Behavioral validation of a wireless low-power neurostimulation technology in a conditioned place preference task

Abstract

Objective: Neurostimulation technologies are important for studying neural circuits and the connections that underlie neurological and psychiatric disorders. However, current methods come with limitations such as the restraint on movement imposed by the wires delivering stimulation. The objective of this study was to assess whether the e-Particle (EP), a novel wireless neurostimulator, could sufficiently stimulate the brain to modify behavior without these limitations.

Approach: Rats were implanted with the EP and a commercially available stimulating electrode. Animals received rewarding brain stimulation, and performance in a conditioned place preference (CPP) task was measured. To ensure stimulation-induced neuronal activation, immediate early gene c-fos expression was also measured.

Main results: The EP was validated in a commonly used CPP task by demonstrating that (1) wireless stimulation via the EP induced preference behavior that was comparable to that induced by standard wired electrodes and (2) neuronal activation was observed in projection targets of the stimulation site.

Significance: The EP may help achieve a better understanding of existing brain stimulation methods while overcoming their limitations. Validation of the EP in a behavioral model suggests that the benefits of this technology may extend to other areas of animal research and potentially to human clinical applications.

Figure 1.

E-particle (EP) design and implantation. (A) The EP is a wireless neurostimulator with a terminal platinum wire to penetrate the neural tissue. A coil (L) is used for inductive power, a capacitor (C_res) to tune the coil to 10.7 MHz, a diode (D) to rectify the received signals, a capacitor (C_shunt) to enhance rectification, and two electrodes (adapted from Freeman et al (2017)). A roughened platinum disc electrode is used as the anode, and a roughened platinum wire is used as the cathode. (B) The injector tool for EP implantation is used by loading the EP into the loading sleeve, and the injector pin is inserted and attached to complete the lever assembly (left). An assembled introducer attached to a stereotaxic cannula holder arm is illustrated on the right. The injector is lowered into the brain, and the lever is pressed to release the EP into the target brain tissue. (C) This diagram illustrates how each animal was implanted with a Plastics One electrode into the medial forebrain bundle (MFB) on one side of the brain and the EP on the other side of the brain.

Figure 2.

CPP field and experimental timeline. (A) Rats in this study were placed into an open field with four quadrants. When the animals entered a predetermined stimulation quadrant (with coil attached), they received stimulation. The three square coils (in red) are attached to bottom left corner of the arena. The rat in the diagram is in quadrant 3 scale bar = 6 in. (B) The CPP experiment consisted of four sessions: habituation, stimulation day 1, stimulation day 2, and test. These sessions took place four times (each quadrant served as a stimulation quadrant). Two rounds of the four sessions were run for each of the two stimulation types for a total of 64 d of testing. Animals were sacrificed at the end of behavioral testing, following 60 min of continuous EP stimulation in the same arena.

Figure 3.

Block diagram of computer setup for behavior. A computer and attached video camera track rats in real time as they explore the arena. When the animal dwells in the target quadrant, stimulation occurs. Pulse sequences sent from LabVIEW through the DAQ are sent through an amplifier to a transmitter coil for EP stimulation and sent through a stimulator for wired stimulation.

Figure 4.

Histological results. (A) Thionine Nissl-stained section illustrating the location of the wired electrode and EP placement in the MFB. Placements of both types of electrodes are shown in the bottom illustration. Red: EP; blue: wired electrode. (B) Magnified image of tissue at the tip of the electrode (40×). Neither the EP-implanted tissue (left) nor the wired electrode (right) showed decreased cellular density suggestive of severe damage. There is some blood present in the right image, suggesting possible microtrauma from the wired electrode insertion; scale bar = 18 μm. (C) This CT-MRI coregistered image of the EP (purple) illustrates its placement in the MFB.

Figure 5.

Quadrant 4 aversion. (A) A subset of animals, new to the CPP field, spent less time in quadrant 4 compared to other quadrants in the absence of any stimulation regardless of tether, *p < 0.05. (B) Heatmaps illustrate the amount of time spent in other quadrants compared to the quadrant 4 (outlined in orange) with more time spent displayed as colors increasing in intensity from black to yellow. Error bars represent SEM.

Figure 6.

CPP behavioral results. (A) Heatmaps demonstrate the formation of a CPP across sessions with time spent in the stimulation quadrant increasing as colors go from black to yellow. Quadrants outlined in red indicate the stimulation quadrant. (B) This bar graph illustrates the percentage of time the animals spent within the stimulation quadrant per session as a result of wired and wireless (EP) stimulation. With wired MFB stimulation, the rats preferred the stimulation quadrant (increased time spent in stimulation quadrant) during the first day of stimulation, and this preference remained during the test session. EP stimulation induced conditioned preference behavior by the second day of stimulation, which was also maintained during the test session, *p < 0.05, compared to baseline; ^§p < 0.05, compared to baseline. Error bars represent SEM.

Figure 7.

Coil warmth tests. (A) To assess whether animals were attracted to heat potentially generated by the coil attached to a corner of the open field, a subset of animals without an implanted EP were placed into the open field with the trigger for the coil turned on as long as the rat remained in the selected stimulating quadrant. There was no difference between time spent in the stimulation quadrant compared to the non-stimulation quadrants. n.s. =not significant, p > 0.05. (B) Heatmaps illustrate the time spent in the quadrants and demonstrate that rats did not increase time spent in the stimulation quadrant. Quadrants outlined in orange indicate the stimulation quadrant. Error bars represent SEM.

Figure 8.

C-fos induced by EP and wired stimulation. (A) Representative confocal images (40×) of DAPI, c-fos, and merged DAPI and c-fos-stained images within the NAcc and MC in the left hemisphere (EP side) only; scale bar = 50 μm. (B) EP stimulation produced significantly more c-fos positive cells (shown here as a percentage of DAPI-stained cells) in the nucleus accumbens (NAcc), a brain region innervated by the MFB, compared to the motor cortex (MC), which does not have direct connections within the MFB. There was a slight increase in NAcc c-fos expression compared to MC with wired stimulation, but it was not statistically significant (n = 2, both animals received EP and wired stimulation). *p < 0.05. Error bars represent SEM.