Adult male Sprague-Dawley rats (n = 10, weighing 250–300 g; Envigo, IN, United States) were used for this study. Rats were kept in group housing in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited vivarium following a standard 12-h light/dark cycle at constant temperature (21°C) and humidity (45%) with ad libitum food and water. Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC), Mayo Clinic, Rochester. The NIH Guide for the Care and Use of Laboratory Animals guidelines (Department of Health and Human Services, NIH publication No. 86-23, revised 1985) were followed for all aspects of animal care.

CFMs were fabricated using an established standardized CFM design at Mayo Clinic (Chang et al., 2013; Oh et al., 2016). Further details, including preparation of the Ag/AgCl counter-reference electrode can be found in our group’s previous publications (Yuen et al., 2021b; Yuen et al., 2022b). CFMs were pretested in TRIS buffer prior to coating deposition with a PEDOT:Nafion deposition solution (Vreeland et al., 2015), which minimized the effect of biofouling in vivo.

Each rat was anesthetized with urethane (1.5 g/kg i.p.; Sigma-Aldrich, St Louis, MO, United States) and administered buprenorphine (0.05–0.1 mg/kg s.c., Par Pharmaceutical, Chestnut Ridge, NY, United States) for analgesia. Following anesthesia, the rat was placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, United States). Respiratory rate (RespiRAT, Intuitive Measurement Systems, AZ, United States) and hind-paw and tail pinch were used to monitor the physiological state and depth of anesthesia, respectively. RespiRAT is a pressure monitor that only counts the peaks of pressure to mitigate interference from noise, and automatically adjusts the detection threshold with the amplitude of the incoming data. Using a standard rat brain atlas (Paxinos and Watson, 2007), three trephine holes were drilled, the first for placement of a CFM into the NAcc (all coordinates from bregma: AP 1.2 mm, ML 2.0 mm, DV 6.5–7.5 mm from dura), the second for a stimulating electrode into the VTA (twisted bipolar stimulating electrode—Plastics One, MS 303/2, Roanoke, VA, United States, with the tips separated by ∼1 mm; AP −5.3 mm, ML 0.9 mm, DV 7.5–9 mm from dura), and a third for an Ag/AgCl reference electrode into the contralateral cortex (Clark et al., 2010).

The depths of the stimulating electrode in the VTA and CFM in the NAcc were first adjusted to obtain a robust stimulation-evoked dopamine signal as measured by fast scan cyclic voltammetry (FSCV) (−0.4 to 1.3 V sweep; 10 Hz). Stimulation parameters were biphasic pulses at 60 Hz, 2 ms pulse width, 0.2 mA, and 2 s duration. Stimulation and FSCV were both performed using the WINCS Harmoni system (Lee et al., 2017), a wireless stimulation and neurochemical sensing system.

Once the optimal electrode depths were identified, the system was switched to the M-CSWV sensing technique (Oh et al., 2018). After at least 60 min of stabilization, the rats were treated according to one of the two experiments. For Experiment 1 (N = 5), i.v. saline (1 mL/kg) was first administered as a negative control. After 30 min, i.v. oxycodone (provided by National Institute on Drug Abuse, Bethesda, MD; 2.5 mg/kg dissolved in 0.5 mL of normal saline) was administered (infused over 2 min via a cannula inserted into the tail vein) and the rat was further observed for another 30 min.

Experiment 2 consisted of the stimulation group (separate set of animals; N = 5), VTA biphasic pulse stimulation was applied at 130 Hz (0.2 ms pulse width, 0.2 mA) continuously for 60 min. The delivered stimulation was interleaved with the M-CSWV recording to minimize artifacts. Once the signal was restabilized to a new plateau (after 30 min of stimulation), i.v. oxycodone was administered (dose and method as above) while stimulation and recording continued. After another 30 min of observation, the stimulation was turned off. The animal was observed for another 30 min post-stimulation before being euthanized using Fatalplus injection (pentobarbital 390 mg/mL i.p.; 10 mL).

Saline administration during VTA stimulation was shown to have no effect on tonic dopamine levels in our previous study and was therefore not repeated here [Figures 4B, 5B of (Yuen et al., 2023)].

After experimentation, changes in dopamine release recorded from individual CFMs were calibrated in vitro with dopamine solutions of different known concentrations. This was performed in a similar manner to previously described procedures (Oh et al., 2018).

For comparison between different dopamine concentrations, repeated-measures one-way (Experiment 1) and two-way (Experiment 2) ANOVA tests were performed. The levels for comparison were all calculated by averaging over 10 data points, i.e., 100 s. In Experiment 1, the levels include: “Baseline” (centered at 5 min prior to saline injection), “Saline” (same post-administration time interval as the “Oxycodone” measurement for fair comparison), and “Oxycodone (Peak)” (centered at the peak of dopamine levels after oxycodone was administered). Post-hoc two-tailed paired t-tests were also conducted in the Experiment 1 between sequential measurements (Baseline vs. Saline; Saline vs. Oxycodone).

In Experiment 2, two-way ANOVA test was performed across: “Baseline” (centered at 5 min prior to start of stimulation), “Stimulation” (centered at 5 min prior to administering oxycodone); “Oxycodone” (centered at the peak of dopamine levels after oxycodone was administered), and “post-stimulation” (centered at 15 min after stimulation stops).

For comparison between respiratory rates, two-tailed paired t-tests were also conducted. Respiratory rates are measured by calculating the moving average over 30 s. Measurements were taken approximately 1/54 s apart. The levels for comparison were calculated by averaging over 10 data points. In Experiment 1, a comparison was made between the levels post-saline (same post-administration time interval as the post-oxycodone measurement for fair comparison) and the levels post-oxycodone (centered at troughs). Likewise, in Experiment 2, a comparison was made between the levels post-stimulation (10 min prior to oxycodone administration) and the levels post-oxycodone (centered at troughs).

Statistical analyses were performed and graphs were constructed using PRISM 8 (GraphPad). All error bars and shaded areas are represented as S.E.M. Statistical significance was set at p < 0.05. No Bonferroni corrections to t-tests were applied as sequential comparisons were set up for testing a pre-planned hypothesis (sequential changes).

Continuous application of VTA DBS resulted in a consistent suppression of NAcc tonic dopamine levels. This also attenuated the facilitatory effects of oxycodone on tonic dopamine levels, with dopamine levels returning to baseline after cessation of DBS. The depletion of presynaptic dopamine vesicular stores may cause this reversible effect. In this case, an initial peak would be expected. Although this was not apparent in the averaged data (Figure 2B), an initial and brief peak increase was indeed seen in three out of five rats (60%) that underwent stimulation. We defined an initial peak to be one that occurs within 1 min (6 MCSWV readings) that is at least more than 2 standard deviations of the 10 averaged points used to calculate the baseline prior to stimulation. The average increase in dopamine among the three rats was 14.4 ± 3.7 mM. This is consistent with the phasic (evoked) increases of dopamine in the NAcc (measured by FSCV) that were seen with brief (2 s) stimulation of the VTA during the targeting progress. This may not be seen universally as the M-CSWV technique has a time resolution of 10 s and if the release of dopamine occurred prior to the following measurement, the peak may be much smaller. Previous studies have shown that prolonged intermittent VTA stimulation was associated with reduced dopamine levels in the NAc, likely due to depletion of accumbal presynaptic dopamine vesicular stores or autoreceptor activation (Hernandez et al., 2006; Hernandez and Shizgal, 2009). This is consistent with the phenomenon seen in the current study. Another explanation may be activation or inhibition of other inhibitory and excitatory neurotransmitters. Examples include GABA (inhibitory), dopamine (inhibitory), glutamate (excitatory), and acetylcholine (facilitatory), all of which have been found in the VTA in microdialysis studies (Westerink et al., 1996; Lester et al., 2010). It is well known that opiates increase firing activity of dopamine neurons at least in part by decreasing the inhibitory effect of GABA via activation of µ-opiate receptors on GABAergic interneuron terminals in the VTA, and glutamate transmission is required for morphine’s effect on dopamine cells (Barrot, 2015; Saigusa et al., 2021). Therefore, DBS may exert its effect on extracellular dopamine levels via modulation of these other neurotransmitters.

One further possibility is that continuous electrical stimulation of the VTA may induce a relatively rapid depolarization block (DB) similar to chronic antipsychotic treatments that, in turn, decrease tonic DA levels in the NAcc (Lane and Blaha, 1987; Grace et al., 1997; Boye and Rompre, 2000; Hernandez et al., 2016). These hypotheses can be investigated by further studies using other pharmacological tests and measuring techniques such as amperometry.

VTA DBS is currently being used as an experimental treatment for treatment-refractory cluster headache (Akram et al., 2016). Interestingly, it has been shown that elevation of peripheral dopamine levels is implicated in cluster headache pathophysiology, suggesting cluster headaches may be a consequence of overactivity of the dopaminergic and autonomic systems (D'Andrea et al., 2006; D'Andrea et al., 2019). Therefore, the therapeutic effect of VTA DBS on cluster headache may be explained by its suppressive effect on forebrain dopamine levels.

With regards to the potential applicability of DBS for substance use disorder, no human studies have been reported using VTA DBS. In preclinical studies, one study demonstrated continuous optogenetic stimulation of VTA dopaminergic neurons can reduce ethanol self-administration in mice (Bass et al., 2013). Although optogenetic and electrical stimulation have different underlying mechanisms of action, one study demonstrated that both modalities could activate similar brain regions (Weidner et al., 2020).

In the current study, the NAc core was chosen as the main target for dopamine measurements for two main reasons. First, from microdialysis studies, extracellular dopamine in the core was higher than that in the shell, whereas norepinephrine levels (a potential electroactive interferent) were higher in the shell (McKittrick and Abercrombie, 2007). Second, these are acute experiments where the animals are exposed to the drug for the first time. It has been suggested that the NAcc underlies the phenomenon of initial acquisition of drug taking, cue-elicited drug seeking behavior, and impulsive choices (Di Chiara, 2002; Salgado and Kaplitt, 2015). Although we did not perform comparison studies using the accumbens shell subregion, it remains an interesting target for future studies using opioids in combination with VTA DBS. VTA DBS may modulate drug-seeking behavior by suppressing dopamine levels in the NAcc. This may also be applicable to hyperdopaminergic pathologies such as mania, schizophrenia, and dopamine dysregulation syndrome (Berk et al., 2007; Grace, 2016; Ashok et al., 2017).

Consistent with previous microdialysis studies (Westerink et al., 1996; Lester et al., 2010), oxycodone induced significant increases in NAcc dopamine levels, which likely account for the initiating steps in the pathophysiology of opioid use disorder. The suppression of NAcc dopamine levels appeared reversible within 60 min of DBS but this may not be the case for longer stimulation duration or repeated stimulations. Given dopamine is associated with both drug-related and pharmacological rewards, depression of dopamine levels in a normal dopaminergic state may theoretically lead to anhedonia and depression. Therefore, more studies need to be conducted before widely implementing this potential therapeutic option. In addition, the present study suggests that intermittent or continuous measurement of tonic dopamine levels may serve as a potential biomarker for closed-loop DBS for substance use disorder. However, the risks of implanting a separate sensing electrode and the ethics of such interventions should be considered (Lo et al., 2023).

The cases of opioid overdose in the United States have more than quadrupled between 2000 and 2020 and, without further intervention, is expected to double by 2029 (Humphreys et al., 2022). Therefore, it is of interest that the current study demonstrated that VTA DBS may be used to reduce the effects of oxycodone and therefore potentially prevent overdose deaths. Given the volume of tissue affected by electrical stimulation, it is highly likely that other brain structures, such as descending white matter fibers that control the motor aspect of respiratory effort, were co-stimulated, leading to the observed attenuating effect on oxycodone-associated respiratory suppression. Several studies using intracranial self-stimulation also showed that DBS can alter the physiological parameters such as the respiratory rate, demonstrating the complex interplay between brain stimulation and the autonomic nervous system (Malmo, 1963; Angyan, 1981; Burgess et al., 1993). One more possible explanation is that stimulation of the VTA would likely also stimulate the substantia nigra (SN) given its proximity to the VTA. There are known anatomical connections between the SN compacta and respiratory control centers, including a direct SNc pathway to the pre-Bötzinger complex (Zhou et al., 2011). The pre-Bötzinger complex interneurons have direct connections to caudal areas containing bulbospinal neurons that are directly involved in generating rhythm to the premotor neurons that transmit oscillatory drive to spinal respiratory motoneurons (Mahoney et al., 2021). Indeed, endogenous activation of D1Rs acts against opioid depression of the respiratory network (Qu et al., 2019). Nevertheless, to our knowledge, this is the first study that demonstrated DBS can attenuate the respiratory effect of opioids. Further optogenetic studies that focus on particular neurons such as dopamine or glutamate neurons, may be used to further discern the target that led to this effect. Also, tractography, particularly in larger animal studies, may be used to map the relationship between the volume of tissue activated by DBS and white matter tracts near the VTA.

Limited historical precedence or evidence implicates DBS as a potential procedure for respiration modulation. One study showed subthalamic nucleus stimulation may improve upper respiratory and laryngeal control in Parkinson’s patients (Hammer et al., 2010), but no lower respiratory effect (intercostal muscles and diaphragm) was measured. Our results suggest that, in addition to opioid overdose, diseases characterized by respiratory insufficiency or failure, such as intractable sleep apnea, may be treated by DBS.

This proof-of-concept preclinical study utilized anesthetized, naïve rats as subjects rather than a chronic addiction model. Although a relatively high acute dose of oxycodone was administered that induced a clinically corollary of respiratory depression, there were no measures for addictive behaviors such as drug-seeking and compulsion. Future studies in awake animals, particularly in drug self-administration paradigms, will be required to validate the dopaminergic effects, as well as behavioral and respiratory benefits.

While dopamine plays a key role in the process of drug addiction, there are also studies which showed opioids may have a dopamine-independent reward mechanism (Ettenberg et al., 1982; Pettit et al., 1984; Nader and van der Kooy, 1997; Hnasko et al., 2005). Therefore, how much VTA DBS may alter the behavioral changes should be further studied.