The nucleus accumbens (NAc), which forms the main part of the ventral striatum, belongs to the basal ganglia of the brain. It is located where the head of the caudate and the anterior portion of the putamen meet just rostral to the anterior commissure. NAc plays an important role in motivation and emotional processes, and it is involved in many neurologic psychiatric disorders, such as Alzheimer’s disease, Parkinson’s disease, depression, schizophrenia, obsessive–compulsive disorder (OCD), anxiety disorders, and addiction 1. In recent years, NAc has become a novel target in functional neurosurgery, and deep brain stimulation (DBS) of NAc has been considered as a potential treatment for many psychiatric disorders, such as major depression, addiction, and OCD 2.
Although DBS of NAc has shown a promising therapeutic effect in some otherwise intractable diseases, many issues related to it need to be addressed. A proportion of patients did not benefit from the treatment 3–5, as suitable indications and predictors for good outcomes are still unknown. The optimal parameters of NAc DBS for treating certain psychiatric disorders are still unclear and require to be explored. Furthermore, the mechanism of NAc DBS is far from clear and calls for further studies. Nonhuman primate models are essential and irreplaceable in this kind of neuroscience research. The model of DBS for Parkinson’s disease helps to clarify the mechanism underlying improvement in the symptoms of parkinsonian disease by high-frequency electrical stimulation of the subthalamic nucleus 6–8. Another study found that high-frequency DBS of the ventromedial hypothalamus induced a moderate increase in food consumption in monkeys 9. Most of the studies involved stereotactic targeting and manipulation of the subthalamic nucleus. However, DBS of NAc in nonhuman primates has not been described to the best of our knowledge. Here, we report how to identify NAc in nonhuman primate stereotactic surgery and implant the DBS to stimulate it.
Materials and methods
Experiments were carried out on four 5-year-old male rhesus monkeys weighing 5–6.5 kg. They were housed in individual cages for the duration of the study. Animal care was provided in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1996), and all procedures were performed with the prior approval of the Animal Research Committee, Fourth Military Medical University.
The animals’ behavioral observations were made before and after surgery, and during the DBS period, which consisted of motor features including movement of limbs, speed of movement, facial expression, posture and stability, and affective features including tameness, appetite, and changes in emotion. After adaption in the home cages for at least 6 months, these features of all animals were observed individually for 7 days before surgery as the baseline and subsequently for 4 weeks after surgery. Then the DBS was programmed and observations continued for 7 days. Meanwhile, the spontaneous locomotor activity of the animals was recorded using an infrared beam motion detector in the cage between 9:00 a.m. and 17:00 p.m. on consecutive days. Postsurgery assessment was carried out in the fourth week. Activity counts per hour were calculated for each stage and compared.
Stereotactic targeting of the nucleus accumbens
Traditionally, identification of the anatomical target location in nonhuman primate stereotactic surgery is based on coordinates of the standard brain atlases 10. This approach is prone to error because the brain size, shape, and location of subcortical structures can vary between animals 8. Recent studies have resorted to the integration of brain atlases and MRI in stereotactic surgery of large animals 11–13, which is already used in clinical practice. Here, we also targeted NAc under the guidance of MRI with the clinical Leksell stereotactic system (Elekta AB, Stockholm, Sweden).
The head of monkeys is much smaller than that of human beings and the forehead is narrow. Therefore, the clinical Leksell Coordinate Frame (Elekta AB) could not be affixed to the monkeys’ head firmly. We developed an annular adapter made of Plexiglas, which has screw holes and can be fixed to a monkey’s head with screws pressing on temporal and occipital bone (Fig. 1). It is like enlarging the head circumference of monkey, which makes the Leksell frame suitable for use.
Each animal was initially anesthetized using ketamine (10 mg/kg, intramuscularly), followed by the administration of atropine sulfate (0.04 mg/kg, intramuscularly), maintenance doses of anesthetics (propofol 0.2 mg/kg/min, through the saphenous vein), and intubation. The frame adapter was fixed to the monkey’s head, followed by affixing the Leksell frame to the adaptor (Fig. 1). A Leksell localizer (six axial fiducial markers) was attached to the frame before the animal was transported to the MRI scanner (1.0 T; Philips Medical Systems, Best, the Netherlands), where it was subjected to preoperative MRI to obtain contiguous T1-weighted and T2-weighted axial slices (1.5 mm slice thickness) of a volume, which included the fiducials and the brain. For the T1 imaging, the parameters were as follows: TR 329 ms, TE 15 m, field of view 28×28 cm, and matrix size 256×256. For the T2 imaging, the parameters were as follows: TR 2246 ms, TE 110 ms, field of view 28×28 cm, and matrix size 256×256. MRI data were imported to the stereotactic planning software (Leksell Surgiplan; Elekta AB) and reconstructed in three dimensions.
MRI is introduced in nonhuman primate stereotactic neurosurgery for more accuracy. Although NAc has no clear identification and exact delimitation in either T1-weighted or T2-weighted MR images, it could be identified by adjacent anatomic structures. We also referred to a monkey brain atlas with combined MRI and histology images 14. NAc can be recognized as the area of continuity between the caudate nucleus and the putamen in the reconstructed coronal sections, which is beneath the internal capsule, and the gray matter nucleus between the ventromedial prefrontal cortex and anterior commissure in axial sections, which is medial to the putamen (Fig. 2). NAc is located 2–5 mm lateral to the midline, and its dimensions are 6.5×3×2 mm (anterior–posterior, medial–lateral, and superior–inferior). The target is pointed at the posterior part of the NAc because this part is more prominent.
Deep brain stimulation implantation
Following MRI, the animal was transferred to the operation room. The Leksell human stereotactic arc was attached to the frame affixed to the animal’s head for subsequent stereotactic surgical manipulations. After making a midline incision, 5 mm-diameter burr holes were drilled anterior to the coronal suture and 1 cm lateral to the midline suture on each side for the approach to the target. The four-contact electrode (Medtronic Inc., Minneapolis, Minnesota, USA) was stereotactically inserted through the guide tube and the deepest contact was placed in the target according to the Leksell stereotactic planning software (Fig. 3). The proximal part of the electrode was secured to the skull with acrylic and medical-grade silicone cements. For the first monkey, the bilateral electrodes were implanted in two stages with an interval of 1 week. Furthermore, in other animals, the electrodes were implanted in the same operation. Brain MRI was performed after the surgery to determine the accuracy of electrode position. If the electrode deviated from the target, we would adjust and implant it again. Afterwards, the electrodes were connected to the implantable pulse generator (IPG) (Kinetra; Medtronic) with the extension tunneled subcutaneously to the back of the animal. IPG was subcutaneously implanted below the scapula or the lower abdomen, allowing unrestricted and free movement of the animal. Nonabsorbable sutures were used to secure the extension leads and IPG.
Deep brain stimulation programming
Following the animal’s full recovery from surgery (4 weeks after the implantation), the IPG was programmed with different parameter settings (frequency, pulse width, and amplitude) in the monopolar mode. Contacts 0 and 4, the most ventral contacts in the left and right NAc leads, were programmed as the cathode and the IPG case as the anode. Although there are literally thousands of possible combinations of stimulator parameters, only a specific range of combinations was trialed. Varying voltages from 1 to 5 V were tried with increments of 0.5 V. Pulse width was varied from 30 to 240 μs. A range of frequencies was used from 30 to 160 Hz.
The coronal sections of MRI are more suitable for identifying NAc in stereotactic surgery, and anterior commissure could be a useful landmark. The mean and maximum error on the stereotactic system, calculated by the SurgiPlan system during import of the monkey stereotactic MRI images, were 0.4–0.6 and 0.9–1.3 mm, respectively. These values are similar to those obtained during human stereotaxy. After careful target recognition in the stereotactic planning software by an experienced surgeon, the coordinates of NAc was mainly at a point 2.0–3.0 mm inferior, 3.0–4.0 mm anterior, and 4.5–5.5 mm lateral to the anterior commissure. The animals tolerated the general anesthesia well during the entire surgical procedure, and they regained consciousness within 3 h after the end of surgery. Postoperative MRI confirmed the accurate placement of the DBS electrodes within the NAc (Fig. 4). The motor and affective behaviors of the animals reverted gradually to the presurgery status in 7 days. Intracranial hemorrhage, paralysis, and other obvious abnormal neurological symptoms did not occur. However, subcutaneous effusion of the internal pulse generator pocket was encountered, and was handled with suction and pressure dressing. Infection and skin ulceration were likely to emerge over time because the animals scratched their skin. Consequently, the internal pulse generator was removed 3 months after the operation.
Effects of stimulation
Stimulation trials began at the fifth week after the surgery. First, the amplitude was increased gradually from 1 to 5 V with a fixed pulse width (90 μs) and frequency (160 Hz). The animals were observed to have no obvious discomfort and abnormal behaviors. Nevertheless, the animals became dull when the amplitude was immediately increased from 0 to 3 V or above, but revived soon probably because of tolerance. Then, a 3.5 V stimulation with varying pulse width and frequency was tried, and the animals’ behaviors seemed unchanged. Eventually, continuous stimulation at 90 μs, 3.5 V, 160, or 60 Hz was individually administered for 7 days to determine the long-term effects. The motor and affective behaviors of all animals remained unchanged throughout the stimulation period.
Spontaneous locomotor activity
The average hourly activity count recorded before surgery was 117±14.7 (mean±SD, n=4). Because of the surgery, the spontaneous activity decreased significantly, but gradually returned to normal in a week. After the animals recovered fully, the stimulator was switched on with a low and a high frequency (60 and 160 Hz) sequentially. Neither low-frequency nor high-frequency stimulation elicited significant changes in activity counts (113±13.1 and 118±15.1) compared with that in the stimulation ‘off’ state (109±13.4, paired t-test, P>0.05, n=4).
Historically, stereotaxy was based on the reproducibility of the relationships between landmarks on the skull and intracerebral reference points visualized by ventriculography. Clinical stereotactic techniques have advanced considerably since the introduction of computed tomography and MRI, which made the brain structures visible directly. The commercially available stereotactic system could enable surgeons to maintain the mechanical accuracy within 1 mm. However, in the animal stereotaxy, similar image-guided systems for research are lacking. Compared with stereotactic manipulation based only on the brain atlas, subject-specific image-guided stereotaxy has the advantage that one can identify anatomical target points with certainty, while ensuring that implant trajectories avoid important brain structure and vital blood vessels 11. Therefore, we utilized the clinical stereotactic system to implant DBS in NAc with the help of the frame adapter, which is the equivalent of enlarging the head circumference of the monkey. NAc was identified in the axial and the reconstructed coronal sections. Postoperative MRI showed the deepest contact of the DBS electrode located in the NAc. Although image-guided targeting in a monkey’s brain is better than targeting relying on the atlas only, the coordinates of NAc to the anterior commissure in our work might be a reference to further studies about the functions, electrophysiology, and clinical application of this nucleus.
Subcutaneous effusion, skin infection, and injury were common complications involved in IPG implantation, which resulted in removal at last. The human IPG used in our study is large for the monkey, which leads to high skin tension and discomfort. Custom scale-down IPG might avoid this problem. An alternative way could be the use of a jacket with an external pocket designed to hold the internal pulse generator with a percutaneous extension wire during the experiment.
As the interface between the limbic and the motor system on the basis of its inputs from the limbic cortical structures and its outputs to structures involved in motor control, NAc has been considered as a promising surgical target for intractable psychiatric disorders in the last decade. It was reported that ablation and DBS of NAc could alleviate heroin addiction 15,16. Kuhn et al.17,18 found smoking cessation and remission of alcohol dependence after NAc DBS, and a consistent result was obtained from other studies 19,20. In addition to substance abuse and addiction, DBS of NAc was applied in some carefully selected patients with OCD, refractory major depression, Tourette syndrome, and anorexia nervosa 3–5,21. Almost all of the studies reported alleviation of the disorders with high-frequency (>100 Hz) stimulation. Okun et al.22 reported that DBS of NAc was associated with mood responses in some OCD patients, but it was inconsistent with others 3,16. In our study, chronic stimulation of NAc in monkeys did not lead to detectable changes in behaviors. The reasons for this might be inadequate expression from animals and tolerance to the stimulation. Also, subjects may have shown different responses because of different health status or disorders.
Behaviors and spontaneous locomotor activity did not alter during high-frequency and low-frequency stimulation of NAc, which hint at the safety of NAc DBS. More validated nonhuman primate models for mental diseases could be studied further, such as a self-administration model for addiction. DBS of NAc could attenuate cocaine priming-induced reinstatement of drug seeking in rats 23, but with incommensurable stimulation parameters from clinical practice. DBS has been a well-established treatment for Parkinson’s disease, and its success relies on accurate targeting and ‘programming’, which means selecting the best parameters of electrical pulse after surgery. The nonhuman primate model may provide more details about stimulation parameters and be more helpful for addiction treatment in human beings.
This is the first report on DBS of NAc in nonhuman primates to the best of our knowledge. Although NAc has no clear identification and exact delimitation in MR images, it can be distinguished from adjacent anatomic structures in axial and coronal sections. Electrical stimulation of bilateral NAc did not alter the behavior and spontaneous activity in monkeys. This model could be helpful in further studies on the clinical use of NAc stimulation for psychiatric disorders and for better understanding the functions of this nucleus.
This research was supported by a grant from the Science and Technology Department of Shaanxi Province in China (2011KTCL03-08).
Conflicts of interest
There are no conflicts of interest.
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