Elsevier

Neuroscience

Volume 150, Issue 4, 19 December 2007, Pages 829-840
Neuroscience

Cellular neuroscience
Optical and electrophysiological recordings of corticospinal synaptic activity and its developmental change in in vitro rat slice co-cultures

https://doi.org/10.1016/j.neuroscience.2007.10.010Get rights and content

Abstract

Electrophysiological recordings and optical imaging with a fast voltage-sensitive dye (di-4-ANNEPS) were used to directly examine the spatiotemporal properties of in vitro corticospinal synapses formed in co-cultures of cerebral cortex and spinal cord slices. Whole cell recordings from spinal cord cells showed both monosynaptic and polysynaptic excitatory postsynaptic currents (EPSCs) in response to stimulation of corticospinal axons. Monosynaptic EPSCs and excitatory postsynaptic potentials (EPSPs) were isolated in artificial cerebrospinal fluid containing high concentrations of divalent cations. Optical imaging and extracellular recordings were done simultaneously. Both EPSPs and optically recorded excitatory postsynaptic potentials (optEPSPs) lasted 300–500 ms and were almost always positive. The major component of these long-lasting potentials was blocked by ifenprodil, a specific antagonist of the NR2B subunit-containing N-methyl-d-aspartate receptor (NMDAR). The spatial distribution of corticospinal optEPSPs paralleled that of the corticospinal field excitatory postsynaptic potentials (fEPSPs), suggesting that positive fEPSP amplitude is a reliable indicator of the distribution of corticospinal synapses. Corticospinal optEPSPs spread into the ventrolateral region by 6–7 days in vitro (DIV), but were restricted to the dorsomedial area by 11–13 DIV, suggesting synapses were eliminated from the ventrolateral side of the spinal cord. After the recordings were complete, corticospinal fibers were often anterogradely labeled with biocytin to assess the relation between presynaptic fiber distribution and the optical signals (optically-recorded presynaptic fiber volley (opt-prevolley) and optEPSP). The distributions of the opt-prevolleys and optEPSPs correlated well with the distribution of presynaptic fibers, suggesting the opt-prevolley reflects corticospinal fiber activity and that the fibers made synapses relatively evenly along their axons. The NR2B-mediated component of the corticospinal synaptic response declined during the interval between 6 and 7 DIV and 11–13 DIV, suggesting that a shift in the NMDAR subtype from NR2B to something else (perhaps NR2A) may be involved in regulating developmental plasticity in the rat spinal cord and the process of corticospinal synapse elimination.

Section snippets

Organotypic slice culture

The methods used for slice co-cultures of sensorimotor cortex and spinal cord were essentially the same as described elsewhere (Takuma et al., 2002). Briefly, blocks of brain and spinal cord were excised from Wistar rats at postnatal day 0 or 1. Coronal slices (330 μm) of the sensorimotor cortex and axial slices (430 μm) of the cervical cord were sectioned with a Microslicer (Dosaka EM, Kyoto, Japan) in a chilled “cutting solution” (120 mM choline chloride, 3 mM KCl, 28 mM NaHCO3, 1.25 mM NaH2PO

Whole cell recordings of CS excitatory postsynaptic currents (EPSCs)

Initially, we stimulated the deep layer of the cerebral cortex, while spinal cord cells were patch clamped in the whole-cell mode. Fig. 1A shows the evoked postsynaptic currents. Putative polysynaptic and spontaneous synaptic currents, as well as direct monosynaptic currents, were recorded in the normal ACSF. When traces were averaged several tens of times, the multiple peaks of the putative polysynaptic currents were diminished somewhat as a result of variation of their location within the

Discussion

In this study, we found CS-optEPSPs had durations of ∼500 ms, which was not detected with the usual field recordings processed through a low cut filter. However, when recorded with DC mode, field potentials also had a relatively long duration (∼300 ms). Furthermore, our finding that ifenprodil reduced the amplitudes and durations of EPSPs at 6–7 DIV indicated that NMDARs containing the NR2B subunit contribute substantially to the long duration of optEPSPs (Vicini et al., 1998).

fEPSPs recorded

Acknowledgments

We would like to thank Mr. Haruyoshi Takaki for statistical analysis, Dr. Michinori Ichikawa for useful discussions. This study was supported by Grant-in-Aid for Scientific Research for Priority Area (Elucidation of neural network function in the brain) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18021034) and Mitsubishi Foundation to M.S. and the Nakajima Foundation to T.O.

References (53)

  • E.M. Quinlan et al.

    A molecular mechanism for stabilization of learning-induced synaptic modifications

    Neuron

    (2004)
  • S.L. Stegenga et al.

    Developmental regulation of N-methyl-D-aspartate- and kainate-type glutamate receptor expression in the rat spinal cord

    Neuroscience

    (2001)
  • H. Takuma et al.

    In vitro formation of corticospinal synapses in an organotypic slice co-culture

    Neuroscience

    (2002)
  • T. Tominaga et al.

    Quantification of optical signals with electrophysiological signals in neural activities of Di-4-ANEPPS stained rat hippocampal slices

    J Neurosci Methods

    (2000)
  • Y. Tsau et al.

    Dye screening and signal-to-noise ratio for retrogradely transported voltage-sensitive dyes

    J Neurosci Methods

    (1996)
  • P. Wenner et al.

    Voltage-sensitive dye recording using retrogradely transported dye in the chicken spinal cord: staining and signal characteristics

    J Neurosci Methods

    (1996)
  • W. Bao et al.

    Propagating wave and irregular dynamics: spatiotemporal patterns of cholinergic theta oscillations in neocortex in vitro

    J Neurophysiol

    (2003)
  • A.L. Barth et al.

    NMDAR EPSC kinetics do not regulate the critical period for LTP at thalamocortical synapses

    Nat Neurosci

    (2001)
  • G. Carmignoto et al.

    Activity-dependent decrease in NMDA receptor responses during development of the visual cortex

    Science

    (1992)
  • G.C. Castellani et al.

    A biophysical model of bidirectional synaptic plasticity: dependence on AMPA and NMDA receptors

    Proc Natl Acad Sci U S A

    (2001)
  • L.B. Cohen et al.

    Optical measurement of membrane potential

    Rev Physiol Biochem Pharmacol

    (1978)
  • L.B. Cohen et al.

    Optical methods for monitoring neuron activity

    Annu Rev Neurosci

    (1978)
  • M. Constantine-Paton et al.

    Patterned activity, synaptic convergence, and the NMDA receptor in developing visual pathways

    Annu Rev Neurosci

    (1990)
  • S.G. Cull-Candy et al.

    Role of distinct NMDA receptor subtypes at central synapses

    Sci STKE 2004

    (2004)
  • R. Demir et al.

    Voltage imaging of epileptiform activity in slices from rat piriform cortex: onset and propagation

    J Neurophysiol

    (1998)
  • A. Erisir et al.

    Decline of the critical period of visual plasticity is concurrent with the reduction of NR2B subunit of the synaptic NMDA receptor in layer 4

    J Neurosci

    (2003)

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