This rodent study explores the effects of harmine treatment on chronic unpredictable stress (CUS)-induced depressive-like behaviors and astrocytic dysfunctions. The results demonstrated that the development of depression is critically contributed by astrocytic dysfunction as a potential mechanism and harmine induces antidepressant-like effects likely via restoration of the said astrocytic functions.
Abstract
“Depression is a world-wide disease with no effective therapeutic methods. Increasing evidence indicates that astrocytic pathology contributes to the formation of depression. In this study, we investigated the effects of harmine, a natural β-carboline alkaloid and potent hallucinogen, known to modulate astrocytic glutamate transporters, on chronic unpredictable stress (CUS)-induced depressive-like behaviors and astrocytic dysfunctions. Results showed that harmine treatment (10, 20 mg/kg) protected the mice against the CUS-induced increases in the immobile time in the tail suspension test (TST) and forced swimming test (FST), and also reversed the reduction in sucrose intake in the sucrose preference experiment. Harmine treatment (20 mg/kg) prevented the reductions in brain-derived neurotrophic factor (BDNF) protein levels and hippocampal neurogenesis induced by CUS. In addition, harmine treatment (20 mg/kg) increased the protein expression levels of glutamate transporter 1 (GLT-1) and prevented the CUS-induced decreases in glial fibrillary acidic protein (GFAP) protein expressions in the prefrontal cortex and hippocampus, suggesting that restoration of astrocytic functions may be a potential mechanism underlying the antidepressant-like effects of harmine. This opinion was proved by the results that administration of mice with l-Alpha-Aminoadipic Acid (L-AAA), a gliotoxin specific for astrocytes, attenuated the antidepressant-like effects of harmine, and prevented the improvement effects of harmine on BDNF protein levels and hippocampal neurogenesis. These results provide further evidence to confirm that astrocytic dysfunction contributes critically to the development of depression and that harmine exerts antidepressant-like effects likely through restoration of astrocytic functions.”
Authors: Fengguo Liu, Jingjing Wu, Yu Gong, Peng Wang, Lei Zhu, Lijuan, Tong, Xiangfan Chen, Yong Ling & ChaoHuang
Summary
Tong, Xiangfan Chen, Yong Ling, Chao Huang,* Department of Neurology, Danyang People’s Hospital, Department of Cardiology, Suzhou Kowloon Hospital of Shanghai Jiaotong University School of Medicine, #118 Wansheng Street, Suzhou 215021, Jiangsu, China
ALS, CUS, doublecortin, FITC, fluorescein isothiocyanate, GAPDH, glial fibrillary acidic protein, GLAST, glutamate transporter- 1, GLT- 1, glutamine synthetase, L- AAA, tail suspension test.
Depression is a world- wide disease with no effective therapeutic methods. Harmine, a natural carboline alkaloid, can modulate astrocytic glutamate transporters. Harmine treatment prevented depression-like effects in mice by increasing glutamate transporter 1 (GLT- 1) and preventing depression-like effects in mice by decreasing BDNF protein levels and hippocampal neurogenesis. These results suggest that restoration of astrocytic functions may be a potential mechanism underlying the antidepressant- like effects of harmine.
Major depression is a common disease affecting numerous persons in the world- wide. Traditional antidepressants exhibit numerous limitations, and novel mechanism- based antidepressants are necessary to improve the present status of drug therapy of depression.
Decreased numbers of astrocytes in the brain have been observed in rodents treated with chronic stresses or maternal deprivation, and in post- mortem studies of tissues from depressed patients. This suggests that inhibition of astrocyte dysfunction may be a potential strategy for depression therapy.
Harmine is a hallucinogenic alkaloid found in the seed of Peganum harmala and Banisteriopsis caapi. It has been shown to have antidepressant-like effects in acute and chronic depression models, and may also have an inverse-agonistic mechanism located in the benzodiazepine receptors. Harmine was shown to increase GLT- 1 gene and protein expression and glutamate uptake activity in animal models of ALS and cerebral ischemia, suggesting that harmine may exert neuroprotective effects via enhancement of GLT- 1 functions.
Mice were housed five per cage under standard conditions for 1 week with free access to food and water. Behavioral experiments were conducted during the light phase.
Harmine, fluoxetine and L- Alpha- Aminoadipic Acid were administered intraperitoneally and intracerebroventricularly, respectively, in a volume of 10 mL/kg. Antibodies against doublecortin, GFAP, GS, GLAST, and GAPDH were purchased from Cell Signaling Technology.
The mice were placed in a glass cylinder filled with water at 25°C for 6 min or suspended 50 cm above the floor for 6 min. The immobile time was recorded during the last 4 min by an investigator blind to the study.
C57BL/6J mice were subjected to a combination of stressors for 5 weeks, which included cage shaking, lights on during the night, cold room, mild restraint, 45° cage tilt, wet cage, and noise in the room.
At day 36, mice were given the choice to drink from two bottles, one with 1% sucrose solution and the other with water. The position of two bottles was changed every 6 h to prevent side preference. In this experiment, mice were anaesthetized with pentobarbital sodium and placed in a stereotaxic frame. L-AAA was injected into the left lateral brain ventricle using an osmotic minipump and the speed of injection was 1 L/ h.
Hippocampal and prefrontal cortical tissues were dissected and stored at – 70°C. BDNF protein levels were measured by anti-BDNF sandwich-ELISA according to manufacturer’s protocols, and a direct correlation between Optical Density and BDNF protein concentrations was demonstrated. Brain tissues were dissected and homogenized in lyses buffer containing 50 mM Tris – HC l (pH 7.4), 1 mM EDTA, 100 mM NaCl, 20 mM NaF, 3 mM Na 3VO 4, 1 mM PMSF, 1% NP- 40 and protease inhibitor cocktail.
Animals were anesthetized, perfused with saline and paraformaldehyde, and their brains were removed, frozen, and sectioned at 25 m. The sections were then incubated with goat anti- DC X antibody and FITC- labeled horse anti- rabbit IgG for 2 h at room temperature.
All analyses were performed using SPSS 13.0 software. ANOVA and Bonferroni’s post hoc test were used to assess isolated comparisons.
In this study, we used a widely-used animal model of depression, CUS, to detect the antidepressant-like activities of harmine. The results showed that harmine reduced the immobile time of mice in the FST, similar to fluoxetine treatment.
Stress and drug treatment affected the immobile time of mice in the TST, but not stress-drug treatment interaction. Harmine treatment reduced the immobile time of naive mice in the TST.
Harmine attenuates the CUS-induced reduction s in BDNF protein levels and hippocampal neurogenesis, suggesting that harmine may be used to treat depression.
Adult normal mice were received daily injections of harmine (10, 20 mg/kg) for 10 days, and the protein levels of BDNF in their hippocampus and prefrontal cortex were detected. The treatment of harmine increased the protein level of BDNF in the hippocampus.
Stress and drug treatment affected the protein level of BDNF in the prefrontal cortex of mice, but not stress – drug treatment interaction. Chronic harmine treatment increased the protein level of BDNF in naive mice.
Since stress and BDNF can modulate hippocampal neurogenesis, we explored whether harmine affects hippocampal neurogenesis. We found that stress and drug treatment affected DCX protein expression levels, but not stress – treatment interaction. CUS decreased the number of DCX positive cells in the hippocampus and the expression level of DCX protein. Harmine substantially reversed these effects. We investigated the effects of C US and/or harmine on astrocyte-associated markers in mice hippocampus and prefrontal cortex. We found that GFAP and GLT- 1 expressions were altered, but not by stress or stress- drug treatment interaction. Two- way ANOVA revealed no significant effects for stress, drug treatment and stress – drug treatment interaction for hippocampal GS and GLAST.
The effects of drug treatment and stress were significant, but the effects of stress – drug treatment interaction were not. Stress, drug treatment and stress-drug treatment interaction did not affect prefrontal cortical GS and GLAST expression levels. Chronic harmine treatment prevented the CUS-induced decreases in GFAP protein expressions in the hippocampus and prefrontal cortex, demonstrating that the architecture and/or function of astrocytes may be altered by CUS and that chronic harmine treatment may attenuate glutamatergic hyperactivity.
To investigate the potential role of astrocytes in the antidepressant-like effects of harmine, mice were co-injected with harmine and L- AAA for 10 days. The results showed that harmine prevented the CUS-induced increases in immobile time and decrease in sucrose intake. For FST and TST, stress and drug treatment had significant effects, but not for stress-drug treatment interaction. For sucrose preference, stress and drug treatment had significant effects, but not for stress-drug treatment interaction.
L- AAA co-administration abolished the improvement effect of harmine on brain BDNF protein levels and hippocampal neurogenesis in stressed mice, but not on stress – drug treatment interaction.
Further studies showed that the astrocyte was essential for the effect of harmine on hippocampal neurogenesis, and that L- AAA co- administration blocked the increases in hippocampal neurogenesis and DCX protein expressions in harmine- treated mice.
The major contribution of this study is the identification of the antidepressant- like effects of harmine, which have been reported in previous studies. However, the stress environment helps evaluate the ir antidepressant- like activities properly.
Harmine, a compound found in ayahuasca, has been found to have antidepressant-like effects. In this study, chronic daily injections of harmine (10 days) improved the behavioral impairments induced by CUS, and the antidepressant-like effects of harmine were similar to that of fluoxetine. Ayahuasca, a harmine-containing plant, has been shown to produce an antidepressant-like effect in depressed patients, establishing stronger correlations between harmine and depression. However, because ayahuasca also contains other substances, such as dimethyltryptamine, evaluation of the correlation between harmine and depression should be cautious.
We showed that chronic stress impaired hippocampal neurogenesis, and harmine treatment markedly reversed this impairment. Hippocampal BDNF levels were reduced in depressed patients, and harmine may be able to regulate depressive-like behaviors via activation of BDNF signals.
Numerous studies employing depressed patients and animals show that astrocyte pathology mediates the pathogenesis of depression. The integrity of hippocampal astroglial plasticity mediates the antidepressant- like effects of some clinical antidepressants, and the rapid antidepressant- like effect of ketamine correlates astroglial plasticity.
CUS induces the brain incapable of clearing extracellular glutamate, which results in an altered ratio of synaptic to extra- synaptic glutamate content and altered neurotransmission. The regulating effect of harmine on GFAP expressions indicates that astrocytes may be involved in the antidepressant- like effect s of harmine. Harmine, a compound that increases glutamate transport, is a potential candidate for the treatment of depression. It has been shown to improve depression-like behaviors in several studies.
The involvement of brain astrocytes in the antidepressant-like effects of harmine was further ascertained by the astrocyte inhibition experiment, which showed that the inhibition of astrocytic functions prevented the amelioration effects of harmine on depressive- like behaviors. This finding would help understand the in-depth mechanism of action of harmine in major depression. Reactive astrocytes can produce the inhibitory gliotransmitter GABA, which reduces spike probability of granule cells by acting on presynaptic GABA receptors. BDNF may be necessary for the regulation of depressive- like behaviors by endogenous astrocytes, as shown by the blockade of harmine-induced increases in hippocampal and prefrontal cortical BDNF protein levels by L- AAA treatment.
Previous studies have shown that BDNF promotes local neurogenesis and anxiolytic activities in hippocampal astrocytes, and thus improves depressive-like behaviors and hippocampal neurogenesis.
Harmine, a natural-derived drug reported to increase GLT- 1 expression, reversed the behavioral deficits induced by CUS, and restored BDNF protein levels and hippocampal neurogenesis, supporting the hypothesis that astrocyte dysfunction contributes critically to the development of depression.
This work was supported by the Natural Science Foundation of China and the Science and Technology Project of Nantong City. All experiments were conducted according to ethical standards.
Harmine (10 or 20 mg/kg) inhibited CUS-induced behavioral abnormalities, including increased immobile time, decreased sucrose intake, and decreases in BDNF protein levels, in both the hippocampus and prefrontal cortex of mice.
A study using harmine (20 mg/kg) showed that it restored hippocampal DC X+ cells and DCX protein expressions in rats after chronic ethanol stimulation (CUS).
The effects of harmine (20 mg/kg) on GFAP, GLT-1, GS, and GLAST protein expressions in the hippocampus and prefrontal cortex were examined with and without CUS treatment. L- AAA attenuated the restoration effect of harmine (20 mg/kg) on CUS-induced behavioral abnormalities.
CUS-induced increases in immobile time and TST were attenuated by L- AAA co-administration. This attenuated the decrease in sucrose intake that was induced by CUS.
L- AAA, a specific inhibitor of astrocytic functions, prevented the decrease of BDNF protein levels and hippocampal neurogenesis in mice hippocampus and prefrontal cortex following CUS treatment.