Pharmacological analysis of excitatory and inhibitory synaptic transmission in horizontal brainstem slices preserving three subnuclei of spinal trigeminal nucleus

Abstract

Spinal trigeminal nucleus (Vsp) consists of three subnuclei: oralis (Vo), interpolaris (Vi) and caudalis (Vc). Previous anatomical studies using antero-/retro-grade tracers have suggested that intersubnuclear ascending/descending synaptic transmissions exist between subnuclei. However, pharmacological properties of the intersubnuclear synaptic transmission have not been studied yet. Since three subnuclei are located in Vsp along rostro-caudal axis, it will be necessary to prepare horizontal brainstem slices to perform pharmacological analysis of the intersubnuclear synaptic transmission. We here show horizontal brainstem slices retaining three subnuclei, and that, using blind whole-cell recordings in the slices, synaptic transmission may be abundantly retained between subnuclei in the horizontal slices, except for the transmission from Vo to Vc. Finally, pharmacological analysis shows that excitatory and inhibitory synaptic responses, respectively, are mediated by AMPA and NMDA receptors and by GABA_A and glycine receptors, with a differential contribution to the synaptic responses between subnuclei. We therefore conclude that horizontal brainstem slices will be a useful preparation for studies on intersubnuclear synaptic transmission, modulation and plasticity between subnuclei, as well as, further, other brainstem nuclei.

Introduction

Somatosensory signals are frequently generated in oral and perioral tissues during food intake. Trigeminal sensory neurons primarily relay the signals from the periphery to second-order neurons existing in trigeminal sensory nuclei (Vsn). Vsn comprises mesencephalic nucleus, principal sensory nucleus (Vp) and spinal trigeminal nucleus (Vsp); the Vsp is further sub-divided, from rostrally to caudally, into oralis (Vo), interpolaris (Vi) and caudalis (Vc) (Olszewski, 1950). Vsn integrates the somatosensory signals, including pain, and further transmits the signals into the higher brain areas via projection neurons in Vp and Vsp (Waite and Tracey, 1995). In Vsp, primary afferents associated with oral receptive fields including tooth pulp are preferentially terminated in the rostral nuclei above the obex such as Vp, Vo and Vi, while afferents with facial receptive fields primarily in Vc (Arvidsson and Gobel, 1981, Marfurt and Turner, 1984, Shigenaga et al., 1986, Takemura et al., 1991, Takemura et al., 1993, Takemura et al., 2006). Since all or part of the somatosensory signals from different peripheral areas are processed and integrated in the Vsn before being transmitted into the higher brain centers, the signals assigned in various areas of Vsn, particularly subnuclei of the Vsp, should be intimately interrelated (Takemura et al., 2006, Woda, 2003).

Interconnections supporting the notion for the intimate interrelationship between subnuclei of Vsp have been suggested by various early observations using histological (Gobel and Purvis, 1972, Khayyat et al., 1975) and antero- and retro-grade tracing methods (Falls, 1984a, Falls, 1984b, Ikeda et al., 1982, Ikeda et al., 1984, Jacquin et al., 1990, Li et al., 2000, Lovick and Wolstencroft, 1983, Nasution and Shigenaga, 1987, Panneton and Burton, 1982, Voisin et al., 2002). In addition, anatomical (Voisin et al., 2002) or functional (Chiang et al., 2002, Dallel et al., 1998, Hu et al., 1981, Park et al., 2001, Woda et al., 2001) studies have recently demonstrated an ascending connection from Vc to Vo which may be required for C-fiber-mediated nociceptive transmission from Vc to Vo (Pajot et al., 2000). In spite of these studies above, pharmacological analysis of the intersubnuclear synaptic transmission have not been tried yet.

For pharmacological analysis of the synaptic transmission in Vsp, it is necessary to perform whole-cell voltage-clamp recordings in a live slice preparation that would retain all three subnuclei and allow visual guidance of the recording patch pipettes on each subnucleus targeted. Although transverse or horizontal slices of rat trigeminal nucleus have been used for electrophysiological recordings of, especially, Vc neurons (Grudt and Williams, 1994, Hamba and Onimaru, 1998, Hamba et al., 2000, Jennings, 2001, Jennings et al., 2003, Jennings et al., 2004, Jennings et al., 2006, Liang et al., 2004, Onodera et al., 2000, Wang et al., 2001), it has not been tried to prepare a single brainstem slice that preserves all three subnuclei because of their anatomical separation along the rostro-caudal axis. Therefore, we attempted to produce live slices that could retain all three subnuclei of Vsp by horizontally cutting brainstem tissue in a rostro-caudal direction, and performed blind whole-cell voltage-clamp recordings to investigate the pharmacological properties of excitatory and inhibitory synaptic transmission in the Vsp.