
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39251806
71841
10.1038/s41598-024-71841-1
Article
Non-invasive temporal interference brain stimulation reduces preference on morphine-induced conditioned place preference in rats
Mojiri Zohre 1
Rouhani Ehsan 1
Akhavan Amir aakhavan@iut.ac.ir

1
Jokar Zahra 2
Alaei Hojjatallah 2
1 https://ror.org/00af3sa43 grid.411751.7 0000 0000 9908 3264 Department of Electrical and Computer Engineering, Isfahan University of Technology, Isfahan, 84156-83111 Iran
2 https://ror.org/04waqzz56 grid.411036.1 0000 0001 1498 685X Department of Physiology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
9 9 2024
9 9 2024
2024
14 210408 11 2023
31 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Long-term use of opioid drugs such as morphine can induce addiction in the central nervous system through dysregulation of the reward system of the brain. Deep brain stimulation (DBS) is a non-pharmacological technique capable of attenuating behavioral responses associated with opioid drug consumption and possesses the capability to selectively activate and target localized brain regions with a high spatial resolution. However, long-term implantation of electrodes in brain tissue may limit the effectiveness of DBS due to changes in impedance, position, and shape of the tip of the stimulation electrode and the risk of infection of nerve tissue around the implanted electrode. The main objective of the current study is to evaluate the effect of temporal interference (TI) brain stimulation on addictive behaviors of morphine-induced conditioned place preference (CPP) in rats. TI stimulation is a non-invasive technique used transcranially to modulate neural activity within targeted brain regions. It involves applying two high-frequency currents with slightly different frequencies, resulting in interference and targeted stimulation of different brain areas with the desired spatial resolution. The results indicated that TI stimulation with the amplitude of I1=I2=0.5 mA, carrier frequency of 2 kHz, frequency difference of 25 Hz, ON–OFF stimulation frequency of 0.25 Hz, and total duration of 10 min in three consecutive days resulted in a significant reduction of morphine preference in the morphine-stimulation group in comparison with the morphine group (p < 0.001). These findings highlight the potential of TI stimulation as a modulatory intervention in mitigating the addictive properties of morphine and provide valuable insights into the therapeutic implications of this stimulation paradigm for treatment of opioid drugs in human subjects.

Subject terms

Biomedical engineering
Behavioural methods
http://dx.doi.org/10.13039/100012330 Cognitive Sciences and Technologies Council 11927 issue-copyright-statement© Springer Nature Limited 2024
==== Body
pmcIntroduction

Drug addiction is a persistent and recurring disorder that is characterized by an uncontrollable urge to use drugs, an inability to regulate drug consumption, and the presence of negative emotions during the withdrawal phase1–3. Long-term use of opioid drugs such as morphine can induce addiction in the central nervous system through disruption and dysregulation of the reward system of the brain. The interaction between the ventral tegmental area (VTA) and nucleus accumbens (NAc), through the release and modulation of dopamine, forms a vital circuit within the reward system4,5. Furthermore, the medial prefrontal cortex (mPFC), in connection with the VTA and NAc actively participates in the modulation of the reward system by regulating the excitability of VTA dopaminergic neurons through the dopamine pathways in the mesocorticolimbic dopamine system4,6. Dysfunction of the mPFC can contribute to alterations in reward processing, leading to psychiatric disorders such as morphine addiction. The pleasurable effects of morphine are derived from the stimulation of µ-opioid receptors in the VTA, which subsequently activates dopaminergic neural pathways in the prefrontal cortex4. Pharmacological interventions, particularly opioid agonists can normalize the activity of the mPFC and restore its regulatory role in reward processing by stabilizing the µ-opioid receptors. In the literature7–9, the researchers have shown that Corydaline, L-tetrahydropalmatine, and Mitragynine attenuated the reinforcing effects of morphine in rats. Jiang et al. showed that Corydaline could reduce the effect of morphine by reversing the decreased expression of dopamine D2 receptors in mPFC7. However, the body's tolerance to drugs, toxic and unwanted side effects, difficulties with abstinence, and dosage can limit the clinical applications of pharmacological approaches.

Electrical stimulation of the brain as an alternative to pharmacological interventions is an effective technique for the treatment of various diseases including depression10–12, Alzheimer13,14, Parkinson15–17, and addiction18–25. This approach can be categorized into transcranial electrical stimulation (tES)26–31 and deep brain stimulation (DBS)18,19,21–24,32–34 methods. The tES is a noninvasive brain stimulation technique that involves the application of weak electrical currents to the scalp to modulate neuronal excitability in cortical regions of the brain26. Pedron et al. demonstrated that repeated transcranial direct current stimulation (tDCS)28,30 of the frontal cortex of Swiss female mice is an effective approach to decrease behavioral responses to cocaine over several weeks29. An investigation has been addressed in31 to evaluate the effectiveness of bimodal tDCS on alcohol consumption in adult male Wistar rats. The results showed that bimodal tDCS could be a valuable non-pharmacological adjuvant technique to reduce voluntary alcohol consumption over time induced by stimulation. In all aforementioned studies, although tES serves as a non-pharmacological technique capable of attenuating behavioral responses associated with opioid drug consumption, it cannot stimulate specific regions within the mesocorticolimbic dopamine system associated with dopamine release selectively, thereby lacking precise spatial resolution.

Deep brain stimulation (DBS) possesses the capability to selectively activate and target localized brain regions with a high spatial resolution. To access the specific brain regions, the electrodes must be surgically positioned at the desired locations within the brain tissue. Fakhrieh-Asl et al. investigated the effectiveness of low frequency- and high frequency-DBS in the orbitofrontal cortex (OFC) for controlling addictive behaviors on morphine-induced CPP in rats33. The results revealed that high frequency-DBS (130Hz) of OFC during the conditioning phase decreased morphine preference while low frequency-DBS (13Hz) during the extinction phase did not elicit any significant alterations in addictive behaviors. In our previous studies, we evaluated the effect of DBS on reward-related nuclei mPFC18,35, VTA19, NAc21, central amygdala (CeA)24, basolateral amygdala (BLA)22, and Lateral habenula (LHB)23 on morphine-induced rats by using the behavioral CPP test. The results showed that DBS with a current intensity of 100–150 μA (200 pulses with 0.4 ms duration and a frequency of 25Hz every 5 s) for 10 min on three consecutive days, decreased morphine preference in the CPP model. The results of18 indicated that electrical stimulation of the prelimbic cortex (PL) of mPFC with intensity of 100 μA suppressed morphine-induced CPP. All of the above studies18,19,23,24 involved surgery and implantation of the stimulation electrodes in the desired nuclei and a CPP test (morphine injection + DBS) performed over a period of several weeks. Long-term implantation of electrodes in brain tissue may limit the effectiveness of DBS due to changes in impedance, position, and shape of the tip of the stimulation electrode and the risk of infection of nerve tissue around the implanted electrode.

Temporal interference (TI) stimulation is a non-invasive technique used transcranially to modulate neural activity within targeted brain regions by applying two high-frequency sinusoidal currents simultaneously with slightly different frequencies36–40. The interference of two generated fields can follow the envelope modulation that oscillates at the frequency difference to stimulate different brain areas with the desired spatial resolution. Nevertheless, some challenges facing the clinical application of TI to focally activate the brain regions with high spatial resolution are determining the optimal placement of electrodes and adjusting stimulation parameters including frequency, amplitude, and current ratio. To overcome the challenges of TI stimulation in clinical use, in the literature, there are some attempts to evaluate the performance of TI stimulation by computational modeling36,41,42 and experimental approaches36,43 to show how the field is distributed in the brain through TI stimulation. To take advantage of the TI stimulation method and also overcome the previously mentioned limitations of the tES technique, the main objective of the current study is to evaluate the effect of TI electrical stimulation of the brain on addictive behaviors of morphine-induced CPP in rats. The results of the study demonstrated a noteworthy decrease in morphine preference within the morphine-stimulation group when compared to the morphine group. These findings highlight the potential of TI stimulation as a modulatory intervention in mitigating the addictive properties of morphine and provide valuable insights into the therapeutic implications of this stimulation paradigm for the treatment of opioid drugs in human subjects.

The paper is organized as follows. "Materials and methods" provides details on the methods including the TI stimulator, stimulation protocol, electrode implantation, and CPP paradigm. "Experimental results" presents the results of the effect of TI stimulation on the morphine-induced CPP. "Discussion and conclusion" discusses and concludes the study.

Materials and methods

In this research, results from thirty-two adult male Wistar rats (250–300 g body weight) are reported to evaluate the effect of TI stimulation on addictive behaviors. All surgical procedures were approved by the Research and Ethics Committee of Isfahan University of Medical Sciences, Isfahan, Iran (No. IR.MUI.AEC.1401.010). The current study was carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org/). All experimental protocols adhered to the ethical and methodological standards of Laboratory Animals (National Institutes of Health Publication No. 85-23). The experiments were performed in accordance with relevant guidelines and regulations. The animals were housed in cages (three animals per cage) and maintained under standard environmental conditions that featured a 12-h light/dark cycle. The rats had ad libitum access to water and rodent chow. The Rats were divided into five groups: the morphine group that received saline and morphine throughout the experiment without any TI stimulation and surgery (n = 6), the morphine-sham group that underwent surgery and received saline and morphine and no active stimulation during the experiment (n = 6), the morphine-stimulation group (main group) that underwent surgery and was administered saline and morphine, with active TI stimulation during every experiment phase (n = 8), the saline-sham group that received saline and no active stimulation was performed during every phase of the experiment (n = 6), the saline-stimulation group that underwent surgery and was administered saline, with active TI stimulation during every experiment phase (n = 6). In this study, the G*Power 3.1 software was used to choose the appropriate total sample size. We used an “a priori” method for power analysis to estimate the number of animals in each group44. To do so, the α level, power, and the expected value for the effect size should be adjusted. To align with previous studies utilizing invasive DBS, in accordance with the results of prior study24, we aimed for an effect size of d = 0.694. Based on the result of G*Power 3.1 software, for the expected effect size of d = 0.694 in a one-way ANOVA test with α = 0.05, power = 0.8, and the number of groups 5, a minimum sample size of n = 6 per group was required. Since the main objective of the current study is to evaluate the efficacy of TI stimulation in reducing morphine preference, in order to increase the statistical validity of the experiment, the number of animals in the morphine-stimulation group has been increased to 8.

TI stimulator and stimulation protocol

TI is a non-invasive brain stimulation technique employing two or more electrode pairs to target deep regions of the brain. TI involves the interference of two or more high-frequency electric fields within the brain to generate a pattern of low-frequency interference. To do this, two electric fields are applied at high frequencies (f1 and f2 = f1  + Δf) outside the normal neural activity range with a small frequency difference. The interference of two applied fields can be able to follow the envelope modulation that oscillates at the frequency difference. The amplitude of the envelope modulation is regulated by the magnitudes of the two applied sinusoidal electric currents, while the localization of the envelope modulation is controlled by the arrangement of the stimulation electrodes and the ratio of current amplitudes between the two stimulations36. In this study, a custom-made four-channel isolated TI stimulator is used to deliver TI stimuli pulses through the electrodes implanted on the skulls of the animals. In this study, only two channels are applied to stimulate the brain regions. Each channel of the stimulator incorporates a Howland current source (HCS). The HCS is a grounded-load circuit that uses an operational amplifier (op-amp) to generate a precise and stable output current that is proportional to the input voltage45. The stimulator is powered by a direct current (DC) power source, and MINMAX converters (mau203, Ultra Miniature High Isolation SIP DC/DC Converter) are employed for electrical isolation of the voltage supplies in each channel of the stimulator. The sinusoidal voltage signals are applied to the input of the stimulating device via signal generators (Leader LAG-125 Low Distortion Audio Generator, India), and the sinusoidal current signals are produced at the output of the channels. The output of each channel can generate either a continuous or pulsed signal. The pulse amplitude ([−3 to 3] mA), ON–OFF stimulation frequency ([0.1‒1] Hz), and carrier frequency ([1‒5000] Hz,) of the stimulation signal can be adjusted manually through the selectors and push buttons embedded on the panel of the stimulator. In the current study, the current I1=I2=0.5 mA36, carrier frequency of 2 kHz36, frequency difference of 25 Hz18,19,23,24, ON–OFF stimulation frequency of 0.25 Hz36, duty cycle of 50% (two seconds of ON stimulation followed by two seconds of OFF stimulation)36, and total duration of 10 min18,19,23,24 were used to stimulate the tissue of the brain.

Electrode implantation

During the initial phase of the experimental procedure, a one-week period (0–6 days in Fig. 1) was allowed for the rats to adapt to the new conditions. Subsequently, surgical procedures were performed on day 7 to implant electrodes onto the skulls of the animals. The rats in morphine-sham, morphine-TI stimulation, saline-sham, and saline-stimulation groups were anesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). The incision area of the head has been shaved and fixed in a stereotaxic apparatus (Stoelting, USA) and the scalp was opened along the midline. In order to deliver TI stimulation to the brain tissue of awake animal through the electrodes, a 4-pin connector was attached surgically to the head of the animal using dental cement to connect the electrodes to the skull of the animal. The central two pins of the connector were linked to the screw on the skull using copper wire with 0.1 mm diameter as two positive head electrodes (red circles in Fig. 1, surgery). The position of the positive stimulation electrodes is anterior–posterior (AP) = 3 mm, medial–lateral (ML)1 = −2.3 mm, ML2 = 0.7 mm relative to bregma based on the coordinates from the Paxinos atlas46. The connector's side pins were attached to the bilateral cheeks of the animals so that one end of the copper wire insulated with polyurethane (Round Polyurethane-Lacquered copper wire, diameter 0.1 mm, SIM LAKI FARS, Shiraz, Iran) was connected to pins housed within a 4-pin connector. The other end of the wire was attached to a copper piece with a 5 mm diameter and was cautiously passed under the scalp and secured to the cheek area using sutures (black circles in Fig. 1, surgery). After surgery, the rats were allowed to recover in a five- to seven-day period (7–13 Days in Fig. 1) prior to being subjected to CPP behavioral tests (Days 14–18 in Fig. 1).Fig. 1 Experimental setup and proposed CPP paradigms with timeline. The CPP experimental protocol spans a duration of five consecutive days and encompasses three distinct phases: the preconditioning stage, the conditioning stage, and the postconditioning stage (test day).

Exploring the distribution of the TI-induced electric field within the brain

In order to evaluate the distribution of the TI-induced voltage within the brain, an experiment was conducted on five male Wistar rats. The animals were deeply anesthetized with urethane (1.6 g/kg). The hairs of the head of each animal were shaved and the animal was positioned on a stereotaxic device (Stoelting, USA) to surgically expose the scalp. The stimulation electrodes were positioned as expressed in "Electrode implantation". The position of the recording electrode was changed in the DV direction from 0 to 5 mm, the AP direction from −5 to 5 mm, and the ML direction from −3.8 to 2.2 mm, and the mean amplitude of the envelope oscillation was investigated. To do this, the dorsal surface of the skull was drilled using a drill (Marathon Champion, South Korea) to access the deep regions of the brain. Two stainless-steel recording and reference electrodes (Stainless steel wire AISI316LVM, BarewireØ0.125 mm, Advent Research Materials Ltd, Witney, England) insulated (Nominal O.D coated wire 0.175 mm) with quadruple Polytetrafluoroethylene (Advent Research Materials Ltd, Witney, England) were twisted and implanted intracortically to record the electrical interference generated by TI stimulation. The tip of the reference electrode is positioned 0.1 mm away from the recording electrode, and the uncoated tips of the electrodes are placed within each hole by adjusting the positions of the electrodes in the DV direction using the micromanipulator of the stereotaxic device. Figure 2 shows the positions of the recording locations in three directions AP = [−5 5] mm with a step size of 2 mm, ML = [−3.8 2.2] mm with a step size of 1.5 mm, and dorsal–ventral (DV) = [0 5] mm with a step size of 0.5 mm relative to bregma. The electric signal during TI stimulation with the parameters described in TI stimulator and stimulation protocol" was acquired using a recorder (eWave, Sciencebeam Co, Tehran, Iran) at a sampling rate of 10 kHz, and the signal was monitored using the eProb software (version 8.1, Sciencebeam Co, Tehran, Iran). The recorded signal was filtered using a Butterworth band-pass filter with cutoff frequencies of 1.9 kHz and 2.1 kHz, implemented in MATLAB R2019a software to effectively eliminate unwanted noise and artifacts.Fig. 2 Schematic of the trapezoidal arrangement of the stimulation electrodes and the position of the recording electrodes in three directions AP from −5 to 5 mm, ML from −3.8 to 2.2 mm, and DV from 0 to 5 mm relative to the bregma.

Conditioned place preference (CPP) paradigm

CPP is a behavioral experiment used to assess the rewarding effects of drugs or other stimuli on animal models. It involves training animals to associate a specific environment (such as a particular chamber or box) with the administration of a rewarding stimulus. The CPP experiments are widely used to facilitate a comprehensive understanding of the underlying processes involved in substance abuse and addiction-related behaviors47. The CPP apparatus comprises three chambers. The first two chambers are of identical dimensions (30 cm × 30 cm × 40 cm) and exhibit distinct environmental features. One chamber is characterized by black floor and walls, while the other presents white walls and a checkered floor pattern. These chambers are separated by a guillotine door. Additionally, a third chamber (28 cm × 12.5 cm × 40 cm), referred to as the “red tunnel” is connected to both the white and black chambers via two separate doors. The ANY-maze Video Tracking software (ANY-maze, version 5.14, Stoelting Co., USA) is employed to monitor and track the precise positioning of the animal within each compartment. The data is collected through a camera (480p Computer Webcam Accessories USB 2.0 Camera, China) positioned on top of the CPP. This camera serves as an integral component in the capture of relevant behavioral information, providing visual observations of the animal's activities within the experimental environment (black and white chambers). The CPP experimental protocol spans a duration of five consecutive days and encompasses three distinct phases: the preconditioning stage, the conditioning stage, and the postconditioning stage (test day). Each phase plays a crucial role in the comprehensive evaluation and characterization of CPP.

Preconditioning stage

During the preconditioning stage, each rat is introduced into the red tunnel, with the guillotine doors separating the compartments being in an open position and the animal is allowed to move freely for 15 min. The duration spent by the animal in each compartment was recorded by ANY-maze software to determine the preferred and non-preferred (exhibiting less preference) compartments. The non-preferred chamber is selected as the compartment to be paired with morphine (day 14 in Fig. 1).

Conditioning stage

The conditioning phase is a three-day period (days 15–17 in Fig. 1) and entails the closure of the guillotine doors to impose restrictions on the animal's locomotion within the chambers. Daily injections were administered using a two-step protocol, with a five-hour interval between each step. The animals in the morphine-stimulation (-sham) group received a total of three subcutaneous injections of saline and three subcutaneous injections of morphine with a dose of 7.5 mg/kg18,23,33,34,48. Note that in all five experimental groups, both TI (sham) stimulation and morphine (saline) injection are performed outside the CPP. The animal on the first day (day 15 in Fig. 1) undergoes real TI (sham) stimulation for a duration of 10 min at 8:00 a.m., prior to the administration of morphine at 8:10 a.m. Following the injection of morphine, the animals are placed within the non-preferred chamber for a duration of 30 min19. After a period of five hours, at 13:00 p.m., the animals are administered a saline injection, followed by placement within their preferred compartments for a duration of 30 min. On the second day of the experiment (referred to as day 16 in Fig. 1), the stimulation protocol consists of saline injection at 8:00 a.m., positioning in the preferred chamber for a duration of 30 min, TI (sham) stimulation at 13:00 p.m., morphine injection at 13:10 p.m. and finally positioning in the non-preferred chamber for a duration of 30 min. The procedures implemented on the third day (day 17 in Fig. 1) are identical to those conducted on the first day. The details of the conditioning stage for morphine, saline-stimulation, and saline sham groups are provided in Appendix A of the Supplementary Materials file.

Postconditioning stage

During the final stage of the proposed CPP paradigms, each animal is positioned within the red tunnel and is allowed to roam freely in each chamber for a duration of 15 min, while the ANY-maze Video Tracking software continuously monitors the behavioral state of the animal. The score of place preference in percentage for each animal is computed as follows:1 CPPscore=Tpost,npTpost,np+Tpost,p-Tpre,npTpre,np+Tpre,p×100

where in Eq. (1), Tpost,np and Tpost,p indicates the duration spent in the non-preferred and preferred chambers, respectively during the postconditioning phase (day 18 in Fig. 1), and Tpre,np and Tpre,p denote the time spent in the non-preferred and preferred chambers, during the preconditioning phase (day 14 in Fig. 1), respectively. Moreover, the distance traveled by the rats in all CPP chambers during the postconditioning phase is considered as the total distance traveled index.

The statistical analysis is performed using GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA) and SPSS 27.0 (SPSS Inc., Chicago, IL, USA). The Shapiro–Wilk and Kolmogorov–Smirnov tests are employed to verify that the data adhered to a normal distribution and that the variances across groups are approximately consistent (homogeneity of variances). In order to analyze the results of the CPP scores, a one-way ANOVA was utilized, followed by the least significant difference (LSD) post hoc test to compare the means of different groups. Moreover, in the analysis, the Effect sizes based on Cohen's guidelines are used to determine whether the observed difference between groups is statistically significant (quantitative evaluation of the strength of the differences between groups). In the current study, to interpret the results, the effect sizes correspond to Cohen's d values less than 0.2 are considered small and non-significant, medium effect sizes correspond to d values between 0.2 and 0.5, a large d is represented between 0.5 and 0.8, and very large and strong effect sizes correspond to d values greater than 0.849,50.

Experimental results

Distribution of the induced voltage during TI stimulation

In this section, the results of the mean amplitude of envelope oscillation during TI stimulation were analyzed. Figure 3 shows the variation of the mean amplitude of the envelope oscillation along the AP and ML directions for different DV coordinates. According to Fig. 3A, B, the highest amplitude of envelope oscillations is reached at AP = 3 mm and ML = −0.8 mm, respectively. Consequently, the TI stimulation is localized at this point in the frontal plane. However, the various curves in Fig. 3 demonstrate that as we get closer to the surface layers of the brain, the envelope amplitude of the induced voltage increases. This occurs due to the close proximity of the two positive stimulation electrodes on the skull.Fig. 3 The mean amplitude and standard deviation of envelope oscillation through TI stimulation for five rats. The positions of the recording electrodes were selected as in Fig. 2 and the positions of the positive stimulation electrodes are AP = 3 mm, ML1 = −2.3 mm, and ML2 = 0.7 mm.

Results of temporal interference electrical stimulation on morphine-induced CPP

In this section, the effect of TI stimulation on morphine-induced CPP is evaluated. The CPP score and the total distance traveled index are used as the performance indexes for assessing the attenuation of morphine preference. The result of the CPP score index confirms the adherence to assumptions of normality and homogeneity with the Shapiro–Wilk and Kolmogorov–Smirnov tests. Figure 4 shows the mean CPP score and the standard error of the mean (Mean ± SEM) obtained from all rats across the five experimental groups using a one-way ANOVA test and LSD post hoc test. The results revealed a statistically significant difference in the means of the groups (F (4,27) = 7.357, p < 0.001, d = 0.794). The results show that morphine injections over a three-day period induced a pronounced preference on morphine in the animals. The larger the value of the CPP score, the more preference on morphine. Furthermore, the results of the LSD test showed that no statistically significant difference was observed between the CPP score of the morphine group and the morphine-sham group (p = 0.739, d = 0.144). However, a significant difference between the CPP score of the saline-sham group and the morphine group indicated the preference on the chamber paired with morphine in the morphine group (LSD test, p < 0.01, d = 1.236). The statistical analysis demonstrated that the saline-stimulation group exhibited significant differences when compared to the morphine group (Fig. 4, p < 0.01, d = 1.528). Additionally, the results showed that 10 min TI stimulation of the brain tissue before morphine administration for three consecutive days reduced the effects of morphine preference in morphine-stimulation group compared to the morphine (Fig. 4, p < 0.001, d = 1.689) and the morphine-sham groups (Fig. 4, p < 0.001, d = 1.545). The results illustrated that there was no significant difference in CPP score between morphine-stimulation and the saline-stimulation groups (p = 0.69, d = 0.161). Despite a decrease in the mean CPP score from 4.6% in the saline-sham group to −5.351% in the morphine-stimulation group, no statistically significant difference was observed between the CPP scores of these groups (p = 0.267, d = 0.453). The statistical analysis showed that the saline stimulation group exhibited significant differences when compared to both the morphine group (Fig. 4, p < 0.01, d = 1.528) and the morphine-sham group (Fig. 4, p < 0.01, d = 1.384). A statistically significant difference was also observed between the morphine-sham group and the saline-sham group (Fig. 4, p < 0.05, d = 1.098).Fig. 4 The comparative statistical analysis of mean CPP score and the standard error of the mean (mean ± SEM) for the different experimental groups using one-way ANOVA (p < 0.001) and LSD post hoc tests. *p < 0.05, **p < 0.01, ***p < 0.001.

For analyzing the total distance traveled index, the Shapiro–Wilk and Kolmogorov–Smirnov tests confirmed the data normality and homogeneity assumption within the experimental groups. Figure 5 shows the mean total distance traveled and standard error of the mean for all rats across the five experimental groups using a one-way ANOVA test and LSD post-hoc analysis. The findings showed that there was a statistically significant difference in the means of all the groups (F (4,27) = 3.164, p < 0.05, d = 0.62). The results of the LSD test showed a significant difference between the total distance traveled index of the morphine group and the saline-sham group (p < 0.05, d = 1.223). The statistical analysis demonstrated that there was a statistically significant difference between the morphine-sham group and the saline-sham group (Fig. 5, p < 0.05, d = 1.34). Furthermore, the morphine-stimulation group exhibited significant differences when compared to the saline-sham group (Fig. 5, p < 0.01, d = 1.5).Fig. 5 The statistical analysis of mean total distance traveled and the standard error of the mean for the different experimental groups using one-way ANOVA and LSD post hoc tests. Morphine-stimulation and saline-sham groups (p < 0.01), morphine-sham and saline-sham groups (p < 0.05), and morphine and saline-sham groups (p < 0.05). *p < 0.05, **p < 0.01.

Discussion and conclusion

In this paper, we investigated the effect of TI stimulation of the brain tissue of rats on morphine-induced CPP. In the literature 47, several indexes including dosage, duration of stimulation, and other variables might influence the attenuating behavioral responses associated with morphine consumption. In the current study, two indexes, i.e., the total distance traveled and the CPP score were evaluated. In the context of CPP score, the findings of this study revealed that TI stimulation over a period of three consecutive days resulted in a significant reduction of CPP score in morphine preference within the morphine-stimulation group in comparison with the morphine and morphine-sham groups (p < 0.001). While the exact mechanism underlying the effect of TI stimulation remains unknown, the results of the proposed TI stimulation in reducing morphine preference are consistent with previous studies investigating DBS targeting PL18, BLA22, mPFC51, lateral hypothalamic area52, and orbitofrontal cortex33 in the context of morphine-induced CPP. In the context of the total distance traveled index, the results of Fig. 5 indicated that there was no significant difference between the saline-stimulation and saline-sham groups. Consequently, the stimulation does not influence the distance traveled index. Additionally, the results showed that there was no significant difference between the morphine, morphine-sham, and morphine-stimulation groups. This finding aligns with the previous research presented in19 which demonstrated no significant difference in locomotion activity during the post-conditioning phase among these three groups through the DBS of VTA. However, there was a statistically significant difference between the saline-sham group and these three groups with morphine injection. The effects of morphine on locomotor activity arise from a complex interplay of neurochemical, molecular, and behavioral mechanisms. Morphine binds to mu-opioid receptors, influencing various neurotransmitter systems, and can either stimulate or inhibit locomotor activity based on the dose and individual differences4,53. The previous study54 has demonstrated that the low doses of morphine (1.9, 3.8, 7.5 mg/kg) decrease locomotion activity in the morphine group compared with the saline group which is consistent with the findings of the current study (morphine dose, 7.5 mg/kg) in decreasing the locomotion activity. It is worth noting that although the morphine-sham and morphine-stimulation groups do not have a significant difference in terms of total distance traveled, they differ significantly in their CPP score. Therefore, the difference in CPP score between the two groups is not mediated by the difference in locomotor activities. Similar results have been observed between morphine and morphine-stimulation groups.

The mesocorticolimbic dopamine system plays a crucial role in the rewarding, reinforcing, and motivational aspects of morphine addiction. The nuclei VTA, NAc, mPFC, BLA, and LHb collectively contribute to the complex functioning of the mesocorticolimbic dopamine system in addiction. The VTA is a key origin of dopaminergic neurons in the system and sends projections to various brain regions, including the NAc, mPFC, and amygdala. The NAc is a major target of dopaminergic projections from the VTA and plays a central role in reward processing and reinforcement. The mPFC receives dopaminergic inputs from the VTA and is involved in the regulation of dopamine release in the NAc. The BLA is interconnected with the mesocorticolimbic pathway to modulate dopamine release in the NAc by influencing the activity of dopaminergic neurons in the VTA. The LHb is a diminutive neural structure that receives afferent inputs from the VTA and modulates reward processing through the regulation of dopaminergic neuronal activity. In the literature, invasive electrical stimulation of different nuclei within the mesocorticolimbic system, including the VTA19, NAc21, mPFC18,33,35, BLA22, and LHb23, can be considered as a therapeutic approach for morphine addiction in animal models. In the current study, we examined the potential of TI stimulation to attenuate the reinforcing effects of morphine in rats. Our results showed that non-invasive TI stimulation of the brain tissue could significantly reduce the effects of morphine preference in the morphine-stimulation group compared to the morphine group (Fig. 4, p < 0.001). In this work, only two pairs of electrodes were used for the stimulation of the brain tissue to attenuate the effect of morphine preference. The experimental tests in Fig. 3 showed that the focus of the applied TI stimulation in the frontal plane was at AP = 3 mm and ML = −0.8 mm. It was shown that the more dorsal regions including the cingulate and motor cortex experienced higher interfering signal amplitudes. Moreover, since the stimulation is periodically on and off, the onset response effect55 can activate dorsal cortical tissue and axons innervating cheek muscle. However, the statistical tests for considering the effect of stimulation on total distance traveled (Fig. 5) do not support the hypothesis that the stimulation significantly affects the motor activity (p = 0.25 for saline-sham and saline stimulation groups and p = 0.74 for morphine-sham and morphine-stimulation groups).

Our previous studies demonstrated that electrical stimulation of the mPFC18, VTA19, and BLA22, with different current intensities and frequency of 25 Hz resulted in a reduction of morphine-induced CPP in rats. In18, it was observed that electrical stimulation of the mPFC with an intensity of 100 µA inhibited morphine-induced CPP while the results of19 showed that 5 mg/kg morphine-induced CPP was suppressed by 150 μA electrical stimulation of VTA. A similar investigation has been addressed in33 to evaluate the effect of low- and high-frequency DBS of OFC on morphine-induced CPP. These findings suggest that DBS at specific stimulation parameters can modulate the rewarding effects of morphine and provide valuable insights into the effects of DBS stimulation on morphine-induced CPP. In the current study, the stimulation parameters were set at the intensity of I1 = I2 = 0.5 mA, carrier frequency of 2 kHz, and frequency difference of 25 Hz. Another parameter in the TI stimulation is the positioning of the electrodes. Grossman et al.36 evaluated the distribution of TI stimulation in a tissue phantom consisting of a plastic cylinder filled with saline. They discovered that by strategically controlling the position of the electrodes, the peak of the interference envelope could be achieved at specific locations. Their findings indicated that when the electrodes were positioned in a trapezoidal geometry, the focal stimulation area was located between the two electrodes and near the surface of the cortex. By increasing the distance between the positive electrodes and transforming the arrangement into a rectangular geometry, the position of the peak envelope modulation could be directed deeper into the tissue. It is worth noting that, our experimental results in Fig. S1 indicated that increasing the distance between the two positive stimulation electrodes resulted in a reduction of the amplitude of the envelope oscillation. Consequently, in this study, the distance between the two positive electrodes was chosen to be as close to each other as possible (according to the size of the screws on the skull). However, this is at the cost of losing localized stimulation in deep regions along the DV axis. For more information, see the Appendix B of the Supplementary Materials file.

One limitation of the current study is that the effect of TI stimulation on reduction of the morphine preference is evaluated only with behavioral CPP-related indexes and the neuronal activity of neurons was not recorded. Further research should focus on the efficacy of TI stimulation in the context of mitigating morphine preference by varying the stimulation parameters and evaluating the neural activity of the target region. Such investigations would contribute to a deeper understanding of how TI stimulation can be optimized to achieve desired outcomes in reducing morphine preference and enhance the capability of TI stimulation as a prospective therapeutic modality for attenuating drug-seeking preference.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71841-1.

Acknowledgements

This research was supported by the Cognitive Sciences and Technologies Council under Grant 11927. The authors would like to thank the anonymous reviewers for their careful reading of the manuscript and their many insightful comments and suggestions. We truly appreciate their time, effort, and dedication in reviewing our work and providing invaluable suggestions that have greatly enhanced the manuscript.

Author contributions

A.A., E.R., and H.A. administrated the project and designed the method. Z.M. and Z.J. performed the experimental setup and prepared the results. Z.M. prepared Figs. 1, 2, 3, 4 and 5 and S1. Z.M., E.R., and A.A. prepared the primary version of the manuscript, discussed the results, and reviewed the manuscript.

Data availability

All data analyzed during this study are included in the Appendix C of the Supplementary Materials file.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
==== Refs
References

1. Koob GF Volkow ND Neurobiology of addiction: A neurocircuitry analysis Lancet Psychiatry 2016 3 760 773 10.1016/S2215-0366(16)00104-8 27475769
Koob, G. F. & Volkow, N. D. Neurobiology of addiction: A neurocircuitry analysis. Lancet Psychiatry 3, 760–773 (2016).27475769 10.1016/S2215-0366(16)00104-8
2. Pergolizzi JV Raffa RB Rosenblatt MH Opioid withdrawal symptoms, a consequence of chronic opioid use and opioid use disorder: Current understanding and approaches to management J. Clin. Pharm. Ther. 2020 45 903 10.1111/jcpt.13114
Pergolizzi, J. V., Raffa, R. B. & Rosenblatt, M. H. Opioid withdrawal symptoms, a consequence of chronic opioid use and opioid use disorder: Current understanding and approaches to management. J. Clin. Pharm. Ther. 45, 903. 10.1111/jcpt.13114 (2020).10.1111/jcpt.13114
3. Badshah I Anwar M Murtaza B Khan MI Molecular mechanisms of morphine tolerance and dependence; Novel insights and future perspectives Mol. Cell. Biochem. 2023 10.1007/s11010-023-04810-3 37470850
Badshah, I., Anwar, M., Murtaza, B. & Khan, M. I. Molecular mechanisms of morphine tolerance and dependence; Novel insights and future perspectives. Mol. Cell. Biochem.10.1007/s11010-023-04810-3 (2023).37470850 10.1007/s11010-023-04810-3
4. Listos J The mechanisms involved in morphine addiction: An overview Int. J. Mol. Sci. 2019 20 122 10.3390/ijms20174302
Listos, J. et al. The mechanisms involved in morphine addiction: An overview. Int. J. Mol. Sci. 20, 122 (2019).10.3390/ijms20174302
5. Doyle MA Mazei-Robison MS Opioid-induced molecular and cellular plasticity of ventral tegmental area dopamine neurons Cold Spring Harb. Perspect. Med. 2021 11 1 15 10.1101/cshperspect.a039362
Doyle, M. A. & Mazei-Robison, M. S. Opioid-induced molecular and cellular plasticity of ventral tegmental area dopamine neurons. Cold Spring Harb. Perspect. Med. 11, 1–15 (2021).10.1101/cshperspect.a039362
6. Tzschentke TM The medial prefrontal cortex as a part of the brain reward system Amino Acids 2000 19 211 219 10.1007/s007260070051 11026491
Tzschentke, T. M. The medial prefrontal cortex as a part of the brain reward system. Amino Acids 19, 211–219. 10.1007/s007260070051 (2000).11026491 10.1007/s007260070051
7. Jiang WN Corydaline and l-tetrahydropalmatine attenuate morphine-induced conditioned place preference and the changes in dopamine D2 and GluA1 AMPA receptor expression in rats Eur. J. Pharmacol. 2020 884 32 10.1016/j.ejphar.2020.173397
Jiang, W. N. et al. Corydaline and l-tetrahydropalmatine attenuate morphine-induced conditioned place preference and the changes in dopamine D2 and GluA1 AMPA receptor expression in rats. Eur. J. Pharmacol. 884, 32 (2020).10.1016/j.ejphar.2020.173397
8. Frankowska M Treatment with dopamine β-hydroxylase (DBH) inhibitors prevents morphine use and relapse-like behavior in rats Pharmacol. Rep. 2021 73 1694 1711 10.1007/s43440-021-00307-2 34236605
Frankowska, M. et al. Treatment with dopamine β-hydroxylase (DBH) inhibitors prevents morphine use and relapse-like behavior in rats. Pharmacol. Rep. 73, 1694–1711 (2021).34236605 10.1007/s43440-021-00307-2
9. Hassan R Mitragynine attenuates morphine withdrawal effects in rats—A comparison with methadone and buprenorphine Front. Psychiatry 2020 11 32 10.3389/fpsyt.2020.00411 32116847
Hassan, R. et al. Mitragynine attenuates morphine withdrawal effects in rats—A comparison with methadone and buprenorphine. Front. Psychiatry 11, 32 (2020).32116847 10.3389/fpsyt.2020.00411
10. Moffa AH Efficacy and acceptability of transcranial direct current stimulation (tDCS) for major depressive disorder: An individual patient data meta-analysis Prog. Neuro-Psychopharmacol. Biol. Psychiatry 2020 99 29 10.1016/j.pnpbp.2019.109836
Moffa, A. H. et al. Efficacy and acceptability of transcranial direct current stimulation (tDCS) for major depressive disorder: An individual patient data meta-analysis. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 99, 29 (2020).10.1016/j.pnpbp.2019.109836
11. Zhang Y Deep brain stimulation in the lateral habenula reverses local neuronal hyperactivity and ameliorates depression-like behaviors in rats Neurobiol. Dis. 2023 180 106069 10.1016/j.nbd.2023.106069 36893902
Zhang, Y. et al. Deep brain stimulation in the lateral habenula reverses local neuronal hyperactivity and ameliorates depression-like behaviors in rats. Neurobiol. Dis. 180, 106069 (2023).36893902 10.1016/j.nbd.2023.106069
12. Sharma G Preclinical validation of electrodes for single anodal transcranial direct current stimulation on rat model with chronic stress-induced depression IEEE Sens. J. 2023 23 12133 12145 10.1109/JSEN.2023.3266235
Sharma, G. et al. Preclinical validation of electrodes for single anodal transcranial direct current stimulation on rat model with chronic stress-induced depression. IEEE Sens. J. 23, 12133–12145 (2023).10.1109/JSEN.2023.3266235
13. Luo Y Deep brain stimulation for Alzheimer’s disease: Stimulation parameters and potential mechanisms of action Front. Aging Neurosci. 2021 13 34 10.3389/fnagi.2021.619543
Luo, Y. et al. Deep brain stimulation for Alzheimer’s disease: Stimulation parameters and potential mechanisms of action. Front. Aging Neurosci. 13, 34 (2021).10.3389/fnagi.2021.619543
14. Liu H Deep brain stimulation of the nucleus basalis of Meynert in an experimental rat model of dementia: Stimulation parameters and mechanisms Neurobiol. Dis. 2022 171 105797 10.1016/j.nbd.2022.105797 35738477
Liu, H. et al. Deep brain stimulation of the nucleus basalis of Meynert in an experimental rat model of dementia: Stimulation parameters and mechanisms. Neurobiol. Dis. 171, 105797 (2022).35738477 10.1016/j.nbd.2022.105797
15. Lin Z Zhang C Li D Sun B Lateralized effects of deep brain stimulation in Parkinson’s disease: evidence and controversies npj Park. Dis. 2021 7 64 10.1038/s41531-021-00209-3
Lin, Z., Zhang, C., Li, D. & Sun, B. Lateralized effects of deep brain stimulation in Parkinson’s disease: evidence and controversies. npj Park. Dis. 7, 64 (2021).10.1038/s41531-021-00209-3
16. Helf C Subthalamic nucleus deep brain stimulation induces nigrostriatal dopaminergic plasticity in a stable rat model of Parkinson’s disease Neuroreport 2023 34 506 511 10.1097/WNR.0000000000001917 37270842
Helf, C. et al. Subthalamic nucleus deep brain stimulation induces nigrostriatal dopaminergic plasticity in a stable rat model of Parkinson’s disease. Neuroreport 34, 506–511 (2023).37270842 10.1097/WNR.0000000000001917
17. Xie J Deep brain stimulation on the external segment of the globus pallidus improves the electrical activity of internal segment of globus pallidus in a rat model of Parkinson’s disease Brain Res. 2022 1797 148115 10.1016/j.brainres.2022.148115 36202223
Xie, J. et al. Deep brain stimulation on the external segment of the globus pallidus improves the electrical activity of internal segment of globus pallidus in a rat model of Parkinson’s disease. Brain Res. 1797, 148115 (2022).36202223 10.1016/j.brainres.2022.148115
18. Kargari AA Ramshini E Alaei HA Sedighi M Oryan S Different current intensities electrical stimulation of prelimbic cortex of mPFC produces different effects on morphine-induced conditioned place preference in rats Behav. Brain Res. 2012 231 187 192 10.1016/j.bbr.2012.03.016 22465169
Kargari, A. A., Ramshini, E., Alaei, H. A., Sedighi, M. & Oryan, S. Different current intensities electrical stimulation of prelimbic cortex of mPFC produces different effects on morphine-induced conditioned place preference in rats. Behav. Brain Res. 231, 187–192 (2012).22465169 10.1016/j.bbr.2012.03.016
19. Alaei H Ghobadi Pour M Stimulation and transient inactivation of ventral tegmental area modify reinstatement of acquisition phase of morphine-induced conditioned place preference in male rats Brain Res. Bull. 2021 176 130 141 10.1016/j.brainresbull.2021.08.014 34480979
Alaei, H. & Ghobadi Pour, M. Stimulation and transient inactivation of ventral tegmental area modify reinstatement of acquisition phase of morphine-induced conditioned place preference in male rats. Brain Res. Bull. 176, 130–141 (2021).34480979 10.1016/j.brainresbull.2021.08.014
20. Kallupi M Deep brain stimulation of the nucleus accumbens shell attenuates cocaine withdrawal but increases cocaine self-administration, cocaine-induced locomotor activity, and GluR1/GluA1 in the central nucleus of the amygdala in male cocaine-dependent rats Brain Stimul. 2022 15 13 22 10.1016/j.brs.2021.11.003 34742997
Kallupi, M. et al. Deep brain stimulation of the nucleus accumbens shell attenuates cocaine withdrawal but increases cocaine self-administration, cocaine-induced locomotor activity, and GluR1/GluA1 in the central nucleus of the amygdala in male cocaine-dependent rats. Brain Stimul. 15, 13–22 (2022).34742997 10.1016/j.brs.2021.11.003
21. Radahmadi M Ramshini E Hosseini N Karimi S Alaei H Effect of electrical stimulation of nucleus accumbens with low, median and high currents intensities on conditioned place preference induced by morphine in rats Adv. Biomed. Res. 2014 3 14 10.4103/2277-9175.124643 24600598
Radahmadi, M., Ramshini, E., Hosseini, N., Karimi, S. & Alaei, H. Effect of electrical stimulation of nucleus accumbens with low, median and high currents intensities on conditioned place preference induced by morphine in rats. Adv. Biomed. Res. 3, 14 (2014).24600598 10.4103/2277-9175.124643
22. Rezaei Z Alaei H Reisi P Effects of electrical stimulation and temporary inactivation of basolateral amygdala on morphine-induced conditioned place preference in rats Neurosci. Lett. 2022 774 136519 10.1016/j.neulet.2022.136519 35151827
Rezaei, Z., Alaei, H. & Reisi, P. Effects of electrical stimulation and temporary inactivation of basolateral amygdala on morphine-induced conditioned place preference in rats. Neurosci. Lett. 774, 136519 (2022).35151827 10.1016/j.neulet.2022.136519
23. Amohashemi E Reisi P Alaei H Lateral habenula electrical stimulation with different intensities in combination with GABAB receptor antagonist reduces acquisition and expression phases of morphine-induced CPP Neurosci. Lett. 2021 759 135996 10.1016/j.neulet.2021.135996 34062194
Amohashemi, E., Reisi, P. & Alaei, H. Lateral habenula electrical stimulation with different intensities in combination with GABAB receptor antagonist reduces acquisition and expression phases of morphine-induced CPP. Neurosci. Lett. 759, 135996 (2021).34062194 10.1016/j.neulet.2021.135996
24. Jokar Z Khatamsaz S Alaei H Shariati M The electrical stimulation of the central nucleus of the amygdala in combination with dopamine receptor antagonist reduces the acquisition phase of morphine-induced conditioned place preference in male rat Res. Pharm. Sci. 2023 18 430 438 10.4103/1735-5362.378089 37614617
Jokar, Z., Khatamsaz, S., Alaei, H. & Shariati, M. The electrical stimulation of the central nucleus of the amygdala in combination with dopamine receptor antagonist reduces the acquisition phase of morphine-induced conditioned place preference in male rat. Res. Pharm. Sci. 18, 430–438 (2023).37614617 10.4103/1735-5362.378089
25. Jokara Z Khatamsaz S Alaei HA Shariati M Effect of electrical stimulation of central nucleus of the amygdala on morphine conditioned place preference in male rats Iran. J. Basic Med. Sci. 2022 25 604 610 35911639
Jokara, Z., Khatamsaz, S., Alaei, H. A. & Shariati, M. Effect of electrical stimulation of central nucleus of the amygdala on morphine conditioned place preference in male rats. Iran. J. Basic Med. Sci. 25, 604–610 (2022).35911639
26. Reed T CohenKadosh R Transcranial electrical stimulation (tES) mechanisms and its effects on cortical excitability and connectivity J. Inherit. Metab. Dis. 2018 41 1123 1130 10.1007/s10545-018-0181-4 30006770
Reed, T. & CohenKadosh, R. Transcranial electrical stimulation (tES) mechanisms and its effects on cortical excitability and connectivity. J. Inherit. Metab. Dis. 41, 1123–1130 (2018).30006770 10.1007/s10545-018-0181-4
27. Regner GG Preclinical to clinical translation of studies of transcranial direct-current stimulation in the treatment of epilepsy: A systematic review Front. Neurosci. 2018 12 189 10.3389/fnins.2018.00189 29623027
Regner, G. G. et al. Preclinical to clinical translation of studies of transcranial direct-current stimulation in the treatment of epilepsy: A systematic review. Front. Neurosci. 12, 189 (2018).29623027 10.3389/fnins.2018.00189
28. Pedron S Monnin J Haffen E Sechter D Van Waes V Repeated transcranial direct current stimulation prevents abnormal behaviors associated with abstinence from chronic nicotine consumption Neuropsychopharmacology 2014 39 981 988 10.1038/npp.2013.298 24154668
Pedron, S., Monnin, J., Haffen, E., Sechter, D. & Van Waes, V. Repeated transcranial direct current stimulation prevents abnormal behaviors associated with abstinence from chronic nicotine consumption. Neuropsychopharmacology 39, 981–988 (2014).24154668 10.1038/npp.2013.298
29. Pedron S Transcranial direct current stimulation produces long-lasting attenuation of cocaine-induced behavioral responses and gene regulation in corticostriatal circuits Addict. Biol. 2017 22 1267 1278 10.1111/adb.12415 27265728
Pedron, S. et al. Transcranial direct current stimulation produces long-lasting attenuation of cocaine-induced behavioral responses and gene regulation in corticostriatal circuits. Addict. Biol. 22, 1267–1278 (2017).27265728 10.1111/adb.12415
30. Pedron S Transcranial direct current stimulation (tDCS) reduces motivation to drink ethanol and reacquisition of ethanol self-administration in female mice Sci. Rep. 2022 12 198 10.1038/s41598-021-03940-2 34997004
Pedron, S. et al. Transcranial direct current stimulation (tDCS) reduces motivation to drink ethanol and reacquisition of ethanol self-administration in female mice. Sci. Rep. 12, 198 (2022).34997004 10.1038/s41598-021-03940-2
31. Santos DS Bimodal transcranial direct current stimulation reduces alcohol consumption and induces long-term neurochemical changes in rats with neuropathic pain Neurosci. Lett. 2021 759 1360 10.1016/j.neulet.2021.136014
Santos, D. S. et al. Bimodal transcranial direct current stimulation reduces alcohol consumption and induces long-term neurochemical changes in rats with neuropathic pain. Neurosci. Lett. 759, 1360 (2021).10.1016/j.neulet.2021.136014
32. Krauss JK Technology of deep brain stimulation: Current status and future directions Nat. Rev. Neurol. 2021 17 75 87 10.1038/s41582-020-00426-z 33244188
Krauss, J. K. et al. Technology of deep brain stimulation: Current status and future directions. Nat. Rev. Neurol. 17, 75–87 (2021).33244188 10.1038/s41582-020-00426-z
33. Fakhrieh-Asl G Sadr SS Karimian SM Riahi E Deep brain stimulation of the orbitofrontal cortex prevents the development and reinstatement of morphine place preference Addict. Biol. 2020 25 12780 10.1111/adb.12780
Fakhrieh-Asl, G., Sadr, S. S., Karimian, S. M. & Riahi, E. Deep brain stimulation of the orbitofrontal cortex prevents the development and reinstatement of morphine place preference. Addict. Biol. 25, 12780 (2020).10.1111/adb.12780
34. Yang C The effect of high-frequency electrical stimulation of bilateral nucleus accumbens on the behavior of morphine-induced conditioned place preference rats at extinction and reinstatement phases Evid.-Based Complement. Altern. Med. 2020 2020 8232809 10.1155/2020/8232809
Yang, C. et al. The effect of high-frequency electrical stimulation of bilateral nucleus accumbens on the behavior of morphine-induced conditioned place preference rats at extinction and reinstatement phases. Evid.-Based Complement. Altern. Med. 2020, 8232809 (2020).10.1155/2020/8232809
35. Keramatian A Alaei H Eidi A Radahmadi M Electrical stimulation mPFC affects morphine addiction by changing glutamate concentration in the ventral tegmental area Metab. Brain Dis. 2019 34 1171 1180 10.1007/s11011-019-00426-z 31115726
Keramatian, A., Alaei, H., Eidi, A. & Radahmadi, M. Electrical stimulation mPFC affects morphine addiction by changing glutamate concentration in the ventral tegmental area. Metab. Brain Dis. 34, 1171–1180 (2019).31115726 10.1007/s11011-019-00426-z
36. Grossman N Noninvasive deep brain stimulation via temporally interfering electric fields Cell 2017 169 1029 1041.e16 10.1016/j.cell.2017.05.024 28575667
Grossman, N. et al. Noninvasive deep brain stimulation via temporally interfering electric fields. Cell 169, 1029-1041.e16 (2017).28575667 10.1016/j.cell.2017.05.024
37. Guo W A novel non-invasive brain stimulation technique: “Temporally interfering electrical stimulation” Front. Neurosci. 2023 10.3389/fnins.2023.1092539 38577033
Guo, W. et al. A novel non-invasive brain stimulation technique: “Temporally interfering electrical stimulation”. Front. Neurosci.10.3389/fnins.2023.1092539 (2023).38577033 10.3389/fnins.2023.1092539
38. Zhang Z Designing and pilot testing a novel transcranial temporal interference stimulation device for neuromodulation IEEE Trans. Neural Syst. Rehabil. Eng. 2022 30 1483 1493 10.1109/TNSRE.2022.3179537 35657852
Zhang, Z. et al. Designing and pilot testing a novel transcranial temporal interference stimulation device for neuromodulation. IEEE Trans. Neural Syst. Rehabil. Eng. 30, 1483–1493 (2022).35657852 10.1109/TNSRE.2022.3179537
39. Violante I Non-invasive temporal interference electrical stimulation of the human hippocampus Brain Stimul. 2023 16 408 10.1016/j.brs.2023.01.833
Violante, I. et al. Non-invasive temporal interference electrical stimulation of the human hippocampus. Brain Stimul. 16, 408 (2023).10.1016/j.brs.2023.01.833
40. Alania K Rhodes E Suhayl IB Violante I Grossman N Investigating the direct effect of temporal interference (TI) stimulation on cortical activity with simultaneous EEG recording in humans Brain Stimul. 2023 16 343 10.1016/j.brs.2023.01.655
Alania, K., Rhodes, E., Suhayl, I. B., Violante, I. & Grossman, N. Investigating the direct effect of temporal interference (TI) stimulation on cortical activity with simultaneous EEG recording in humans. Brain Stimul. 16, 343 (2023).10.1016/j.brs.2023.01.655
41. Mirzakhalili E Barra B Capogrosso M Lempka SF Biophysics of temporal interference stimulation Cell Syst. 2020 11 557 572.e5 10.1016/j.cels.2020.10.004 33157010
Mirzakhalili, E., Barra, B., Capogrosso, M. & Lempka, S. F. Biophysics of temporal interference stimulation. Cell Syst. 11, 557-572.e5 (2020).33157010 10.1016/j.cels.2020.10.004
42. Karimi F Attarpour A Amirfattahi R Nezhad AZ Computational analysis of non-invasive deep brain stimulation based on interfering electric fields Phys. Med. Biol. 2019 64 122 10.1088/1361-6560/ab5229
Karimi, F., Attarpour, A., Amirfattahi, R. & Nezhad, A. Z. Computational analysis of non-invasive deep brain stimulation based on interfering electric fields. Phys. Med. Biol. 64, 122 (2019).10.1088/1361-6560/ab5229
43. Mojiri Z Akhavan A Rouhani E Non-invasive deep electrical stimulation of the primary motor cortex of the rat by temporal interference method Iran. J. Biomed. Eng. 2022 16 201 210
Mojiri, Z., Akhavan, A. & Rouhani, E. Non-invasive deep electrical stimulation of the primary motor cortex of the rat by temporal interference method. Iran. J. Biomed. Eng. 16, 201–210 (2022).
44. Kang H Sample size determination and power analysis using the G*Power software J. Educ. Eval. Health Prof. 2021 18 23 10.3352/jeehp.2021.18.17 34551510
Kang, H. Sample size determination and power analysis using the G*Power software. J. Educ. Eval. Health Prof. 18, 23 (2021).34551510 10.3352/jeehp.2021.18.17
45. Ghorbani R Nahvi M Analysis of performance of Howland AC current source for electrical impedance spectro-tomography Sens. Imaging 2019 20 1 10.1007/s11220-019-0251-1
Ghorbani, R. & Nahvi, M. Analysis of performance of Howland AC current source for electrical impedance spectro-tomography. Sens. Imaging 20, 1 (2019).10.1007/s11220-019-0251-1
46. Paxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates: Hard Cover Edition (Elsevier, 2006).
47. Buccafusco JJ Methods of behavior analysis in neuroscience Methods Behav. Anal. Neurosci. 2000 10.1201/9781420041811
Buccafusco, J. J. Methods of behavior analysis in neuroscience. Methods Behav. Anal. Neurosci.10.1201/9781420041811 (2000).10.1201/9781420041811
48. Chang H Continuous high frequency deep brain stimulation of the rat anterior insula attenuates the relapse post withdrawal and strengthens the extinction of morphine seeking Front. Psychiatry 2020 11 223 10.3389/fpsyt.2020.577155 32265763
Chang, H. et al. Continuous high frequency deep brain stimulation of the rat anterior insula attenuates the relapse post withdrawal and strengthens the extinction of morphine seeking. Front. Psychiatry 11, 223 (2020).32265763 10.3389/fpsyt.2020.577155
49. J. C. Statistical Power Analysis for the Behavioural Science. Statistical Power Analysis for the Behavioral Sciences. 2nd Ed. 110–116 (1988).
50. Schäfer T Schwarz MA The meaningfulness of effect sizes in psychological research: Differences between sub-disciplines and the impact of potential biases Front. Psychol. 2019 10 1 13 10.3389/fpsyg.2019.00813 30713512
Schäfer, T. & Schwarz, M. A. The meaningfulness of effect sizes in psychological research: Differences between sub-disciplines and the impact of potential biases. Front. Psychol. 10, 1–13 (2019).30713512 10.3389/fpsyg.2019.00813
51. Esfahlani MA Arabmoazzen S Badini F Mirshekar MA Arezoomandan R The effects of deep brain stimulation in medial prefrontal cortex on morphine dependency and electrical recording of the nucleus accumbens Int. J. High Risk Behav. Addict. 2023 12 85
Esfahlani, M. A., Arabmoazzen, S., Badini, F., Mirshekar, M. A. & Arezoomandan, R. The effects of deep brain stimulation in medial prefrontal cortex on morphine dependency and electrical recording of the nucleus accumbens. Int. J. High Risk Behav. Addict. 12, 85 (2023).
52. Fattahi M Ashabi G Karimian SM Riahi E Preventing morphine reinforcement with high-frequency deep brain stimulation of the lateral hypothalamic area Addict. Biol. 2019 24 685 695 10.1111/adb.12634 29737638
Fattahi, M., Ashabi, G., Karimian, S. M. & Riahi, E. Preventing morphine reinforcement with high-frequency deep brain stimulation of the lateral hypothalamic area. Addict. Biol. 24, 685–695 (2019).29737638 10.1111/adb.12634
53. van Steenbergen H Eikemo M Leknes S The role of the opioid system in decision making and cognitive control: A review Cogn. Affect. Behav. Neurosci. 2019 19 3
van Steenbergen, H., Eikemo, M. & Leknes, S. The role of the opioid system in decision making and cognitive control: A review. Cogn. Affect. Behav. Neurosci. 19, 3 (2019).
54. Vindenes V Handal M Ripel Å Boix F Mørland J Conditioned place preference induced by morphine and morphine-6-glucuronide in mice Pharmacol. Biochem. Behav. 2006 85 292 10.1016/j.pbb.2006.08.010 17011617
Vindenes, V., Handal, M., Ripel, Å., Boix, F. & Mørland, J. Conditioned place preference induced by morphine and morphine-6-glucuronide in mice. Pharmacol. Biochem. Behav. 85, 292 (2006).17011617 10.1016/j.pbb.2006.08.010
55. Peña E Pelot NA Grill WM Spatiotemporal parameters for energy efficient kilohertz-frequency nerve block with low onset response J. Neuroeng. Rehabil. 2023 20 72 10.1186/s12984-023-01195-8 37271812
Peña, E., Pelot, N. A. & Grill, W. M. Spatiotemporal parameters for energy efficient kilohertz-frequency nerve block with low onset response. J. Neuroeng. Rehabil. 20, 72 (2023).37271812 10.1186/s12984-023-01195-8
