Characterization of Superficial Dorsal Horn Neurons from “Tamamaki” Mice and Stability of their GAD67-EGFP Phenotype in Defined-Medium Organotypic Culture

Defined medium organotypic cultures (DMOTC) containing spinal dorsal horn neurons are especially useful in studying the etiology and pharmacology of chronic pain. We made whole-cell recordings from neurons in acutely isolated mouse spinal cord slices or from those maintained in DMOTC for up to 6 weeks. In acute slices, neurons in the substantia gelatinosa exhibited 7 different firing patterns in response to 800-ms depolarizing current commands; delay (irregular), delay (tonic), tonic, regular firing, phasic, initial bursting and single spiking. Initial bursting and regular firing neurons are not found in rat substantia gelatinosa. In acute slices from “Tamamaki” mice that express enhanced green fluorescent protein (EGFP) under the control of the glutamic acid decarboxylase 67 (GAD67) promotor, tonic, phasic and regular firing neurons exhibited the strongest GABAergic (GAD67-EGFP+) phenotype. Delay (tonic) and delay (irregular) neurons almost never expressed GAD67 (GAD67-EGFP−) and are likely glutamatergic. All seven phenotypes were preserved in mouse spinal cord neurons in DMOTC prepared from e12 embryos and the GAD67-EGFP+ phenotype continued to associate with phasic and regular firing neurons. Only 3 out of 51 GAD67-EGFP+ neurons exhibited a delay (tonic) firing pattern. Modifications to the mouse genome thus continue to be expressed when embryonic neurons develop in vitro in DMOTC. However, analysis of the amplitude and interevent interval of spontaneous EPSCs (sEPSCs) indicated substantial re-arrangement of synaptic connections within the cultures. Despite this, the characteristics and age-dependence of asynchronous oscillatory activity, as monitored by multiphoton Ca2+ imaging, were similar in acute slices and in DMOTC.