Botulinum toxin suppression of CNS network activity in vitro

Abstract

The botulinum toxins are potent agents which disrupt synaptic transmission. While the standard method for BoNT detection and quantification is based on the mouse lethality assay, we have examined whether alterations in cultured neuronal network activity can be used to detect the functional effects of BoNT. Murine spinal cord and frontal cortex networks cultured on substrate integrated microelectrode arrays allowed monitoring of spontaneous spike and burst activity with exposure to BoNT serotype A (BoNT-A). Exposure to BoNT-A inhibited spike activity in cultured neuronal networks where, after a delay due to toxin internalization, the rate of activity loss depended on toxin concentration. Over a 30 hr exposure to BoNT-A, the minimum concentration detected was 2 ng/mL, a level consistent with mouse lethality studies. A small proportion of spinal cord networks, but not frontal cortex networks, showed a transient increase in spike and burst activity with exposure to BoNT-A, an effect likely due to preferential inhibition of inhibitory synapses expressed in this tissue. Lastly, prior exposure to human-derived antisera containing neutralizing antibodies prevented BoNT-A induced inhibition of network spike activity. These observations suggest that the extracellular recording from cultured neuronal networks can be used to detect and quantify functional BoNT effects.

Figure 1

Average network spike production per minute as a function of time. All activity changes were determined as percent activity decreases relative to the native activity (Reference).

Figure 2

Simulated RC integration with a rise time constant of 70 ms. BD: burst duration; BI: burst interval; BP: burst period. Note the stretching of the burst duration by the slower RC decay. A 10 ms adjustment was made to provide a BD closer to the spike profile.

Figure 3

Temporal evolution of spike and burst rates per minute under the influence of 50 ng/mL BoNT-A. Each data point represents a one-minute average of spike rate (top trace, left ordinate) and burst rate (lower trace, right ordinate). 40 μM bicuculline was added at 83 min to increase activity and reduce minute-to-minute activity fluctuations. A stable reference state was established at 860 spikes/min and all decreases were expressed as percent of this reference state. BoNT-A was added at 124 min and resulted in an irreversible decay of activity starting after a delay of approximately 80 min. Bursting ceased at 400 min with only low levels of residual spiking remaining.

Figure 4

Characteristic response profiles from two different CNS tissues and two different concentrations of BoNT-A. (a) Spinal cord network exposed to 12 ng/mL showing a latency of 100 min and a 90% decrease in 500 min. (b) Frontal cortex network exposed to 100 ng/mL showing a latency of 110 min and a 90% activity decrease in 290 min. (c) Dual networks from age- and maintenance-matched frontal cortex tissue exposed to 50 and 100 ng/mL simultaneously. The higher BoNT-A concentration decreased activity more rapidly, reaching 50% inhibition at 110 min and 175 min for 50 and 100 ng/mL, respectively.

Figure 5

Quantification of frontal cortex (a) and spinal cord (b) network responses to BoNT-A concentrations (x-axis; ng/mL). The time required to reach 10, 50, and 90 percent activity decreases is displayed on the y-axis. Both tissues generate approximate linear power function trend lines over the concentration range from 2 to 100 ng/mL (data points represent mean ± standard deviation).

Figure 6

Average spike (S) and burst (B) rate plot per minute of SC network activity exposed to 50 ng/mL BoNT-A at 138 minutes into the experiment (age: 30 d.i.v.; 34 discriminated units). A sharp increase in spike counts and bursts occurs after a delay of 62 min.

Figure 7

Protection of a frontal cortex network activity with antisera pretreatment. BoNT-A (50 ng/mL) was added 20 min after application of 2% antiserum. The network maintained spontaneous activity for 40 hrs despite increases in BoNT-A concentrations from 100 to 200 ng/mL. Activity was finally stopped by 250 ng/mL. Without the antiserum, 90% of the activity would have been lost at 300 min (white arrow). T: time base switch from 1 min to 2 min.