Correspondence address: HaoweiShen Department of Psychiatry, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Email: email@example.com
Abstract The antidepressive actions of electroconvulsive shock (ECS) therapy are considered to involve altered neurotransmission of serotonin. In this study, we investigated the effects of acute and chronic ECS on 5-hydroxytryptamine (5-HT) transporter mRNA expression in rat raphe nucleus. We found that serotonin transporter (5-HTT) mRNA expression was decreased in 9 and 24 h after acute ECS and in 3, 9, 24 h and 2 weeks after chronic ECS in rat raphe nucleus. We presume that the adaptive change in 5-HTT mRNA expression is possibly related to the therapeutic efficacy of electroconvulsive therapy (ECT) on medication-resistant depression.
The antidepressive actions of electroconvulsive shock (ECS) therapy are considered to involve enhanced neurotransmission of serotonin (5-hydroxytryptamine, 5-HT).1,2 After its release and action on a receptor, 5-HT is reabsorbed into presynaptic terminals by serotonin transporter (5-HTT) and 5-HT neurotransmission will be subsequently terminated. Therefore, 5-HTT possibly plays an important role in maintaining a steady state of enhanced 5-HT neurotransmission after ECS. However, the results of previous studies concerning the effects of ECS on 5-HTT binding sites are inconsistent. Hayakawa et al.3 reported that single and repeated ECS increased 5-HTT binding sites in rat frontal cortex, and other studies demonstrated no change in 5-HTT binding sites.4,5
Although 5-HTT protein is widely distributed throughout the rat brain, with the highest density in the forebrain region, 5-HTT mRNA has been localized exclusively in serotonergic neuron of raphe nucleus.6–9 It can be supposed that 5-HTT protein synthesis occurs mainly in raphe nucleus. Thus, 5-HTT mRNA expression in raphe nucleus should relate to 5-HT reabsorption sites in various regions of the brain. In the present study, we examine the effects of acute and chronic ECS on 5-HTT mRNA expression in rat raphe nucleus.
MATERIALS AND METHODS
Adult male rats, weighing 190–210 g at the beginning of experiments, were group-housed (8 rats per cage) with food and water ad libitum in a room maintained at 22 ± 2°C and 65 ± 5% humidity under a 12-h L:D cycle. Electroconvulsive shock was administered via ear clip electrodes (70 mA, 0.5 s), and a typical tonic– clonic seizure lasting 5–10 s was induced. In acute ECS studies, a single shock was administered, and the rats were decapitated after 3, 9 and 24 h. In chronic ECS studies, shock was administered every other day, totalling seven shocks. In 3, 9, 24 h and 2 weeks after the last ECS, the rats were decapitated for analysis. The control rats received sham ECS treatment.
We extracted total RNA using RNAzolTM B (TEL-TEST Inc., TX, USA) from midbrain raphe complex, corresponding to a midline sagittal section 2 mm in width and 3 mm in length including the dorsal and median raphe nucleus. Total RNA (20 μg/lane) was fractionated on 1.2% agarose/2.2 M formaldehyde gels and transferred to nylon membrane (HyBond-N; Amersham, Bucks, UK) by capillary blotting.
Single-stranded complementary DNA (cDNA) was synthesized from the total RNA by reverse transcriptase (SUPERSCRIPT First-Strand Synthesis System for reverse transcriptase–polymerase chain reaction (RT-PCR) kit; GIBCO-BRL, MD, USA). Using oligonucleotide primer corresponding to nucleotides 534–555 (5′-CATCTGGCGGTTTCCTTACATA-3′) and 1209–1229 (5′-ATCTGAGCGGCGGCATCTAC-3′), a 696-bp cDNA fragment of rat 5-HTT gene was generated by polymerase chain reaction. This cDNA fragment was subcloned into pGEM-Teasy (Promega, WI, USA). The 5-HTT cDNA inserts were digested by EcoRI and labelled with α-[32P]-dCTP (Amersham) and Rediprime DNA labelling system (Amersham).
For northern blot analysis, hybridization was performed with 1 × 106 c.p.m. of 5HTT cDNA probe/mL at 42°C for 16 h. Blots were then washed stringently in 0.1 × SSC/0.1% SDS at 60°C for 20 min. After hybridization, the residual cDNA was removed and rehybridized with beta-actin cDNA probe, the results of which were used for standardization. Radioactivity associated with each hybridization signal was estimated using a Fujix Bio-Image Analyser BAS2000 (Fuji Photo Film Co., Kanagawa, Japan).
Statistical analysis of the data was carried out using one-way ANOVA followed by the least significant difference test. In all cases, P values less than 0.05 were considered statistically significant.
An approximately 1.4 kb 5-HTT mRNA, corresponding to previous reports, was identified by northern blot analysis.10 Preliminary analysis revealed that the expression of beta actin mRNA was not affected by ECS (data not shown).
Figures 1 and 2 show alterations in 5-HTT mRNA (standardized by beta-actin mRNA) induced by acute or chronic ECS. The 5-HTT mRNA expression in raphe nucleus was not altered after 3 h, but significantly decreased 9 and 24 h after the single ECS, compared with the sham treated group (Fig. 1). Chronic ECS significantly decreased the 5-HTT mRNA expression in 3, 9, 24 h and 2 weeks after the last ECS (Fig. 2).
In the present study, we observed that 5-HTT mRNA expression was decreased by acute and chronic ECS. In contrast, previous reports showed that ECS increased 5-HTT binding sites in rat frontal cortex and resulted in an increased tendency in human platelet imipramine binding sites.3,11 Precise reasons for the discrepancy between alterations in 5-HTT mRNA and binding by ECS remain unclear. Following transcription, 5-HTT mRNA is translated to 5-HTT protein in serotonergic cell bodies of raphe nucleus, and 5-HTT protein is transported to the serotonergic presynaptic terminal in forebrain to affect optimal 5-HT clearance. We suppose that 5-HTT protein may be preferentially transported to the frontal cortex rather than other regions after ECS. It is necessary to investigate 5-HTT protein expression in frontal cortex and other regions after ECS.
Selective serotonin re-uptake inhibitors (SSRI) have no or only transient effects on 5-HTT mRNA and the delayed therapeutic effect of SSRI in major depression is regarded as relating to desensitized 5-HT1A autoreceptor and decreased 5-HT neuronal firing activity.12–17 ECS does not affect the sensitivity of either the 5-HT1A or 5-HT1B autoreceptor but increases post-synaptic 5-HT1A receptor sensitivity and 5-HT2A receptor levels.1,2 Therefore, it is generally thought that the effects of ECS on serotonergic transmission are principally exerted at the post-synaptic level. Our results reveal another distinct effect on 5-HTT expression between ECS and antidepressant medication. It is a possible explanation of the efficacy of ECS on medication-resistant depression.
After chronic ECS, the downregulation of 5-HTT expression had continued for at least 2 weeks. It is interesting that to obtain a sufficient and therapeutic efficacy, a repeated ECS course (7–12 times) is often performed in the clinical treatment of depression.18 Perhaps the persistent downregulation of 5-HTT expression induced by chronic ECS is conducive for maintaining an available and steady increase of 5-HT in the synaptic cleft. It is possibly related to the therapeutic efficacy of repeated ECS on refractory depression.
In conclusion, acute and chronic ECS decreased 5-HTT mRNA expression in rat raphe nucleus. After chronic ECS, the downregulation of 5-HTT expression had continued for at least 2 weeks. The adaptive change in 5-HTT mRNA expression is possibly related to the therapeutic efficacy of ECT on medication-resistant depression.
This study was partly supported by a Grant-in-Aid for Scientific Research [(B)(2) No. 10470200 (YN)] from the Japanese Ministry of Education, Science and Culture, and Health Sciences Research Grants for Research on Brain Science from the Ministry of Health and Welfare [No. 100109 (MS)].