Transcranial Direct Current Stimulation (tDCS) in Mice

In recent years, neurostimulation techniques have been entering clinical practice as a promising procedure to treat neuropsychiatric disorders²³. To reduce the constraint imposed by the lack of knowledge of the mechanisms of neurostimulation, we presented here a tDCS mouse model carrying an electrode that can target brain regions. Since the electrode is chronically implantable, this animal model enables the investigation of long-lasting biological effects evoked by tDCS (for at least 1 month) in complex stimulation patterns. The described tDCS animal model presents high implant tolerance and little chance of infection if executed correctly. Overall, the surgery steps to place the implant are of quick and straightforward execution (30 min/animal). One additional advantage of this tDCS model is that it is possible to track the electrode contact quality and the actual current stimulation values.

The main drawback of this animal model is the proper implant fixation on the mouse's cranium. During surgery, it is essential to restrict the animal's head in a manner in which no lateral head shifting is possible (the head will only move vertically). This will assure that the animal's scalp is wholly aligned with the implant's base, allowing proper fixation with the dental cement, and higher precision in modulating the intended target area. It is critical to make the incision large enough to receive the implant. A larger cut may be necessary for some tDCS implants. Using two to four surgical hooks made from hypodermic needles will increase the cemented area. However, avoid placing the hooks too close to the animal's eyes to remove any possibility of lesions. Whereas gently scratching the scalp will improve the adhesion of the super-glue and the cement onto the cranium, any residual debris may prevent good implant adherence. Furthermore, when applying the dental cement, prepare the first layer with a higher viscosity, which avoids the cement of running down the animal's skull. Each cement layer must be allowed to dry for at least 4 min because applying cement over wet layers will delay the hardening of the bottom layers and may cause the implant to shift or even fall. From experience, there must be no more than 3 layers of cement around the implant to avoid obstruction of the screw thread. For both the glue and the cement, be sure to maintain their application restricted to the implant's base. Avoid allowing residue to spread within the implant, which will decrease the surface conductivity and lower the tDCS effects.

The implantable electrode used in this procedure was not fabricated in-house but acquired from a medical research company specialized in producing neuromodulation devices. The implants are made out of polypropylene with 9 mm of height and an outer and inner diameter of 5.7 mm and 3.5 mm, respectively. It can hold a total saline volume of 80 µL. The superior portion of the implant is prepared with a screw thread to receive a pin-type electrode holder. The pin-type electrode holder's outer body is also made of polypropylene measuring 4 mm high with a 5.3 mm outer diameter and a 3.75 mm inner diameter. The electrode pin is made from Ag/AgCl, an inert material used due to its non-dissolving properties (Figure 2). Since the implant location is a critical factor for effective tDCS, it is essential to select a proper electrode size according to the region of interest. The implant used in this animal model occupies a surface area of 9.61 cm², spreading the electrical field over a 1.75 mm radius from the intended brain coordinate resulting in a 36,3967 μA/cm² current density. Possibly, the tDCS stimulus executed in this protocol was mostly directed to the M1 and M2 cortices.

Usually, the electrode configuration varies according to the intended excitatory or inhibitory stimulation effects (anodal versus cathodal). Although currents will always flow out of the anode in the direction of the cathode, by placing the electrode in inverted terminal positions enables different electrophysiology effects. For example, when ions flow from the cathode in the direction of the anode, the procedure is usually defined as a cathodal stimulation²⁴. In this experiment, we performed anodal stimulation in which the anode was placed over the M1/M2 cortices, and the cathode was placed down on the animal's thorax. Thus, in our tDCS setup, it is expected that stimulation produces excitatory potentials²⁵. The tDCS effect can also be regulated through the changes in current intensity and duration. Most studies in rodents have used currents varying from 0.2 to 1.0 mA. tDCS currents are expected to generate concentrated heat traveling through the electrode. The direct contact of the tDCS electrode to the animal's head must be avoided. The use of conducting mediums stretches the distance between the electrode and the cranium and prevents the harmful effects of local chemical reactions on the biological tissue. It is possible that a high ionic concentration in liquid conduct media may cause gas formation and bubbles resulted from electrolysis^23,24. However, this is unlikely to have happened in our tDCS model since isotonic saline solution and low current delivery may decrease the chance of such complications²⁴. Nevertheless, other conducting media can also be used with similar efficiencies, such as gelatinous and cream-like conductors²⁴.

When choosing the tDCS stimulator, it is vital to consider flexible configuration capabilities. For this protocol, we used a stimulator powered by two 9 V alkaline batteries, which render an expected duration of 1 h of stimulation at 0.35 mA. This stimulator possesses a 0.02 to 1 mA current range with a 10 µA resolution, ideal for rodent stimulation. It is critical that the tDCS stimulator is equipped with an actual current indicator and contact quality (CQ) feedback system to verify optimal stimulation conditions. The current indicator assures when the programmed stimulation intensity is being met. In this tDCS model, the most common factor for faulty current is the presence of bubbles in the saline solution. This issue can be indicated by the CQ feedback system, which measures the contact of both electrodes through the conducting medium and the animal's body. The tDCS stimulator used throughout this experiment displays CQ (SMARTscan) values varying from 1 to 10 on a led scale. This scale is based on voltage values that can infer resistance according to Ohm's law. Led 1 indicates little or atypical low resistance, led 2 indicates open circuit and led 3 through 10 indicates poor to optimal quality (Figure 2-item C). The CQ was registered daily for both tDCS and sham groups to verify the electrode viability. It is noteworthy that the average CQ value during stimulation session was higher than 7, meaning that desired currents are being delivered. Overall, no statistical differences were observed for CQ among the groups or day of stimulation (Figure 3A). To further validate our tDCS model, we performed quantitative polymerase chain reactions (qPCR) to investigate whether five tDCS sessions (10 min, 350 μA) change cortical gene expression. We found that mRNA levels of BDNF and GFAP were increased in the M1/M2 cortex of tDCS groups, relative to the Sham mice (Figure 3B). These results are consistent with other studies^19,25.

Neurostimulation studies in experimental animals can provide new insights regarding the brain mechanisms with relevance to neuropsychiatric disorders. Depending on the experimental configuration, the tDCS assembly in this animal model can also be combined with an existing optogenetic or electrophysiology headstage to produce a setup for simultaneous recording and stimulation, alongside a multitude of brain sample experiments. These approaches would be challenging to carry out in humans. Therefore, the opportunity of inserting flexible additions to the currently reported animal tDCS offers a prominent contribution for the understanding of the neural subtracts of tDCS and the rationale for its therapeutic use.