Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons

Abstract

Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of in vivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the in vitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.

Keywords: botulinum toxins; electrophysiology; neurons; spontaneous postsynaptic currents; synaptic transmission; tetanus toxin.

FIG. 1.

Intoxication of diverse neuron cultures with BoNT/A or BoNT/B blocks spontaneous neurotransmission and results in SNARE protein cleavage. (A) Representative whole-cell recordings, cumulative probability distributions of inter-event intervals and mean mEPSC frequencies demonstrating spontaneous synaptic activity in ESNs, hSNs, CtNs, and CNs (n = 14–30 for each). Scale bar = 2 s × 60 pA for ESNs and 20 s × 60 pA for others. (B) ICC confirming expression and localization of neurotypic markers NeuN (orange and MAP2 (magenta) as well as CNT target proteins SNAP-25 (green) and Syb2 (white). Scale = 20 µm (C) Representative mEPSC recordings and (D) normalized mEPSC frequencies demonstrating reduced spontaneous release in cultures treated with BoNT/A or BoNT/B but not formalin-inactivated toxoid (n = 8–16 each). Scale is same as panel A. Data are presented as mean ± SEM. *P < 0.05. ***P < 0.001. (E) Immunoblot demonstrating changes in SNAP-25, Syb2 and Stx1 following treatment with BoNT/A or BoNT/B but not formalin-inactivated toxoid.

FIG. 2.

BoNT/A-G or TeNT intoxication blocks synaptic neurotransmission in ESNs. Treatment with BoNT/A-G or TeNT reduces or eliminates spontaneous release after 20 h, demonstrated in (A) representative whole-cell recordings and (B) mean normalized mEPSC frequencies. Scale bar = 2.5 s × 60 pA (n = 8–16 each). ***P < 0.001. (C) Representative immunoblot demonstrating cleavage of SNAP-25 (S25) and loss of immunoreactivity of Stx1 in cultures treated with BoNT/A, /C or /E (n ≥ 4). (D) Representative immunoblot demonstrating loss of Syb2 immunoreactivity following treatment with BoNT/B, /D, /F, /G, and TeNT (n ≥ 4).

FIG. 3.

Concentration–response comparisons of synaptic neurotransmission and SNARE protein cleavage in ESNs exposed to BoNT/A, BoNT/B or TeNT. Representative whole-cell recordings (A–C) and immunoblots (D–F) demonstrating impaired synaptic neurotransmission and SNARE protein cleavage following exposure to BoNT/A, BoNT/B or TeNT. (G–I) Concentration-response curves of normalized mEPSC frequencies (n = 12–20 each) and SNARE protein cleavage (n = 4–5 each) to each CNT. Scale bar = 5 s × 60 pA. Data are presented as mean ± SEM. (J) Tabular summary of median concentrations for each toxin determined from whole-cell recordings and immunoblot data.

FIG. 4.

Use of synaptic neurotransmission to determine toxin serotypes and evaluate antitoxin specificity. Pre-incubation of toxin with serotype-specific antitoxin protects spontaneous release frequencies and SNARE protein integrity at 20 h after intoxication. A, B, Representative whole-cell recordings. C, D, Normalized mEPSC frequencies. E, F, Representative immunoblots. Scale bar = 2.5 s × 60 pA. Normalized data are from 12 to 20 recordings and presented as mean ± SEM. ***P < 0.001.

FIG. 5.

Exposure to clinical concentrations of BOTOX block spontaneous release and produce cleaved SNAP-25 in ESNs. Representative whole-cell recordings (A), quantitation of normalized mEPSC frequencies (B) and representative immunoblot (C) demonstrating that exposure to 1 or 2 U/mL of BOTOX results in reduced neurotransmission and production of cleaved SNAP-25 after 20 h. Scale bar = 2 s × 60 pA. Normalized data are from 12 to 20 recordings and presented as mean ± SEM. ** P < 0.01, *** P < 0.001.

FIG. 6.

Detection of BoNT/A prepared in nutritive samples using synaptic activity. Representative whole-cell recordings and normalized mEPSC frequencies demonstrating reduced synaptic transmission and cSNAP-25 production at 20 h after treatment of ESNs with final concentrations of 55 fM BoNT/A and 1% of CMs. (A) Vehicle, (B) whole milk, (C) green bean liquid or (D) apple cider. Scale bar = 5 s × 50 pA. Normalized data are from 12 to 20 recordings and presented as mean ± SEM. *P < 0.05, ** indicates P < 0.01. (E) Representative immunoblot.

FIG. 7.

Detection of BoNT/A prepared in human serum using synaptic activity. Representative whole-cell recordings (A) and normalized mEPSC frequencies (B) demonstrating reduced synaptic neurotransmission at 20 h after addition of final concentrations of 55 fM BoNT/A and 10% human serum to ESNs. Scale bar = 1 s × 60 pA. Normalized data are from 12 to 20 recordings and presented as mean ± SEM. *** indicates P < 0.001. (C) Representative immunoblot demonstrating production of cSNAP-25 for above treatments.