Regional differences in nerve terminal Na+ channel subtype expression and Na+ channel-dependent glutamate and GABA release in rat CNS

Abstract

We tested the hypothesis that expression of pre-synaptic voltage-gated sodium channel (Na(v)) subtypes coupled to neurotransmitter release differs between transmitter types and CNS regions in a nerve terminal-specific manner. Na(v) coupling to transmitter release was determined by measuring the sensitivity of 4-aminopyridine (4AP)-evoked [(3)H]glutamate and [(14)C]GABA release to the specific Na(v) blocker tetrodotoxin (TTX) for nerve terminals isolated from rat cerebral cortex, hippocampus, striatum and spinal cord. Expression of various Na(v) subtypes was measured by immunoblotting using subtype-specific antibodies. Potencies of TTX for inhibition of glutamate and GABA release varied between CNS regions. However, the efficacies of TTX for inhibition of 4AP-evoked glutamate release were greater than for inhibition of GABA release in all regions except spinal cord. The relative nerve terminal expression of total Na(v) subtypes as well as of specific subtypes varied considerably between CNS regions. The region-specific potencies of TTX for inhibition of 4AP-evoked glutamate release correlated with greater relative expression of total nerve terminal Na(v) and Na(v)1.2. Nerve terminal-specific differences in the expression of specific Na(v) subtypes contribute to transmitter-specific and regional differences in pharmacological sensitivities of transmitter release.

Figure 1

Effects of tetrodotoxin on neurotransmitter release in various CNS regions. Data for inhibition of L-[³H]glutamate and [¹⁴C]GABA release evoked by 1 mM 4AP from rat cortical, hippocampal, striatal, and spinal cord synaptosomes were fitted to sigmoidal concentration-effect curves (mean±SEM; n=24–33). IC₅₀ values and maximal inhibition values are shown in Fig 2. Control data are presented as mean±SD (n=10–18).

Figure 2

Potency and efficacy for inhibition of glutamate and GABA release from isolated nerve terminals by tetrodotoxin. a: IC₅₀ values (±SEM) derived by fitting data to sigmoidal concentration-effect curves (Fig. 1) for release from cerebral cortex (Cx), hippocampus (Hip), striatum (Str) and spinal cord (SC). Statistical comparisons between cerebral cortex and other CNS regions (*P < 0.05) and between glutamate and GABA release (†P < 0.05; †††P < 0.001) were analyzed by comparing logIC₅₀ values between sigmoidal curve fits by F-tests. b: Maximal inhibition by TTX determined as the lower plateau of sigmoidal concentration-effect curve fits (Fig. 1) expressed as I₀ − I_max/I₀ × 100 (±SEM). Statistical comparisons between cerebral cortex and other CNS regions (***P < 0.001) and between glutamate and GABA release (††P < 0.01; †††P < 0.001) were analyzed by comparing lower plateaus of curves by F-tests.

Figure 3

Effects of tetrodotoxin and A-803467 on 4AP-evoked glutamate and GABA release from cortex and spinal cord nerve terminals. Ca²⁺-independent 4AP-evoked glutamate release from cortex, but not spinal cord, was inhibited by 1 μM tetrodotoxin (TTX). TTX inhibited Ca²⁺-independent 4AP-evoked GABA release from both cortex and spinal cord. The selective Na_v1.8 blocker A-803467 had no effect on TTX-insensitive glutamate or GABA release from cortical nerve terminals in the presence of Ca²⁺, but inhibited TTX-insensitive glutamate, but not GABA, release from spinal cord nerve terminals. *P < 0.05, **P < 0.01 by unpaired Student t-test with Welch correction (mean±SEM). Group sizes (n) for GABA are the same as for glutamate.

Figure 4

Neurotransmitter release evoked by 4-aminopyridine or veratridine from various CNS regions. Release of L-[³H]glutamate and [¹⁴C]GABA from rat cortical (Cx), hippocampal (Hip), striatal (Str) and spinal cord (SC) nerve terminals evoked by 2 min pulses of 1 mM 4AP (a, n=14–22) or 10 μM VTD (b, n=10–31). Statistical comparisons between cortex and other CNS regions (***P < 0.001), and between glutamate and GABA (†P < 0.05; ††P < 0.01; †††P < 0.001) were analyzed by one-way ANOVA using Tukey’s post-hoc testing (mean±SD).

Figure 5

Effects of tetrodotoxin on neurotransmitter release evoked by veratridine. A: Inhibition by tetrodotoxin of L-[³H]glutamate and [¹⁴C]GABA release evoked by 10 μM VTD from rat cortical synaptosomes (n=20). B: IC₅₀ values for inhibition by tetrodotoxin of transmitter release form cortical nerve terminals determined by fitting data to sigmoidal concentration-effect curves. Statistical comparisons between cortex and other CNS regions (***P < 0.001) and between glutamate and GABA (††P < 0.01) release curves were analyzed by comparing log IC₅₀ values between sigmoidal curve fits by F-test (mean±SEM).

Figure 6

Relative expression of total voltage-gated Na⁺ channel immunoreactivity in rat CNS nerve terminal preparations. Representative immunoblot shown for pan-specific Na_v1 antibody labeling of cerebral cortical (Cx), hippocampal (Hip), striatal (Str), and spinal cord (SC) nerve terminals (upper panel). Relative expression of immunoreactive bands (260–280 kDa; arrowheads) was compared to cortex by repeated measures one-way ANOVA. **P < 0.01, ***P < 0.001 vs. cortex (mean±SEM; n=14).

Figure 7

Relative expression of voltage-gated Na⁺ channel subtype immunoreactivity in rat CNS nerve terminal preparations. Representative immunoblots (upper panels) shown for a: Na_v1.1 (n=16), b: Na_v1.2 (n=16), c: Na_v1.6 (n=13), and d: Na_v1.7 (n=7) labeling of cortical (Cx), hippocampal (Hip), striatal (Str), and spinal cord (SC) nerve terminals. Relative expression of immunoreactive bands (260–280 kDa; arrowheads) was compared to cortex by repeated measures one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 vs. cortex (mean±SEM).