Role of extracellular sialic acid in regulation of neuronal and network excitability in the rat hippocampus

Abstract

The extracellular membrane surface contains a substantial amount of negatively charged sialic acid residues. Some of the sialic acids are located close to the pore of voltage-gated channel, substantially influencing their gating properties. However, the role of sialylation of the extracellular membrane in modulation of neuronal and network activity remains primarily unknown. The level of sialylation is controlled by neuraminidase (NEU), the key enzyme that cleaves sialic acids. Here we show that NEU treatment causes a large depolarizing shift of voltage-gated sodium channel activation/inactivation and action potential (AP) threshold without any change in the resting membrane potential of hippocampal CA3 pyramidal neurons. Cleavage of sialic acids by NEU also reduced sensitivity of sodium channel gating and AP threshold to extracellular calcium. At the network level, exogenous NEU exerted powerful anticonvulsive action both in vitro and in acute and chronic in vivo models of epilepsy. In contrast, a NEU blocker (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid) dramatically reduced seizure threshold and aggravated hippocampal seizures. Thus, sialylation appears to be a powerful mechanism to control neuronal and network excitability. We propose that decreasing the amount of extracellular sialic acid residues can be a useful approach to reduce neuronal excitability and serve as a novel therapeutic approach in the treatment of seizures.

Figure 1.

Tissue-specific effects of NEU treatment in the hippocampal formation of rat brain. A, Probing control sections with SNA-I showed an intense vascular staining together with a weak parenchymal staining. B, C, In sections treated with NEU alone (B), the overall SNA-I staining in the pyramidal layer (double asterisks) was greatly reduced compared with sections treated with NEU blocker before NEU treatment (C). Comparable staining patterns were obtained in six independent experiments. Scale bar, 50 μm. so, Stratum oriens; sp, pyramidal layer; sr, stratum radiatum.

Figure 2.

Effect of desialylation of the cellular membrane on the steady-state activation and inactivation of neuronal voltage-gated sodium channels. Nucleated patch (A, B) and whole-cell (C, D) recordings were made from CA3 pyramidal neurons correspondingly from P10–P17 and P2–P5 rats. Summarized voltage dependences of the normalized conductance (A, C) and steady-state inactivation (B, D) of sodium channels recorded from CA3 pyramidal neurons from control (filled circles), NEU-treated (open circles), and NEU/NEU blocker-treated (open squares) hippocampal slices fitted by a Boltzmann function are shown. Values are mean ± SEM.

Figure 3.

Effect of NEU treatment on epileptiform activity in vitro. An extracellular field potential was recorded from the CA3 pyramidal cell layer of brain slices from P8–P21 rats. Tonic-like and clonic-like epileptiform activity was induced by 10 mm [K⁺]_o/low[Mg²⁺]_o ACSF, and recordings were simultaneously obtained from control (top) and NEU-treated (bottom) slices. Examples of initial bursting and tonic-like and clonic-like discharges are shown on an expanded time scale.

Figure 4.

Comparison of effects of NEU treatment versus NEU blocker treatment on high-potassium-induced seizures in vivo. Hippocampal seizures were induced by 10 mm [K⁺]_o/low [Mg²⁺]_o ACSF in P8–P21 rats in vivo. Extracellular field potentials were recorded from the CA3 pyramidal cell layer. A, Responses to repetitive microinjections (arrows) of 10 mm [K⁺]_o/low [Mg²⁺]_o ACSF (shown at the same voltage scale for all traces). Note that epileptiform events are induced from the fifth injection in NEU-treated and control rats and from the second injection in NEU blocker-treated rats. Examples of ictal-like events (asterisks) are shown below on an expanded time scale to reveal phases: (1) tonic- and (2) clonic-like activity. B, Summary plot of seizure probability as a function of the number of injections. C, D, Duration of ictal-like activity (C) and maximal PS frequency (D) for control (black bar; n = 10), NEU-treated (gray bar; n = 8), and NEU blocker-treated (white bar; n = 10) rats at P8–P21. Statistical comparisons were performed using the t test, *p < 0.05; ***p < 0.005.

Figure 5.

Effect of NEU treatment in kindling model of epilepsy. Comparison of effect of NEU on AD (A) and kindling stage (B) in fully kindled rats before (black bars) and after (white bars) NEU was injected in rats not treated with the NEU blocker and before (dark gray bars) and after (light gray bars) NEU was administrated in rats that received NEU blocker injection. NEU significantly reduced AD duration and kindling stage in control rats, whereas the NEU administered after the NEU blocker had no significant effect on kindling stage or AD.