Blocker-resistant presynaptic voltage-dependent Ca²⁺ channels underlying glutamate release in mice nucleus tractus solitarii

Abstract

The visceral sensory information from the internal organs is conveyed via the vagus and glossopharyngeal primary afferent fibers and transmitted to the second-order neurons in the nucleus of the solitary tract (NTS). The glutamate release from the solitary tract (TS) axons to the second-order NTS neurons remains even in the presence of toxins that block N- and P/Q-type voltage-dependent Ca²⁺ channels (VDCCs). The presynaptic VDCC playing the major role at this synapse remains unidentified. To address this issue, we examined two hypotheses in this study. First, we examined whether the remaining large component occurs through activation of a ω-conotoxin GVIA (ω-CgTX)-insensitive variant of N-type VDCC by using the mice genetically lacking its pore-forming subunit α_1B. Second, we examined whether R-type VDCCs are involved in transmitter release at the TS–NTS synapse. The EPSCs evoked by stimulation of the TS were recorded in medullary slices from young mice. ω-Agatoxin IVA (ω-AgaIVA; 200 nM) did not significantly affect the EPSC amplitude in the mice genetically lacking N-type VDCC. SNX-482 (500 nM) and Ni²⁺ (100 μM) did not significantly reduce EPSC amplitude in ICR mice. These results indicate that, unlike in most of the brain synapses identified to date, the largest part of the glutamate release at the TS–NTS synapse in mice occurs through activation of non-L, non-P/Q, non-R, non-T and non-N (including its posttranslational variants) VDCCs at least according to their pharmacological properties identified to date.

Introduction

The brain controls the internal environment of the body by generating optimum sympathetic, parasympathetic and respiratory outputs from the central nervous system (CNS). This control system relies on continuous processing of sensorial information coming from a variety of types of visceral receptors that monitor the unceasingly varying internal environment of the organism. The caudal part of the nucleus of the solitary tract (NTS) is the “gateway” of such information (Andresen and Kunze, 1994) because the primary afferent fibers of the tractus solitarius (TS) from these receptors converge to and form the first synapses in the NTS (called here as TS–NTS synapses). These primary afferent fibers release glutamate and activate non-NMDA and NMDA receptors of the second-order neurons (Andresen and Yang, 1990, Aylwin et al., 1997).

The most critical process of action potential-dependent synchronous transmitter release is Ca²⁺ entry into the presynaptic terminal through voltage-dependent Ca²⁺ channels (VDCCs) (Mochida, 2000). In most of CNS synapses analyzed to date, N-type (Ca_V2.2) and P/Q-type (Ca_V2.1) VDCCs underlie transmitter release (Reid et al., 2003). For example, Iwasaki et al. (2000) compared the type of presynaptic VDCCs using two selective inhibitors, ω-conotoxin GVIA (ω-CgTX) and ω-agatoxin IVA (ω-AgaIVA) for N- and P/Q-types, respectively, in various CNS synapses at various developmental stages and found that postsynaptic current is abolished by application of either of or both of these toxins. In addition to these two types, the most recently identified R-type VDCCs (Ca_V2.3) has been shown to play a role in transmitter release in some but limited CNS structures (Bao et al., 1998, Dietrich et al., 2003, Gasparini et al., 2001, Sochivko et al., 2003, Wu et al., 1998). Our current general understanding is that these three types (N-, P/Q- and R-) of Ca_V2 families expressed at axon terminals underlie the Ca²⁺-dependent transmitter release in the central nervous system (Catterall, 2000). The L- and T-type VDCCs, which play important roles in somatic Ca²⁺ entry and intrinsic regulation of the membrane potential, play little role in the action potential-dependent transmitter release in the central synapses (Catterall, 2000, Mochida, 2000, Reid et al., 2003).

At the TS–NTS synapse of the rat, approximately 56% (Mendelowitz et al., 1995) or 70% (Glaum and Miller, 1995) of evoked EPSP or EPSC is sensitive to ω-CgTX (1 μM), and the remaining component is not affected by addition of ω-AgaIVA. Since these reports published 10 years ago, no attempt has been made to identify this toxin-resistant VDCC underlying the ∼30–45% component of the glutamate release at the TS–NTS synapse, which is rather exceptional among most of the CNS synapses (Iwasaki et al., 2000). Especially in these 10 years, there have been a number of reports describing toxin-insensitive posttranslational isoforms of N-type VDCCs (Adams et al., 2003, Bell et al., 2004, Coppola et al., 1994) and functional presynaptic expression of R-type VDCCs in the CNS (Bao et al., 1998, Dietrich et al., 2003, Gasparini et al., 2001, Kamp et al., 2005, Sochivko et al., 2003, Wu et al., 1998). Identification of the VDCC involved in the TS–NTS synaptic transmission is a requisite to develop pharmacological strategies based on the selective blockade of distinct types of VDCCs involved specifically in various distinct functions such as pain signaling and cardiovascular regulation (Bell et al., 2004, Hatakeyama et al., 2001, Minami et al., 1998, Wright et al., 2000). The goal of this study is to identify the type of VDCCs involved in the synaptic transmission between the visceral primary afferents and NTS neurons with a more developed techniques and pharmacological tools.

In this study, we examined two hypotheses to address this issue. First, as the largest portion yet identified of the transmitter release from the TS terminal occurs through presynaptic Ca²⁺ entry through N-type VDCC, we examined whether the remaining large release component occurs through activation of a ω-CgTX-insensitive variant of N-type VDCC. N-type VDCC is now known for many distinct posttranslational variants of its pore-forming subunit α_1B (Adams et al., 2003, Bell et al., 2004, Coppola et al., 1994). These variants show distinct pharmacological characteristics, especially the sensitivity to conotoxin families (Favreau et al., 2001, Feng et al., 2001, Lewis et al., 2000, Nielsen et al., 2000), and are expressed and operational in the central and peripheral neurons. However, due to their distinct sensitivity to distinct toxin blockers (Favreau et al., 2001), it has been difficult to pharmacologically demonstrate involvement of these N-type variants in physiological processes. To directly address this issue, we analyzed the effect of VDCC blockers on the TS–NTS transmission in brainstem from the mice genetically lacking the pore-forming subunit of the N-type VDCC (Ino et al., 2001). Because this transgenic mouse strain was generated by backcrossing with ICR mice, we analyzed the contribution of VDCCs in ICR mice, which provide a control to this transgenic strain (Ino et al., 2001, Hatakeyama et al., 2001, Mori et al., 2002). Second, we examined whether “R-type” VDCCs, of which the molecular identities and pharmacological characteristics became clearer than they had been 10 years ago, are involved in the transmitter release at the TS–NTS synapse by a use of selective blockers.