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Chronic deep brain stimulation of the lateral habenula nucleus in a rat model of depression
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Research Report
Chronic deep brain stimulation of the lateral habenula nucleus in a rat model of depression Hongmei Meng a , Yanan Wang a , Min Huang b , Weihong Lin a,⁎, Shao Wang b , Baimin Zhang c,⁎ a
Department of Neurology, First Hospital of Jilin University, Changchun 130021, Jilin Province, China Department of Physiology, School of Basic Medical Science, Jilin University, Changchun 130021, Jilin Province, China c Department of Cardiac Surgery, China-Japan Union Hospital, Jilin University, Changchun 130021, Jilin Province, China b
A R T I C LE I N FO
AB S T R A C T
Article history:
In the present study, we aim to determine the antidepressant effects of chronic deep brain
Accepted 16 August 2011
stimulation (DBS) of the lateral habenula nucleus (LHb) in a rat model of depression and to
Available online 22 August 2011
explore the potential mechanism of DBS induced improvement of depressive symptoms. To establish the rat depression model, animals were repeatedly exposed to a set of chronic mild stressors for four consecutive weeks. The open-field and sucrose consumption tests were
Keywords:
used as measures of depression. For DBS treatment, rats were stereotaxically implanted
Deep brain stimulation
with electrodes into the LHb and stimulated over a course of 28 d. A separate positive control
Lateral habenula nucleus
group was given pharmacotherapy with clomipramine hydrochloride. Open-field testing was
Depression
used to determine behavioral changes following DBS treatment. Monoamine concentrations
Behavior
in blood and brain tissues were determined by fluorescence spectrophotometry. This study
Monoamine
demonstrates that DBS of the LHb region significantly improved depressive-like symptoms in the rat model. These improvements manifested as elevated numbers of crossings and rearings during the open-field test in DBS-treated depressed rats compared to controls. In addition, concentrations of monoamines including norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in blood serum and brain tissues were also increased by DBS of the LHb. Therefore, significant improvements in all outcomes were detected following chronic DBS treatment. These results indicate that long-term DBS treatment of the LHb region effectively improved depressive symptoms in rats, likely as a result of elevated monoamine levels. LHb DBS may therefore provide a valuable therapeutic strategy for the clinical treatment of depression. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Depression is an important public-health issue with a high incidence; it has also been recognized as a major cause of disability and death, both by suicide and from high rates of physical disorders (Paykel, 2006). The prevalence of depression that is associated with chronic disease is increasing, and depression is ⁎ Corresponding author. Fax: +86 431 88782378. E-mail address: [email protected] (W. Lin). 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.08.041
predicted to be the second leading cause of disease burden by the year 2020 (Moussavi et al., 2007). With the adequate care provided by available antidepressant therapies, the majority of patients with major depressive disorders may achieve remission (Rakofsky et al., 2009). However, 20 to 30% of depressed patients fail to respond significantly to standard treatments, such as multiple medications, psychotherapies, and electroconvulsive
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therapies (Mayberg et al., 2005). This cohort is characterized as patients with treatment-resistant depression (TRD). Deep brain stimulation (DBS) is achieved through the stereotactic surgical placement of stimulating electrodes into the brain, and has emerged as an effective and safe treatment for patients with Parkinson's disease (PD), essential tremor, dystonia, cluster headaches, and chronic pain (Benabid, 2003; Leone et al., 2005; Morrell, 2006; Wallace et al., 2004). Mayberg et al. for the first time, demonstrated that chronic electrical stimulation of the subgenual cingulate white matter effectively reversed symptoms in four out of six patients with TRD (Mayberg et al., 2005). Following these findings, the application of DBS in the treatment of depression has been widely investigated. A variety of promising DBS targets in the brain have been proposed for the treatment of depressed patients, including the lateral habenula nucleus (LHb) (Li et al., 2011; Sartorius and Henn, 2007), the anterior limb of the anterior internal capsule (Gutman et al., 2009), the nucleus accumbens (NAc) (Schlaepfer et al., 2008) and the thalamic peduncle ( Jimenez et al., 2005). Accumulating lines of evidences indicate that the LHb, which innervates multiple brain regions and directly influences the serotonergic, noradrenergic and dopaminergic brain system, exhibits hyperactivity during depressed states (Hikosaka et al., 2008; Sartorius and Henn, 2007). Pharmacological inhibition of the LHb by the stereotaxic application of the gammaaminobutyric acid A (GABAA) receptor agonist muscimol improved depressive-like behavior in a rat model of TRD (Winter et al., 2011). Electrical stimulation of the LHb can result similar to pharmacological inhibition (Friedman et al., 2011). A recent study demonstrated that bilateral DBS of the major afferent bundle of the LHb resulted in the efficient antidepressant therapy of patients with TRD (Sartorius et al., 2010). As the electrical stimulation of LHb leads to elevated levels of norepinephrine (NE) in the hippocampus and medial prefrontal cortex (mPFC), as well as increased serotonin (5-HT) in striatum (Cenci et al., 1992; Kalen et al., 1989a,b), it is possible that DBS-induced antidepressant effects are due to the enhanced expression of 5-HT and NE by LHb inhibition. High-frequency DBS enhances the levels of inhibitory neurotransmitters released by presynapic neurons (Li et al., 2004), and may therefore reduce the activity of habenula nucleus. Nevertheless, the mechanism involved is not yet clearly understood. In the present study, we investigated the antidepressant effects of chronic electrical stimulation of the LHb in a rat model of depression and found that this treatment led to efficient improvements in depressive symptoms in the treated rats. This effect was likely via the elevation of brain monoamine levels. These observations help to elucidate the mechanisms underlying LHb inhibition-induced antidepressant responses in TRD patients after DBS treatment.
2.
Results
2.1.
Treatment outcomes
Depressive behavior was observed at the second time point of behavioral testing, after the 28 days of stressors. A variety of behavioral changes were observed in the experimental animals, including irritability, hyperactivity, and increased aggressiveness (Table 1). With the increase in exposure time, rats exhibited symptoms of depression, such as anhedonia and decreased activity (Table 1). Following chronic stress exposure, animals were further assigned to one of eight groups as follows: depressed control, DBS treatment (lasting for 7, 14, 21, or 28 d), sham stimulation, drug treatment group and placebo treatment group (Table 2). During testing, one animal in the non-depressed control group died of infection, and one animal died in the depressed control group. Among the DBStreated rats, two animals died in the 7-day stimulation group and one died in the 14-day stimulation group. In the drug treatment group, one rat died from complications from oral gavage. None died in the placebo treatment control group. Data for analysis was therefore obtained from the seven surviving rats in the non-depressed control group and the 59 remaining rats in the depressed groups.
2.2. Chronic mild stress induces depressive behavior in animals As shown in Fig. 3, depressed rats demonstrated significantly reduced horizontal activity (number of crossings) as well as vertical activity (number of rearings) compared with controls. These results confirmed the induction of depressive behavior in these animals, demonstrating that chronic stress reduced both locomotor and exploratory activities. In addition, the sucrose consumption test showed that the total intake of the sucrose solution was decreased in rats following the chronic stress paradigm, confirming that the animal model of depression had been successfully established.
2.3. DBS into the LHb alleviates depressive behavior in the open-field test We then investigated the effects of DBS of the LHb upon the behavior of rats with depression. The locus of DBS was confirmed by Nissl staining (Fig. 2). As shown in Fig. 3, sham stimulation did not result in any changes in either horizontal or vertical activity. Twenty-one days of DBS treatment significantly increased horizontal activity in depressed animals compared to untreated and sham-stimulated controls (P < 0.05). The effect of DBS was even more apparent after
Table 1 – Behavioral changes caused by chronic stress exposure. Preexposure Behavior
+
Post-exposure Day 1
Day 7
Day 14
Day 21
Day 28
++
+++
++
−
−−
+, normal activity; + +, irritability; + + +, hyperactivity; -, decreased activity; − −, anhedonia.
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Table 2 – Time course of experimental tests. Day 1d 1 d, 8 d, 15 d, 22 d 2 d, 9 d, 16 d, 23 d 3 d, 10 d, 17 d, 24 d 4 d, 11 d, 18 d, 25 d 5 d, 12 d, 19 d, 26 d 6 d, 13 d, 20 d, 27 d 7 d, 14 d, 21 d, 28 d 28 d 29 d 35 d 42 d 49 d 56 d
Experimental tests Sucrose consumption test Open-field test Water deprivation Tail clipping Food deprivation Ice water swimming Shaking Reversal of day and night Pyretic fumigation Sucrose consumption test Open-field test Beginning therapeutic treatment Open-field test Open-field test Open-field test Open-field test
28 days of treatment, when both horizontal and vertical activity was dramatically increased in depressed rats compared to controls (P < 0.01). These results suggest DBS into the LHb may alleviate symptoms in a rat model of depression.
2.4. DBS elevates monoamine levels in blood serum and brain tissue of depressed rats A significant reduction was detected in the monoamine transmitter levels in both blood serum and brain tissues obtained from depressed rats compared to controls (P < 0.01; Tables 3 and 4). However, treatment with clomipramine hydrochloride tablets remarkably enhanced the levels of NE, DA, and 5-HT in the drug treatment group compared to depressed control groups (P < 0.01). In addition, DBS of the LHb led to gradual increases in the concentrations of monoamines in the blood serum and brain of depressed rats (Tables 3 and 4). Twentyeight days of DBS treatment led to the most significant elevations in monoamine levels, suggesting that DBS into the LHb can ameliorate depression by promoting the monoamine levels in blood and brain tissues. Therefore, long-term (chronic) DBS
treatment may contribute to a better outcome in the treatment of depression.
3.
Discussion
Depression is a highly prevalent and disabling psychiatric disease that often requires long-term treatment (Paykel, 2006). Major depressive disorder is believed to be a stress-related disorder (Fava and Kendler, 2000; Plotsky et al., 1998). A variety of depression models has been established in rodents, including stress (learned helplessness, chronic mild stress, and social stress), lesion (olfactory bulbectomy), pharmacological (reserpine, tryptophan, psychostimulant withdrawal) and genetic (genetically engineered mice, selective breeding) models (Deussing, 2006). In the current study, the chronic mild stress paradigm, which is sensitive to antidepressant treatment (Willner, 2005), establishes a rat model of depression. After repeated exposure to a series of chronic mild stressors for four consecutive weeks, rats exhibited significantly reduced locomotor activity and exploratory behavior, accompanied by a reduction in sucrose consumption (Fig. 1). These observations confirmed the successful establishment of the chronic mild stress in the present study. DBS, targeted to particular brain areas specific to particular neurological disorders, has been broadly accepted and applied to relieve symptoms and improve the overall functioning of patients with involuntary movement or psychiatric diseases (Benabid, 2003; DeLong, 2011; Leone et al., 2005; Morrell, 2006; Wallace et al., 2004). The application of implantable electrical stimulation for the treatment of essential tremor and Parkinson's disease (PD) was approved by the US Food and Drug Administration (FDA) in 1997, and has since been widely used as an alternative to ablative procedures for refractory PD and other movement disorders (Coffey, 2009). Mayberg et al. (2005) demonstrated that chronic electrical stimulation of the subgenual cingulate white matter effectively relieved symptoms in four out of six patients with TRD (Mayberg et al., 2005). A recent study further demonstrated that bilateral DBS of the major afferent bundle of the LHb results in a case study of TRD (Sartorius et al., 2010).
Fig. 1 – Changes in rat behavior and sucrose intake after the establishment of the depression model. (A) The number of crossings and rearings were counted on the day when the depression model was established. (B) Sucrose consumption (g) was determined within 1 h after water deprived for 24 h. *P < 0.01 compared with control.
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Fig. 2 – The locus of DBS was proven by Nissl staining. Brain tissues obtained from normal animals (A) or animals with DBS treatment (B) were sectioned and stained with Nissl. Arrow in (A) indicated the location of LHb; arrow in (B) indicated the location of stimulating electrode in the LHb.
However, the mechanism by which DBS improved depression is still poorly understood. In the current study, we found that DBS into the LHb area significantly ameliorated depressive symptoms in rats. These results are consistent with previous reports that have found DBS into the LHb to relieve symptoms in patients with TRD (Sartorius et al., 2010). The monoaminergic theory of depression, which predicts that major depressive disorder results from the deregulation of neurotransmission by the monoamines DA, 5-HT, and NE, has been widely applied to the understanding of the pathophysiology and pharmacological treatment of this disease (Skolnick, 2002). Symptom relief in depressed patients may be achieved by increasing and normalizing the levels of these neurotransmitters (Charney,
Fig. 3 – Behavioral changes following DBS. The number of crossings and rearings in rats from different experimental groups were counted within 3 min of horizontal or vertical activity. The number of crossings and rearings in mice from depressed control group were counted on Day 56. *P < 0.05 or **P < 0.01 compared with depressed control; △P < 0.05 or △△ P < 0.01 compared with sham stimulation group.
1998). In agreement, in the current study, rats in the depressed control group presented significant reductions in monoamine transmitter levels in both blood serum and brain tissue. In contrast, following DBS treatment, the levels of NE, DA and 5-HT in depressed rats increased over the course of treatment. Interestingly, a significant elevation of 5-HT levels was found after DBS into the LHb, as compared with levels of NE and DA, suggesting a close correlation between the activity of the LHb and serotonergic neurotransmission. We found that chronic (28 d) DBS treatment yielded better outcomes both in the relief of depressive symptoms and the up-regulation of monoamine concentrations compared with 7, 14, or 21 day DBS treatment. Future studies will focus on the long-term (longer than one month) effects of DBS into the LHb in the rat model of depression. In addition, the administration of clomipramine hydrochloride tablets promoted the concentrations of monoamines in the brain and blood serum of depressed rats to a significantly greater degree than did DBS treatment. This disparity may result from the relatively mild DBS paradigm used, in which rats were given DBS treatment for of 30 min each day. It will be interesting, in future studies, to determine whether the antidepressant effects of DBS can be increased by continuous chronic stimulation of the LHb. In summary, the present study demonstrates that chronic DBS of the LHb efficiently relieved symptoms of depression in rats, and this symptom relief correlated with elevations in monoamine transmitter levels in both blood serum and brain tissues. These observations provide valuable insight into the mechanisms underlying the positive effects of DBS into the LHb upon outcomes in patients with TRD.
4.
Experimental procedures
4.1.
Reagents and chemicals
Clomipramine hydrochloride tablets were purchased from Jiangsu Nhwa Pharmaceutical Co., Ltd. (Xuzhou, China). Monoamine standards including norepinephrine (NE), dopamine (DA), and serotonin (5-HT) were obtained from Sigma. L-
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Table 3 – Concentrations of monoamines: norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat blood serum.
Control Depressed control Sham stimulation Drug treatment 7-day DBS 14-day DBS 21-day DBS 28-day DBS
n
NE
DA
5-HT
7 7 8 7 6 7 8 8
964.33 ± 154.86 240.26 ± 100.39** 426.69 ± 193.23** 913.51 ± 287.64△△## 364.69 ± 122.23*▲ 401.80 ± 174.43**▲ 499.88 ± 345.21*▲ 795.42 ± 323.99△△#
630.36 ± 117.78 265.44 ± 75.45** 410.33 ± 95.37 869.10 ± 244.77*△△## 431.16 ± 51.52▲▲ 563.74 ± 115.81△△▲ 629.00 ± 118.19△△#▲ 782.33 ± 203.98△△##
1005.61 ± 198.96 299.31 ± 96.73** 480.12 ± 255.44* 1465.97 ± 262.70*△△## 350.32 ± 127.71*▲▲ 709.39 ± 301.00▲▲ 974.29 ± 306.85△△#▲ 1142.35 ± 489.88△△##▲
*P < 0.05 or **P < 0.01 compared with control; △P < 0.05 or △△P < 0.01 compared with depression model; #P < 0.05 or ##P < 0.01 compared with sham stimulation group; ▲P < 0.05 or ▲▲P < 0.01 compared with drug treatment group.
cysteine was purchased from Shanghai Xinxing Chemical Reagent Research Institute (Shanghai, China). O-Phthalaldehyde (OPT) was bought from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Na2S2O3 and 1-butanol were purchased from the No.1 Shanghai Reagent Factory (Shanghai, China). 4.2.
Animals
A total of 72 male Wistar rats, weighing 256.8 ± 18.7 g, were provided by Experimental Animal Center, Norman Bethune College of Medicine, Jilin University. Animals were routinely housed under controlled conditions of lighting (12 h light/12 h dark cycle), humidity (50–60%), and temperature (18–22 °C) with ad libitum access to food and water. Rats were acclimated to the laboratory environment for one week before behavioral testing. 4.3.
Experimental design
Prior to the start of the experiments, rats were randomly assigned to one of nine groups (n= 8 for each group). These included a control group, a depression model group, DBS treatment (lasting for 7, 14, 21, or 28 d) groups, a sham stimulation group, drug treatment group and placebo treatment group. To establish the rat depression model, animals were individually housed and repeatedly exposed to a set of chronic mild stressors for 4 consecutive weeks as follows: 1 min tail clamp, 24 h of food or water deprivation, exposure to an experimental room at 45 °C for 5 min, 24 h of reversed day and night, 30 min horizontal vibration (160 times/min), 5 min cold swim at 4 °C. Animals received one stressor each day. The same stressor was not applied successively so that rats could not anticipate
the occurring of stress. In the chronic DBS groups, rats received DBS for 7, 14, 21, or 28 d after electrode implantation. In the sham stimulation group, the electrode was implanted in the lateral habenula nucleus, but animals were not given electrical stimulation. Mice in the sham stimulation group were kept until the end of experiment. In the drug treatment group, rats were administered intragastrically with 6.32 mg/kg clomipramine hydrochloride tablets at the beginning of the experiment. Drugs were administrated when DBS treatment groups were tested. Drug was given once daily, with an increment of 0.53 mg/kg each day. The dosage was increased over 28 days to a final of 21.07 mg/kg. Control rats were received placebo treatment (normal saline). These animals were measured at the same times post-surgery (7, 14, 21, 28 d) as the DBS animals in the behavioral tests. All animal experiments followed a protocol approved by the Animal Research Committee of Jilin University, China, and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (China Ministry of Health). 4.4.
Open-field test
Animals were subjected to open-field testing immediately prior to, or after, the establishment of the depression model. Testing was carried out as previously described (Katz et al., 1981), with slight modifications. Briefly, the apparatus was a four-sided 100 cm × 100 cm × 50 cm wooden enclosure, with the inside walls painted black. The apparatus floor was divided by black lines into 25 equal squares to assess locomotion. During testing, the movements of rats in the open-field were quantified by counting the number of crossings (four paws in a square) and the number of rearings (lifting front legs
Table 4 – Concentrations of monoamines: norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat brain tissue.
Control Depressed control Sham stimulation Drug treatment 7-day DBS 14-day DBS 21-day DBS 28-day DBS
n
NE
DA
5-HT
7 7 8 7 6 7 8 8
18.94 ± 4.22 3.04 ± 1.32** 4.12 ± 1.05** 14.43 ± 2.42**△△## 3.81 ± 1.01**▲▲ 8.27 ± 1.29**△△##▲▲ 9.61 ± 1.45**△△##▲▲ 13.46 ± 3.04**△△##
55.50 ± 17.73 30.60 ± 4.66** 33.22 ± 4.46** 59.52 ± 7.83△△ 43.80 ± 4.62 44.65 ± 8.61 46.98 ± 5.31△ 55.87 ± 13.76△△##
1.98 ± 0.73 0.46 ± 0.17** 0.46 ± 0.11** 1.83 ± 0.86△△## 0.67 ± 0.21* 0.77 ± 0.18*▲ 0.84 ± 0.31**▲ 1.54 ± 0.95△#
*P < 0.05 or **P < 0.01 compared with control; △P < 0.05 or △△P < 0.01 compared with depression model; #P < 0.05 or ##P < 0.01 compared with sham stimulation group; ▲P < 0.05 or ▲▲P < 0.01 compared with drug treatment group.
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≥1 cm above the floor). Each rat was tested three minutes and the apparatus was cleaned after each test. Horizontal activity (number of crossings) is a measure of locomotor activity, while vertical activity (number of rearings) represents exploratory behavior. Behavioral testing was performed on the first and the last day after rats were exposed to the set of chronic mild stressors. Animals with reduced number of crossings and rearings were recognized as depressed rats here (Han et al., 2010; Huang et al., 2007). 4.5.
Sucrose consumption test
Sucrose test was performed on the same day as the open-field test. Sucrose consumption was measured on days 1, 8, 15, and 22 after the beginning of the chronic stress paradigm. Animals were water deprived for 24 h before the sucrose consumption test, and were exposed to both the test solution (1% sucrose) and regular drinking water for a 1 h period. Sucrose consumption was evaluated by changes in the weight of the test solution bottle. Alterations in the consumption of sucrose represent a measure of anhedonia, or the inability to experience anhedonia which is a symptom of depression (Shalaev and Steponkus, 2001). 4.6.
37
levels in brain tissue, rats were sacrificed and the hippocampus was dissected. After weighing, the hippocampal tissue was flash frozen in liquid nitrogen and stored at −80 °C until use. Standard solutions of 5-HT, NE or DA (500 μg/mL) were prepared in 0.01 N HCl and were diluted to 1.25 μg/mL before use. Samples were supplemented with 10X n-butanol followed by shaking and centrifugation. After samples were heated in a 100 °C boiling water bath, the monoamine concentrations were determined by fluorescence spectrophotometry (RF-5310PC, Shimadzu Co., Japan). The concentration of 5-HT, NE or DA was calculated by the following formulas: Concentration of monoamine ðμg=LÞ Fsample −Fblank contents of standard 100 ¼ contents of sample ðFstandard −Fblank Þ 4.9.
Statistical analysis
Statistical analyses were performed using the SPSS 14.0 statistical software package. Data are presented as mean ± standard error of the mean (SEM). Results were analyzed using one-way ANOVA with Fisher's Least Significant Difference (LSD) test. P values < 0.05 were considered statistically significant.
Surgery and electrode implantation REFERENCES
Bipolar stimulating electrodes were made from pairs of Ni–Cr enamel-insulated wires with a 0.5 mm vertical tip separation. Rats were anesthetized with intraperitoneal injections of 10% chloral hydrate (0.3 ml/100 g) and placed in a stereotaxic apparatus (Narishige SR-6N, Japan). The scalp was retracted and a hole was drilled in the skull for the placement of the stimulating electrode into the LHb (anterior, 3.3 mm; lateral, 0.5 mm; ventral, 4.0 mm from Bregma). The electrode was secured to the skull with screws and dental acrylic cement. Penicillin (2 × 105 U/day) was injected intraperitoneally for three days post-operatively. Rats were given 5 mg/kg carprofen (q 12 h) as pain reliever by subcutaneous injection. The locus of DBS was proven by Nissl staining. Briefly, formalin-fixed, paraffin-embedded brain tissues were sectioned and stained with Nissl. Sections were mounted and evaluated via microscope. 4.7.
Electrical stimulation procedure
Rats in the DBS treatment groups were given high-frequency stimulation every day using an electrical stimulator (SEN3201, Nihon-Kohden Inc., Japan). Electrical stimulation, with 150 Hz square-wave pulses, was given at 80–100 μA for 0.3 ms. Each rat received high frequency stimulation for a duration of 30 min. Following 28 days of stimulation, animal behavior was monitored every 7 days. 4.8.
Determination of monoamine concentrations
Rats were sacrificed by cervical dislocation. Cardiac blood samples (1.5–2 ml collected from the left ventricle) were added to chilled heparin-containing tubes. After centrifugation at 2000 rpm for 10 min, the supernatants were stored at −20 °C until monoamine determinations. To evaluate monoamine
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Paykel, E.S., 2006. Depression: major problem for public health. Epidemiol. Psichiatr. Soc. 15, 4–10. Plotsky, P.M., Owens, M.J., Nemeroff, C.B., 1998. Psychoneuroendocrinology of depression. Hypothalamic-pituitary-adrenal axis. Psychiatr. Clin. North Am. 21, 293–307. Rakofsky, J.J., Holtzheimer, P.E., Nemeroff, C.B., 2009. Emerging targets for antidepressant therapies. Curr. Opin. Chem. Biol. 13, 291–302. Sartorius, A., Henn, F.A., 2007. Deep brain stimulation of the lateral habenula in treatment resistant major depression. Med. Hypotheses 69, 1305–1308. Sartorius, A., Kiening, K.L., Kirsch, P., von Gall, C.C., Haberkorn, U., Unterberg, A.W., Henn, F.A., Meyer-Lindenberg, A., 2010. Remission of major depression under deep brain stimulation of the lateral habenula in a therapy–refractory patient. Biol. Psychiatry 67, e9–e11. Schlaepfer, T.E., Cohen, M.X., Frick, C., Kosel, M., Brodesser, D., Axmacher, N., Joe, A.Y., Kreft, M., Lenartz, D., Sturm, V., 2008. Deep brain stimulation to reward circuitry alleviates anhedonia in refractory major depression. Neuropsychopharmacology 33, 368–377. Shalaev, E.Y., Steponkus, P.L., 2001. Depression of the glass transition temperature of sucrose confined in a phospholipid mesophase. Langmuir 17, 5137–5140. Skolnick, P., 2002. Beyond monoamine-based therapies: clues to new approaches. J. Clin. Psychiatry 63 (Suppl 2), 19–23. Wallace, B.A., Ashkan, K., Benabid, A.L., 2004. Deep brain stimulation for the treatment of chronic, intractable pain. Neurosurg. Clin. N. Am. 15, 343–357 (vii). Willner, P., 2005. Chronic mild stress (CMS) revisited: consistency and behavioural–neurobiological concordance in the effects of CMS. Neuropsychobiology 52, 90–110. Winter, C., Vollmayr, B., Djodari-Irani, A., Klein, J., Sartorius, A., 2011. Pharmacological inhibition of the lateral habenula improves depressive-like behavior in an animal model of treatment resistant depression. Behav. Brain Res. 216, 463–465.
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Research Report
Chronic deep brain stimulation of the lateral habenula nucleus in a rat model of depression Hongmei Meng a , Yanan Wang a , Min Huang b , Weihong Lin a,⁎, Shao Wang b , Baimin Zhang c,⁎ a
Department of Neurology, First Hospital of Jilin University, Changchun 130021, Jilin Province, China Department of Physiology, School of Basic Medical Science, Jilin University, Changchun 130021, Jilin Province, China c Department of Cardiac Surgery, China-Japan Union Hospital, Jilin University, Changchun 130021, Jilin Province, China b
A R T I C LE I N FO
AB S T R A C T
Article history:
In the present study, we aim to determine the antidepressant effects of chronic deep brain
Accepted 16 August 2011
stimulation (DBS) of the lateral habenula nucleus (LHb) in a rat model of depression and to
Available online 22 August 2011
explore the potential mechanism of DBS induced improvement of depressive symptoms. To establish the rat depression model, animals were repeatedly exposed to a set of chronic mild stressors for four consecutive weeks. The open-field and sucrose consumption tests were
Keywords:
used as measures of depression. For DBS treatment, rats were stereotaxically implanted
Deep brain stimulation
with electrodes into the LHb and stimulated over a course of 28 d. A separate positive control
Lateral habenula nucleus
group was given pharmacotherapy with clomipramine hydrochloride. Open-field testing was
Depression
used to determine behavioral changes following DBS treatment. Monoamine concentrations
Behavior
in blood and brain tissues were determined by fluorescence spectrophotometry. This study
Monoamine
demonstrates that DBS of the LHb region significantly improved depressive-like symptoms in the rat model. These improvements manifested as elevated numbers of crossings and rearings during the open-field test in DBS-treated depressed rats compared to controls. In addition, concentrations of monoamines including norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in blood serum and brain tissues were also increased by DBS of the LHb. Therefore, significant improvements in all outcomes were detected following chronic DBS treatment. These results indicate that long-term DBS treatment of the LHb region effectively improved depressive symptoms in rats, likely as a result of elevated monoamine levels. LHb DBS may therefore provide a valuable therapeutic strategy for the clinical treatment of depression. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Depression is an important public-health issue with a high incidence; it has also been recognized as a major cause of disability and death, both by suicide and from high rates of physical disorders (Paykel, 2006). The prevalence of depression that is associated with chronic disease is increasing, and depression is ⁎ Corresponding author. Fax: +86 431 88782378. E-mail address: [email protected] (W. Lin). 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.08.041
predicted to be the second leading cause of disease burden by the year 2020 (Moussavi et al., 2007). With the adequate care provided by available antidepressant therapies, the majority of patients with major depressive disorders may achieve remission (Rakofsky et al., 2009). However, 20 to 30% of depressed patients fail to respond significantly to standard treatments, such as multiple medications, psychotherapies, and electroconvulsive
33
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therapies (Mayberg et al., 2005). This cohort is characterized as patients with treatment-resistant depression (TRD). Deep brain stimulation (DBS) is achieved through the stereotactic surgical placement of stimulating electrodes into the brain, and has emerged as an effective and safe treatment for patients with Parkinson's disease (PD), essential tremor, dystonia, cluster headaches, and chronic pain (Benabid, 2003; Leone et al., 2005; Morrell, 2006; Wallace et al., 2004). Mayberg et al. for the first time, demonstrated that chronic electrical stimulation of the subgenual cingulate white matter effectively reversed symptoms in four out of six patients with TRD (Mayberg et al., 2005). Following these findings, the application of DBS in the treatment of depression has been widely investigated. A variety of promising DBS targets in the brain have been proposed for the treatment of depressed patients, including the lateral habenula nucleus (LHb) (Li et al., 2011; Sartorius and Henn, 2007), the anterior limb of the anterior internal capsule (Gutman et al., 2009), the nucleus accumbens (NAc) (Schlaepfer et al., 2008) and the thalamic peduncle ( Jimenez et al., 2005). Accumulating lines of evidences indicate that the LHb, which innervates multiple brain regions and directly influences the serotonergic, noradrenergic and dopaminergic brain system, exhibits hyperactivity during depressed states (Hikosaka et al., 2008; Sartorius and Henn, 2007). Pharmacological inhibition of the LHb by the stereotaxic application of the gammaaminobutyric acid A (GABAA) receptor agonist muscimol improved depressive-like behavior in a rat model of TRD (Winter et al., 2011). Electrical stimulation of the LHb can result similar to pharmacological inhibition (Friedman et al., 2011). A recent study demonstrated that bilateral DBS of the major afferent bundle of the LHb resulted in the efficient antidepressant therapy of patients with TRD (Sartorius et al., 2010). As the electrical stimulation of LHb leads to elevated levels of norepinephrine (NE) in the hippocampus and medial prefrontal cortex (mPFC), as well as increased serotonin (5-HT) in striatum (Cenci et al., 1992; Kalen et al., 1989a,b), it is possible that DBS-induced antidepressant effects are due to the enhanced expression of 5-HT and NE by LHb inhibition. High-frequency DBS enhances the levels of inhibitory neurotransmitters released by presynapic neurons (Li et al., 2004), and may therefore reduce the activity of habenula nucleus. Nevertheless, the mechanism involved is not yet clearly understood. In the present study, we investigated the antidepressant effects of chronic electrical stimulation of the LHb in a rat model of depression and found that this treatment led to efficient improvements in depressive symptoms in the treated rats. This effect was likely via the elevation of brain monoamine levels. These observations help to elucidate the mechanisms underlying LHb inhibition-induced antidepressant responses in TRD patients after DBS treatment.
2.
Results
2.1.
Treatment outcomes
Depressive behavior was observed at the second time point of behavioral testing, after the 28 days of stressors. A variety of behavioral changes were observed in the experimental animals, including irritability, hyperactivity, and increased aggressiveness (Table 1). With the increase in exposure time, rats exhibited symptoms of depression, such as anhedonia and decreased activity (Table 1). Following chronic stress exposure, animals were further assigned to one of eight groups as follows: depressed control, DBS treatment (lasting for 7, 14, 21, or 28 d), sham stimulation, drug treatment group and placebo treatment group (Table 2). During testing, one animal in the non-depressed control group died of infection, and one animal died in the depressed control group. Among the DBStreated rats, two animals died in the 7-day stimulation group and one died in the 14-day stimulation group. In the drug treatment group, one rat died from complications from oral gavage. None died in the placebo treatment control group. Data for analysis was therefore obtained from the seven surviving rats in the non-depressed control group and the 59 remaining rats in the depressed groups.
2.2. Chronic mild stress induces depressive behavior in animals As shown in Fig. 3, depressed rats demonstrated significantly reduced horizontal activity (number of crossings) as well as vertical activity (number of rearings) compared with controls. These results confirmed the induction of depressive behavior in these animals, demonstrating that chronic stress reduced both locomotor and exploratory activities. In addition, the sucrose consumption test showed that the total intake of the sucrose solution was decreased in rats following the chronic stress paradigm, confirming that the animal model of depression had been successfully established.
2.3. DBS into the LHb alleviates depressive behavior in the open-field test We then investigated the effects of DBS of the LHb upon the behavior of rats with depression. The locus of DBS was confirmed by Nissl staining (Fig. 2). As shown in Fig. 3, sham stimulation did not result in any changes in either horizontal or vertical activity. Twenty-one days of DBS treatment significantly increased horizontal activity in depressed animals compared to untreated and sham-stimulated controls (P < 0.05). The effect of DBS was even more apparent after
Table 1 – Behavioral changes caused by chronic stress exposure. Preexposure Behavior
+
Post-exposure Day 1
Day 7
Day 14
Day 21
Day 28
++
+++
++
−
−−
+, normal activity; + +, irritability; + + +, hyperactivity; -, decreased activity; − −, anhedonia.
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Table 2 – Time course of experimental tests. Day 1d 1 d, 8 d, 15 d, 22 d 2 d, 9 d, 16 d, 23 d 3 d, 10 d, 17 d, 24 d 4 d, 11 d, 18 d, 25 d 5 d, 12 d, 19 d, 26 d 6 d, 13 d, 20 d, 27 d 7 d, 14 d, 21 d, 28 d 28 d 29 d 35 d 42 d 49 d 56 d
Experimental tests Sucrose consumption test Open-field test Water deprivation Tail clipping Food deprivation Ice water swimming Shaking Reversal of day and night Pyretic fumigation Sucrose consumption test Open-field test Beginning therapeutic treatment Open-field test Open-field test Open-field test Open-field test
28 days of treatment, when both horizontal and vertical activity was dramatically increased in depressed rats compared to controls (P < 0.01). These results suggest DBS into the LHb may alleviate symptoms in a rat model of depression.
2.4. DBS elevates monoamine levels in blood serum and brain tissue of depressed rats A significant reduction was detected in the monoamine transmitter levels in both blood serum and brain tissues obtained from depressed rats compared to controls (P < 0.01; Tables 3 and 4). However, treatment with clomipramine hydrochloride tablets remarkably enhanced the levels of NE, DA, and 5-HT in the drug treatment group compared to depressed control groups (P < 0.01). In addition, DBS of the LHb led to gradual increases in the concentrations of monoamines in the blood serum and brain of depressed rats (Tables 3 and 4). Twentyeight days of DBS treatment led to the most significant elevations in monoamine levels, suggesting that DBS into the LHb can ameliorate depression by promoting the monoamine levels in blood and brain tissues. Therefore, long-term (chronic) DBS
treatment may contribute to a better outcome in the treatment of depression.
3.
Discussion
Depression is a highly prevalent and disabling psychiatric disease that often requires long-term treatment (Paykel, 2006). Major depressive disorder is believed to be a stress-related disorder (Fava and Kendler, 2000; Plotsky et al., 1998). A variety of depression models has been established in rodents, including stress (learned helplessness, chronic mild stress, and social stress), lesion (olfactory bulbectomy), pharmacological (reserpine, tryptophan, psychostimulant withdrawal) and genetic (genetically engineered mice, selective breeding) models (Deussing, 2006). In the current study, the chronic mild stress paradigm, which is sensitive to antidepressant treatment (Willner, 2005), establishes a rat model of depression. After repeated exposure to a series of chronic mild stressors for four consecutive weeks, rats exhibited significantly reduced locomotor activity and exploratory behavior, accompanied by a reduction in sucrose consumption (Fig. 1). These observations confirmed the successful establishment of the chronic mild stress in the present study. DBS, targeted to particular brain areas specific to particular neurological disorders, has been broadly accepted and applied to relieve symptoms and improve the overall functioning of patients with involuntary movement or psychiatric diseases (Benabid, 2003; DeLong, 2011; Leone et al., 2005; Morrell, 2006; Wallace et al., 2004). The application of implantable electrical stimulation for the treatment of essential tremor and Parkinson's disease (PD) was approved by the US Food and Drug Administration (FDA) in 1997, and has since been widely used as an alternative to ablative procedures for refractory PD and other movement disorders (Coffey, 2009). Mayberg et al. (2005) demonstrated that chronic electrical stimulation of the subgenual cingulate white matter effectively relieved symptoms in four out of six patients with TRD (Mayberg et al., 2005). A recent study further demonstrated that bilateral DBS of the major afferent bundle of the LHb results in a case study of TRD (Sartorius et al., 2010).
Fig. 1 – Changes in rat behavior and sucrose intake after the establishment of the depression model. (A) The number of crossings and rearings were counted on the day when the depression model was established. (B) Sucrose consumption (g) was determined within 1 h after water deprived for 24 h. *P < 0.01 compared with control.
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35
Fig. 2 – The locus of DBS was proven by Nissl staining. Brain tissues obtained from normal animals (A) or animals with DBS treatment (B) were sectioned and stained with Nissl. Arrow in (A) indicated the location of LHb; arrow in (B) indicated the location of stimulating electrode in the LHb.
However, the mechanism by which DBS improved depression is still poorly understood. In the current study, we found that DBS into the LHb area significantly ameliorated depressive symptoms in rats. These results are consistent with previous reports that have found DBS into the LHb to relieve symptoms in patients with TRD (Sartorius et al., 2010). The monoaminergic theory of depression, which predicts that major depressive disorder results from the deregulation of neurotransmission by the monoamines DA, 5-HT, and NE, has been widely applied to the understanding of the pathophysiology and pharmacological treatment of this disease (Skolnick, 2002). Symptom relief in depressed patients may be achieved by increasing and normalizing the levels of these neurotransmitters (Charney,
Fig. 3 – Behavioral changes following DBS. The number of crossings and rearings in rats from different experimental groups were counted within 3 min of horizontal or vertical activity. The number of crossings and rearings in mice from depressed control group were counted on Day 56. *P < 0.05 or **P < 0.01 compared with depressed control; △P < 0.05 or △△ P < 0.01 compared with sham stimulation group.
1998). In agreement, in the current study, rats in the depressed control group presented significant reductions in monoamine transmitter levels in both blood serum and brain tissue. In contrast, following DBS treatment, the levels of NE, DA and 5-HT in depressed rats increased over the course of treatment. Interestingly, a significant elevation of 5-HT levels was found after DBS into the LHb, as compared with levels of NE and DA, suggesting a close correlation between the activity of the LHb and serotonergic neurotransmission. We found that chronic (28 d) DBS treatment yielded better outcomes both in the relief of depressive symptoms and the up-regulation of monoamine concentrations compared with 7, 14, or 21 day DBS treatment. Future studies will focus on the long-term (longer than one month) effects of DBS into the LHb in the rat model of depression. In addition, the administration of clomipramine hydrochloride tablets promoted the concentrations of monoamines in the brain and blood serum of depressed rats to a significantly greater degree than did DBS treatment. This disparity may result from the relatively mild DBS paradigm used, in which rats were given DBS treatment for of 30 min each day. It will be interesting, in future studies, to determine whether the antidepressant effects of DBS can be increased by continuous chronic stimulation of the LHb. In summary, the present study demonstrates that chronic DBS of the LHb efficiently relieved symptoms of depression in rats, and this symptom relief correlated with elevations in monoamine transmitter levels in both blood serum and brain tissues. These observations provide valuable insight into the mechanisms underlying the positive effects of DBS into the LHb upon outcomes in patients with TRD.
4.
Experimental procedures
4.1.
Reagents and chemicals
Clomipramine hydrochloride tablets were purchased from Jiangsu Nhwa Pharmaceutical Co., Ltd. (Xuzhou, China). Monoamine standards including norepinephrine (NE), dopamine (DA), and serotonin (5-HT) were obtained from Sigma. L-
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Table 3 – Concentrations of monoamines: norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat blood serum.
Control Depressed control Sham stimulation Drug treatment 7-day DBS 14-day DBS 21-day DBS 28-day DBS
n
NE
DA
5-HT
7 7 8 7 6 7 8 8
964.33 ± 154.86 240.26 ± 100.39** 426.69 ± 193.23** 913.51 ± 287.64△△## 364.69 ± 122.23*▲ 401.80 ± 174.43**▲ 499.88 ± 345.21*▲ 795.42 ± 323.99△△#
630.36 ± 117.78 265.44 ± 75.45** 410.33 ± 95.37 869.10 ± 244.77*△△## 431.16 ± 51.52▲▲ 563.74 ± 115.81△△▲ 629.00 ± 118.19△△#▲ 782.33 ± 203.98△△##
1005.61 ± 198.96 299.31 ± 96.73** 480.12 ± 255.44* 1465.97 ± 262.70*△△## 350.32 ± 127.71*▲▲ 709.39 ± 301.00▲▲ 974.29 ± 306.85△△#▲ 1142.35 ± 489.88△△##▲
*P < 0.05 or **P < 0.01 compared with control; △P < 0.05 or △△P < 0.01 compared with depression model; #P < 0.05 or ##P < 0.01 compared with sham stimulation group; ▲P < 0.05 or ▲▲P < 0.01 compared with drug treatment group.
cysteine was purchased from Shanghai Xinxing Chemical Reagent Research Institute (Shanghai, China). O-Phthalaldehyde (OPT) was bought from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Na2S2O3 and 1-butanol were purchased from the No.1 Shanghai Reagent Factory (Shanghai, China). 4.2.
Animals
A total of 72 male Wistar rats, weighing 256.8 ± 18.7 g, were provided by Experimental Animal Center, Norman Bethune College of Medicine, Jilin University. Animals were routinely housed under controlled conditions of lighting (12 h light/12 h dark cycle), humidity (50–60%), and temperature (18–22 °C) with ad libitum access to food and water. Rats were acclimated to the laboratory environment for one week before behavioral testing. 4.3.
Experimental design
Prior to the start of the experiments, rats were randomly assigned to one of nine groups (n= 8 for each group). These included a control group, a depression model group, DBS treatment (lasting for 7, 14, 21, or 28 d) groups, a sham stimulation group, drug treatment group and placebo treatment group. To establish the rat depression model, animals were individually housed and repeatedly exposed to a set of chronic mild stressors for 4 consecutive weeks as follows: 1 min tail clamp, 24 h of food or water deprivation, exposure to an experimental room at 45 °C for 5 min, 24 h of reversed day and night, 30 min horizontal vibration (160 times/min), 5 min cold swim at 4 °C. Animals received one stressor each day. The same stressor was not applied successively so that rats could not anticipate
the occurring of stress. In the chronic DBS groups, rats received DBS for 7, 14, 21, or 28 d after electrode implantation. In the sham stimulation group, the electrode was implanted in the lateral habenula nucleus, but animals were not given electrical stimulation. Mice in the sham stimulation group were kept until the end of experiment. In the drug treatment group, rats were administered intragastrically with 6.32 mg/kg clomipramine hydrochloride tablets at the beginning of the experiment. Drugs were administrated when DBS treatment groups were tested. Drug was given once daily, with an increment of 0.53 mg/kg each day. The dosage was increased over 28 days to a final of 21.07 mg/kg. Control rats were received placebo treatment (normal saline). These animals were measured at the same times post-surgery (7, 14, 21, 28 d) as the DBS animals in the behavioral tests. All animal experiments followed a protocol approved by the Animal Research Committee of Jilin University, China, and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (China Ministry of Health). 4.4.
Open-field test
Animals were subjected to open-field testing immediately prior to, or after, the establishment of the depression model. Testing was carried out as previously described (Katz et al., 1981), with slight modifications. Briefly, the apparatus was a four-sided 100 cm × 100 cm × 50 cm wooden enclosure, with the inside walls painted black. The apparatus floor was divided by black lines into 25 equal squares to assess locomotion. During testing, the movements of rats in the open-field were quantified by counting the number of crossings (four paws in a square) and the number of rearings (lifting front legs
Table 4 – Concentrations of monoamines: norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat brain tissue.
Control Depressed control Sham stimulation Drug treatment 7-day DBS 14-day DBS 21-day DBS 28-day DBS
n
NE
DA
5-HT
7 7 8 7 6 7 8 8
18.94 ± 4.22 3.04 ± 1.32** 4.12 ± 1.05** 14.43 ± 2.42**△△## 3.81 ± 1.01**▲▲ 8.27 ± 1.29**△△##▲▲ 9.61 ± 1.45**△△##▲▲ 13.46 ± 3.04**△△##
55.50 ± 17.73 30.60 ± 4.66** 33.22 ± 4.46** 59.52 ± 7.83△△ 43.80 ± 4.62 44.65 ± 8.61 46.98 ± 5.31△ 55.87 ± 13.76△△##
1.98 ± 0.73 0.46 ± 0.17** 0.46 ± 0.11** 1.83 ± 0.86△△## 0.67 ± 0.21* 0.77 ± 0.18*▲ 0.84 ± 0.31**▲ 1.54 ± 0.95△#
*P < 0.05 or **P < 0.01 compared with control; △P < 0.05 or △△P < 0.01 compared with depression model; #P < 0.05 or ##P < 0.01 compared with sham stimulation group; ▲P < 0.05 or ▲▲P < 0.01 compared with drug treatment group.
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≥1 cm above the floor). Each rat was tested three minutes and the apparatus was cleaned after each test. Horizontal activity (number of crossings) is a measure of locomotor activity, while vertical activity (number of rearings) represents exploratory behavior. Behavioral testing was performed on the first and the last day after rats were exposed to the set of chronic mild stressors. Animals with reduced number of crossings and rearings were recognized as depressed rats here (Han et al., 2010; Huang et al., 2007). 4.5.
Sucrose consumption test
Sucrose test was performed on the same day as the open-field test. Sucrose consumption was measured on days 1, 8, 15, and 22 after the beginning of the chronic stress paradigm. Animals were water deprived for 24 h before the sucrose consumption test, and were exposed to both the test solution (1% sucrose) and regular drinking water for a 1 h period. Sucrose consumption was evaluated by changes in the weight of the test solution bottle. Alterations in the consumption of sucrose represent a measure of anhedonia, or the inability to experience anhedonia which is a symptom of depression (Shalaev and Steponkus, 2001). 4.6.
37
levels in brain tissue, rats were sacrificed and the hippocampus was dissected. After weighing, the hippocampal tissue was flash frozen in liquid nitrogen and stored at −80 °C until use. Standard solutions of 5-HT, NE or DA (500 μg/mL) were prepared in 0.01 N HCl and were diluted to 1.25 μg/mL before use. Samples were supplemented with 10X n-butanol followed by shaking and centrifugation. After samples were heated in a 100 °C boiling water bath, the monoamine concentrations were determined by fluorescence spectrophotometry (RF-5310PC, Shimadzu Co., Japan). The concentration of 5-HT, NE or DA was calculated by the following formulas: Concentration of monoamine ðμg=LÞ Fsample −Fblank contents of standard 100 ¼ contents of sample ðFstandard −Fblank Þ 4.9.
Statistical analysis
Statistical analyses were performed using the SPSS 14.0 statistical software package. Data are presented as mean ± standard error of the mean (SEM). Results were analyzed using one-way ANOVA with Fisher's Least Significant Difference (LSD) test. P values < 0.05 were considered statistically significant.
Surgery and electrode implantation REFERENCES
Bipolar stimulating electrodes were made from pairs of Ni–Cr enamel-insulated wires with a 0.5 mm vertical tip separation. Rats were anesthetized with intraperitoneal injections of 10% chloral hydrate (0.3 ml/100 g) and placed in a stereotaxic apparatus (Narishige SR-6N, Japan). The scalp was retracted and a hole was drilled in the skull for the placement of the stimulating electrode into the LHb (anterior, 3.3 mm; lateral, 0.5 mm; ventral, 4.0 mm from Bregma). The electrode was secured to the skull with screws and dental acrylic cement. Penicillin (2 × 105 U/day) was injected intraperitoneally for three days post-operatively. Rats were given 5 mg/kg carprofen (q 12 h) as pain reliever by subcutaneous injection. The locus of DBS was proven by Nissl staining. Briefly, formalin-fixed, paraffin-embedded brain tissues were sectioned and stained with Nissl. Sections were mounted and evaluated via microscope. 4.7.
Electrical stimulation procedure
Rats in the DBS treatment groups were given high-frequency stimulation every day using an electrical stimulator (SEN3201, Nihon-Kohden Inc., Japan). Electrical stimulation, with 150 Hz square-wave pulses, was given at 80–100 μA for 0.3 ms. Each rat received high frequency stimulation for a duration of 30 min. Following 28 days of stimulation, animal behavior was monitored every 7 days. 4.8.
Determination of monoamine concentrations
Rats were sacrificed by cervical dislocation. Cardiac blood samples (1.5–2 ml collected from the left ventricle) were added to chilled heparin-containing tubes. After centrifugation at 2000 rpm for 10 min, the supernatants were stored at −20 °C until monoamine determinations. To evaluate monoamine
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