Serotonergic neurons are found in several areas of the central nervous system, including the rostroventromedial medulla (RVM) in the brain stem, where they play an important role in the modulation of nociceptive information. In an attempt to visualize serotoninergic neurons, we generated a bacterial artificial chromosome (BAC) transgenic mouse in which enhanced green fluorescent protein (EGFP) is expressed in serotonergic neurons under the control of the tryptophan hydroxylase 2 (TPH2) gene promoter. TPH2 is the rate-limiting enzyme that synthesizes serotonin.

To label spinally projecting serotonergic neurons in the RVM, 1'-dioactadecyl-3,3,3',3'-tetramethylindocarbodyanine perchlorate (DiI) was injected into the spinal dorsal horn in the BAC transgenic mice. To characterize spinally projecting EGFP fluorescent neurons in the RVM, tight-seal whole-cell recording was made from the DiI labeled GFP fluorescent neurons in the brain stem slices prepared from the mice.

To investigate their membrane properties, the DiI labeled GFP fluorescent neurons were stimulated with suprathreshold current steps under current clamp. Substitution of the internal K⁺ with Cs⁺ inhibited the majority of K⁺ currents and evoked a large increase in a plateau component of the action potential. Under voltage clamp, a depolarizing pulse also induced an action current with a pronounced plateau component in the Cs⁺-filled neuron. The plateau component was obviously reduced in the external solution without Ca²⁺ or was blocked with external Cd²⁺, indicating that the component was dependent on Ca²⁺ entry. In contrast, in DiI labeled neurons without GFP fluorescence, the substitution of the internal K⁺ with Cs⁺ did not induce the plateau component of the action potential. The plateau component of an action potential recorded from the Cs⁺-filled neuron may be one of the useful criteria for identification of a serotonergic neuron in spinally projecting neurons in the RVM. Further electrophysiological analyses are currently underway to characterize serotonergic neurons in the RVM.