Involvement of pre- and postsynaptic NMDA receptors at local circuit interneuron connections in rat neocortex

Abstract

To investigate the involvement of N-Methyl-D-aspartate (NMDA) receptors in local neocortical synaptic transmission, dual whole-cell recordings - combined with biocytin labelling - were obtained from bitufted adapting, multipolar adapting or multipolar non-adapting interneurons and pyramidal cells in layers II-V of rat (postnatal days 17-22) sensorimotor cortex. The voltage dependency of the amplitude of Excitatory postsynaptic potentials (EPSPs) received by the three types of interneuron appeared to coincide with the interneuron subclass; upon depolarisation, EPSPs received by multipolar non-adapting interneurons either decreased in amplitude or appeared insensitive, multipolar adapting interneuron EPSP amplitudes increased or appeared insensitive, whereas bitufted interneuron EPSP amplitudes increased or decreased. Connections were challenged with the NMDA receptor antagonist d-(-)-2-amino-5-phosphonopentanoic acid (d-AP5) (50μM) revealing NMDA receptors to contribute to EPSPs received by all cell types, this also abolished the non-conventional voltage dependency. Reciprocal connections were frequent between pyramidal cells and multipolar interneurons, and inhibitory postsynaptic potentials (IPSPs) elicited in pyramidal cells by both multipolar adapting and multipolar non-adapting interneurons were sensitive to a significant reduction in amplitude by d-AP5. The involvement of presynaptic NMDA receptors was indicated by coefficient of variation analysis and an increase in the failures of transmission. Furthermore, by loading MK-801 into the pre- or postsynaptic neurons, we observed that a reduction in inhibition requires presynaptic and not postsynaptic NMDA receptors. These results suggest that NMDA receptors possess pre- and postsynaptic roles at selective neocortical synapses that are probably important in governing spike-timing and information flow.

Fig. 1

EPSPs received by bitufted adapting, multipolar adapting and multipolar non-adapting interneurons, and their voltage dependency. (A) Biocytin-labelled bitufted adapting, multipolar adapting and multipolar non-adapting interneurons with their respective firing pattern and presynaptic pyramidal cell. Interneuron soma and dendrites in red, axon in blue and pyramidal cell dendrites in grey. (B) EPSPs received by the three interneurons shown in A. Examples of single data are superimposed (grey traces) with average trace (black trace) for each class of synapse studied. Inserts show examples of apparent synaptic failures observed (asterisk denotes a failure), similarly five single sweep traces (grey) are superimposed with the averaged trace (black). (C) The voltage ratio (change in amplitude dependent on the holding potential of the cell, see “Data analysis”) of EPSPs received by bitufted adapting, multipolar adapting and multipolar non-adapting interneurons. Each circle represents one pair.

Fig. 2

NMDA receptors contribute to EPSPs received by bitufted adapting, multipolar adapting and multipolar non-adapting interneurons. (A) Average EPSPs received by all three interneuron groups were significantly reduced by d-AP5 (50 μM, paired t test, p < 0.05). Each circle represents a pair, while the bars represent the group average. Error bars denote SD. (B) The change in the failure rate with d-AP5 shown for individual pairs and (C) the paired-pulse ratio (PPR) during control and with d-AP5 shown for individual pairs. The group average is shown for each interneuron group with bars representing SD. (D) Normalised (d-AP5/control) plot of the CV⁻² against the normalised mean for the three cell types. A greater change in the mean than CV⁻² indicates a postsynaptic mechanism. (E) The voltage ratio with d-AP5 for the three types of connection (individual circles represent a pair). (F) EPSPs elicited in bitufted and multipolar adapting interneurons at depolarised and hyperpolarised potentials during control and with d-AP5. The voltage relation becomes conventional when NMDA receptors are blocked with d-AP5. In B–D, circles represent bitufted pairs, diamonds represent multipolar adapting pairs and triangles represent multipolar non-adapting pairs. (G) EPSPs elicited in a mulitipolar adapting in control conditions, after repatching the presynaptic pyramidal cells with the NMDA receptor antagonist, MK-801 (1 mM, grey traces), and then subsequently repatching the postsynaptic interneurons with MK-801 (blue traces). (H) The average EPSP amplitudes (% of control) of each individual pair with MK-801 in the pipette of the pyramidal cells and interneurons.

Fig. 3

NMDA receptors enhance inhibition between multipolar interneurons and pyramidal cells. (Ai) Example of a biocytin-labelled multipolar adapting interneuron and pyramidal cell that were reciprocally connected. Interneuron soma and dendrites in red, axon in blue and pyramidal cell dendrites in grey. (Aii) Plot of an experiment showing peak amplitude of IPSPs in control and after bath application of D-AP5 (50 μM) against time, and (Aiii) the average IPSP. (Aiv) EPSPs were reduced in the presence of d-AP5. (B) The effect of d-AP5 on the amplitude of multipolar adapting and multipolar non-adapting interneuron containing pairs. Each circle represents a pair, while the bars represent the group average. Error bars denote SD. Both groups were significantly reduced from control (paired t test, p < 0.05). (C) d-AP5 (50 μM) increased the IPSP failure rate in seven out of the eight pairs. Multipolar adapting pairs marked by a black circle, multipolar non-adapting interneurons by a hollow circle. (D) Plot of the normalised (d-AP5/control) CV⁻² against the normalised (d-AP5/control) mean. A greater change in the CV⁻² compared with that of the mean indicates a presynaptic mechanism of action. Each point represents a single pair. As before, multipolar adapting pairs represented by a black circle, multipolar non-adapting pairs by a hollow circle.

Fig. 4

Presynaptic NMDA receptors enhance inhibition between multipolar interneurons and pyramidal cells. (Ai and Aii) IPSPs elicited by a mulitipolar non-adapting and adapting interneuron in control conditions, after repatching the postsynaptic pyramidal cells with the NMDA receptor antagonist, MK-801 (1 mM, grey traces), and then subsequently repatching the presynaptic interneurons with MK-801 (blue traces). (Aiii) Plot of an example experiment shown in (Aii), during control, repatching the pyramidal cell with Mk-801 and subsequent repatching the presynaptic interneuron with MK-801 (vertical lines indicate the onset of MK-801 application). (B) The average IPSP amplitudes (% of control) and failure rates of each individual pair with MK-801 in the pipette of the pyramidal cells and interneurons. Pre and post refer to the presynaptic interneuron and postsynaptic pyramidal cell, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)