Accepted Manuscript Title: Lateral habenula as a link between dopaminergic and serotonergic systems contributes to depressive symptoms in Parkinson’s disease Author: Xiao Feng Luo Bei Lin Zhang Ji Cheng Li Ying Ying Yang Yan Fei Sun Hua Zhao PII: DOI: Reference:
S0361-9230(14)00189-0 http://dx.doi.org/doi:10.1016/j.brainresbull.2014.11.006 BRB 8811
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
28-8-2014 26-11-2014 29-11-2014
Please cite this article as: X.F. Luo, B.L. Zhang, J.C. Li, Y.Y. Yang, Y.F. Sun, H. Zhao, Lateral habenula as a link between dopaminergic and serotonergic systems contributes to depressive symptoms in Parkinson’s disease, Brain Research Bulletin (2014), http://dx.doi.org/10.1016/j.brainresbull.2014.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights 1. We examined whether LHb lesions improve depressive symptoms of PD rats by increasing 5-HT levels of RN
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2. LHb showed an obviously elevated cytochrome c oxidase in PD rats with
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depressive-like behavior
3. LHb lesions decreased the immobility time of FST in PD rats with depressive-like
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behavior
4. LHb lesions increased the climbing time of FST in PD rats with depressive-like
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behavior
5. LHb lesions increased the 5-HT levels of raphe nuclei in PD rats with
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depressive-like behavior
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Lateral habenula as a link between dopaminergic and serotonergic
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systems contributes to depressive symptoms in Parkinson’s disease
Xiao Feng Luo, Bei Lin Zhang, Ji Cheng Li, Ying Ying Yang, Yan Fei Sun and
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Hua Zhao*
Department of Physiology, Key Laboratory of Pathobiology, Ministry of
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Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China
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Xiao Feng Luo and Bei Lin Zhang contributed equally to this work.
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*Corresponding Author:
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Professor Hua Zhao, PhD
Abbraviations:Parkinson's disease (PD); Parkinson's disease with depressive-like behavior (PDD); The lateral habenula (LHb); substantia nigra (SN); horseradish peroxidase (HRP); forced swim test (FST); open-field test (OFT); cytochrome c oxidase (CCO); medial forebrain bundle (MFB); 5-hydroxytryptamine(5-HT); raphe nuclei(RN).
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Department of Physiology, College of Basic Medical Sciences, Jilin University, 126 Xinmin Street, Changchun 130021, P. R. China
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Telephone: +86(431)85619472
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E-mail: [email protected]
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Lateral habenula as a link between dopaminergic and serotonergic
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systems contributes to depressive symptoms in Parkinson’s disease
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Xiao Feng Luo, Bei Lin Zhang, Ji Cheng Li, Ying Ying Yang, Yan Fei
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Sun and Hua Zhao*
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Department of Physiology, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University,
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Changchun 130021, China
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Xiao Feng Luo and Bei Lin Zhang contributed equally to this work.
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*Corresponding Author:
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Professor Hua Zhao, PhD
Abbraviations:Parkinson's disease (PD); Parkinson's disease with depressive-like behavior (PDD); The lateral habenula (LHb); substantia nigra (SN); horseradish peroxidase (HRP); forced swim test (FST); open-field test (OFT); cytochrome c oxidase (CCO); medial forebrain bundle (MFB); 5-hydroxytryptamine(5-HT); raphe nuclei(RN).
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Department of Physiology, College of Basic Medical Sciences, Jilin University, 126 Xinmin Street, Changchun 130021, P. R. China
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Telephone: +86(431)85619472
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E-mail: [email protected]
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Abstract Degeneration of substantia nigra dopaminergic neurons is a key of Parkinson’s disease (PD), and its motor
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pathological change
consequences have been widely recognized. Recently, mood disorders
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associated with PD have begun to attract a great deal of interest, however,
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their pathogenesis remains unclear. PD is associated with not only degenerative changes in dopaminergic neurons in the substantia nigra but
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also changes in serotonergic neurons in the raphe nuclei. The abnormalities in central 5-hydroxytryptamine (5-HT) neurotransmission
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are thought to play a key role in the pathogenesis of depression. The
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lateral habenula (LHb) is closely related to the substantia nigra and raphe
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nuclei, and its hyperactivity is closely related to the pathogenesis of depression. In this study, we screened rats with
depressive-like
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behaviors from PD model animals and found that cytochrome c oxidase activity in the LHb of these rats was twice that seen in the control rats. In the forced swim test,
LHb lesions caused a decrease in depressive-like
behavior of PD rats as indexed by decreased immobility times and increased climbing times. Additionally, LHb lesions caused an enhance in
5-HT levels in the raphe nuclei. These results suggest that LHb lesions may improve depressive-like behavior in PD rats by increasing 5-HT levels in the raphe nuclei. Thus, LHb contributes to the depressive-like 3
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behavior in PD rats via mediating the effects of dopaminergic neurons in the substantia nigra on serotonergic neurons in the raphe nuclei.
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behavior; Raphe nuclei ; Substantia nigra; Serotonin
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Keywords: Lateral habenula; 6-hydropamine rat model; Depressive-like
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1. Introduction Parkinson’s disease (PD) is a degenerative disease of the central
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nervous system that is common in the elderly population. The motor symptoms of PD are well recognized and have been the focus of much
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research. However, the mood disorders observed in most PD patients,
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such as depression (Shulman et al., 2001; McDonald et al., 2003), should not be neglected because they seriously affect the quality of life and
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prognosis of PD patients; furthermore, they can be a direct cause of death
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in these patients (Carod-Artal et al., 2007; Rahman et al., 2008). Numerous studies have shown that movement disorders result from
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reductions in dopamine levels in the striatum, which are caused by
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degenerative pathological changes in midbrain substantia nigra pars
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compacta dopaminergic neurons. Therefore, the clinical application of levodopa can obviously improve movement disorders in PD patients (Zibetti et al., 2013). However, PD-related affective disorders are associated with not only disrupted dopamine systems but also nondopamine system changes, such as central 5-hydroxytryptamine (5-HT) system disorders (Scatton et al., 1983). It is widely known that a decreased 5-HT level in the raphe nuclei is related to the onset of depression (Stockmeier et al., 1998; Gray et al., 2013). Interestingly, Mayeux et al. (1984) have shown that 5-hydroxyindoleacetic acid levels 5
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in the cerebrospinal fluid of PD patients with depression were lower than those in the normal controls and nondepressed PD patients. In addition, serotonin reuptake inhibitors can markedly improve depressive-like
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symptoms in PD patients (Devos et al., 2008). Consequently, depression
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symptoms in PD patients may be the result of the interaction of the two systems. Recently, Bastide et al. (2013) have reported that immediate
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early gene levels, such as ΔFosB, change not only in basal ganglia
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structures but also in other brain areas of PD-model animals given levodopa to induce hyperactive behavior, including the lateral habenula
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(LHb). The structure may link the substantia nigra dopamine system with the raphe nucleus serotonin system, thus playing important roles in PD
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patients with depressive-like behavior (PDD). The LHb directly projects into the substantia nigra and raphe nucleus
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and it is involved in a wide range of functions related to the two structures, including cognitive and emotional functions, pain sensitivity and sleep and circadian rhythm regulation (Lecourtier et al., 2004; Shelton et al., 2012; Zhao and Rusak. 2005; Aizawa et al., 2013). In recent years, increasing attention has been paid to the role of the LHb in depression (Yang et al., 2008). It has been reported that LHb activity is enhanced in animals with depressive-like behaviors as well as in depressed patients (Caldecott-Hazard et al., 1988; Morris et al., 1999).
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Moreover, high-frequency deep-brain stimulation of the habenula to inhibit its activity was able to alleviate depressive symptoms in patients who could not be improved by antidepressants (Sartorius et al., 2010).
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Additionally, in animal models, LHb lesions were shown to improve
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depressive-like behavior by increasing raphe 5-HT levels (Yang et al.,
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2008).
To date, the relationships between the LHb and substantia nigra
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dopaminergic neurons, especially in reward processing, have been an area of focus as these two systems inhibit each other and act in distinct ways
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(Matsumoto et al., 2007). Lesions of the substantia nigra (SN) have been
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shown to increase LHb glucose use rates, whereas efferent signals from
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the medial globus pallidus to the LHb in 6-hydroxydopamine (6-OHDA)-induced PD animals were found to be increased (Wooten et al.,
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1981). Therefore, the LHb not only directly controls substantia-nigra and raphe-nuclei activities, but it also acts as a bridge for communication between the substantia nigra dopamine system and the raphe nuclei serotonin system. Accordingly, substantia nigra dopaminergic neuron losses in PD rats were hypothesized to reverse its inhibitory effect on LHb, thereby increasing LHb suppression on the raphe neurons and decreasing 5-HT levels in the raphe nuclei. In this study, the effects of LHb lesions on depressive-like behavior and the 5-HT concentration in
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the raphe nuclei in PD rats were observed to determine whether the hyperactivity of LHb is involved in the pathogenesis of depression associated with PD by enhancing its inhibition on raphe nuclei 5-HT
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neurons.
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2. Materials and methods
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2.1. Animals
Male Wistar rats (Department of Experimental Animals, Jilin
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University, Changchun, China) were used in this study. Rats were housed
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in a standard cage, with free access to food and water, in a
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temperature-controlled room (23±1°C) on a 12-h light/dark cycle (lights on at 8:00 am).
A total of 130 Wistar rats, 9 to 10 weeks of age and weighing 200-230g,
were used in this study. Of all these rats injected with 6-OHDA into brains, two died before the rotation test (a mortality rate of 1.5%). We obtained 61 PD rats from 128 rats injected with 6-OHDA and screened 26 rats with depression-like behavior from 61 PD rats by using the forced swimming test. Animals were randomly divided into four experimental
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groups: The control group (control, n = 10), PD with depressive-like behavior group (PDD, n = 7), PDD and electrical bilateral lesions of the LHb group (PDD+LHb lesion, n = 13), and PDD and sham lesions of the
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LHb group (PDD+LHb sham, n = 6).
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All procedures used in this study were conducted in accordance with
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the international standards for animal welfare and were approved by the local Committee for Animal Care Research at Jilin University. All efforts
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were made to minimize the number of animals used and their suffering.
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Apomorphine and 6-OHDA were obtained from Sigma (St. Louis, MO,
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USA).
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2.2. Animal model of PD
Animals were anesthetized with sodium pentobarbital (60 mg/kg,
intraperitoneally). To protect noradrenergic neurons, rats were pretreated with desipramine (25 mg/kg, intraperitoneally) 30 min before 6-OHDA was injected. The pretreated rats were fixed in a stereotaxic instrument, then the scalp was incised to expose the skull, and a single hole was drilled over the right medial forebrain bundle (MFB) (4.0 mm posterior to the bregma; 1.65 mm lateral to the midline; 8.0 mm ventral to the dura surface; Paxinos & Watson, 1998). Four microliters of 6-OHDA
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hydrochloride solution (4µg/µL) containing 0.2% ascorbic acid was injected into the right MFB with a microinjector. The microinjector was
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left in place for an additional 8 min before being slowly withdrawn. After the operation, the rats were monitored until they reached
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consciousness and then they were returned to their cages. Other than only
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being injected with the solvent, the sham-operated rats were treated in an identical manner to the model rats. The rats were submitted to a rotational
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test induced by apomorphine (0.1 mg/kg, intraperitoneally) at the end of the 4th week after the 6-OHDA injection. Only the rats that exhibited a
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contralateral rotation rate greater than 7/min were considered to have
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hemiparkinsonism, and these rats were selected for further study.
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2.3. Behavioral experiments
In this study, we used the forced swim test (FST) and open-field test (OFT) to assess depressive-like behavior in the PD model. The animals in the control and the PDD groups were submitted only once to the behavioral tests, and the other two groups (PDD+LHb lesion and PDD+LHb sham) were submitted twice to the behavioral tests. We did not find a significant difference between twice behavioral tests, which is consistent with the results in our previous experiments (Yang et al., 2008)
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and also the results reported by Armario (Armario et al,.1988). Since we had an additional PDD+LHb sham group as control, the repeated tests should not have affected the behavioral results.
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2.4. Forced swim test
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The rats were placed in a clear cylinder (20-cm internal diameter, 50
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cm high) filled with 24±1ºC water to a depth of 30 cm, according to a previous report (Porsolt et al., 1978). To avoid interference from feces
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and other floating objects, the water was changed after each experiment. A 15-min pretest followed by a 5-min test was conducted the next day.
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Only the times spent immobile and climbing during the 5-min test were
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measured to determine the behavioral score. A digital video camera was
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mounted above the cylinder, and recorded files were used for future analyses. After the pretest and test sessions, the animals were taken out of
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the water, dried with a paper towel, and returned to their cages.
2.5. Open-field test
According to a previous report (Katz et al., 1981), the animals were
placed in the center of an open-field chamber (100 cm × 100 cm × 50 cm) with the inside walls painted black. The chamber floor was divided by black lines into 25 equal squares to assess locomotion. Each rat was 1
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tested over 3 min. The movements of the rats in the open field were quantified by counting the number of crossings (all four paws in a square) and the number of rearings (lifting front legs ≥ 1 cm above the floor). The
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horizontal movement assessed by the number of crossings was considered
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as locomotor activity, whereas the vertical movement quantified by the
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number of rearings was deemed as exploratory behavior.
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2.6. LHb lesions
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After the behavioral experiments, the PDD rats were anesthetized with sodium pentobarbital intraperitoneally at a dose of 60 mg/kg. For the
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electrical lesion of the LHb, a direct current of 0.2 mA was applied for 30
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s through electrodes inserted into the LHb bilaterally (3.6–3.8 mm
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posterior to the bregma; 0.7 mm lateral to the midline; 4.5 mm ventral to the dura surface). Sham rats were treated as above except that no current was passed. Rats were placed in a recovery chamber until the anesthetic dissipated. The FST and OFT were again performed 7 days later to observe the influence of LHb damage on depressive-like behavior. Rats were decapitated under anesthesia with sodium pentobarbital given intraperitoneally, and each brain was removed and fixed in 10% formalin. The fixed brains were embedded in optimal cutting temperature
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compound. The brains were then cut into 30-μm-thick coronal cryostat sections, and the site of the LHb lesions was verified by crystal violet staining. The subjects that were shown to have complete lesions were
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included in the statistical analysis (Fig. 1), while the rats with failed
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lesions were discarded.
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2.7. Measurement of cytochrome c oxidase (CCO) activity
After all of the behavioral experiments, the rats were injected with an
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overdose of sodium pentobarbital and the LHb extracted from the brain tissue was removed. For the cytochrome c oxidase measurement, the
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tissue was placed in ice-cold buffer (10 mM Tris, 0.2 mM EDTA, and
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0.25 M sucrose; pH 7.6) and homogenized. The homogenates were
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centrifuged at 1500×g and 4°C for 10 min, and the pellet was discarded. Next, the supernatant was centrifuged at 8000×g for 30 min, and the new supernatant was discarded. The pellet was resuspended in the same buffer and centrifuged at 8000×g for 30 min again. Then, the new pellet was resuspended with enzyme dilution buffer (10 mM Tris, 0.2 mM EDTA, 0.25 M sucrose; pH 7.0). The CCO activity was analyzed using a CCO Assay Kit (Sigma, St. Louis, MO, USA), according to the manufacturer’s instructions. The CCO assay measures the decrease in absorbance at 550
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nm of ferrocytochrome c caused by its oxidation to ferricytochrome c by CCO; and one unit of CCO activity is defined as the oxidization of 1.0
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μmol of ferrocytochrome c per minute at pH 7.0 and 25°C.
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2.8. Neurochemical measurements
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Tissues were weighed and sonically disrupted in 10 volumes (v/w) of
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ice-cold 0.1 M HClO4, and homogenates were centrifuged at 13,000×g and 4°C for 40 min to remove cellular debris. A 10-μL aliquot of the
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supernatant was injected into a high-performance liquid chromatography (HPLC) column (Waters C18, Milford, MA, USA; 150 mm × 4.6 mm,
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particle size 5 µm) protected with a Brownlee precolumn (3 cm × 5 µm).
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The mobile phase for the elution (at a flow rate of 0.7 mL/min) consisted
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of 0.1 mM sodium acetate, 0.1 mM disodium EDTA, and 10% methanol, which was adjusted to pH 5.1 with glacial acetic acid. Serotonin was detected by fluorescence (HPLC/FD) at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. Absolute tissue concentrations of serotonin were determined by comparison with external standard curves and corrected for tissue weight.
2.9. Statistical analysis 1
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All data are presented as the mean ± standard error. Intergroup variation was measured by one-way analysis of variance, and the behavioral changes before and after LHb lesions were compared using
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paired t tests in the PDD+LHb lesion group. Statistical analysis was
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conducted using SPSS Statistical Software version 13.0. A P-value < 0.05
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indicated statistical significance.
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3. Results
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3.1. Effects of LHb lesions on depressive-like behavior in PD rats
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A total of 140 rats were used for all of the experiments, of which 130
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rats were injected with 6-OHDA unilaterally in the MFB and 10 rats were injected with saline as a control group. Four weeks after the injection, 61 PD rats showed a positive response to apomorphine in the rotation test. In the FST, only 26 of 61 PD rats (42.6%) exhibited depressive-like behavior. These rats were divided into PDD (n = 7), PDD+LHb lesion (n = 13), and PDD+LHb sham (n = 6) groups. Immobility and climbing times were measured in these three groups as well as the control group by using the FST. Rats in the PDD group showed significantly longer immobility times compared with those in the control group (P0.05) (Fig. 3A-B).
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Furthermore, the numbers of horizontal (n = 13, t = 10.6, P < 0.01) and
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vertical (n = 13, t = 4.6, P < 0.01) (Fig. 3C) movements were
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significantly different before and after the induction of LHb lesions in the PDD+LHb lesion group. These results suggest that LHb lesions
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significantly improved the exploratory behavior in PDD rats.
3.3. Changes of CCO activity in the LHb of PDD rats Differences in CCO activity between the control group and the PDD
group were compared. The COO activity in the PDD group (1.56 ± 0.05μmol/mg/min) was significantly increased compared with the control group (0.78 ± 0.08μmol/mg/min, P < 0.01, Fig. 4) suggesting that the 17
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activity of LHb neurons in the PDD rats was significantly increased.
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3.4. Effects of LHb lesions on raphe nuclei 5-HT content in PDD rats
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When the 5-HT levels in the raphe nuclei among the groups were compared, it was found that the 5-HT levels in the PDD group were
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significantly less than those in the control group (P < 0.01), whereas the
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5-HT levels in the PDD+LHb lesion group were significantly higher than those in the PDD group (P < 0.01). These results indicate that LHb
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lesions significantly elevated the 5-HT levels in the raphe nuclei. Similarly, the 5-HT levels in the PDD+LHb lesion group were
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significantly higher than those in the sham group (P < 0.01), whereas
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there was no significant difference between the PDD+LHb sham and
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PDD groups (P > 0.05) (Fig. 5).
4. Discussion
Mood disorders associated with PD are related to not only dysfunction of dopaminergic neurons in the substantia nigra but also to changes in 5-HT neurons in the raphe nuclei (Paulus et al., 1991). The LHb is one of 1
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the few areas in the brain that contributes to the regulation of both DAand 5-HT-containing neurons (Hikosaka et al., 2010) In the experiments described herein, the CCO activity in the LHb increased more in the PDD
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group compared with the control group. In addition, LHb lesions
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significantly improved depressive-like behaviors in PDD rats, as shown by the longer climbing times and shorter immobility times in the FST in
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the lesion group than in the sham group. Consistent with these changes in
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depressive-like behaviors, the lesion group showed significantly increased 5-HT levels in the raphe nuclei, indicating that the
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hyperactivity of the LHb contributes to the pathogenesis of PD-associated
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nuclei 5-HT neurons.
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depressive-like behavior by enhancing its inhibitory effect on the raphe
The classical PD model involves the injection of 6-OHDA directly into
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the MFB (Truong et al., 2006), and this model has been used extensively to study the pathogenesis of PD. Likewise, depressive-like behaviors in rats are commonly evaluated by the FST and OFT, which separately reflect core symptoms of depression with behavioral despair and decreased exploratory behaviors in a new environment. Our results are consistent with those reported by Tadaiesky et al. (2008), who showed that 40–50% of PD rats exhibited depressive-like behaviors, as indexed by prolonged immobility times and shortened climbing times in the FST
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as well as reduced horizontal and vertical activity in the OFT. The importance of the LHb in the pathogenesis of depression has been
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demonstrated in previous animal experiments from our group and in depressed clinical patients (Yang et al., 2008; Sartorius et al., 2010).
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Furthermore, the LHb has shown an obviously elevated metabolism in
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four different animal models of depression (Caldecott-Hazard et al., 1988; Shumake et al., 2003). Considering that CCO is an endogenous metabolic
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marker of neurons that plays a key role in oxidation in the mitochondria, is positively related to neuronal activities, and is commonly used to
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evaluate neuronal function (Wong-Riley, 1989), CCO activity was
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measured to determine the change in activity of the LHb when midbrain
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dopaminergic neurons were damaged in PD rats with depressive-like behavior. It was found that the CCO activity of the LHb in PDD rats was
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elevated, suggesting that PD also leads to functional changes in other areas of the brain outside of the basal ganglia and thus providing a basis for studying the role of the LHb in PD with depression. Moreover, McCulloch et al. (1980) found that the systemic administration of dopamine receptor agonists reduced the glucose use of the LHb, whereas antagonists increased this process. Similarly, Wooten et al. (1981) reported that substantia nigra lesions increased the glucose use of the LHb and that the administration of levodopa reversed this increased LHb
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glucose use (Trugman et al., 1991). Consistent with these results, our previous study indicated that single-pulse stimulation that was delivered to midbrain dopaminergic structures triggered transient inhibition of
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firing of LHb neurons (Shen et al., 2012). In recent years, increasing
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attention has been paid to functional connections between LHb and midbrain dopaminergic neurons in reward and motivation (Matsumoto et
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al., 2007) because they have different responses to reward processes. On
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the one hand, during reward presentation, substantia nigra dopaminergic neuron firing rates increase, followed by decreases in LHb neuron firing
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rates. On the other hand, during a lack of reward presentation or during aversive stimulation, LHb neurons are excited and substantia nigra
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dopaminergic neurons are inhibited. These results imply that these two
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areas influence each other in terms of function and may inhibit each other
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during distinct reward-related processes, which may be the reason that the LHb activity is increased when dopaminergic neurons are damaged in PD. In addition, during a lack of reward presentation, the medial globus pallidus is excited, which is considered to be an important structure in the control of LHb neuronal activity (Hong et al., 2008, 2013). As such, increased LHb activity may be due to indirect excitement of the globus pallidus, which is a consequence of dysfunction of substantia nigra dopaminergic neurons.
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In this study, coincident with improved depressive-like behavior, including increased climbing times and decreased immobility times in the FST in PD rats after the induction of LHb lesions, 5-HT levels in the
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raphe nuclei were higher than those in the control group. These results
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suggest that improvements in depressive-like behavior following LHb
lesion induction may be a result of increased 5-HT levels in the raphe
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nuclei. Currently, it is still widely believed that decreases in 5-HT levels (Jacobsen et al.,
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are associated with the pathogenesis of depression
2012; Tanabe et al., 2007). Moreover, 5-HT levels in plasma and
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cerebrospinal fluid are relatively low in PD patients with depression, suggesting that depressive-like behavior is related to functional changes
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in 5-HT systems (Mayeux., 1984, 1986). Furthermore, numerous studies
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have shown that the raphe nuclei is the main region involving 5-HT
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synthesis and release in the brain (Adell et al., 2002). For example, dorsal raphe nucleus lesions can significantly decrease brain 5-HT levels (Yalcin et al., 2008). Much research has reported that the LHb is an important region in regulating the activity of the raphe nuclei, since there are close anatomic and functional connections between these two areas (Aghajanian et al., 1977; Herkenham et al., 1979). Morphologically, injections of the retrograde neuronal tracer horseradish peroxidase (HRP) in the dorsal raphe nuclei labeled more somata in the LHb than in any
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other brain regions projecting to the dorsal raphe nuclei (Aghajanian et al., 1977). Functionally, electrical stimulation of the LHb inhibited firing of 5-HT neurons in the dorsal raphe nuclei thereby decreasing the release of
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5-HT (Stern et al., 1979). It is generally thought that glutamatergic
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neurons of the LHb inhibit the raphe nuclei activity through exciting
γ-aminobutyric acid interneurons in the dorsal raphe nucleus (Varga et al.,
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2003). Thus, hyperactivity of LHb in PD rats with depression likely
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increases the inhibitory output of the LHb-raphe pathway, which may be one cause of depressive-like behaviors in PD rats. Recently, Sourani et al.
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(2012) have reported similar results that the raphe nuclei is involved in the pathogenesis of PD with depression. However, in their research, PD
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rats were examined rather than PD rats with depressive-like behavior and
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the changes in the raphe nucleus were indirectly inferred by nuclear
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magnetic resonance methods. Moreover, the entire habenula complex was lesioned, rather than selective lesioning of the LHb. Of note, the medial habenula and LHb differ in many respects, including their ontogeny, cytoarchitecture, neurochemistry, efferent and afferent connectivity, and functional roles (Aizawa et al., 2012).
Conclusion
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The primary pathology of PD is dopaminergic neuron degeneration in
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the midbrain structures, and the main clinical symptom is dyskinesia. In this study, 42.6% of PD rats exhibited depressive-like behaviors and the
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activity of the LHb, a structure outside of the basal ganglia, was increased.
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In addition, the depressive-like behaviors of the PD rats were significantly improved after the induction of LHb lesions, and the
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mechanism underlying this recovery appeared to be related to increased 5-HT levels in the raphe nuclei. Thus, the LHb as a link between the
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dopaminergic and serotonergic systems contributes to depressive
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Conflict of interest
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symptoms with PD.
The authors declare that they have no duality or conflict of interest.
Acknowledgments
We thank Shao Jun Yang and Jin Yu Xiao for the assistance on the experiment. This study was supported by the National Natural Science Foundation of China (Nos, 30970956 and 91332117) and Jilin provincial 2
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Science & technology Department (No, 20090928).
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Figure legends
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Fig. 1. A crystal violet staning photomicrograph of lateral habenula lesion. Scale bar=200μm. LHb, Lateral habenula; MHb, medial habenula; PVP,
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posterior paraventricular nucleus of the thalamus; MD, mediodorsal
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third ventricle.
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nucleus of the thalamus; GrDG, granular layer dentate gyrus; D3V, dorsal
Fig. 2. Effects of lateral habenula lesions on the forced swimming test in
PD model rats with depressive-like behavior. A. The immobile time in four different animal groups. B. The climbing time in four different animal groups. C. The immobile time and climbling time changes before and after rat lateral habenula lesions in the PDD+LHb lesion group. Bars represent the mean value of immobile time and climbing time (±S.E.M). *p＜0.01. PDD, Parkinson's disease model rats with depressive-like
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behavior; LHb, Lateral habenula.
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Fig. 3. Effects of lateral habenula lesions on spontaneous locomotor
activity in PD model rats with depressive-like behavior. A. The number of
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crossing in four different animal groups. B. The number of rearing in four
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different animal groups. C.The number of crossing and the number of rearing changes before and after lateral habenula lesions in the PDD+LHb
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lesion group. Bars represent the mean value of crossing number and
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rearing number (±S.E.M). *p＜0.01. PDD, Parkinson's disease model
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rats with depressive-like behavior; LHb, Lateral habenula.
Fig. 4. Changes of cytochrome c oxidase activity in lateral habenula of
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PD model rats with depressive-like behavior. Bars represent the mean value of cytochrome c oxidase activity (±S.E.M). * p＜0.01. PDD, Parkinson's disease model rats with depressive-like behavior; LHb, Lateral habenula; CCO, Cytochrome c oxidase.
Fig. 5. Effects of lateral habenula lesions on 5-HT level in raphe nuclei of PD model rats with depressive-like behavior. Bars represent the mean 3
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value of 5-HT level (±S.E.M). *p＜0.01. PDD, Parkinson's disease model rats with depressive-like behavior; LHb, Lateral habenula; 5-HT,
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S0361-9230(14)00189-0 http://dx.doi.org/doi:10.1016/j.brainresbull.2014.11.006 BRB 8811
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
28-8-2014 26-11-2014 29-11-2014
Please cite this article as: X.F. Luo, B.L. Zhang, J.C. Li, Y.Y. Yang, Y.F. Sun, H. Zhao, Lateral habenula as a link between dopaminergic and serotonergic systems contributes to depressive symptoms in Parkinson’s disease, Brain Research Bulletin (2014), http://dx.doi.org/10.1016/j.brainresbull.2014.11.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights 1. We examined whether LHb lesions improve depressive symptoms of PD rats by increasing 5-HT levels of RN
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2. LHb showed an obviously elevated cytochrome c oxidase in PD rats with
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depressive-like behavior
3. LHb lesions decreased the immobility time of FST in PD rats with depressive-like
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behavior
4. LHb lesions increased the climbing time of FST in PD rats with depressive-like
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behavior
5. LHb lesions increased the 5-HT levels of raphe nuclei in PD rats with
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depressive-like behavior
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1
Lateral habenula as a link between dopaminergic and serotonergic
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systems contributes to depressive symptoms in Parkinson’s disease
Xiao Feng Luo, Bei Lin Zhang, Ji Cheng Li, Ying Ying Yang, Yan Fei Sun and
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Hua Zhao*
Department of Physiology, Key Laboratory of Pathobiology, Ministry of
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Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China
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Xiao Feng Luo and Bei Lin Zhang contributed equally to this work.
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*Corresponding Author:
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Professor Hua Zhao, PhD
Abbraviations:Parkinson's disease (PD); Parkinson's disease with depressive-like behavior (PDD); The lateral habenula (LHb); substantia nigra (SN); horseradish peroxidase (HRP); forced swim test (FST); open-field test (OFT); cytochrome c oxidase (CCO); medial forebrain bundle (MFB); 5-hydroxytryptamine(5-HT); raphe nuclei(RN).
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Department of Physiology, College of Basic Medical Sciences, Jilin University, 126 Xinmin Street, Changchun 130021, P. R. China
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Telephone: +86(431)85619472
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E-mail: [email protected]
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2
Lateral habenula as a link between dopaminergic and serotonergic
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systems contributes to depressive symptoms in Parkinson’s disease
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Xiao Feng Luo, Bei Lin Zhang, Ji Cheng Li, Ying Ying Yang, Yan Fei
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Sun and Hua Zhao*
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Department of Physiology, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University,
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Changchun 130021, China
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Xiao Feng Luo and Bei Lin Zhang contributed equally to this work.
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*Corresponding Author:
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Professor Hua Zhao, PhD
Abbraviations:Parkinson's disease (PD); Parkinson's disease with depressive-like behavior (PDD); The lateral habenula (LHb); substantia nigra (SN); horseradish peroxidase (HRP); forced swim test (FST); open-field test (OFT); cytochrome c oxidase (CCO); medial forebrain bundle (MFB); 5-hydroxytryptamine(5-HT); raphe nuclei(RN).
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Department of Physiology, College of Basic Medical Sciences, Jilin University, 126 Xinmin Street, Changchun 130021, P. R. China
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Telephone: +86(431)85619472
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E-mail: [email protected]
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Abstract Degeneration of substantia nigra dopaminergic neurons is a key of Parkinson’s disease (PD), and its motor
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pathological change
consequences have been widely recognized. Recently, mood disorders
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associated with PD have begun to attract a great deal of interest, however,
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their pathogenesis remains unclear. PD is associated with not only degenerative changes in dopaminergic neurons in the substantia nigra but
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also changes in serotonergic neurons in the raphe nuclei. The abnormalities in central 5-hydroxytryptamine (5-HT) neurotransmission
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are thought to play a key role in the pathogenesis of depression. The
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lateral habenula (LHb) is closely related to the substantia nigra and raphe
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nuclei, and its hyperactivity is closely related to the pathogenesis of depression. In this study, we screened rats with
depressive-like
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behaviors from PD model animals and found that cytochrome c oxidase activity in the LHb of these rats was twice that seen in the control rats. In the forced swim test,
LHb lesions caused a decrease in depressive-like
behavior of PD rats as indexed by decreased immobility times and increased climbing times. Additionally, LHb lesions caused an enhance in
5-HT levels in the raphe nuclei. These results suggest that LHb lesions may improve depressive-like behavior in PD rats by increasing 5-HT levels in the raphe nuclei. Thus, LHb contributes to the depressive-like 3
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behavior in PD rats via mediating the effects of dopaminergic neurons in the substantia nigra on serotonergic neurons in the raphe nuclei.
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behavior; Raphe nuclei ; Substantia nigra; Serotonin
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Keywords: Lateral habenula; 6-hydropamine rat model; Depressive-like
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1. Introduction Parkinson’s disease (PD) is a degenerative disease of the central
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nervous system that is common in the elderly population. The motor symptoms of PD are well recognized and have been the focus of much
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research. However, the mood disorders observed in most PD patients,
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such as depression (Shulman et al., 2001; McDonald et al., 2003), should not be neglected because they seriously affect the quality of life and
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prognosis of PD patients; furthermore, they can be a direct cause of death
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in these patients (Carod-Artal et al., 2007; Rahman et al., 2008). Numerous studies have shown that movement disorders result from
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reductions in dopamine levels in the striatum, which are caused by
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degenerative pathological changes in midbrain substantia nigra pars
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compacta dopaminergic neurons. Therefore, the clinical application of levodopa can obviously improve movement disorders in PD patients (Zibetti et al., 2013). However, PD-related affective disorders are associated with not only disrupted dopamine systems but also nondopamine system changes, such as central 5-hydroxytryptamine (5-HT) system disorders (Scatton et al., 1983). It is widely known that a decreased 5-HT level in the raphe nuclei is related to the onset of depression (Stockmeier et al., 1998; Gray et al., 2013). Interestingly, Mayeux et al. (1984) have shown that 5-hydroxyindoleacetic acid levels 5
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in the cerebrospinal fluid of PD patients with depression were lower than those in the normal controls and nondepressed PD patients. In addition, serotonin reuptake inhibitors can markedly improve depressive-like
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symptoms in PD patients (Devos et al., 2008). Consequently, depression
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symptoms in PD patients may be the result of the interaction of the two systems. Recently, Bastide et al. (2013) have reported that immediate
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early gene levels, such as ΔFosB, change not only in basal ganglia
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structures but also in other brain areas of PD-model animals given levodopa to induce hyperactive behavior, including the lateral habenula
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(LHb). The structure may link the substantia nigra dopamine system with the raphe nucleus serotonin system, thus playing important roles in PD
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patients with depressive-like behavior (PDD). The LHb directly projects into the substantia nigra and raphe nucleus
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and it is involved in a wide range of functions related to the two structures, including cognitive and emotional functions, pain sensitivity and sleep and circadian rhythm regulation (Lecourtier et al., 2004; Shelton et al., 2012; Zhao and Rusak. 2005; Aizawa et al., 2013). In recent years, increasing attention has been paid to the role of the LHb in depression (Yang et al., 2008). It has been reported that LHb activity is enhanced in animals with depressive-like behaviors as well as in depressed patients (Caldecott-Hazard et al., 1988; Morris et al., 1999).
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Moreover, high-frequency deep-brain stimulation of the habenula to inhibit its activity was able to alleviate depressive symptoms in patients who could not be improved by antidepressants (Sartorius et al., 2010).
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Additionally, in animal models, LHb lesions were shown to improve
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depressive-like behavior by increasing raphe 5-HT levels (Yang et al.,
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2008).
To date, the relationships between the LHb and substantia nigra
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dopaminergic neurons, especially in reward processing, have been an area of focus as these two systems inhibit each other and act in distinct ways
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(Matsumoto et al., 2007). Lesions of the substantia nigra (SN) have been
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shown to increase LHb glucose use rates, whereas efferent signals from
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the medial globus pallidus to the LHb in 6-hydroxydopamine (6-OHDA)-induced PD animals were found to be increased (Wooten et al.,
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1981). Therefore, the LHb not only directly controls substantia-nigra and raphe-nuclei activities, but it also acts as a bridge for communication between the substantia nigra dopamine system and the raphe nuclei serotonin system. Accordingly, substantia nigra dopaminergic neuron losses in PD rats were hypothesized to reverse its inhibitory effect on LHb, thereby increasing LHb suppression on the raphe neurons and decreasing 5-HT levels in the raphe nuclei. In this study, the effects of LHb lesions on depressive-like behavior and the 5-HT concentration in
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the raphe nuclei in PD rats were observed to determine whether the hyperactivity of LHb is involved in the pathogenesis of depression associated with PD by enhancing its inhibition on raphe nuclei 5-HT
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neurons.
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2. Materials and methods
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2.1. Animals
Male Wistar rats (Department of Experimental Animals, Jilin
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University, Changchun, China) were used in this study. Rats were housed
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in a standard cage, with free access to food and water, in a
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temperature-controlled room (23±1°C) on a 12-h light/dark cycle (lights on at 8:00 am).
A total of 130 Wistar rats, 9 to 10 weeks of age and weighing 200-230g,
were used in this study. Of all these rats injected with 6-OHDA into brains, two died before the rotation test (a mortality rate of 1.5%). We obtained 61 PD rats from 128 rats injected with 6-OHDA and screened 26 rats with depression-like behavior from 61 PD rats by using the forced swimming test. Animals were randomly divided into four experimental
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groups: The control group (control, n = 10), PD with depressive-like behavior group (PDD, n = 7), PDD and electrical bilateral lesions of the LHb group (PDD+LHb lesion, n = 13), and PDD and sham lesions of the
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LHb group (PDD+LHb sham, n = 6).
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All procedures used in this study were conducted in accordance with
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the international standards for animal welfare and were approved by the local Committee for Animal Care Research at Jilin University. All efforts
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were made to minimize the number of animals used and their suffering.
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Apomorphine and 6-OHDA were obtained from Sigma (St. Louis, MO,
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USA).
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2.2. Animal model of PD
Animals were anesthetized with sodium pentobarbital (60 mg/kg,
intraperitoneally). To protect noradrenergic neurons, rats were pretreated with desipramine (25 mg/kg, intraperitoneally) 30 min before 6-OHDA was injected. The pretreated rats were fixed in a stereotaxic instrument, then the scalp was incised to expose the skull, and a single hole was drilled over the right medial forebrain bundle (MFB) (4.0 mm posterior to the bregma; 1.65 mm lateral to the midline; 8.0 mm ventral to the dura surface; Paxinos & Watson, 1998). Four microliters of 6-OHDA
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hydrochloride solution (4µg/µL) containing 0.2% ascorbic acid was injected into the right MFB with a microinjector. The microinjector was
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left in place for an additional 8 min before being slowly withdrawn. After the operation, the rats were monitored until they reached
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consciousness and then they were returned to their cages. Other than only
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being injected with the solvent, the sham-operated rats were treated in an identical manner to the model rats. The rats were submitted to a rotational
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test induced by apomorphine (0.1 mg/kg, intraperitoneally) at the end of the 4th week after the 6-OHDA injection. Only the rats that exhibited a
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contralateral rotation rate greater than 7/min were considered to have
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hemiparkinsonism, and these rats were selected for further study.
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2.3. Behavioral experiments
In this study, we used the forced swim test (FST) and open-field test (OFT) to assess depressive-like behavior in the PD model. The animals in the control and the PDD groups were submitted only once to the behavioral tests, and the other two groups (PDD+LHb lesion and PDD+LHb sham) were submitted twice to the behavioral tests. We did not find a significant difference between twice behavioral tests, which is consistent with the results in our previous experiments (Yang et al., 2008)
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and also the results reported by Armario (Armario et al,.1988). Since we had an additional PDD+LHb sham group as control, the repeated tests should not have affected the behavioral results.
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2.4. Forced swim test
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The rats were placed in a clear cylinder (20-cm internal diameter, 50
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cm high) filled with 24±1ºC water to a depth of 30 cm, according to a previous report (Porsolt et al., 1978). To avoid interference from feces
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and other floating objects, the water was changed after each experiment. A 15-min pretest followed by a 5-min test was conducted the next day.
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Only the times spent immobile and climbing during the 5-min test were
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measured to determine the behavioral score. A digital video camera was
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mounted above the cylinder, and recorded files were used for future analyses. After the pretest and test sessions, the animals were taken out of
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the water, dried with a paper towel, and returned to their cages.
2.5. Open-field test
According to a previous report (Katz et al., 1981), the animals were
placed in the center of an open-field chamber (100 cm × 100 cm × 50 cm) with the inside walls painted black. The chamber floor was divided by black lines into 25 equal squares to assess locomotion. Each rat was 1
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tested over 3 min. The movements of the rats in the open field were quantified by counting the number of crossings (all four paws in a square) and the number of rearings (lifting front legs ≥ 1 cm above the floor). The
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horizontal movement assessed by the number of crossings was considered
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as locomotor activity, whereas the vertical movement quantified by the
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number of rearings was deemed as exploratory behavior.
an
2.6. LHb lesions
M
After the behavioral experiments, the PDD rats were anesthetized with sodium pentobarbital intraperitoneally at a dose of 60 mg/kg. For the
d
electrical lesion of the LHb, a direct current of 0.2 mA was applied for 30
te
s through electrodes inserted into the LHb bilaterally (3.6–3.8 mm
Ac ce p
posterior to the bregma; 0.7 mm lateral to the midline; 4.5 mm ventral to the dura surface). Sham rats were treated as above except that no current was passed. Rats were placed in a recovery chamber until the anesthetic dissipated. The FST and OFT were again performed 7 days later to observe the influence of LHb damage on depressive-like behavior. Rats were decapitated under anesthesia with sodium pentobarbital given intraperitoneally, and each brain was removed and fixed in 10% formalin. The fixed brains were embedded in optimal cutting temperature
1
Page 15 of 47
compound. The brains were then cut into 30-μm-thick coronal cryostat sections, and the site of the LHb lesions was verified by crystal violet staining. The subjects that were shown to have complete lesions were
ip t
included in the statistical analysis (Fig. 1), while the rats with failed
us
cr
lesions were discarded.
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2.7. Measurement of cytochrome c oxidase (CCO) activity
After all of the behavioral experiments, the rats were injected with an
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overdose of sodium pentobarbital and the LHb extracted from the brain tissue was removed. For the cytochrome c oxidase measurement, the
d
tissue was placed in ice-cold buffer (10 mM Tris, 0.2 mM EDTA, and
te
0.25 M sucrose; pH 7.6) and homogenized. The homogenates were
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centrifuged at 1500×g and 4°C for 10 min, and the pellet was discarded. Next, the supernatant was centrifuged at 8000×g for 30 min, and the new supernatant was discarded. The pellet was resuspended in the same buffer and centrifuged at 8000×g for 30 min again. Then, the new pellet was resuspended with enzyme dilution buffer (10 mM Tris, 0.2 mM EDTA, 0.25 M sucrose; pH 7.0). The CCO activity was analyzed using a CCO Assay Kit (Sigma, St. Louis, MO, USA), according to the manufacturer’s instructions. The CCO assay measures the decrease in absorbance at 550
1
Page 16 of 47
nm of ferrocytochrome c caused by its oxidation to ferricytochrome c by CCO; and one unit of CCO activity is defined as the oxidization of 1.0
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μmol of ferrocytochrome c per minute at pH 7.0 and 25°C.
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2.8. Neurochemical measurements
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Tissues were weighed and sonically disrupted in 10 volumes (v/w) of
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ice-cold 0.1 M HClO4, and homogenates were centrifuged at 13,000×g and 4°C for 40 min to remove cellular debris. A 10-μL aliquot of the
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supernatant was injected into a high-performance liquid chromatography (HPLC) column (Waters C18, Milford, MA, USA; 150 mm × 4.6 mm,
d
particle size 5 µm) protected with a Brownlee precolumn (3 cm × 5 µm).
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The mobile phase for the elution (at a flow rate of 0.7 mL/min) consisted
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of 0.1 mM sodium acetate, 0.1 mM disodium EDTA, and 10% methanol, which was adjusted to pH 5.1 with glacial acetic acid. Serotonin was detected by fluorescence (HPLC/FD) at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. Absolute tissue concentrations of serotonin were determined by comparison with external standard curves and corrected for tissue weight.
2.9. Statistical analysis 1
Page 17 of 47
All data are presented as the mean ± standard error. Intergroup variation was measured by one-way analysis of variance, and the behavioral changes before and after LHb lesions were compared using
ip t
paired t tests in the PDD+LHb lesion group. Statistical analysis was
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conducted using SPSS Statistical Software version 13.0. A P-value < 0.05
an
us
indicated statistical significance.
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3. Results
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3.1. Effects of LHb lesions on depressive-like behavior in PD rats
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A total of 140 rats were used for all of the experiments, of which 130
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rats were injected with 6-OHDA unilaterally in the MFB and 10 rats were injected with saline as a control group. Four weeks after the injection, 61 PD rats showed a positive response to apomorphine in the rotation test. In the FST, only 26 of 61 PD rats (42.6%) exhibited depressive-like behavior. These rats were divided into PDD (n = 7), PDD+LHb lesion (n = 13), and PDD+LHb sham (n = 6) groups. Immobility and climbing times were measured in these three groups as well as the control group by using the FST. Rats in the PDD group showed significantly longer immobility times compared with those in the control group (P0.05) (Fig. 3A-B).
M
Furthermore, the numbers of horizontal (n = 13, t = 10.6, P < 0.01) and
d
vertical (n = 13, t = 4.6, P < 0.01) (Fig. 3C) movements were
te
significantly different before and after the induction of LHb lesions in the PDD+LHb lesion group. These results suggest that LHb lesions
Ac ce p
significantly improved the exploratory behavior in PDD rats.
3.3. Changes of CCO activity in the LHb of PDD rats Differences in CCO activity between the control group and the PDD
group were compared. The COO activity in the PDD group (1.56 ± 0.05μmol/mg/min) was significantly increased compared with the control group (0.78 ± 0.08μmol/mg/min, P < 0.01, Fig. 4) suggesting that the 17
Page 20 of 47
activity of LHb neurons in the PDD rats was significantly increased.
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3.4. Effects of LHb lesions on raphe nuclei 5-HT content in PDD rats
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When the 5-HT levels in the raphe nuclei among the groups were compared, it was found that the 5-HT levels in the PDD group were
us
significantly less than those in the control group (P < 0.01), whereas the
an
5-HT levels in the PDD+LHb lesion group were significantly higher than those in the PDD group (P < 0.01). These results indicate that LHb
M
lesions significantly elevated the 5-HT levels in the raphe nuclei. Similarly, the 5-HT levels in the PDD+LHb lesion group were
d
significantly higher than those in the sham group (P < 0.01), whereas
te
there was no significant difference between the PDD+LHb sham and
Ac ce p
PDD groups (P > 0.05) (Fig. 5).
4. Discussion
Mood disorders associated with PD are related to not only dysfunction of dopaminergic neurons in the substantia nigra but also to changes in 5-HT neurons in the raphe nuclei (Paulus et al., 1991). The LHb is one of 1
Page 21 of 47
the few areas in the brain that contributes to the regulation of both DAand 5-HT-containing neurons (Hikosaka et al., 2010) In the experiments described herein, the CCO activity in the LHb increased more in the PDD
ip t
group compared with the control group. In addition, LHb lesions
cr
significantly improved depressive-like behaviors in PDD rats, as shown by the longer climbing times and shorter immobility times in the FST in
us
the lesion group than in the sham group. Consistent with these changes in
an
depressive-like behaviors, the lesion group showed significantly increased 5-HT levels in the raphe nuclei, indicating that the
M
hyperactivity of the LHb contributes to the pathogenesis of PD-associated
te
nuclei 5-HT neurons.
d
depressive-like behavior by enhancing its inhibitory effect on the raphe
The classical PD model involves the injection of 6-OHDA directly into
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the MFB (Truong et al., 2006), and this model has been used extensively to study the pathogenesis of PD. Likewise, depressive-like behaviors in rats are commonly evaluated by the FST and OFT, which separately reflect core symptoms of depression with behavioral despair and decreased exploratory behaviors in a new environment. Our results are consistent with those reported by Tadaiesky et al. (2008), who showed that 40–50% of PD rats exhibited depressive-like behaviors, as indexed by prolonged immobility times and shortened climbing times in the FST
1
Page 22 of 47
as well as reduced horizontal and vertical activity in the OFT. The importance of the LHb in the pathogenesis of depression has been
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demonstrated in previous animal experiments from our group and in depressed clinical patients (Yang et al., 2008; Sartorius et al., 2010).
cr
Furthermore, the LHb has shown an obviously elevated metabolism in
us
four different animal models of depression (Caldecott-Hazard et al., 1988; Shumake et al., 2003). Considering that CCO is an endogenous metabolic
an
marker of neurons that plays a key role in oxidation in the mitochondria, is positively related to neuronal activities, and is commonly used to
M
evaluate neuronal function (Wong-Riley, 1989), CCO activity was
d
measured to determine the change in activity of the LHb when midbrain
te
dopaminergic neurons were damaged in PD rats with depressive-like behavior. It was found that the CCO activity of the LHb in PDD rats was
Ac ce p
elevated, suggesting that PD also leads to functional changes in other areas of the brain outside of the basal ganglia and thus providing a basis for studying the role of the LHb in PD with depression. Moreover, McCulloch et al. (1980) found that the systemic administration of dopamine receptor agonists reduced the glucose use of the LHb, whereas antagonists increased this process. Similarly, Wooten et al. (1981) reported that substantia nigra lesions increased the glucose use of the LHb and that the administration of levodopa reversed this increased LHb
2
Page 23 of 47
glucose use (Trugman et al., 1991). Consistent with these results, our previous study indicated that single-pulse stimulation that was delivered to midbrain dopaminergic structures triggered transient inhibition of
ip t
firing of LHb neurons (Shen et al., 2012). In recent years, increasing
cr
attention has been paid to functional connections between LHb and midbrain dopaminergic neurons in reward and motivation (Matsumoto et
us
al., 2007) because they have different responses to reward processes. On
an
the one hand, during reward presentation, substantia nigra dopaminergic neuron firing rates increase, followed by decreases in LHb neuron firing
M
rates. On the other hand, during a lack of reward presentation or during aversive stimulation, LHb neurons are excited and substantia nigra
d
dopaminergic neurons are inhibited. These results imply that these two
te
areas influence each other in terms of function and may inhibit each other
Ac ce p
during distinct reward-related processes, which may be the reason that the LHb activity is increased when dopaminergic neurons are damaged in PD. In addition, during a lack of reward presentation, the medial globus pallidus is excited, which is considered to be an important structure in the control of LHb neuronal activity (Hong et al., 2008, 2013). As such, increased LHb activity may be due to indirect excitement of the globus pallidus, which is a consequence of dysfunction of substantia nigra dopaminergic neurons.
2
Page 24 of 47
In this study, coincident with improved depressive-like behavior, including increased climbing times and decreased immobility times in the FST in PD rats after the induction of LHb lesions, 5-HT levels in the
ip t
raphe nuclei were higher than those in the control group. These results
cr
suggest that improvements in depressive-like behavior following LHb
lesion induction may be a result of increased 5-HT levels in the raphe
us
nuclei. Currently, it is still widely believed that decreases in 5-HT levels (Jacobsen et al.,
an
are associated with the pathogenesis of depression
2012; Tanabe et al., 2007). Moreover, 5-HT levels in plasma and
M
cerebrospinal fluid are relatively low in PD patients with depression, suggesting that depressive-like behavior is related to functional changes
d
in 5-HT systems (Mayeux., 1984, 1986). Furthermore, numerous studies
te
have shown that the raphe nuclei is the main region involving 5-HT
Ac ce p
synthesis and release in the brain (Adell et al., 2002). For example, dorsal raphe nucleus lesions can significantly decrease brain 5-HT levels (Yalcin et al., 2008). Much research has reported that the LHb is an important region in regulating the activity of the raphe nuclei, since there are close anatomic and functional connections between these two areas (Aghajanian et al., 1977; Herkenham et al., 1979). Morphologically, injections of the retrograde neuronal tracer horseradish peroxidase (HRP) in the dorsal raphe nuclei labeled more somata in the LHb than in any
2
Page 25 of 47
other brain regions projecting to the dorsal raphe nuclei (Aghajanian et al., 1977). Functionally, electrical stimulation of the LHb inhibited firing of 5-HT neurons in the dorsal raphe nuclei thereby decreasing the release of
ip t
5-HT (Stern et al., 1979). It is generally thought that glutamatergic
cr
neurons of the LHb inhibit the raphe nuclei activity through exciting
γ-aminobutyric acid interneurons in the dorsal raphe nucleus (Varga et al.,
us
2003). Thus, hyperactivity of LHb in PD rats with depression likely
an
increases the inhibitory output of the LHb-raphe pathway, which may be one cause of depressive-like behaviors in PD rats. Recently, Sourani et al.
M
(2012) have reported similar results that the raphe nuclei is involved in the pathogenesis of PD with depression. However, in their research, PD
d
rats were examined rather than PD rats with depressive-like behavior and
te
the changes in the raphe nucleus were indirectly inferred by nuclear
Ac ce p
magnetic resonance methods. Moreover, the entire habenula complex was lesioned, rather than selective lesioning of the LHb. Of note, the medial habenula and LHb differ in many respects, including their ontogeny, cytoarchitecture, neurochemistry, efferent and afferent connectivity, and functional roles (Aizawa et al., 2012).
Conclusion
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Page 26 of 47
The primary pathology of PD is dopaminergic neuron degeneration in
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the midbrain structures, and the main clinical symptom is dyskinesia. In this study, 42.6% of PD rats exhibited depressive-like behaviors and the
cr
activity of the LHb, a structure outside of the basal ganglia, was increased.
us
In addition, the depressive-like behaviors of the PD rats were significantly improved after the induction of LHb lesions, and the
an
mechanism underlying this recovery appeared to be related to increased 5-HT levels in the raphe nuclei. Thus, the LHb as a link between the
M
dopaminergic and serotonergic systems contributes to depressive
Ac ce p
Conflict of interest
te
d
symptoms with PD.
The authors declare that they have no duality or conflict of interest.
Acknowledgments
We thank Shao Jun Yang and Jin Yu Xiao for the assistance on the experiment. This study was supported by the National Natural Science Foundation of China (Nos, 30970956 and 91332117) and Jilin provincial 2
Page 27 of 47
Science & technology Department (No, 20090928).
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Figure legends
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Fig. 1. A crystal violet staning photomicrograph of lateral habenula lesion. Scale bar=200μm. LHb, Lateral habenula; MHb, medial habenula; PVP,
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posterior paraventricular nucleus of the thalamus; MD, mediodorsal
Ac ce p
te
third ventricle.
d
nucleus of the thalamus; GrDG, granular layer dentate gyrus; D3V, dorsal
Fig. 2. Effects of lateral habenula lesions on the forced swimming test in
PD model rats with depressive-like behavior. A. The immobile time in four different animal groups. B. The climbing time in four different animal groups. C. The immobile time and climbling time changes before and after rat lateral habenula lesions in the PDD+LHb lesion group. Bars represent the mean value of immobile time and climbing time (±S.E.M). *p＜0.01. PDD, Parkinson's disease model rats with depressive-like
3
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behavior; LHb, Lateral habenula.
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Fig. 3. Effects of lateral habenula lesions on spontaneous locomotor
activity in PD model rats with depressive-like behavior. A. The number of
cr
crossing in four different animal groups. B. The number of rearing in four
us
different animal groups. C.The number of crossing and the number of rearing changes before and after lateral habenula lesions in the PDD+LHb
an
lesion group. Bars represent the mean value of crossing number and
M
rearing number (±S.E.M). *p＜0.01. PDD, Parkinson's disease model
te
d
rats with depressive-like behavior; LHb, Lateral habenula.
Fig. 4. Changes of cytochrome c oxidase activity in lateral habenula of
Ac ce p
PD model rats with depressive-like behavior. Bars represent the mean value of cytochrome c oxidase activity (±S.E.M). * p＜0.01. PDD, Parkinson's disease model rats with depressive-like behavior; LHb, Lateral habenula; CCO, Cytochrome c oxidase.
Fig. 5. Effects of lateral habenula lesions on 5-HT level in raphe nuclei of PD model rats with depressive-like behavior. Bars represent the mean 3
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value of 5-HT level (±S.E.M). *p＜0.01. PDD, Parkinson's disease model rats with depressive-like behavior; LHb, Lateral habenula; 5-HT,
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