Closed-loop neural stimulation for pentylenetetrazole-induced seizures in zebrafish

Abstract

Neural stimulation can reduce the frequency of seizures in persons with epilepsy, but rates of seizure-free outcome are low. Vagus nerve stimulation prevents seizures by continuously activating noradrenergic projections from the brainstem to the cortex. Cortical norepinephrine then increases GABAergic transmission and increases seizure threshold. Another approach, responsive nervous stimulation, prevents seizures by reactively shocking the seizure onset zone in precise synchrony with seizure onset. The electrical shocks abort seizures before they can spread and manifest clinically. The goal of this study was to determine whether a hybrid platform in which brainstem activation triggered in response to impending seizure activity could prevent seizures. We chose the zebrafish as a model organism for this study because of its ability to recapitulate human disease, in conjunction with its innate capacity for tightly controlled high-throughput experimentation. We first set out to determine whether electrical stimulation of the zebrafish hindbrain could have an anticonvulsant effect. We found that pulse train electrical stimulation of the hindbrain significantly increased the latency to onset of pentylenetetrazole-induced seizures, and that this apparent anticonvulsant effect was blocked by noradrenergic antagonists, as is also the case with rodents and humans. We also found that the anticonvulsant effect of hindbrain stimulation could be potentiated by reactive triggering of single pulse electrical stimulations in response to impending seizure activity. Finally, we found that the rate of stimulation triggering was directly proportional to pentylenetetrazole concentration and that the stimulation rate was reduced by the anticonvulsant valproic acid and by larger stimulation currents. Taken as a whole, these results show that that the anticonvulsant effect of brainstem activation can be efficiently utilized by reactive triggering, which suggests that alternative stimulation paradigms for vagus nerve stimulation might be useful. Moreover, our results show that the zebrafish epilepsy model can be used to advance our understanding of neural stimulation in the treatment of epilepsy.

Fig. 1.

Latency to onset of Stage III behavioral seizure activity. Onset of Stage III behavioral seizure activity was measured in response to 15 mM PTZ in fish that had stimulator wire insertion and no stimulation (SHAM), stimulation (STIM) or stimulation in the presence of 25 μM POB. Hindbrain stimulation increases seizure onset latency. The effect was partially blocked by the α-noradrenergic antagonist POB. Bars indicate the data median. Boxes and whiskers indicate the 25th to 75th and 5th to 95th percentiles, respectively. Crosses indicate outliers.

Fig. 2.

Changes in zebrafish cerebral field potential in response to PTZ. (A) Probability densities of 20 consecutive 10-second forebrain field potential recordings from before (red) and after (blue) PTZ exposure. The field potential was sampled at 64 Hz. Samples are normalized on a scale of 0 to 1 and then individual points sorted into one of seven equidistant probability bins. Blue traces are obtained in the baseline state (prior to PTZ application). Red traces are obtained in the pre-seizure state (after PTZ application, but before electrographic seizure activity). The horizontal blue dashed line represents a theoretical probability density function in which all points in the sample are different and the entropy is maximal and equal to log₂N where N is the number of points in the sample. The vertical red dashed line represents a probability density function in which all points are the same and the entropy is minimal and equal to zero. The blue traces more closely approximate the blue horizontal dashed line, indicating a wider dispersion and higher entropy in the baseline state. The red traces more closely approximate the red vertical dashed line, indicating a narrower dispersion and lower entropy in the pre-seizure state. (B) Simultaneous forebrain field potential and scalar entropy for a PTZ-induced seizure. PTZ was applied at the down arrow and then removed at the up arrow. Following a latency (bracket), an electrographic seizure occurs (over-score), as evidenced by the marked increase in the amplitude of the cerebral field potential (black). The scalar entropy (blue) estimated on the field potential data decreases following PTZ exposure and prior to seizure onset. Thus, a 3 s.d. reduction in scalar entropy (red asterisk) from the mean of a rolling baseline average can be used as a trigger for reactive hindbrain stimulation.

Fig. 3.

Reactive hindbrain stimulator. (A) Scheme of the reactive neural stimulation circuit. Forebrain (FB) field potential is recorded via high-gain amplifier (upper left) and fed into the control element consisting of an analog-digital converter (ADC) and a central processing unit (CPU). When the CPU detects seizure activity, it triggers the hindbrain (HB) stimulator (upper right; 0.1 mA/0.1 milliseconds) in the locus coeruleus (LC) via a digital to analog converter (DAC) resulting in norepinephrine (NE) delivery to the FB and an anticonvulsant effect as discussed (MB, midbrain). (B) Flow diagram for control and triggering of reactive hindbrain stimulation. Following the acquisition of a 3-minute baseline, the hindbrain stimulator was armed. Scalar entropy was estimated on consecutive 10-second epochs of cerebral field potential data. Stimulation was triggered when the entropy was greater than 3 s.d. below the baseline mean. Data acquisition was paused for 5 milliseconds to allow stimulator artifact to dissipate so that entropy estimates would not be contaminated and skewed by stimulator artifact.

Fig. 4.

Reactive hindbrain stimulation in the presence of PTZ. (A) Simultaneous recording of forebrain field potential (black) and scalar entropy (blue). PTZ was applied continuously (down arrow). Following a latency, there was a decrease in scalar entropy that results in a volley of hindbrain stimulations (red asterisks). A total of three stimulation volleys with 21 total hindbrain stimulations occurred over a 30-minute interval. Despite more than 30 minutes of exposure to PTZ, no seizure occurred. The amplifier registered a stimulation artifact in the EEG tracing with each stimulation. (B) Histogram showing number of stimulations per 30-minute PTZ exposure time. The stimulation rate was directly dependent on PTZ concentration and was reduced by the addition of valproic acid (VA). Use of a stimulator pulse that delivers 10× the charge per stimulation (10XSTIM) resulted in significantly fewer hindbrain stimulations over a 30-minute period in the presence of 15 mM PTZ. Bars indicate the data median. Boxes and whiskers indicate the 25th to 75th and 5th to 95th percentiles, respectively.

Fig. 5.

Anatomical correlation. (A) Histologic locations of distal-most aspect of stimulating electrode as shown on necropsy with a representative section shown in the insert. In each instance, the stimulating electrode was located within 1 mm of the area deemed to be most consistent with the LC. Symbols on the left side of the templates represent experiments done in the presence of 7.5 mM PTZ and symbols on the right side of the templates represent experiments done in the presence of 15 mM PTZ. Open circles represent experiments done in the presence of PTZ alone. Filled circles represent experiments done in the presence of PTZ and 10 mM valproic acid. Red dots represent experiments done in the presence of 15 mM PTZ using 0.1 μC of charge (0.5 mM/0.2 milliseconds) per stimulation. All other experiments were performed using 0.01 μC of charge (0.1 mM/0.1 milliseconds) per stimulation. OB, olfactory bulb; TEL, telencephalon; CER, cerebellum; MED, medulla; SC, spinal cord; IL, inferior lobe; LC, locus coeruleus. (B) Reactive hindbrain stimulation using a far caudal stimulation site. Following a latency after PTZ application (down arrow) the scalar entropy (blue) fell and stimulation was triggered (red asterisks). Despite stimulation, an electrographic seizure occurred as evidenced by the increased amplitude of the cerebral field potential (black). A second round of stimulation terminated this seizure. These results imply that more caudal stimulation sites are less effective at attenuating seizures.