International Journal of Neuropsychopharmacology (2014), 17, 1477–1486. doi:10.1017/S1461145714000200
© CINP 2014
ARTICLE
Effects of brief pulse and ultrabrief pulse electroconvulsive stimulation on rodent brain and behaviour in the corticosterone model of depression Sinead O’Donovan1*, Victoria Dalton1*, Andrew Harkin2 and Declan M. McLoughlin3 1
Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin 2, Ireland School of Pharmacy & Pharmaceutical Sciences & Trinity College Institute of Neuroscience, Trinity College Dublin, Dublin 2, Ireland 3 St. Patrick’s University Hospital & Trinity College Institute of Neuroscience, Trinity College Dublin, Ireland 2
Abstract Brief pulse electroconvulsive therapy (BP ECT; pulse width 0.5–1.5 ms) is the most effective treatment available for severe depression. However, its use is associated with side-effects. The stimulus in ultrabrief pulse ECT (UBP ECT; pulse width 0.25–0.3 ms) is more physiological and has been reported to be associated with less cognitive side-effects, but its antidepressant effectiveness is not yet well established. Using electroconvulsive stimulation (ECS), the animal model of ECT, we previously reported UBP ECS to be significantly less effective than wellestablished BP ECS in eliciting behavioural, molecular and cellular antidepressant-related effects in naïve rats. We have now compared the effects of BP and UBP ECS in an animal model of depression related to exogenous supplementation with the stress-induced glucocorticoid hormone, corticosterone. Corticosterone administration resulted in an increase in immobility time in the forced swim test (FST) (p < 0.01) and decreases in the expression of brain-derived neurotrophic factor (BDNF) (p < 0.05) and glial fibrillary acidic protein (GFAP) (p < 0.001) in the hippocampus and frontal cortex. There was no significant difference in the duration or type of seizure induced by BP (0.5 ms) or UBP (0.3 ms) ECS. UBP ECS proved to be as effective as BP ECS at inducing a behavioural antidepressant response in the FST with a significant decrease (p < 0.001) in immobility seen following administration of ECS. Both forms of ECS also induced significant increases in BDNF protein (p < 0.01) expression in the hippocampus. BP ECS (p < 0.05) but not UBP ECS induced a significant increase in GFAP levels in the hippocampus and frontal cortex. Overall, UBP ECS effectively induced antidepressant-related behavioural and molecular responses in the corticosterone supplementation model, providing the first preclinical data on the potential role of this form of ECS to treat a depression phenotype related to elevated corticosterone. Received 13 January 2014; Reviewed 21 January 2014; Revised 30 January 2014; Accepted 3 February 2014; First published online 10 March 2014 Keywords: Antidepressant, corticosterone, electroconvulsive stimulation, pulse width.
Introduction Electroconvulsive therapy (ECT) is a highly effective treatment for severe depression, although its use is associated with a number of side-effects, in particular cognitive side-effects (UK ECT Review Group, 2003; Eranti et al., 2007; Semkovska and McLoughlin, 2010). Developments in ECT treatment are focussed on minimising its cognitive side-effect profile while maintaining treatment effectiveness. In the past, changes in electrical stimulus from a sine wave pulse form to a square wave pulse form reduced cognitive side-effects while preserving
Address for correspondence: D. M. McLoughlin, Dept of Psychiatry & Trinity College Institute of Neuroscience, Trinity College Dublin, St Patrick’s University Hospital, James’s Street, Dublin 8, Ireland. Tel.: +353 (0) 1 2493343 Fax: +353 (0)1 2493428 Email:
[email protected] * These authors contributed equally to the work.
treatment efficacy (Weiner et al., 1986). Electrode placement can also impact on the severity of the side-effects experienced following treatment. There is little difference between bitemporal and bifrontal electrode placement (Dunne and McLoughlin, 2012) while right unilateral electrode placement results in fewer cognitive side-effects but, unless higher stimulus doses are used, is not as effective as bitemporal ECT (Sackeim et al., 1993; Semkovska et al., 2011). Another mechanism to minimise the side-effects of ECT is to reduce the pulse width at which ECT is delivered from a brief pulse (BP) width of 0.5–1.5 ms to an ultrabrief pulse (UBP) width of 0.3 ms that is closer to the minimum pulse width required for neuronal depolarisation (0.1–0.2 ms) (Ranck, 1975). Although UBP ECT has been administered in a number of randomised controlled trials, its effectiveness compared to the well-established BP ECT is still not clear. Although it has been reported that right unilateral UBP ECT led to exceptionally high
1478 S. O’Donovan et al. rates of remission (77%) compared to other forms of ECT (Sackeim et al., 2008), this has not been repeated in other studies. The effectiveness of UBP ECT as an alternative antidepressant therapy to BP ECT has, therefore, yet to be determined. Recent systematic reviews suggest that the heterogeneity of the studies conducted to date to examine the effects of UBP ECT make it difficult to draw conclusions about its efficacy (Loo et al., 2012; Spaans et al., 2013). To understand the different effects of BP and UBP ECT we previously compared the two treatments using the animal analogue of ECT, electroconvulsive stimulation (ECS) in rats (O’Donovan et al., 2012). There was no difference in the type or form of seizure induced by either pulse width. BP ECS was administered at 0.5 ms pulse width and effectively reduced immobility times in the forced swim test (FST), a test of antidepressant activity, but UBP ECS, administered at 0.3 ms pulse width, did not. Although BP ECS administration induced an almost three-fold increase in cell proliferation in the dentate gyrus and a significant increase in brain-derived neurotrophic factor (BDNF) levels in the hippocampus, UBP ECS did not result in significant molecular or cellular changes compared to sham-treated animals. Corticosterone is the rodent homologue of cortisol and chronic corticosterone supplementation is a wellestablished model of depression. The model draws its validity from evidence of persistent activation of the hypothalamic–pituitary–adrenal (HPA) axis and raised circulating cortisol concentrations in depressed patients (Sachar et al., 1973; Carroll, 1976; Carroll et al., 1976). In addition, patients with Cushing’s disease, a disorder that results in hypercortisolemia, often develop the symptoms of major depression (Kelly et al., 1983; Feelders et al., 2012). Exposing animals to chronic corticosterone results in physiological and behavioural changes associated with depressive behaviour (Woolley et al., 1990; Watanabe et al., 1992; Johnson et al., 2006). Animals treated with corticosterone have reduced body weight gain and decreased adrenal gland weight when compared to control animals. They also exhibit increased immobility time in the FST (Hellsten et al., 2002; Gregus et al., 2005; Johnson et al., 2006). BDNF and GFAP (glial fibrillary acidic protein), an astrocytic marker, have reduced levels of expression in corticosterone and other depression models in the hippocampus and frontal cortex, effects that are reversed by antidepressant treatment (Dwivedi et al., 2006; Gosselin et al., 2009; Li et al., 2013). The use of this model allows, therefore, the transition of our previous studies of UBP and BP ECS in naïve animals into an animal analogue of depression, providing greater insight into the antidepressant actions of ECS. Currently, there are no preclinical studies investigating the effects of UBP ECS in an animal model of depression. Therefore, we have administered BP and UBP ECS to
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Fig. 1. Study design for brief pulse (BP) and ultrabrief pulse (UBP) pulse electroconvulsive stimulation (ECS) treatment in the corticosterone supplementation model of depression. FST forced swim test.
animals treated with corticosterone to further our understanding of the different ECS treatment parameters. Using behavioural and molecular markers, we compare the effects of BP and UBP ECS in the corticosterone supplementation model of depression to determine if reducing the stimulus pulse width of ECS can influence its efficacy.
Method Animal treatments Male Sprague–Dawley rats (UK), weighing 200–250 g at intake, were housed with access to food and water ad libitum under 12 h light-dark conditions. Experiments were conducted in accordance with EU directive 2010/ 63/EU and guidelines of the Bioresources Ethics Committee, Trinity College Dublin. The experimental design is summarised in Fig. 1. Animals were habituated for one week then housed individually for the duration of the experiment. Animals were randomly allocated, using a computer-generated list, to one of four treatment groups according to the ECS and corticosterone treatment protocol: Sham ECS + Saline, Sham ECS + Corticosterone, BP ECS + Corticosterone and UBP ECS + Corticosterone The Sham ECS + Saline treatment group was included to act as a control for the effects of corticosterone administration. More animals were assigned to the ECS treatment groups to allow for non-completion of a full course of ECS (n = 11–14 per group). Corticosterone was sonicated in a saline and 2% Tween 80 solution until dissolved. Animals were subcutaneously injected with corticosterone (40 mg/kg, Sigma) every morning between 10:00 and 11:00 for 21 d (Gregus et al., 2005; Marks et al., 2009). Saline (0.9%) was administered subcutaneously as a sham treatment to control animals. On day 9 of the experiment ECS administration commenced following corticosterone or saline injection. The parameters of ECS treatment were as follows: BP ECS was administered at 0.5 ms pulse width, 100 pulses per second, 75 milliAmps, 0.7 s duration. The UBP ECS parameters were 0.3 ms pulse width, 100 pulses per second, 75 milliAmps, 0.7 s duration. Sham animals were handled in the same manner as treated animals but no charge was delivered. ECS was administered daily for ten consecutive days via ear-clip electrodes using the ECT Unit 57 800 device (Ugo Basile, Italy).
Effect of UBP ECS in the corticosterone model of depression Animals were only included in the study if a tonic–clonic seizure was successfully induced on each treatment day. Animal weights were monitored throughout the experiment. The durations of the tonic and clonic phases of seizure, defined as the rigid gathering of the animals hind legs and forearms against the body followed by the extension of the limbs away from the body and rapid extension and flexing of the limbs (O’Donovan et al., 2012), were recorded for all ECS sessions. Two hours after the final corticosterone administration on day 21, animals were sacrificed by decapitation and the whole hippocampus, dissected bilaterally, and frontal cortex were dissected from the brain, snap frozen and stored at −80 °C. The adrenal glands were also removed and weighed. Forced swim test The FST (Porsolt et al., 1977) was conducted in two sessions on experiment days 19 and 20 from 08:00, prior to corticosterone administration, as described (O’Donovan et al., 2012). Briefly, it involved a 15 min pre-test followed by a 5 min test 24 h later. A clear cylinder was filled 35 cm from the base with water. The water was changed after each animal’s test session. The length of time animals spent immobile was measured over the 5 min test session on day 20. Immobility behaviour was recorded when animals were making only those movements required to stay afloat. Protein and RNA extraction Protein and RNA were extracted from the left hemisphere of hippocampal and frontal cortex tissue using the mirVana PARIS kit (Ambion, Life Technologies, Ireland). According to the manufacturer’s instructions, tissue was homogenised in the provided cell disruption buffer. A volume of homogenate was removed, centrifuged and the supernatant removed and stored at −80 °C for further protein work. The remaining homogenate was mixed with an equal volume of the supplied 2X Denaturing Buffer and mixed. An equal volume of acid-phenol:chloroform was added to each sample and the mixture was centrifuged. The resulting aqueous phase was removed and 1.25 volumes of ethanol were added and mixed. The sample mixture was pipetted into a filter cartridge. The filters were washed with supplied buffer components and the resulting total RNA was eluted and stored at −80 °C. BDNF levels Hippocampal BDNF protein levels were measured using a sandwich enzyme linked immunosorbent assay (ELISA) kit (Chemikine, Chemicon, Millipore, USA) according to the manufacturer’s instructions and as previously described (O’Donovan et al., 2012). Protein samples were assayed to determine the total protein concentration of each sample.
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The protein homogenate extracted from the hippocampus was used to measure BDNF levels. The amount of BDNF in each sample was analysed in duplicate and determined from the regression line generated by BDNF standards (range 7.8 to 500 pg/ml). BDNF results are presented as pg BDNF per mg of total protein content in each sample. mRNA expression cDNA synthesis Total RNA concentration was determined by Nanodrop (Fisher Scientific, UK) and each sample was adjusted to a standard concentration prior to complementary DNA (cDNA) synthesis. 500 ng of total RNA was reverse transcribed into cDNA using a High Capacity cDNA Archive Kit (Applied Biosystems, Germany) as per manufacturer’s instructions. The resultant cDNA was stored at −20 °C until required for real-time polymerase chain reaction (PCR) in order to measure frontal cortex and hippocampal GFAP levels. Real-time PCR Real-time PCR was performed using Taqman Gene Expression Assays (Applied Biosystems, UK) which contain forward and reverse primers, and a 6-carboxyfluoroscein (FAM)-labelled minor groove binder (MGB) Taqman probe. The assay ID for GFAP is Rn00566603. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as the endogenous control to normalise gene expression data. GAPDH expression was measured using a gene expression assay labelled with a VIC MGB Taqman probe with assay ID: 4352338E. Real-time PCR was conducted on a StepOnePlus™ machine (Applied Biosystems, UK). A 25 μl volume was added to each well (10 μl of diluted cDNA, 1.25 μl of target primer and 1.25 μl of endogenous control primer and 12.5 μl of Taqman® Universal PCR Master Mix). The PCR cycle conditions of an initial polymerase activation step of 95 °C for 10 min followed by 40 cycles of two-step PCR, 95 °C for 15 s (denaturation) and 60 °C for 1 min (transcription). Gene expression was calculated relative to the endogenous control samples and to the control sample giving a relative quantitation (RQ) value (2−∆∆Ct). Statistical analyses Data are presented as mean ± S.E.M. and were analysed using GraphPad Prism version 5.0 (Graphpad Software, USA). Repeated measures analysis of variance (ANOVA) was used to compare tonic and clonic phase seizure duration with between group (ECS treatments; BP, UBP) and within group (Time; experimental day 9–18) factors. Repeated measures ANOVA was applied to compare the effect of ECS treatment on body weight and food weight in groups Sham ECS + Corticosterone, BP ECS + Corticosterone, UBP ECS + Corticosterone.
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Results Animal treatments One animal was excluded from the BP ECS group and two from the UBP ECS group because they did not experience all ten tonic–clonic seizures required for inclusion in the study. The final numbers in each group were therefore: Sham ECS + Saline n = 11, Sham ECS + Corticosterone n = 14, BP ECS + Corticosterone n = 14, UBP ECS + Corticosterone n = 13. Seizure durations Tonic and clonic seizure durations were measured following ECS administration (Fig. 2). Repeated measures ANOVA of tonic seizure duration did not show a significant main effect of ECS treatment (F(1,225) = 0.02, p = 0.889; Fig. 2a) or a treatment × time interaction (F(9,225) = 0.3, p = 0.97) but did reveal a significant effect of time (day) (F(9,225) = 16.25, p < 0.001; Fig. 2a). Repeated measures ANOVA of clonic seizure duration showed no significant main effect of ECS treatment (F(1,225) = 2.62, p = 0.12; Fig. 2b) or treatment × time interaction (F(9,225) = 0.89, p = 0.54), but did reveal a significant effect of time (day) (F(9,225) = 8.02, p < 0.001). Animal weights and food weights Repeated measures ANOVA of body weight showed an effect of corticosterone treatment (F(1,506) = 34.25, p < 0.001; Fig. 3a), time (day) (F(22,506) = 16.52, p < 0.001) and a treatment × time interaction (F(22,506) = 145.53, p < 0.001). Post-hoc analysis showed a significant decrease in body weight between corticosterone treatment and saline treatment (p < 0.01), in line with previous studies (Johnson et al., 2006). Repeated measures ANOVA of body weight showed no effect of ECS treatment (F(2,858) = 0.08, p = 0.9229; Fig. 3a), a significant effect of time
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The factors were: between group (treatment; Sham ECS, BP ECS, UBP ECS) and within group (time, experiment day baseline-21). Repeated measures ANOVA were used to analyse the effect of corticosterone on body weight and food weight, comparing the Sham ECS + Corticosterone and Sham ECS + Saline groups. The factors were: between group (treatment; corticosterone, saline) and within group (time, experiment day baseline-21). Bonferroni was applied as the post-hoc test as necessary. To compare adrenal weights, FST data, ELISA results and changes in GFAP mRNA levels, one-way ANOVA, with Tukey’s multiple comparison post-hoc test as required, was applied to the BP ECS + Corticosterone, UBP ECS + Corticosterone and Sham ECS + Corticosterone groups. Student’s t test was applied to the Sham ECS + Saline and Sham ECS + Corticosterone groups to examine the effect of corticosterone on each measure. Statistical significance was set at p < 0.05.
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Fig. 2. Seizure durations of animals treated with BP or UBP ECS in a corticosterone model of depression. There was no significant difference in either tonic (Fig. 2a) or clonic (Fig. 2b) phase seizure length following BP or UBP ECS treatments, although seizure length did increase over the course of 10 ECS sessions. Data expressed as mean ± S.E.M., n = 13–14/group. BP brief pulse, Cort corticosterone, ECS electroconvulsive stimulation, UBP ultrabrief pulse.
(day) (F(22,858) = 317.19, p < 0.001) and no treatment × time interaction (F(44,858) = 0.49 p = 0.9977) suggesting that, although body weight changed over time, differing ECS parameters did not affect animal weight. Repeated measures ANOVA of the weight of food consumed showed an effect of corticosterone treatment (F(1,506) = 41.31, p < 0.001; Fig. 3b), time (day) (F(22,506) = 7.47, p < 0.001) and a treatment × time interaction (F(22,506) = 4.01, p < 0.001). Post-hoc analysis showed a significant decrease in the weight of food consumed between corticosterone treatment and saline treatment (p < 0.01) from experimental day 3. Repeated measures ANOVA of food consumed showed no effect of ECS treatment (F(2,836) = 0.23, p = 0.7921; Fig. 3b), a significant effect of time (day) (F(22,836) = 26.15, p < 0.001) and a treatment × time interaction (F(44,836) = 2.33, p < 0.01), suggesting that, although the weight of food consumed changed over time, differing ECS parameters did not affect food consumption.
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Fig. 4. Adrenal gland weights of animals treated with corticosterone and ECS. There was a significant decrease in adrenal gland weight following corticosterone treatment compared to saline treatment. One-way analysis of variance (ANOVA) showed a significant difference between ECS- and corticosterone-treated groups. Data expressed as mean ± S.E.M., n = 11–14/group. BP brief pulse, Cort corticosterone, ECS electroconvulsive stimulation, UBP ultrabrief pulse. *p < 0.05 compared to Sham ECS + Corticosterone, ***p < 0.001 compared to Sham ECS + Saline, Tukey’s post-hoc test.
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Adrenal gland weights A comparison of Sham ECS + Saline-treated animals and Sham ECS + Corticosterone-treated animals showed a significant decrease in adrenal weight (p = 0.0004; Fig. 4) following corticosterone treatment, in line with earlier studies (Wennstrom et al., 2006). One-way ANOVA showed an effect of ECS on adrenal weight (F(2,38) = 3.5, p = 0.0414). Forced swim test Immobility behaviour was increased (p = 0.0021) following Sham ECS + Corticosterone treatment of animals compared to Sham ECS + Saline treatment (Fig. 5), in line with other studies of the corticosterone depression model (Lussier et al., 2013). One-way ANOVA showed an effect of ECS on immobility behaviour (F(2,38) = 13,
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Fig. 3. Body weight and weight of food consumed by animals over course of treatment with corticosterone and ECS. Fig. 3a shows the effect of corticosterone administration on body weight throughout 21 days of treatment and three days of habituation prior to commencement of experiment. Body weight was significantly lower in animals administered corticosterone. Food intake also decreased in animals administered corticosterone (Fig. 3b). Data expressed as mean ± S.E.M., n = 11–14/group. BP brief pulse, Cort corticosterone, ECS electroconvulsive stimulation, UBP ultrabrief pulse. *p < 0.05, ***p < 0.001 compared to Sham ECS + Saline, Bonferroni post-hoc test.
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Fig. 5. The effect of BP and UBP ECS treatment on immobility times in the forced swim test. An increase in immobility time was associated with corticosterone administration. BP and UBP ECS administration significantly reduced immobility times. Data expressed as mean ± S.E.M., n = 11–14/group. BP brief pulse, Cort corticosterone ECS electroconvulsive stimulation, UBP ultrabrief pulse. **p < 0.01 compared to Sham + Saline, **p < 0.01 compared to Sham ECS + Corticosterone, Tukey’s post-hoc test.
p < 0.001; Fig. 5). Post-hoc testing showed significant decreases in the immobility times of the BP ECS + Corticosterone group (p < 0.001) and UBP ECS + Corticosterone group (p < 0.01) when compared to the Sham ECS + Corticosterone group. There was no significant difference in immobility times of BP compared to UBP-treated animals.
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Fig. 6. The effects of BP and UBP ECS on hippocampal BDNF expression in a corticosterone model of depression. BDNF concentrations decreased following corticosterone administration. BP and UBP ECS administration significantly increased BDNF expression. Data expressed as mean ± S.E.M., n = 11–14/group. BDNF brain-derived neurotrophic factor, BP brief pulse, Cort corticosterone, ECS electroconvulsive stimulation, UBP ultrabrief pulse. *p < 0.05 compared to Sham ECS + Saline, **p < 0.01 compared to Sham ECS + Corticosterone, Tukey’s post-hoc test.
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BDNF ELISA BDNF expression in the hippocampus was decreased in animals administered Sham ECS + Corticosterone but not Sham ECS + Saline (p = 0.0214; Fig. 6), in line with previous studies (Dwivedi et al., 2006). One-way ANOVA showed an effect of ECS on BDNF expression in corticosteronetreated animals (F(2.36) = 11, p = 0.0003). Post-hoc analysis with Tukey’s multiple comparison test showed the BP ECS + Corticosterone (p < 0.001) and UBP ECS + Corticosterone (p < 0.01) groups differed significantly from the Sham ECS + Corticosterone group. There was no significant statistical difference between BP and UBP treatment.
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Fig. 7. The effect of ECS on GFAP mRNA expression in the brain. GFAP levels decreased following corticosterone administration in both brain regions. BP but not UBP ECS administration increased GFAP mRNA expression in the frontal cortex (Fig. 7a) and hippocampus (Fig. 7b). Data expressed as mean ± S.E.M., n = 11–14/group. BP brief pulse, Cort corticosterone, ECS electroconvulsive stimulation, GFAP glial fibrillary acidic protein, UBP ultrabrief pulse. ***p < 0.001 compared to Sham ECS + Saline, *p < 0.05 compared to Sham ECS + Corticosterone, Tukey’s post-hoc test.
GFAP mRNA Figure 7a shows a significant decrease in GFAP mRNA expression in the frontal cortex following Sham ECS + Corticosterone treatment (p = 0.0002) compared to Sham ECS + Saline treatment, a result that has also been reported in other models of depression (Li et al., 2013). One-way ANOVA showed an effect of ECS on GFAP expression (F(2,38) = 4.1, p = 0.0248). Post-hoc analysis showed that BP ECS (p < 0.05) but not UBP ECS induced a significant increase in GFAP mRNA expression. In the hippocampus (Fig. 7b) Sham ECS + Corticosterone treatment significantly reduced the expression of GFAP mRNA compared to Sham ECS + Saline treatment (p < 0.001). One-way ANOVA showed an effect of ECS on GFAP expression (F(2,35) = 4.4, p = 0.0195). Post-hoc analysis showed that BP ECS but not UBP ECS significantly increased GFAP mRNA expression (p < 0.05).
Discussion UBP (0.3 ms) stimulation provides a potential therapeutic alternative to BP (0.5 ms) stimulation as it is proposed to induce fewer cognitive side-effects (Sienaert et al., 2010). However, its antidepressant efficacy has yet to be confirmed (Loo et al., 2012). In this study, contrasting with our earlier study on naïve rats, we have shown that UBP ECS induces molecular and behavioural changes in an animal model of depression that are comparable in almost all measures to those of the well-established BP stimulation. The corticosterone supplementation model of depression was successfully applied and resulted in the physical and behavioural changes associated with administration of the endogenous glucocorticoid. As reported previously, corticosterone-treated animals did not gain weight in line with the saline-treated controls
Effect of UBP ECS in the corticosterone model of depression (Gregus et al., 2005; Johnson et al., 2006). Adrenal gland wet weight was also significantly impacted with a reduction in the weight of the adrenal glands of corticosterone-treated animals (Hellsten et al., 2002). The significant reduction in adrenal weight may be related to the lower weight gain associated with corticosterone treatment (Ulloa et al., 2010). However, treatment with sertraline selective serotonin reuptake inhibitor (SSRI) in combination with corticosterone also results in higher adrenal weights than corticosterone administration alone. Although this effect was not as significant as that seen following ECS administration, it suggests that antidepressant treatment can effectively reverse the reduction in adrenal gland weight induced by corticosterone treatment (Ulloa et al., 2010). The influence of corticosterone on animal behaviour was also seen in the FST and resulted in a significant increase in immobility, a behavioural result common to a number of depression models (Kalynchuk et al., 2004; Gregus et al., 2005; Gigliucci et al., 2013). In naïve rats, we have previously reported that UBP ECS is not as effective as BP ECS at inducing behavioural, molecular and cellular changes associated with antidepressant treatment (O’Donovan et al., 2012). Interestingly, in the corticosterone supplementation model of depression, UBP ECS appears to be almost as effective as BP ECS treatment at bringing about such changes. In the FST, UBP and BP ECS reduced the amount of time animals spent immobile compared to sham ECS-treated animals. These results suggest that UBP ECS treatment effectively ameliorated the depressive effects of corticosterone, inducing a robust behavioural antidepressant response. The expression of BDNF, a widely distributed neurotrophin in the brain, is implicated in the stress response and antidepressant actions in the brain (Duman and Monteggia, 2006). In post-mortem studies of hippocampal BDNF expression levels, depressed suicide patients that did not receive antidepressant treatment had reduced BDNF expression while antidepressant-treated patients had increased levels of expression (Chen et al., 2001; Dwivedi et al., 2003; Karege et al., 2005). Investigation of the role of BDNF in animal models of depression has also shown reductions in BDNF expression. The chronic mild stress model (Gersner et al., 2010) and exposure to immobilisation stress for at least 5 h for one day or 2 h per day for one week (Nooshinfar et al., 2011) resulted in a decrease in hippocampal BDNF levels. It has previously been shown that chronic corticosterone administration is sufficient to induce a decrease in BDNF levels in the frontal cortex, hippocampus and dentate gyrus of the rat brain (Chao and McEwen, 1994; Smith et al., 1995; Dwivedi et al., 2006; Mao et al., 2012). Antidepressant treatment can effectively reverse the effects of corticosterone treatment on BDNF expression. A range of antidepressant pharmacotherapies, including desipramine (noradrenergic reuptake inhibitor), phenelzine
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(monoamine oxidase inhibitor) and fluoxetine (SSRI) can increase BDNF expression following corticosterone administration (Dwivedi et al., 2006; Mao et al., 2012). Therefore, although corticosterone administration effectively reduces BDNF expression, this decrease is rescued by treatment with pharmacotherapy. It has also been demonstrated that ECS induces significant increases in the expression of BDNF levels in the hippocampus (Nibuya et al., 1995; Altar et al., 2003; Li et al., 2007; Balu et al., 2008; O’Donovan et al., 2012). We now show that chronic ECS treatment, when administered at BP and UBP widths can also effectively increase the expression of hippocampal BDNF protein levels in the corticosterone model of depression. UBP ECS reversed the decreased BDNF expression observed following treatment with corticosterone as effectively as BP ECS. Interestingly, unlike BP ECS, UBP ECS did not induce a similar result in naïve animals (O’Donovan et al., 2012). Similarly, subconvulsive electrical stimulation, a focal form of stimulation, also increased hippocampal BDNF expression following chronic mild stress (Gersner et al., 2010), although this protocol was designed to imitate transcranial magnetic stimulation. Subconvulsive electrical stimulation, administered using modified ECS parameters, did not induce mossy fibre sprouting in the rat hippocampus, a measure that was significantly up-regulated by ECS (Lamont et al., 2001). Changes in glial cell number and or activation are now thought to play a role in the pathophysiology of depression. There is a decrease in the density and a reduction in the number of glial cells expressed in the brains of patients diagnosed with depression, among other psychiatric disorders (Rajkowska and Miguel-Hidalgo, 2007). Glial cells appear to undergo changes in nucleus size as well as changes in cell morphology. The changes in glial pathology associated with depressed patients have been seen in various brain regions, including the subgenual prefrontal cortex (Ongur et al., 1998), the dorsolateral prefrontal cortex (Rajkowska et al., 1999; Cotter et al., 2002) and dorsal anterior cingulate cortex (Cotter et al., 2001). Changes in glial cell morphology were also decreased in the amygdala of depressed patients (Bowley et al., 2002; Rajkowska, 2002). Astrocytes, the most numerous type of glial cell, express GFAP, a cytoskeletal marker of reactive astrocytes. Altered levels of expression of GFAP have been reported in post-mortem studies of patients with depression (Muller et al., 2001). GFAP levels were also significantly decreased in the prefrontal cortex of depressed patients who were under the age of sixty compared to patients over the age of 60 and older controls, suggesting GFAP may be relevant to the pathophysiology of depression in relatively younger patients (Si et al., 2004). Evidence from animal studies of a role for glial cells in depression includes the induction of depressive-like symptoms following ablation of prefrontal cortex astrocytes in the rat brain (Banasr and Duman, 2008). Using the chronic social defeat stress model, there was a
1484 S. O’Donovan et al. reduction in cytogenesis of glial cells in the prefrontal cortex that was blocked following fluoxetine administration (Czeh et al., 2007). Significant reductions in GFAPimmunoreactive cells in the hippocampus were reported in Wistar Kyoto rats, a genetic strain used as a model of depression (Gosselin et al., 2009). Corticosterone administration has previously been shown to inhibit gliogenesis in the hippocampus, an effect that is counteracted by ECS (Wennstrom et al., 2006). Glial dysfunction in depression was also reported in two studies that used chronic unpredictable stress to model depression. Clomipramine, a tricyclic antidepressant, and magnolol, a herbal antidepressant, reversed the effect of stress on GFAP levels in the hippocampus and frontal cortex (Liu et al., 2009; Li et al., 2013). In the present study, using the corticosterone supplementation model of depression, we found that GFAP mRNA levels were also reduced compared to salinetreated control animals. Additionally, compared to sham ECS, BP ECS significantly increased GFAP mRNA levels in both the frontal cortex and hippocampus, while UBP ECS induced increases were not statistically significant. This suggests that UBP ECS treatment is not as effective as BP ECS at inducing changes in GFAP in this model of depression. Evidence now points to an emerging role for glial cells in the pathology of neuropsychiatric disorders, including depression (Banasr et al., 2010). The antidepressant effectiveness of ECT may also be in part owing to its action on glia (Ongur and Heckers, 2004). A possible limitation of this study is that, although GFAP mRNA expression was examined and significant changes were seen in this marker of astrocyte activity, the direct impact on the numbers and morphology of glial cells expressed following UBP ECS in the corticosterone model has yet to be explored. BP ECS is a robust inducer of antidepressant related behavioural and molecular responses in rats. UBP ECS, while previously shown to be less effective than BP ECS in naïve animals, successfully induced antidepressant effects in corticosterone-treated animals. Although this result is surprising, there is a precedent for antidepressant treatment to lead to a greater response in an animal model than in naïve animals. The SSRI fluoxetine, had a significant effect on neurogenesis, including cell proliferation and survival, in corticosterone-treated but not vehicle-treated control mice (David et al., 2009). Fluoxetine also had a greater effect in corticosteronetreated animals in certain behavioural measures, such as latency to feed in the novelty suppressed feeding test (David et al., 2009). It was suggested that simulation of chronic stress by corticosterone supplementation may result in changes in the serotonin system and a stronger effect of fluoxetine, potentially owing to desensitisation of the 5HT1A autoreceptor (Hensler et al., 2007; David et al., 2009). ECS and ECT administration also impact the serotonin system, modulate serotonin receptors (Burnet et al., 1999; Lanzenberger et al., 2013) and may
allow UBP ECS to induce a more powerful effect in the corticosterone model using similar mechanisms. These results and those of the present study highlight the utility of animal models in the study of antidepressant action. In summary, we report for the first time on the effect of BP and UBP ECS in a model of depression. Contrary to expectations based on our previous work in naïve animals, UBP ECS effectively induced a similar antidepressant effect to BP ECS. This iterates the importance of translational animal models in understanding the subtle mechanisms underlying antidepressant action in the depressed brain and may aid us in understanding why UBP ECS does not act in the same manner in naïve brain. Future work will include investigating UBP ECS in other models of depression to determine if it maintains a comparable antidepressant efficacy to that associated with BP ECS administration. Further study will also be required to determine whether UBP ECS administration results in milder cognitive side-effects, the primary perceived advantage of modifying the pulse width of ECT (Verwijk et al., 2012), while providing a viable therapeutic alternative to BP ECS.
Acknowledgements This work was supported by the Health Research Board (grant no. TRA/2007/5) and the St. Patrick’s Hospital Foundation.
Statement of Interest None
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