GluN2B and GluN2D NMDARs dominate synaptic responses in the adult spinal cord

OPEN SUBJECT AREAS: ION CHANNELS IN THE NERVOUS SYSTEM CELLULAR NEUROSCIENCE Received 19 August 2013 Accepted 30 January 2014 Published 13 February 2014 Correspondence and requests for materials should be addressed to M.W.S. (mike.salter@ utoronto.ca) * Current address: Department of GluN2B and GluN2D NMDARs dominate synaptic responses in the adult spinal cord Michael E. Hildebrand1,2*, Graham M. Pitcher1,2, Erika K. Harding1,2, Hongbin Li1,2, Simon Beggs1,2 & Michael W. Salter1,2 1 Program in Neurosciences & Mental Health, Hospital for Sick Children, Toronto, ON, Canada, 2Department of Physiology, University of Toronto, Toronto, ON, Canada. The composition of the postsynaptic ionotropic receptors that receive presynaptically released transmitter is critical not only for transducing and integrating electrical signals but also for coordinating downstream biochemical signaling pathways. At glutamatergic synapses in the adult CNS an overwhelming body of evidence indicates that the NMDA receptor (NMDAR) component of synaptic responses is dominated by NMDARs containing the GluN2A subunit, while NMDARs containing GluN2B, GluN2C, or GluN2D play minor roles in synaptic transmission. Here, we discovered NMDAR-mediated synaptic responses with characteristics not described elsewhere in the adult CNS. We found that GluN2A-containing receptors contribute little to synaptic NMDAR responses while GluN2B dominates at synapses of lamina I neurons in the adult spinal cord. In addition, we provide evidence for a GluN2D-mediated synaptic NMDAR component in adult lamina I neurons. Strikingly, the charge transfer mediated by GluN2D far exceeds that of GluN2A and is comparable to that of GluN2B. Lamina I forms a distinct output pathway from the spinal pain processing network to the pain networks in the brain. The GluN2D-mediated synaptic responses we have discovered in lamina I neurons provide the molecular underpinning for slow, prolonged and feedforward amplification that is a fundamental characteristic of pain. Neuroscience, Carleton University, Ottawa, ON, Canada. N -methyl-D-aspartate receptors (NMDARs) are a prominent subtype of ionotropic glutamate receptor1, critical for physiological synaptic plasticity in the developing and mature CNS, and for aberrant plasticity and neuronal death in pathological disorders2. The NMDAR is a multiprotein complex, the core of which is a heterotetrameric assembly of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. GluN1 is encoded by a single gene, GRIN1, whereas there are four GluN2-subunit-encoding genes, GRIN2A –D. Because NMDARs comprised of differing GluN2 subunits have distinctive functional properties and are hypothesized to have differing physiological and pathological roles1,3, a major question has been to determine the specific GluN1/GluN2 subunit composition contributing to NMDAR-mediated transmission at a given synapse. GluN2A has emerged as the principal subtype of GluN2 mediating synaptic NMDAR responses in the adult brain4–8. By contrast, the contribution of NMDARs comprised of GluN2B to synaptic responses is less than that of receptors comprised of GluN2A throughout the adult brain, and at many synapses GluN2B contributes much less than does GluN2A. The GluN2B subunit is nevertheless well-expressed in the adult but primarily localized at extrasynaptic sites9. Conversely, during embryonic and early postnatal development synaptic GluN2B is more prominent than is GluN2A6,10,11. GluN2C shows sparse expression in the adult brain, except for the cerebellum and olfactory bulb where GluN2C is highly expressed beginning in the second postnatal week10,12. An enduring mystery has been that, in contrast to the other GluN2 subunits, synaptic NMDAR responses mediated by GluN2D are lacking in the adult brain9. Here, while investigating glutamatergic synaptic responses of neurons in lamina I of the adult spinal dorsal horn we discovered NMDAR-mediated synaptic responses with characteristics not previously described elsewhere in the adult CNS. Lamina I neurons form a critical part of the nociceptive neuronal network in the dorsal horn which processes inputs from primary sensory afferents and transmits the resultant nociceptive signals to the brain13,14. The nociceptive network in the dorsal horn is a highly interconnected matrix comprising monosynaptic inputs from primary afferents as well as polysynaptic and monosynaptic inputs from local circuit neurons15–17. In order to properly characterize the responses from glutamatergic synapses directly on lamina I neurons it was necessary to avoid activating the nociceptive network because such network activation generates prolonged, SCIENTIFIC REPORTS | 4 : 4094 | DOI: 10.1038/srep04094 1 www.nature.com/scientificreports Figure 1 | Whole-cell patch recordings from lamina I neurons in rat parasagittal spinal cord slices. (A) Top, representative image of a parasagittal slice and micropipette during patch-clamp recording of a lamina I neuron. Through visual discrimination, the lamina I neuron was identified in the darker, striated, superficial-most layer of the dorsal horn (LI) compared to the more ventral, lighter substantia gelatinosa (LII), as indicated. Scale bar 5 200 mm. Bottom, a two-photon image was taken from a lamina I neuron that was filled with lucifer yellow (0.1%, green) during whole-cell recording. Separately, an immunohistochemistry image was taken following slice fixation and staining for CGRP (red) and neuronal (NeuN, blue) labeling. The two-photon image was correctly scaled and superimposed onto the epifluorescence image stack to illustrate the location and orientation of the lamina I neuron within the superficial dorsal horn. Note neuronal morphology corresponding to a type Ib fusiform lamina I neuron62 as well as neuronal location within the outermost CGRP-stained dorsal horn region, characteristic of lamina I. Scale bar 5 100 mm. (B) Raster plot of a representative continuous whole-cell recording from a lamina I neuron held at 260 mV. Scale bar x axis 5 1 s, y axis 5 10 pA. largely polysynaptic excitatory responses which contaminate the direct NMDAR-mediated synaptic responses. To this end, in lamina I neurons we studied miniature excitatory postsynaptic currents (mEPSCs), representing the overall population of direct synaptic responses, and unitary primary afferent-evoked EPSCs, representing individual synapses. Surprisingly, we found that GluN2A contributes little to synaptic NMDAR responses while GluN2B dominates at lamina I synapses in adult spinal cord. Moreover, we discovered that there is a GluN2D-mediated synaptic NMDAR component of neurons in spinal lamina I and that the charge transfer mediated by GluN2D far exceeds that of GluN2A and is comparable to that of GluN2B. Results AMPAR and NMDAR components of mEPSCs in lamina I neurons. We made whole-cell recordings from visually identified SCIENTIFIC REPORTS | 4 : 4094 | DOI: 10.1038/srep04 (...truncated)