Endogenous neurotransmitter activates N-methyl-D-aspartate receptors on differentiating neurons in embryonic cortex.

Abstract

Before synapses form in embryonic turtle cerebral cortex, an endogenous neurotransmitter activates N-methyl-D-aspartate (NMDA) channels on neurons in the cortical plate. Throughout cortical development, these channels exhibit voltage-dependent Mg2' blockade and are antagonized by D-2-amino-5-phosphonovaleric acid, a selective NMDA receptor antagonist. The activation in situ of these nonsynaptic NMDA channels demonstrates a potential physiological substrate for control of early neuronal differentiation.N-Methyl-D-aspartate (NMDA) receptors, in addition to their well-recognized role in synaptic plasticity (1-4), may play an equally important role in the control of neuronal growth and differentiation in the central nervous system (5, 6). Results from experiments using neurons growing in culture suggest that NMDA receptor activation can promote neuronal survival (7,8) and, by regulating intracellular Ca2l levels in growth cones during neurite outgrowth (9, 10) can influence neuronal form (7,8). If similar mechanisms are to operate during normal development, then NMDA receptors on young neurons must be activated by endogenously released neurotransmitter. The recent development of techniques for whole-cell voltage-clamp recording in intact embryonic brain (11) made it possible to test directly whether NMDA receptors are activated during early neuronal differentiation.The early anatomical differentiation of the cerebral cortex proceeds along common lines in all higher vertebrate species (12). Neurons are generated in a ventricular zone, migrate radially toward the pia, and collect in a layer to form the cortical plate. We chose turtles for our physiological studies because the intact embryonic turtle forebrain can be maintained in vitro, and whole-cell recordings can be obtained from cortical neurons (11), allowing analysis of the development and properties of functional NMDA receptors in situ. MATERIALS AND METHODSFertilized eggs of red-eared turtles (Pseudemys scripta, from Tangi Turtles, Ponchatoula, LA) were kept moist in an incubator at 30°C; embryos selected for use were staged according to the morphological criteria of Yntema (13). Embryonic turtles were removed from eggs and anesthetized with hypothermia according to Stanford University guidelines for the care and use of animals. Incisions were made in brain hemispheres at rostral and caudal levels and the midline, allowing the cortex to be flattened, ventricular side up.The preparation, secured on the stage of an upright microscope, was superfused with Ringer's solution (1.5 ml/min at 22-24°C) containing 96.5 mM NaCl, 2.6 mM KCI, 2 mM CaC12, 31.5 mM NaHCO3, and 10 mM dextrose. Bathing solution also contained 0.3-0.5 ,uM tetrodotoxin, 5-10 gM bicuculline methiodide, and in most experiments, the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) at 4 uM. NMDA receptor antagonists D-2-amino-5-phosphonovaleric acid (D-APV; ref. 14) and Mg2+ were added to the bathing solution at 50-250 gM and 2 mM, respectively.Patch electrodes (5 Mfl) containe...

Enhanced activation of NMDA receptor responses at the immature retinogeniculate synapse

“…In view of the proposed involvement of NMDA receptors in synaptic plasticity, it has been suggested that the greater plasticity characterizing immature synapses results from an altered NMDA neuronal response at early stages of development (Garthwaite et al, 1987;Tsumoto et al, 1987;Ben-Ari et al, 1988;BodeGreuel and Singer, 1989;Tremblay et al, 1989;Kleckner and Dingledine, 1991;Siviy et al, 1991) although other authors disagree (Blanton et al, 1990). Recording from LGNd cells in the voltage-clamp mode, we have directly examined the properties of the NMDA-mediated activity during development of retinogeniculate pathways.…”

“…Mechanisms that have been proposed for this enhancement include changes in the number of receptors (Garthwaite et al, 1987;Bode-Greuel and Singer, 1989;Trembley et al, 19891, their response properties (Cambray-Deakin et al, 1990; but see Blanton et al, 1990), or late development of GABAergic inhibition (Luhman and Prince, 1990; Agmon and O'Dowd, 1993;Burgard P21 and Hablitz, 1993). For instance, NMDA receptors in young rat brain have been found to be more sensitive to activation by glycine (Kleckner and Dingledine, 1991) and less sensitive to blockade by Mg2+ (Bowe and Nadler, 1990;Morrisett et al, 1990;Kleckner and Dingledine, 1991) than NMDA receptors found in the mature state.…”

Inhibitory Gating Modulation of Small Conductance Ca2+-Activated K+Channels by the Synthetic Compound (R)-N-(Benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) Reduces Afterhyperpolarizing Current in Hippocampal CA1 Neurons

“…Giga-seal whole-cell recordings were obtained from the somata of CA1 pyramidal cells using the "blind" patch-clamp technique (Blanton et al, 1989). Patch pipettes (4.0-6.5 M⍀) were pulled from borosilicate glass capillary tubes in a two-stage vertical puller (PP-830; Narishige, Tokyo, Japan) and were filled with a pipette solution containing 135 mM potassium methylsulfate, 10 mM KCl, 10 mM HEPES, 1 mM MgCl 2 , 2 mM Na 2 -ATP, and 0.4 mM Na 3 -GTP; pH 7.2-7.3 with KOH; osmolarity 280-295 mOsM.…”

Embryonically Expressed GABA and Glutamate Drive Electrical Activity Regulating Neurotransmitter Specification

“…Neurotransmitters are expressed at early stages of development, long before synapse formation (Lauder et al, 1986;Ma et al, 1992;Antal et al, 1994;Berki et al, 1995), and their release can stimulate elevations of intracellular calcium (Blanton et al, 1990;Flint et al, 1999). They regulate proliferation in rat embryonic neocortical progenitor cells and subventricular zone neuroblasts, and zebrafish spinal interneurons (LoTurco et al, 1995;Liu et al, 2005;McDearmid et al, 2006).…”