Background & Aims
Superficial dorsal horn (SDH), comprised of the Rexed laminae I-III, is enriched with projection neurons and interneurons, and serves as a critical region to relay and integrate sensory inputs. SDH circuits represent a feedforward inhibition model as proposed by the gate control theory (1). It is suggested that GABAergic inhibitory interneurons preponderantly present in SDH in contrast to glycinergic inhibitory interneurons. Recently the dysregulation of KCC2, a key regulator of chloride homeostasis, has been linked to SDH disinhibition that contributes to the development of neuropathic pain. However, the neuron type-specific responses in SDH, e.g., inhibitory vs. excitatory interneurons, during such disinhibition are not fully clear which impedes the effort in identifying therapeutic targets in SDH. We aim to investigate the responses of inhibitory and excitatory interneurons during SDH disinhibition by combining pharmacological manipulation of KCC2 and electrophysiological recording.
Methods
We performed in vivo extracellular recording on lumbar spinal cord from Sprague-Dawley rats to assess the neuronal responses of inhibitory and excitatory neurons during SDH disinhibition. Isolated units were characterized by mechanical stimuli applied to the corresponding receptive fields, electrical stimulation delivered to ipsilateral hind paw, and the waveform of unit spikes. VU0463271, a KCC2 antagonist, was applied to induce SDH disinhibition via impaired chloride extrusion while WNK463 was used to potentiate KCC2 function after the application of VU0463271. The spontaneous activity and evoked responses of units before and after the pharmacological manipulation were recorded and compared.
Results
Analysis showed that the characterized inhibitory interneurons were mostly located at depth corresponding to lamina I – lamina IIi while characterized excitatory interneurons were mainly distributed between lamina IIo – lamina III. The excitatory interneurons showed a homogeneous pattern of responses: adaptive bursting pattern during innocuous punctate stimuli and quiescent spontaneous activity. In addition, during VU0463271-induced disinhibition, the excitatory interneurons demonstrated a stronger augmentation in evoked responses while the responses of the inhibitory interneurons were heterogeneous. Application of WNK463 to VU0463271-treated cord partially restored the neuronal activity in both neuron types suggested that the VU0463271-induced KCC2 downregulation might not entirely rely on WNK-dependent pathway.
Conclusions
In present study, we showed that the excitatory and inhibitory interneurons in SDH received differential level of disinhibition upon the application of KCC2 antagonist. The heterogeneity is possibly due to the diverse nature of KCC2-depedent chloride extruding capacity among different types of interneurons that also receive distinctive types and intensities of inhibitory inputs. Taken together, these properties might predispose the excitatory interneurons to be hyperexcitable during the SDH disinhibition and contribute to the development of chronic pain.
References
1. Melzack R, Wall PD. Pain mechanisms: A new theory. Science. 1965;150:971–9.