Abstracts

RATIONALE: Recent studies suggest that neuronal precursors from the medial ganglionic eminence (MGE) tangentially migrate to neocortex and differentiate into a stable population of GABA-, parvalbumin- or somatostatin-expressing interneurons. Previous work has shown that grafted MGE cells can also migrate and differentiate into interneurons throughout the cortical plate in the host animal. However, it is not known whether newly integrated inhibitory interneurons make functional synaptic connections with existing neurons in neocortex. To address this issue we examined GABAergic function in rodents receiving grafted MGE neuronal precursors.
METHODS: For transplantation, MGE regions were dissected from green fluorescent protein (GFP) mice at embryonic day 14. After mechanical dissociation, cells were loaded into a glass micropipette and stereotactically grafted into brains of 1-day-old rats. Animals were sacrificed at postnatal days 7, 14, 21 and 28. For immunohistochemistry, staining was performed on free-floating 50-100 [mu]M vibratome brain slice sections using antibodies against GABA, somatostatin, parvalbumin, calretinin, calbindin and GFP. For elecctrophysiology, IR-DIC visualized whole-cell voltage-clamp recordings were performed on neocortical brain slices from grafted rodents at 3 different ages (P14-P21-P28, hp: 0 mV). To isolate GABAergic synaptic currents, slices were perfused with nACSF supplemented with CNQX and APV. Spontaneous and evoked IPSCs (sIPSCs- eIPSCs) were recorded from neocortical pyramidal cells in Layers II/III. All cells were filled with Lucifer yellow and analyzed post hoc.
RESULTS: MGE GFP-positive grafted cells migrated from injection sites to the ipsilateral neocortex. At P7, GFP+ cells exhibited the typical migratory morphology expected for MGE neurons and were located mainly in cortical layers II/III. From P14 to P21 grafted cells began to differentiate, reaching a mature morphology by postnatal day 28. Grafted GFP+ cells stained with standard interneuron markers including GABA, somatostatin, parvalbumin, calretinin and calbindin. Spontaneous and evoked IPSCs could be recorded from neocortical pyramidal cells in layers II/III of slices from grafted animals. IPSCs were abolished by the addition 10 [mu]M bicuculline to the bathing medium suggesting that they were mediated by GABA-A receptors. Initial analysis of sIPSC kinetics at P14 revealed a frequency ~ 0.8 Hz and an amplitude ~21 pA (n = 20). At P28, closer to a mature adult time-point and nearly a month after grafting, sIPSC kinetic analysis indicated a frequency ~1.3 Hz and an amplitude ~21 pA (n = 10).
CONCLUSIONS: Our results suggest MGE precursors grafted into a host animal differentiate into cells with the anatomical and physiological properties of GABAergic interneurons. If MGE grafted cells can be used to increase the functional level of inhibition in the host animal, then this type of strategy could prove extremely beneficial for the treatment of epilepsy.
[Supported by: Parents Against Childhood Epilepsy (P.A.C.E.).]