) and cingulate cortex (G–I) 60 DAT. Immunohistochemical colocalization of grafted GFP+cells with GABA, PV, CR, SOM, and NPY. Arrowheads show double-positive cells for GFP and specific marker. Scale bar, 50 μm.
Figure 4.
Grafted MGE-derived cells in the dentate gyrus of the hippocampus 60 DAT. Immunohistochemical colocalization of MGE-derived cells expressing GFP with GABA (A–C), PV (D–F), and SOM (G). Arrowheads show double-positive cells. Scale bar, 100 μm.
Figure 5.
Double-immunohistochemical analysis of tissue obtained from grafted animals at 30 DAT failed to identify a significant population of non-neuronal GFP-expressing cells. Sample immunostaining panels are shown for NeuN (A), glial fibrillary acidic protein (B, GFAP), CaM kinase IIα (C, CamK), ChAT (D), TH (E), Olig2 (F), and S100β (G). Sections shown are at least 300 μm from the graft injection site. Note the absence of double-labeled cells. Near the injection site, GFP-positive cells with oligodendrocyte morphology (yellow arrowheads) were observed near putative blood vessels (red arrowheads).
Figure 6.
MGE-derived cells exhibit interneuronal firing properties. A, IR-DIC image overlaid with an epifluorescence image of an acute coronal slice (4 weeks after grafting) containing GFP+MGE-derived cells; epifluorescence image at right of a cell filled with Alexa Red during the patch recording. B, Membrane potential of the GFP+cell shown in **A**recorded under current clamp at the resting potential (approximately −71 mV). Note the small degree of inward rectification with hyperpolarizing current steps (200 ms) and the lack of spike frequency adaptation with long depolarizing current steps (1000 ms) typical of mature cortical interneurons. This cell was classified as an RSNP interneuron. C, Graph of firing frequency of recorded GFP+cells at depolarizing step of 0.2 nA (_n_= 14). D, Plot of the linear frequency–current relationship for a typical GFP-positive interneuron. E, Sample current-clamp traces from two different GFP-positive interneurons demonstrating spontaneous action potential firing in normal ACSF.
Figure 7.
MGE-grafted cells alter synaptic function in the host brain. A, Recording configuration for analysis of inhibitory current in the host brain. Panel shows the acute coronal slice with GFP+cells in layers I–III visualized under IR-DIC and epifluorescence microscope. A recording was obtained from a pyramidal neuron (yellow asterisk; cell filled with Lucifer yellow) in the vicinity of GFP+cells (green arrows). B, Sample traces of spontaneous IPSCs recorded from pyramidal cells (control brain and graft host brain) 4 weeks after grafting. Note the increase in IPSC amplitude and frequency for grafted animals versus age-matched controls. C, Cumulative data plots for all IPSC recordings from control (black bars) and grafted (red bars) animals are shown. Recordings were made at 2, 3, and 4 weeks after grafting. Data represent 7–10 cells for each bar; data are presented as mean ± SEM, and significance is taken as p< 0.05 using one-way ANOVA. D, Sample IPSC traces before and after application of the GABAAreceptor antagonist bicuculline (10 μ
m
BMI). E, Measurement of the total charge transfer for pyramidal cells from control (gray bars) and grafted (black bars) brain. Note the significant increase for grafted brains at 4 weeks. F, Cumulative probability plot of spontaneous IPSC interevent intervals shows higher frequency values for grafted brains (p< 0.05). G, Distribution histograms for IPSC decay time (left), 10–90% rise time (middle), and amplitude (right). Note the shift to larger amplitude IPSC events for pyramidal cells from grafted animals.
Figure 8.
Synaptic inhibitory current is increased in the hippocampus from grafted mice. A, Spontaneous IPSCs of hippocampal pyramidal cells from control grafted mice. B, Measurement of the total charge transfer of IPSCs recorded from CA1 hippocampal pyramidal cells from control (C) and grafted (G) brain. Note the significant increase values for grafted brains at 4 weeks. C, Cumulative probability plots of spontaneous IPSC interevent intervals shown higher frequency values for grafted brains (p< 0.05). Error bars indicate SEM. **p< 0.001 by ANOVA.
Figure 9.
Glutamatergic synaptic excitation is not altered in neocortex and MGE grafted mice. A, Plots of all cortical pyramidal cells sampled for spontaneous EPSC data. Spontaneous EPSC amplitude, decay time, and frequency show no significant difference between controls (C, light gray bars) and grafted (G, black bars) brains. B, Representative traces of spontaneous EPSCs recorded from a GFP+grafted cell at 4 weeks after grafting. Spontaneous EPSCs were abolished by application of CNQX and APV (bottom trace). C, Sample of evoked EPSC recording from GFP+grafted cells at different holding potentials showing the reversal membrane potential at 0 mV (see inset graph).
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