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GABAergic circuitry in the rat hippocampus
Neuropharmacology 62 (2012) 1944e1953
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Long lasting effects of early-life stress on glutamatergic/GABAergic circuitry in the rat hippocampus Eva Martisova a, Maite Solas a, Igor Horrillo b, Jorge E. Ortega b, J. Javier Meana b, Rosa María Tordera a, María Javier Ramírez a, * a b
Department of Pharmacology, University of Navarra, C/ Irunlarrea 1, 31008 Pamplona, Spain Department of Pharmacology, University of the Basque Country (UPV/EHU) and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Leioa, Bizkaia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 August 2011 Received in revised form 14 December 2011 Accepted 19 December 2011
The objective of the present work was to study the effects of an early-life stress (maternal separation, MS) in the excitatory/inhibitory ratio as a potential factor contributing to the ageing process, and the purported normalizing effects of chronic treatment with the antidepressant venlafaxine. MS induced depressive-like behaviour in the Porsolt forced swimming test that was reversed by venlafaxine, and that persisted until senescence. Aged MS rats showed a downregulation of vesicular glutamate transporter 1 and 2 (VGlut1 and VGlut2) and GABA transporter (VGAT) and increased expression of excitatory amino acid transporter 2 (EAAT2) in the hippocampus. Aged rats showed decreased expression of glutamic acid decarboxylase 65 (GAD65), while the excitatory amino acid transporter 1 (EAAT1) was affected only by stress. Glutamate receptor subunits NR1 and NR2A and GluR4 were upregulated in stressed rats, and this effect was reversed by venlafaxine. NR2B, GluR1 and GluR2/3 were not affected by either stress or age. MS, both in young and aged rats, induced an increase in the circulating levels of corticosterone. Corticosterone induced an increase glutamate and a decrease in GABA release in hippocampal slices, which was reversed by venlafaxine. Chronic treatment with corticosterone recapitulated the main biochemical findings observed in MS. The different effects that chronic stress exerts in young and adult animals on expression of proteins responsible for glutamate/GABA cycling may explain the involvement of glucocorticoids in ageing-related diseases. Modulation of glutamate/GABA release may be a relevant component of the therapeutic action of antidepressants, such as venlafaxine. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Venlafaxine Ageing VGAT VGLUT EAAT Corticosterone
1. Introduction Stress is believed to contribute to the variability of the ageing process and to the development of age-related neuro- and psychopathologies (Heim and Nemeroff, 1999; McEwen, 2002; Miller and O’Callaghan, 2005). In fact, the experience of stress or traumatic experience early in life is thought to make an individual more vulnerable for psychiatric problems, such as depression or anxiety, later in life (Gilmer and McKinney, 2003; Heim and Nemeroff, 2001). Abnormalities in glutamate and gamma-aminobutyric acid (GABA) signal transmission have been postulated to play a role in depression (Krystal et al., 2002). Increased glutamate and reduced GABA levels have been observed in the cortex of depressed patients, leading to an enhanced excitatoryeinhibitory ratio (Bhagwagar
* Corresponding author. Tel.: þ34 948425600; fax: þ34 948425649. E-mail address: [email protected] (M.J. Ramírez). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.12.019
et al., 2007; Sanacora et al., 1999). Interestingly, this imbalance is inhibited by chronic treatment with antidepressants (Sanacora et al., 2002). Because the presynaptic pathways regulating the synthesis and cycling of glutamate and GABA are tightly coupled, it has been suggested that alterations in a shared pathway may account for the observed amino acid abnormalities. For instance, post-mortem studies have shown decreased expression of glutamic acid decarboxylase 65 (GAD65), the enzyme that convert glutamate to GABA, in mood disorders (Fatemi et al., 2005). Microarray analysis of cerebral cortex from individuals who had suffered from major depression disorder have demonstrated significant downregulation of the glial excitatory amino acid transporter 1 and 2 (EAAT1 and EAAT2), key members of the glutamate/neutral amino acid transporter protein family (Choudary et al., 2005). At the experimental level, decrease of the vesicular GABA transporter (VGAT), and GAD65 and an upregulation of EAAT1 has been shown in the hippocampus of animals subjected to chronic stress when adults (chronic mild stress, CMS, Garcia-Garcia et al., 2009). Mice heterozygous for the vesicular glutamate transporter 1 (VGLUT1 þ/) showed increased
E. Martisova et al. / Neuropharmacology 62 (2012) 1944e1953
depressive-like behavioural symptoms as well as increased neuronal synthesis of glutamate and decreased hippocampal GABA, VGLUT1, and EAAT1 levels (Garcia-Garcia et al., 2009). Emotional experience during early life has been shown to interfere with the development of excitatory synaptic networks in hippocampus of rodents. Maternal separation (MS) is an animal paradigm designed to mimic repeated exposure to stress during early life, resulting in animals with behavioural and neuroendocrine signs of elevated stress reactivity as adults (Aisa et al., 2007; Heim and Nemeroff, 2001; Lehmann and Feldon, 2000) or senescent (Solas et al., 2010). The peak period of neurogenesis overlaps the stress hyporesponsive period (postnatal days 4e14) in neonatal rats (Sapolsky and Meaney, 1986). Therefore, early stress, such as MS, could be interfering with the normal maturation of excitatory/ inhibitory synapses in the hippocampus, which might ultimately lead to an increased vulnerability for psychiatric diseases. To test this hypothesis, we have studied the lasting consequences of early-life stress exposure on the expression of presynaptic proteins involved in the glutamate/GABA cycle and on the expression of different glutamate receptor subunits in the hippocampus of young and aged rats. In addition, we have checked if the treatment with the antidepressant venlafaxine in adulthood could be effective in preventing the purported interaction between ageing and stress. Finally, as acute stress is known to increase glutamate release (Gould et al., 2000; Lowy et al., 1993; Musazzi et al., 2010), the effects of the stress hormone corticosterone on glutamate/GABA release “in vitro” have been checked. 2. Materials and methods 2.1. Animals All the experiments were carried out in strict compliance with the recommendations of the EU (86/609/EEC) for the care and use of laboratory animals. All efforts were made to minimise animal suffering, to reduce the number of animals, and alternative to in vivo techniques (in vitro release experiments) have been used. Timed-pregnant Wistar rats were provided on gestation day 16 from Charles River Laboratories (Portage, MI, USA), individually housed in a temperature (21 1 C) and humidity (55 5%) controlled room on a 12-h light/dark cycle with food and water freely available. Animals from different litters were evenly spread over the different experimental groups. For the chronic treatment with corticosterone, commercially made pellets (Innovative Research of America, Sarasota, FL, USA) designed for daily administration of 18 mg/kg of corticosterone or placebo were used. Pellets were implanted subcutaneously on the right flank of male rats (3 months old, n ¼ 6) under chloral hydrate anaesthesia. After the surgical procedure, rats were left to recover. Thirtyfive days after pellet implantation, rats were killed by decapitation, brains removed and hippocampal samples frozen at 80 C until use. 2.2. Maternal separation All litters were born within a 2-day period. As previously described in detail (Aisa et al., 2007), on postnatal day (PND) 2, all pups were sexed and randomly assigned to the control group (animal facility rearing, AFR), pups were only briefly manipulated to change the bedding in their cages once weekly, or the separation group (MS), pups separated from their dam for 180 min from PNDs 2e21 inclusive. Rats were weaned on PND 23 and only males were chosen for the present work. Experiments were performed in young (60e75 days) and aged rats (18 months). Rats were not allowed to grow older as mortality increased significantly in the aged MS group. In experiments testing the effects of the antidepressant treatment, saline or venlafaxine (20 mg/kg, p.o.) were administered daily for 15 days, beginning on PND 60. Venlafaxine was dissolved in saline. The last administration of saline/venlafaxine took place 24 h before behavioural testing. 2.3. Forced swimming To study depressive-like behaviour, the Porsolt forced swimming test was used as described by Porsolt et al. (1977). Two swimming sessions were conducted: an initial 15-min pretest followed 24 h later by a 5-min test. Rats were placed individually in a vertical plexiglass cylinder (height: 45 cm, diameter: 19 cm) filled with 28e30 cm of 26 C water. Immobility was considered as rats floating passively,
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making only small movements to keep its nose above the surface. Immobility times were scored by Ethovision XT 5.0 (Noldus, Netherland). 2.4. Plasma corticosterone determinations Rats were sacrificed by decapitation between 08:00 and 10:00 h, and 24 h after the last administration of venlafaxine. Trunk blood was collected into EDTA tubes, centrifuged at 1250 g (15 min, 4 C), and plasma was frozen and stored at 80 C until processing. Plasma corticosterone was measured using a commercially available radioinmmunoassay kit (Coat-A-Count Rat Corticosterone, Siemens, Los Angeles CA, USA). All assays were performed in duplicate. Limit of detection was 5.7 ng/ml and intra- and interassay coefficients of variation were less than 10% for all comparisons. Corticosterone concentration values were expressed as ng/ml. 2.5. Western blotting Hippocampal samples were homogenized in a 50 mM Tris buffer (pH 7.2, 4 C). Each sample was adjusted to a final protein concentration of 4 mg/ml (DC protein assay; Bio-Rad, Hercules, CA). Extracts were mixed with Laemmeli’s sample buffer boiled for 5 min. Assays were performed as described in Table 1. Immunopositive bands were visualized by a chemiluminescent method (ECL; Amersham, Arlington Heights, IL). The optical density of reactive bands visible on x-ray film was determined densitometrically. b-actin was used as internal control. Results were expressed as percentage of OD values of controls. 2.6. “In vitro” depolarization-evoked glutamate and GABA release Male Wistar rats, 3 months old, were used. As previously described (Marcos et al., 2006), the hippocampus was dissected out and cut sagitally into 500 mm slices using a McIlwain tissue chopper (The Mickle Laboratory Engineering Co. Ltd.). Slices were allowed to equilibrate for 20 min at room temperature in gassed KrebsRinger bicarbonate buffer (KRB) made up of (mmol/l): NaCl, 113; KCl, 4.75; CaCl2, 2.52; MgSO4, 1.19; NaH2PO4, 1.18; NaHCO3, 25; glucose, 10; pH, 7.4. Tissue was then transferred into superfusion chambers (Brandel Superfusion-1000 apparatus) and continuously superfused with continuously oxygenated KRB at a rate of 0.35 ml/min. After a 40 min equilibration period, fractions were collected at 5-min intervals for a total of 40 min (eight samples). The first three samples were used to assess a stable basal neurotransmitter release. Slices were depolarized by changing the superfusion fluid for 5-min to a KRB solution containing 30 mM KCl. Corticosterone was added 5, 10 or 30 min before depolarization. Mifepristone, spironolactones or venlafaxine were added 10-min before depolarization. Neurotransmitter levels were measured in the superfusate fractions. Glutamate and GABA content in supernatant were measured using high performance liquid chromatography HPLC with electrochemical detection (Waters SpheriborÒ 5m ODS2 4.6 150 mm) including precolumn derivatization with o-phthalaldehyde (SigmaeAldrich Ltd, Germany) and b-mercaptoethanol (SigmaeAldrich Ltd, Germany). The mobile phase consisted of 72:28 (v/v) mixture of buffer (NaH2PO4 0.1 M, pH ¼ 5.5) and methanol; the mixture was filtered and degassed through a 0.22 mm nitrocellulose membrane (Millipore, UK). Glutamate and GABA content were calculated by comparing with a 2 ng standard. The limit of detection was 20 pg/10 ml for glutamate and 50 pg/10 ml for GABA content. 2.7. Data analysis Data were analysed by SPSS for Windows, release 11.0. Normality was checked by ShapiroeWilks’s test (p > 0.05). Data were analysed by one or two-way analysis
Table 1 Conditions used in Western blotting experiments. Protein
Membrane
SDSpolyacrylamide gel
Primary antibody (dilution)
VOLUTI VGLUT2 VGAT GAD65 EAAT1 EAAT2 NR1 NR2A NR2B GluR1 GluR2/3 GluR4
PVDF PVDF PVDF PVDF Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose
10% 10% 10% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5%
Rabbit anti-VGLUT1 (l:2000)a Mouse anti-VGLUT2 (l:1000)b Rabbit anti-VGAT (l:1000)b Mouse anti-GAD65 (l:5000)c Rabbit anti-EAAT1 (l:2500)d Rabbit anti-EAAT2 (l:2500)d Rabbit anti-NR1 (1:1000)e Rabbit anti-NR2A (1:1000)e Rabbit anti-NR2B (1:1000)e Rabbit anti-GluR1 (1:1000)e Rabbit anti-GluR2/3(1:1000)e Rabbit anti-GluR4 (1:1000)e
Source of antibodies: a) donated by Dr. S. El Mestikawy, Paris, France; b) Chemicon, Temecula, California; c) Abcam Inc., Cambridge, UK; d) Santa Cruz, Heidelberg, Germany; e) Upstate.
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of variance ANOVA (rearing age or rearing treatment), followed by Student’s ttest adjusted by Bonferroni correction. For all analyses, post hoc comparisons were conducted if appropriate, using Tukey protected least significance test.
3. Results 3.1. Depressive-like behaviour In the forced swimming test (Fig. 1A), two-way ANOVA indicated a significant main effect of stress [F1,82 ¼ 17.217, p < 0.001; n ¼ 10 per group]. Venlafaxine treatment (Fig. 1B) reversed the depressive phenotype associated to MS in young rats [significant interaction stress treatment, F1,59 ¼ 3.386, p < 0.05; n ¼ 10 per group]. 3.2. Regulation of presynaptic proteins As shown in Fig. 2, aged MS rats showed decreased expression of VGlut1 [significant interaction stress age, F1,35 ¼ 11.239, p < 0.05; n ¼ 9 per group]. A specific interaction stress age was found in the expression VGlut2 [F1,35 ¼ 15.675, p < 0.001; n ¼ 9 per group], as even though stress or ageing increased the expression of the transporter, the combined effect decreased its expression. EAAT1
expression was affected only by stress [main effect of stress, F1,35 ¼ 4.974, p < 0.05; n ¼ 9 per group], while aged MS rats showed increased expression of EAAT2 [significant interaction stress age, F1,30 ¼ 9.261, p < 0.01; n ¼ 7e8 per group]. An interaction between stress age was shown in VGAT, and aged MS rats showed increased expression of VGAT [F1,34 ¼ 3.725, p < 0.05; n ¼ 7e9 per group]. Aged rats showed decreased expression of GAD65 [main effect of age, F1,35 ¼ 4.428, p < 0.05; n ¼ 9 per group]. 3.3. Regulation of glutamate receptor subunits NMDA receptor subunits NR1 and NR2A were upregulated in stressed rats [main effect of stress, F1,35 ¼ 8.427, p < 0.01 and F1,35 ¼ 9.466, p < 0.01; n ¼ 9 per group, respectively]. GluR4, subunit of the AMPA-type glutamate receptors was also upregulated in stressed rats [main effect of stress F1,35 ¼ 13.102, p < 0.001; n ¼ 9 per group]. These results are shown in Fig. 3. These effects of stress on glutamate receptor subunits were reverted by venlafaxine [F1,35 ¼ 24.015, p < 0.001, F1,35 ¼ 5.287, p < 0.05 and F1,35 ¼ 4.426, p < 0.05; n ¼ 9 per group, for NR1, NR2A and GluR4 respectively]. All these results are depicted in Fig. 4. The expression of NR2B, GluR1 and GluR2/3 subunits was not affected by either stress or age (Supplementary Fig. 1). 3.4. Levels of plasmatic corticosterone There was a significant rearing age interaction in plasma corticosterone levels [two-way ANOVA, F1,50 ¼ 4.689, p < 0.05; n ¼ 8e19]. Further analysis showed a significant increase in corticosterone levels in young MS rats (Student’s t-test; p < 0.05), aged rats (Student’s t-test; p < 0.05) and almost significant effect in aged MS rats (Student’s t-test; p ¼ 0.06) compared with young controls (Fig. 5A). As shown in Fig. 5B, the increase in corticosterone levels (expressed as % control young rats) in young rats was reversed by chronic treatment with venlafaxine [two-way ANOVA, interaction rearing treatment, F(1,54) ¼ 6.594, p ¼ 0.05; chronic treatments; n ¼ 8e10 per group]. 3.5. Glutamate and GABA release “in vitro”
Fig. 1. Effects of ageing (A) or venlafaxine treatment (B) on stress-induced depressivelike behaviour in the Porsolt forced swimming test. In A, two-way ANOVA (stress age), *p < 0.05 main effect of stress. In B, two-way ANOVA (stress treatment), #significant interaction p < 0.05 vs control (3 months) rats, zsignificant interaction p < 0.05 vs. MS (3 months) rats. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats); Ven: venlafaxine.
All this experiments were performed in hippocampal slices obtained from 3 month old male Wistar rats. In a pilot set of experiments, different time points (5, 10 or 30 min) of incubation with corticosterone 10 mM were checked (Fig. 6), and maximal effects were found at 10 min (one-way ANOVA, F3,33 ¼ 4.541, p < 0.05, n ¼ 8 per group and F3,33 ¼ 3.998, p < 0.05, n ¼ 8 per group for glutamate and GABA respectively). Corticosterone (0.1-1-10 mM, 10 min incubation) induced a concentration-dependant increase in Kþ-induced glutamate release in slices of rat hippocampus, and this increase was significant at the higher concentration of corticosterone used (one-way ANOVA, F3,33 ¼ 4.747, p < 0.05, n ¼ 8 per group, Fig. 7). This increase in glutamate release seemed to be receptor mediated, as both the glucocorticoid receptor (GR) antagonist mifepristone, 1 mM [one-way ANOVA F2,23 ¼ 2.905, p < 0.05, n ¼ 8 per group], or the mineralocorticoid receptor (MR) antagonist spironolactone, 1 mM, were able to reverse the increases in glutamate release after corticosterone (one-way ANOVA F2,23 ¼ 3.644, p < 0.05, n ¼ 8 per group, Fig. 8). Venlafaxine, 1 mM, was able to counteract corticosterone (10 mM)-evoked glutamate release (F2,16 ¼ 2.941, p < 0.05, n ¼ 6 per group, Fig. 7). Kþ-induced GABA release was significantly inhibited by corticosterone, 10 mM (one-way ANOVA, F3,33 ¼ 2.944, p < 0.05, n ¼ 8 per group). Both mifepristone and spironolactone were able to
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Fig. 2. Effects of stress and ageing on vesicular glutamate transporter 1 (VGlut1) and 2 (VGlut2), excitatory amino acid transporter 1 (EAAT1) and 2 (EAAT2), GABA transporter (VGAT), and glutamic acid decarboxylase 65 (GAD65) in the hippocampus. Data is shown as % optical density of control (3 months) rats. In each panel, a representative picture of western blot is shown. Two-way ANOVA (stress age), *p < 0.05 main effect of stress, yp < 0.05 main effect of age, #significant interaction p < 0.05 vs control (3 months) rats, zsignificant interaction p < 0.05 vs. MS (3 months) rats. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats).
counteract the corticosterone-induced inhibition on GABA release (one-way ANOVA F2,23 ¼ 4.134, p < 0.05 and F2,23 ¼ 1.206, p < 0.05, n ¼ 8 per group, respectively). As shown in Fig. 7, there was a tendency, although not significant of venlafaxine, 1 mM, to reverse the inhibition.
increased expression of NR2A and GluR4 and no changes in NR2B (in all cases Student’s t-test vs control, p < 0.05). At difference with MS, NR1, GluR1 and GluR2/3 expression was decreased (in all cases Student’s t-test vs control, p < 0.05). All these results are depicted in Fig. 9.
3.6. Effects of a chronic treatment with corticosterone
4. Discussion
To study a causal relationship between elevated corticosterone levels and the biochemical alterations observed after MS, the effects of a chronic treatment with corticosterone in 3 months old rats were checked. At similarity to data from 3 months old MS rats, corticosterone treatment induced increased expression of VGlut2, EAAT1 and EAAT2, no changes in VGlut1, VGAT and GAD65,
Stressful life events are known to precipitate mood/anxiety disorders. In fact, experimental models of chronic stress are considerer nowadays as useful models to study depression. The effects of early-life stress endure and worsen during ageing (Solas et al., 2010), yet the mechanisms for these purportedly clinically important sequelae are poorly understood. It has been suggested
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Fig. 3. Effects of stress and ageing on glutamate receptor subunits in the hippocampus. Data is shown as % optical density of control (3 months) rats. In each panel, a representative picture of western blot is shown. Two-way ANOVA (stress age), *p < 0.05 main effect of stress. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats).
that maladaptive changes in excitatory/inhibitory circuitry have a primary role in the pathophysiology of mood disorders. Therefore, the first question raised in the present work was: 4.1. Stress or ageing affect or interact to affect the balance between the excitatory (glutamatergic)/inhibitory (GABAergic) inputs in the hippocampus? In order to study this question, the maternal separation (MS) model of chronic stress was chosen. Prolonged separations from the dam have been proposed to be emotionally stressful for the rodent
Fig. 4. Effects of venlafaxine treatment on stress-induced changes in glutamate receptor subunits in the hippocampus. Data is shown as % optical density of control saline (3 months) rats. In each panel, a representative picture of western blot is shown. Two-way ANOVA (stress treatment), #significant interaction p < 0.05 vs control saline (3 months) rats, zsignificant interaction p < 0.05 vs. MS (3 months) saline rats. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats), Ven: venlafaxine.
pups (Pryce et al., 2005). The MS model in rat is considered nowadays as a robust model of enhanced stress responsiveness (Aisa et al., 2007; Ladd et al., 2000; Lehmann and Feldon, 2000; Oitzl et al., 2000). Even though several studies have focus in the characterization of the MS model in adult animals, little work has been done on how neonatal stress would impact the ageing process. In our hands, as previously described (Solas et al., 2010) the behavioural (depressive-like as shown by increased immobility time in the Porsolt swimming test) effects of MS persisted until senescence. This behavioural effect was accompanied by increased levels of the stress hormone corticosterone and decreased expression of GRs, supporting the validity of MS as model of enhanced response to stress.
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Fig. 5. Effects of ageing (A) or venlafaxine treatment (B) on stress-induced changes in corticosterone levels (expressed as % of control young rats) in plasma. In A, two-way ANOVA (stress age), #significant interaction p < 0.05 vs control (3 months) rats. In B, #significant interaction p < 0.05 vs control (3 months) rats, zsignificant interaction p < 0.05 vs. MS (3 months) rats. Corticosterone levels of control young rats were 392.7 51.1 ng/mL. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats); Ven: venlafaxine.
It has been suggested that stress induce changes in glutamate synapses and circuitry. The upregulation of hippocampal EAAT1 and EAAT2 found here in aged stressed rats may occur as a neuroprotective response to stress-induced elevations in glutamate in the synaptic cleft (Duan et al., 1999; Liang et al., 2008) as well as in response to increased corticosterone levels (Rauen and Wiessner, 2000). The upregulation of hippocampal EAAT1 supports the idea
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of increased glutamate release in synapses of stressed animals because this glial transporter is regulated by the amount of glutamate released into the synaptic cleft (Duan et al., 1999; Liang et al., 2008). Further evidence of an MS-induced increase in glutamate release was shown also by the upregulation of glutamate transporter GLAST (SLC1A3) in MS animals (Pickering et al., 2006). Previous studies show that VGLUT1 is the major isoform in the hippocampus (Takamori et al., 2000), where it plays a key role in the vesicular uptake and synaptic transmission of glutamate (Wojcik et al., 2004). Decreased levels of this isoform would support the notion of a decreased uptake of glutamate, perhaps as a compensatory change in a situation of chronic stress. It is of interest that mice heterozygous for the vesicular glutamate transporter 1 (VGLUT1 þ/) showed increased depressive-like behavioural symptoms (Garcia-Garcia et al., 2009). A striking result is the upregulation of VGLUT2 found in MS rats, which is completely reversed in aged stressed animals. It could be suggested that the upregulation of VGLUT2 could contribute to the imbalance between excitatory/inhibitory ratio in the hippocampus, favouring the release of glutamate. However, the loss of VGLUT2 in aged rats could be consequence of the retraction of the dendritic arbor associated to chronic sustained corticosterone levels (see below). Because glutamate is required for the major neuronal GABA synthesis pathway (Mathews and Diamond, 2003), alterations in glutamate release, glial reuptake, or both would be expected to affect GABAergic neurotransmission, and therefore, stress-induced changes of excitatory synaptic composition could be counterbalanced by parallel changes of inhibitory synaptic networks. We suggest that the downregulation of VGAT and its coupled GABA synthesizing enzyme GAD65 in response to MS could lead to decreased GABA levels in the hippocampus. Recent studies addressing the effects of chronic mild stress on GABAergic neurotransmission showed results consistent with ours (Garcia-Garcia et al., 2009; Holm et al., 2011). Altogether, these changes could limit GABA synthesis and elevate levels of extracellular glutamate, which in turn could affect the efficiency of signalling. To further investigate this question, the expression of the different glutamate receptor subunits was studied. In a previous study, Pickering et al. (2006) observed a reduction in hippocampal mRNA expression of the NMDA NR2B and AMPA GluR1 and GluR2 subunits in MS, changes that have not been found presently, perhaps due to the fact that these changes in mRNA levels could be counteracted at the post-transcriptional level. Our results show that the subunit composition of glutamate receptors is altered, and it is especially sensitive to stress, hence affect the pharmacokinetic and physiological properties of the receptor. The present findings point to the importance of understanding the subunit composition of individual glutamate receptors
Fig. 6. Time course of glutamate and GABA release from rat hippocampal slices. Slices came form 3 months old male Wistar rats. Effects of 5, 10 or 30 min incubation with corticosterone on glutamate (left panel) or GABA release (right panel). One-way ANOVA, p < 0.05 vs control. CORT: corticosterone.
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Fig. 7. Glutamate and GABA release from hippocampal slices. Slices came from 3 months old male Wistar rats. In A, effects of different concentrations of corticosterone on glutamate (left panel) or GABA release (right panel). In B, effects of venlafaxine (1 mM) on corticosterone (10 mM)-induced changes. One-way ANOVA, *p < 0.05 vs control, yp < 0.05 vs corticosterone. CORT: corticosterone. Ven: venlafaxine.
in the stressed brain prior to the development of drugs targeted towards those receptors. 4.2. Could stress-induced corticosterone secretion be responsible for changes in glutamate/GABA release? The effects of stress are mediated by the activation of the hypothalamicepituitaryeadrenal (HPA) axis, culminating in increased levels of glucocorticoids. Aged rats, as well as elderly humans, show progressive loss of control of the HPA axis, resulting in hypersecretion of glucocorticoids (present results, Born et al., 1995; Sapolsky et al., 1983; Solas et al., 2010). A considerable body of work, carried out with in vivo microdialysis as well as other methodologies, has shown that both stress or corticosterone treatment induce enhancement of glutamate release, as further supported by the present in vitro results, and that the effects of acute stress on glutamate release are mediated by raised levels of corticosterone (Venero and Borrell, 1999; Stein-Behrens et al., 1994). Interestingly, when comparing results from rats with similar age (young 3 months old), chronic treatment with corticosterone was able to recapitulate the main key biochemical findings observed in MS rats. Regarding the differential results found concerning NR1, GluR1 and GluR2/3, there is no consensus in literature regarding the effects of stress/corticosterone treatment on expression of the different glutamate receptor subunits, which depend importantly on time of stress (neonatal vs corticosterone treatment on adulthood). In any case, and altogether, the present
results point to the idea that corticosterone secretion after stress could be responsible for the changes in glutamate/GABA cycling. In turn, GABA and glutamate play a major role in central integration of HPA stress responses (Herman et al., 2004). Even though we cannot exclude that non-specific membrane effect due to the high lipophilicity of corticosteroids could be associated to the effects of coticosterone on glutamate/GABA release, we propose that glucocorticoid receptors (GR or MR) could be involved, as either the GR antagonist mifepristone or the MR antagonist spironolactone were able to reverse the effects of corticosterone on glutamate/GABA release. Another interesting point is the early response to corticosterone (10 min): It has been shown that corticosteroids may stimulate very rapidly glutamate transmission in the hippocampus. The microdialysis technique has local administration of a high dose of corticosterone to rats increased extracellular glutamate levels in CA1 within 15 min (Venero and Borrell, 1999). Recent electrophysiological and live imaging experiments have however provided experimental data which reinforce the hypothesis that beside these delayed effects, corticosteroid hormones may also act rapidly through membrane receptors. Therefore, in addition to their predominant cytosolic location, corticosteroid receptors may also be located at the cell membrane where they mediate rapid (i.e. non-genomic) and transient effects on stimulation (Chaouloff and Groc, 2011). From the present results it could be suggested that the effects of corticosterone on glutamate release could be mediated, at least in part, by non-conventional MRs receptors.
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Fig. 8. Effect of blockade of glucocorticoid receptors on glutamate and GABA release from rat hippocampal slices. In A, effect of glucocorticoid receptor blockade with mifepristone (MIFE, 1 mM) on corticosterone (10 mM)-induced changes. In B, effect of mineralocorticoid receptor blockade with spironolactone (SPIRO, 1 mM) on corticosterone (10 mM)-induced changes. One-way ANOVA, *p < 0.05 vs control, yp < 0.05 vs corticosterone. CORT: corticosterone.
Results from preclinical studies suggest that stress- and glucocorticoid-induced enhancement of glutamate release and transmission plays a main role in the induction of maladaptive cellular effects, in turn responsible for dendritic remodelling. In a recent review, Musazzi et al. (2011) analysed the putative role that enhancement of glutamate release and transmission, induced by different stressors, leading to changes in neuroplasticity linked to pathophysiology of depression. The authors speculated that an inadequate or excessive stress response, through the action of glucocorticoids, may induce an excess of excitatory neurotransmission in select brain areas, including the hippocampus. This in turn could induce retraction of the dendritic arbor at various locations in these areas, with loss of dendritic spines/synapses which could be linked to volumetric changes consistently found in limbic and cortical areas of depressed subjects (Malykhin et al., 2010). In addition, it is worth mentioning that MS rats show decreased cell proliferation in the hippocampus and alteration in synaptic markers (decreased BDNF and synaptophysin mRNA levels, Aisa et al., 2007) which has been suggested to contribute to an increased vulnerability of the hippocampus to subsequent insults. Therefore, altered glutamate transmission in MS rats might be consequence of alterations in normal development in MS rats, but could also contribute to the maintenance of these alterations during ageing. 4.3. Is a pharmacological intervention with the antidepressant venlafaxine able to counteract the effects of stress/ageing? The combined serotoninenorepinephrine reuptake inhibitor, venlafaxine, has demonstrated better short-term efficacy than other antidepressants, including other selective serotonin reuptake inhibitors in epidemiological pooled analyses (Guelfi et al., 1995; Dierick et al., 1996). As venlafaxine treatment modulates corticosterone levels (present results on corticosterone levels and GR expression), the normalizing actions of venlafaxine on
corticosterone-evoked changes in glutamate/GABA release could be related to its actions on corticosterone signalling. However, as venlafaxine could not alter corticosterone levels in vitro, the dampening action of this drug on changes in glutamate/GABA release must be downstream of this process (Musazzi et al., 2010). Venlafaxine treatment not only modified corticosterone and corticosterone-associated changes in glutamate/GABA release, but also counteracted the changes in glutamate receptor subunits expression associated to MS. Therefore, it could be suggested that antidepressant treatment with venlafaxine in adulthood could prevent from changes associated to the interaction between stress and ageing. 4.4. Conclusions Corticosterone secretion associated to chronic stress could alter glutamate/GABA release which in turn, as compensatory mechanism, results in altered expression of proteins responsible for glutamate/GABA cycling. However, some of these changes could be observed only in aged stressed rats. The different neurochemical effects that chronic stress exerts in young and adult animals may explain the involvement of glucocorticoids in ageing-related diseases, including depression (Pariante and Lightman, 2008), memory impairments (Aisa et al., 2007; Lupien et al., 1998; Sapolsky et al., 1986) or AD (Elgh et al., 2006; Hartmann et al., 1997). An additional important issue is that modulation of glutamate release may be a relevant component of the therapeutic action of drugs such as venlafaxine (Musazzi et al., 2010; Reznikov et al., 2007). Indeed, a dampening of glutamate release could improve the signal to noise ratio, when it becomes compromised by excessive neuronal activation and release due to the action of stress. Understanding the action of antidepressant drugs on glutamate transmission could be of great help in developing drugs for the treatment of mood disorders.
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Fig. 9. Effect of a chronic treatment with corticosterone on proteins related to glutamate/GABA cycling in the hippocampus. Data is shown as % optical density of control saline rats. In each panel, a representative picture of western blot is shown. Statistical analysis by Student’s t-test, *p < 0.05. CORT: corticosterone; VGlut1: vesicular glutamate transporter 1; VGlut2: vesicular glutamate transporter 2; EAAT1: excitatory amino acid transporter 1; EAAT2: excitatory amino acid transporter 2; VGAT: GABA transporter; GAD65: glutamic acid decarboxylase 65.
Acknowledgements Authors thank Ms. M. Luz Muro for her excellent technical assistance. This work has been supported by the Newmood integrated project (EC, LSHM-CT-2004-503474) and “Tu eliges, Tu decides” projects of CAN. All authors disclose any actual or potential conflict of interest including any financial, personal or other relationships with both organizations that sponsored the research. Appendix. Supplementary material Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.neuropharm.2011.12. 019. References Aisa, B., Tordera, R., Lasheras, B., Del Río, J., Ramírez, M.J., 2007. Cognitive impairment associated to HPA axis hyperactivity after maternal separation in rats. Psychoneuroendocrinology 32, 256e266. Bhagwagar, Z., Wylezinska, M., Jezzard, P., Evans, J., Ashworth, F., Sule, A., Matthews, P.M., Cowen, P.J., 2007. Reduction in occipital cortex gammaaminobutyric acid concentrations in medication-free recovered unipolar depressed and bipolar subjects. Biol. Psychiatry 61, 806e812. Born, J., Ditschuneit, I., Schreiber, M., Dodt, C., Fehm, H.L., 1995. Effects of age and gender on pituitaryeadrenocortical responsiveness in humans. Eur. J. Endocrinol. 132, 705e711. Chaouloff, F., Groc, L., 2011. Temporal modulation of hippocampal excitatory transmission by corticosteroids and stress. Front. Neuroendocrinol. 32, 25e42.
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Contents lists available at SciVerse ScienceDirect
Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Long lasting effects of early-life stress on glutamatergic/GABAergic circuitry in the rat hippocampus Eva Martisova a, Maite Solas a, Igor Horrillo b, Jorge E. Ortega b, J. Javier Meana b, Rosa María Tordera a, María Javier Ramírez a, * a b
Department of Pharmacology, University of Navarra, C/ Irunlarrea 1, 31008 Pamplona, Spain Department of Pharmacology, University of the Basque Country (UPV/EHU) and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Leioa, Bizkaia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 August 2011 Received in revised form 14 December 2011 Accepted 19 December 2011
The objective of the present work was to study the effects of an early-life stress (maternal separation, MS) in the excitatory/inhibitory ratio as a potential factor contributing to the ageing process, and the purported normalizing effects of chronic treatment with the antidepressant venlafaxine. MS induced depressive-like behaviour in the Porsolt forced swimming test that was reversed by venlafaxine, and that persisted until senescence. Aged MS rats showed a downregulation of vesicular glutamate transporter 1 and 2 (VGlut1 and VGlut2) and GABA transporter (VGAT) and increased expression of excitatory amino acid transporter 2 (EAAT2) in the hippocampus. Aged rats showed decreased expression of glutamic acid decarboxylase 65 (GAD65), while the excitatory amino acid transporter 1 (EAAT1) was affected only by stress. Glutamate receptor subunits NR1 and NR2A and GluR4 were upregulated in stressed rats, and this effect was reversed by venlafaxine. NR2B, GluR1 and GluR2/3 were not affected by either stress or age. MS, both in young and aged rats, induced an increase in the circulating levels of corticosterone. Corticosterone induced an increase glutamate and a decrease in GABA release in hippocampal slices, which was reversed by venlafaxine. Chronic treatment with corticosterone recapitulated the main biochemical findings observed in MS. The different effects that chronic stress exerts in young and adult animals on expression of proteins responsible for glutamate/GABA cycling may explain the involvement of glucocorticoids in ageing-related diseases. Modulation of glutamate/GABA release may be a relevant component of the therapeutic action of antidepressants, such as venlafaxine. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Venlafaxine Ageing VGAT VGLUT EAAT Corticosterone
1. Introduction Stress is believed to contribute to the variability of the ageing process and to the development of age-related neuro- and psychopathologies (Heim and Nemeroff, 1999; McEwen, 2002; Miller and O’Callaghan, 2005). In fact, the experience of stress or traumatic experience early in life is thought to make an individual more vulnerable for psychiatric problems, such as depression or anxiety, later in life (Gilmer and McKinney, 2003; Heim and Nemeroff, 2001). Abnormalities in glutamate and gamma-aminobutyric acid (GABA) signal transmission have been postulated to play a role in depression (Krystal et al., 2002). Increased glutamate and reduced GABA levels have been observed in the cortex of depressed patients, leading to an enhanced excitatoryeinhibitory ratio (Bhagwagar
* Corresponding author. Tel.: þ34 948425600; fax: þ34 948425649. E-mail address: [email protected] (M.J. Ramírez). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.12.019
et al., 2007; Sanacora et al., 1999). Interestingly, this imbalance is inhibited by chronic treatment with antidepressants (Sanacora et al., 2002). Because the presynaptic pathways regulating the synthesis and cycling of glutamate and GABA are tightly coupled, it has been suggested that alterations in a shared pathway may account for the observed amino acid abnormalities. For instance, post-mortem studies have shown decreased expression of glutamic acid decarboxylase 65 (GAD65), the enzyme that convert glutamate to GABA, in mood disorders (Fatemi et al., 2005). Microarray analysis of cerebral cortex from individuals who had suffered from major depression disorder have demonstrated significant downregulation of the glial excitatory amino acid transporter 1 and 2 (EAAT1 and EAAT2), key members of the glutamate/neutral amino acid transporter protein family (Choudary et al., 2005). At the experimental level, decrease of the vesicular GABA transporter (VGAT), and GAD65 and an upregulation of EAAT1 has been shown in the hippocampus of animals subjected to chronic stress when adults (chronic mild stress, CMS, Garcia-Garcia et al., 2009). Mice heterozygous for the vesicular glutamate transporter 1 (VGLUT1 þ/) showed increased
E. Martisova et al. / Neuropharmacology 62 (2012) 1944e1953
depressive-like behavioural symptoms as well as increased neuronal synthesis of glutamate and decreased hippocampal GABA, VGLUT1, and EAAT1 levels (Garcia-Garcia et al., 2009). Emotional experience during early life has been shown to interfere with the development of excitatory synaptic networks in hippocampus of rodents. Maternal separation (MS) is an animal paradigm designed to mimic repeated exposure to stress during early life, resulting in animals with behavioural and neuroendocrine signs of elevated stress reactivity as adults (Aisa et al., 2007; Heim and Nemeroff, 2001; Lehmann and Feldon, 2000) or senescent (Solas et al., 2010). The peak period of neurogenesis overlaps the stress hyporesponsive period (postnatal days 4e14) in neonatal rats (Sapolsky and Meaney, 1986). Therefore, early stress, such as MS, could be interfering with the normal maturation of excitatory/ inhibitory synapses in the hippocampus, which might ultimately lead to an increased vulnerability for psychiatric diseases. To test this hypothesis, we have studied the lasting consequences of early-life stress exposure on the expression of presynaptic proteins involved in the glutamate/GABA cycle and on the expression of different glutamate receptor subunits in the hippocampus of young and aged rats. In addition, we have checked if the treatment with the antidepressant venlafaxine in adulthood could be effective in preventing the purported interaction between ageing and stress. Finally, as acute stress is known to increase glutamate release (Gould et al., 2000; Lowy et al., 1993; Musazzi et al., 2010), the effects of the stress hormone corticosterone on glutamate/GABA release “in vitro” have been checked. 2. Materials and methods 2.1. Animals All the experiments were carried out in strict compliance with the recommendations of the EU (86/609/EEC) for the care and use of laboratory animals. All efforts were made to minimise animal suffering, to reduce the number of animals, and alternative to in vivo techniques (in vitro release experiments) have been used. Timed-pregnant Wistar rats were provided on gestation day 16 from Charles River Laboratories (Portage, MI, USA), individually housed in a temperature (21 1 C) and humidity (55 5%) controlled room on a 12-h light/dark cycle with food and water freely available. Animals from different litters were evenly spread over the different experimental groups. For the chronic treatment with corticosterone, commercially made pellets (Innovative Research of America, Sarasota, FL, USA) designed for daily administration of 18 mg/kg of corticosterone or placebo were used. Pellets were implanted subcutaneously on the right flank of male rats (3 months old, n ¼ 6) under chloral hydrate anaesthesia. After the surgical procedure, rats were left to recover. Thirtyfive days after pellet implantation, rats were killed by decapitation, brains removed and hippocampal samples frozen at 80 C until use. 2.2. Maternal separation All litters were born within a 2-day period. As previously described in detail (Aisa et al., 2007), on postnatal day (PND) 2, all pups were sexed and randomly assigned to the control group (animal facility rearing, AFR), pups were only briefly manipulated to change the bedding in their cages once weekly, or the separation group (MS), pups separated from their dam for 180 min from PNDs 2e21 inclusive. Rats were weaned on PND 23 and only males were chosen for the present work. Experiments were performed in young (60e75 days) and aged rats (18 months). Rats were not allowed to grow older as mortality increased significantly in the aged MS group. In experiments testing the effects of the antidepressant treatment, saline or venlafaxine (20 mg/kg, p.o.) were administered daily for 15 days, beginning on PND 60. Venlafaxine was dissolved in saline. The last administration of saline/venlafaxine took place 24 h before behavioural testing. 2.3. Forced swimming To study depressive-like behaviour, the Porsolt forced swimming test was used as described by Porsolt et al. (1977). Two swimming sessions were conducted: an initial 15-min pretest followed 24 h later by a 5-min test. Rats were placed individually in a vertical plexiglass cylinder (height: 45 cm, diameter: 19 cm) filled with 28e30 cm of 26 C water. Immobility was considered as rats floating passively,
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making only small movements to keep its nose above the surface. Immobility times were scored by Ethovision XT 5.0 (Noldus, Netherland). 2.4. Plasma corticosterone determinations Rats were sacrificed by decapitation between 08:00 and 10:00 h, and 24 h after the last administration of venlafaxine. Trunk blood was collected into EDTA tubes, centrifuged at 1250 g (15 min, 4 C), and plasma was frozen and stored at 80 C until processing. Plasma corticosterone was measured using a commercially available radioinmmunoassay kit (Coat-A-Count Rat Corticosterone, Siemens, Los Angeles CA, USA). All assays were performed in duplicate. Limit of detection was 5.7 ng/ml and intra- and interassay coefficients of variation were less than 10% for all comparisons. Corticosterone concentration values were expressed as ng/ml. 2.5. Western blotting Hippocampal samples were homogenized in a 50 mM Tris buffer (pH 7.2, 4 C). Each sample was adjusted to a final protein concentration of 4 mg/ml (DC protein assay; Bio-Rad, Hercules, CA). Extracts were mixed with Laemmeli’s sample buffer boiled for 5 min. Assays were performed as described in Table 1. Immunopositive bands were visualized by a chemiluminescent method (ECL; Amersham, Arlington Heights, IL). The optical density of reactive bands visible on x-ray film was determined densitometrically. b-actin was used as internal control. Results were expressed as percentage of OD values of controls. 2.6. “In vitro” depolarization-evoked glutamate and GABA release Male Wistar rats, 3 months old, were used. As previously described (Marcos et al., 2006), the hippocampus was dissected out and cut sagitally into 500 mm slices using a McIlwain tissue chopper (The Mickle Laboratory Engineering Co. Ltd.). Slices were allowed to equilibrate for 20 min at room temperature in gassed KrebsRinger bicarbonate buffer (KRB) made up of (mmol/l): NaCl, 113; KCl, 4.75; CaCl2, 2.52; MgSO4, 1.19; NaH2PO4, 1.18; NaHCO3, 25; glucose, 10; pH, 7.4. Tissue was then transferred into superfusion chambers (Brandel Superfusion-1000 apparatus) and continuously superfused with continuously oxygenated KRB at a rate of 0.35 ml/min. After a 40 min equilibration period, fractions were collected at 5-min intervals for a total of 40 min (eight samples). The first three samples were used to assess a stable basal neurotransmitter release. Slices were depolarized by changing the superfusion fluid for 5-min to a KRB solution containing 30 mM KCl. Corticosterone was added 5, 10 or 30 min before depolarization. Mifepristone, spironolactones or venlafaxine were added 10-min before depolarization. Neurotransmitter levels were measured in the superfusate fractions. Glutamate and GABA content in supernatant were measured using high performance liquid chromatography HPLC with electrochemical detection (Waters SpheriborÒ 5m ODS2 4.6 150 mm) including precolumn derivatization with o-phthalaldehyde (SigmaeAldrich Ltd, Germany) and b-mercaptoethanol (SigmaeAldrich Ltd, Germany). The mobile phase consisted of 72:28 (v/v) mixture of buffer (NaH2PO4 0.1 M, pH ¼ 5.5) and methanol; the mixture was filtered and degassed through a 0.22 mm nitrocellulose membrane (Millipore, UK). Glutamate and GABA content were calculated by comparing with a 2 ng standard. The limit of detection was 20 pg/10 ml for glutamate and 50 pg/10 ml for GABA content. 2.7. Data analysis Data were analysed by SPSS for Windows, release 11.0. Normality was checked by ShapiroeWilks’s test (p > 0.05). Data were analysed by one or two-way analysis
Table 1 Conditions used in Western blotting experiments. Protein
Membrane
SDSpolyacrylamide gel
Primary antibody (dilution)
VOLUTI VGLUT2 VGAT GAD65 EAAT1 EAAT2 NR1 NR2A NR2B GluR1 GluR2/3 GluR4
PVDF PVDF PVDF PVDF Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose
10% 10% 10% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5% 7.5%
Rabbit anti-VGLUT1 (l:2000)a Mouse anti-VGLUT2 (l:1000)b Rabbit anti-VGAT (l:1000)b Mouse anti-GAD65 (l:5000)c Rabbit anti-EAAT1 (l:2500)d Rabbit anti-EAAT2 (l:2500)d Rabbit anti-NR1 (1:1000)e Rabbit anti-NR2A (1:1000)e Rabbit anti-NR2B (1:1000)e Rabbit anti-GluR1 (1:1000)e Rabbit anti-GluR2/3(1:1000)e Rabbit anti-GluR4 (1:1000)e
Source of antibodies: a) donated by Dr. S. El Mestikawy, Paris, France; b) Chemicon, Temecula, California; c) Abcam Inc., Cambridge, UK; d) Santa Cruz, Heidelberg, Germany; e) Upstate.
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of variance ANOVA (rearing age or rearing treatment), followed by Student’s ttest adjusted by Bonferroni correction. For all analyses, post hoc comparisons were conducted if appropriate, using Tukey protected least significance test.
3. Results 3.1. Depressive-like behaviour In the forced swimming test (Fig. 1A), two-way ANOVA indicated a significant main effect of stress [F1,82 ¼ 17.217, p < 0.001; n ¼ 10 per group]. Venlafaxine treatment (Fig. 1B) reversed the depressive phenotype associated to MS in young rats [significant interaction stress treatment, F1,59 ¼ 3.386, p < 0.05; n ¼ 10 per group]. 3.2. Regulation of presynaptic proteins As shown in Fig. 2, aged MS rats showed decreased expression of VGlut1 [significant interaction stress age, F1,35 ¼ 11.239, p < 0.05; n ¼ 9 per group]. A specific interaction stress age was found in the expression VGlut2 [F1,35 ¼ 15.675, p < 0.001; n ¼ 9 per group], as even though stress or ageing increased the expression of the transporter, the combined effect decreased its expression. EAAT1
expression was affected only by stress [main effect of stress, F1,35 ¼ 4.974, p < 0.05; n ¼ 9 per group], while aged MS rats showed increased expression of EAAT2 [significant interaction stress age, F1,30 ¼ 9.261, p < 0.01; n ¼ 7e8 per group]. An interaction between stress age was shown in VGAT, and aged MS rats showed increased expression of VGAT [F1,34 ¼ 3.725, p < 0.05; n ¼ 7e9 per group]. Aged rats showed decreased expression of GAD65 [main effect of age, F1,35 ¼ 4.428, p < 0.05; n ¼ 9 per group]. 3.3. Regulation of glutamate receptor subunits NMDA receptor subunits NR1 and NR2A were upregulated in stressed rats [main effect of stress, F1,35 ¼ 8.427, p < 0.01 and F1,35 ¼ 9.466, p < 0.01; n ¼ 9 per group, respectively]. GluR4, subunit of the AMPA-type glutamate receptors was also upregulated in stressed rats [main effect of stress F1,35 ¼ 13.102, p < 0.001; n ¼ 9 per group]. These results are shown in Fig. 3. These effects of stress on glutamate receptor subunits were reverted by venlafaxine [F1,35 ¼ 24.015, p < 0.001, F1,35 ¼ 5.287, p < 0.05 and F1,35 ¼ 4.426, p < 0.05; n ¼ 9 per group, for NR1, NR2A and GluR4 respectively]. All these results are depicted in Fig. 4. The expression of NR2B, GluR1 and GluR2/3 subunits was not affected by either stress or age (Supplementary Fig. 1). 3.4. Levels of plasmatic corticosterone There was a significant rearing age interaction in plasma corticosterone levels [two-way ANOVA, F1,50 ¼ 4.689, p < 0.05; n ¼ 8e19]. Further analysis showed a significant increase in corticosterone levels in young MS rats (Student’s t-test; p < 0.05), aged rats (Student’s t-test; p < 0.05) and almost significant effect in aged MS rats (Student’s t-test; p ¼ 0.06) compared with young controls (Fig. 5A). As shown in Fig. 5B, the increase in corticosterone levels (expressed as % control young rats) in young rats was reversed by chronic treatment with venlafaxine [two-way ANOVA, interaction rearing treatment, F(1,54) ¼ 6.594, p ¼ 0.05; chronic treatments; n ¼ 8e10 per group]. 3.5. Glutamate and GABA release “in vitro”
Fig. 1. Effects of ageing (A) or venlafaxine treatment (B) on stress-induced depressivelike behaviour in the Porsolt forced swimming test. In A, two-way ANOVA (stress age), *p < 0.05 main effect of stress. In B, two-way ANOVA (stress treatment), #significant interaction p < 0.05 vs control (3 months) rats, zsignificant interaction p < 0.05 vs. MS (3 months) rats. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats); Ven: venlafaxine.
All this experiments were performed in hippocampal slices obtained from 3 month old male Wistar rats. In a pilot set of experiments, different time points (5, 10 or 30 min) of incubation with corticosterone 10 mM were checked (Fig. 6), and maximal effects were found at 10 min (one-way ANOVA, F3,33 ¼ 4.541, p < 0.05, n ¼ 8 per group and F3,33 ¼ 3.998, p < 0.05, n ¼ 8 per group for glutamate and GABA respectively). Corticosterone (0.1-1-10 mM, 10 min incubation) induced a concentration-dependant increase in Kþ-induced glutamate release in slices of rat hippocampus, and this increase was significant at the higher concentration of corticosterone used (one-way ANOVA, F3,33 ¼ 4.747, p < 0.05, n ¼ 8 per group, Fig. 7). This increase in glutamate release seemed to be receptor mediated, as both the glucocorticoid receptor (GR) antagonist mifepristone, 1 mM [one-way ANOVA F2,23 ¼ 2.905, p < 0.05, n ¼ 8 per group], or the mineralocorticoid receptor (MR) antagonist spironolactone, 1 mM, were able to reverse the increases in glutamate release after corticosterone (one-way ANOVA F2,23 ¼ 3.644, p < 0.05, n ¼ 8 per group, Fig. 8). Venlafaxine, 1 mM, was able to counteract corticosterone (10 mM)-evoked glutamate release (F2,16 ¼ 2.941, p < 0.05, n ¼ 6 per group, Fig. 7). Kþ-induced GABA release was significantly inhibited by corticosterone, 10 mM (one-way ANOVA, F3,33 ¼ 2.944, p < 0.05, n ¼ 8 per group). Both mifepristone and spironolactone were able to
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Fig. 2. Effects of stress and ageing on vesicular glutamate transporter 1 (VGlut1) and 2 (VGlut2), excitatory amino acid transporter 1 (EAAT1) and 2 (EAAT2), GABA transporter (VGAT), and glutamic acid decarboxylase 65 (GAD65) in the hippocampus. Data is shown as % optical density of control (3 months) rats. In each panel, a representative picture of western blot is shown. Two-way ANOVA (stress age), *p < 0.05 main effect of stress, yp < 0.05 main effect of age, #significant interaction p < 0.05 vs control (3 months) rats, zsignificant interaction p < 0.05 vs. MS (3 months) rats. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats).
counteract the corticosterone-induced inhibition on GABA release (one-way ANOVA F2,23 ¼ 4.134, p < 0.05 and F2,23 ¼ 1.206, p < 0.05, n ¼ 8 per group, respectively). As shown in Fig. 7, there was a tendency, although not significant of venlafaxine, 1 mM, to reverse the inhibition.
increased expression of NR2A and GluR4 and no changes in NR2B (in all cases Student’s t-test vs control, p < 0.05). At difference with MS, NR1, GluR1 and GluR2/3 expression was decreased (in all cases Student’s t-test vs control, p < 0.05). All these results are depicted in Fig. 9.
3.6. Effects of a chronic treatment with corticosterone
4. Discussion
To study a causal relationship between elevated corticosterone levels and the biochemical alterations observed after MS, the effects of a chronic treatment with corticosterone in 3 months old rats were checked. At similarity to data from 3 months old MS rats, corticosterone treatment induced increased expression of VGlut2, EAAT1 and EAAT2, no changes in VGlut1, VGAT and GAD65,
Stressful life events are known to precipitate mood/anxiety disorders. In fact, experimental models of chronic stress are considerer nowadays as useful models to study depression. The effects of early-life stress endure and worsen during ageing (Solas et al., 2010), yet the mechanisms for these purportedly clinically important sequelae are poorly understood. It has been suggested
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Fig. 3. Effects of stress and ageing on glutamate receptor subunits in the hippocampus. Data is shown as % optical density of control (3 months) rats. In each panel, a representative picture of western blot is shown. Two-way ANOVA (stress age), *p < 0.05 main effect of stress. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats).
that maladaptive changes in excitatory/inhibitory circuitry have a primary role in the pathophysiology of mood disorders. Therefore, the first question raised in the present work was: 4.1. Stress or ageing affect or interact to affect the balance between the excitatory (glutamatergic)/inhibitory (GABAergic) inputs in the hippocampus? In order to study this question, the maternal separation (MS) model of chronic stress was chosen. Prolonged separations from the dam have been proposed to be emotionally stressful for the rodent
Fig. 4. Effects of venlafaxine treatment on stress-induced changes in glutamate receptor subunits in the hippocampus. Data is shown as % optical density of control saline (3 months) rats. In each panel, a representative picture of western blot is shown. Two-way ANOVA (stress treatment), #significant interaction p < 0.05 vs control saline (3 months) rats, zsignificant interaction p < 0.05 vs. MS (3 months) saline rats. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats), Ven: venlafaxine.
pups (Pryce et al., 2005). The MS model in rat is considered nowadays as a robust model of enhanced stress responsiveness (Aisa et al., 2007; Ladd et al., 2000; Lehmann and Feldon, 2000; Oitzl et al., 2000). Even though several studies have focus in the characterization of the MS model in adult animals, little work has been done on how neonatal stress would impact the ageing process. In our hands, as previously described (Solas et al., 2010) the behavioural (depressive-like as shown by increased immobility time in the Porsolt swimming test) effects of MS persisted until senescence. This behavioural effect was accompanied by increased levels of the stress hormone corticosterone and decreased expression of GRs, supporting the validity of MS as model of enhanced response to stress.
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Fig. 5. Effects of ageing (A) or venlafaxine treatment (B) on stress-induced changes in corticosterone levels (expressed as % of control young rats) in plasma. In A, two-way ANOVA (stress age), #significant interaction p < 0.05 vs control (3 months) rats. In B, #significant interaction p < 0.05 vs control (3 months) rats, zsignificant interaction p < 0.05 vs. MS (3 months) rats. Corticosterone levels of control young rats were 392.7 51.1 ng/mL. MS: maternal separation; 3 m: 3 months (young rats); 18 m: 18 months (aged rats); Ven: venlafaxine.
It has been suggested that stress induce changes in glutamate synapses and circuitry. The upregulation of hippocampal EAAT1 and EAAT2 found here in aged stressed rats may occur as a neuroprotective response to stress-induced elevations in glutamate in the synaptic cleft (Duan et al., 1999; Liang et al., 2008) as well as in response to increased corticosterone levels (Rauen and Wiessner, 2000). The upregulation of hippocampal EAAT1 supports the idea
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of increased glutamate release in synapses of stressed animals because this glial transporter is regulated by the amount of glutamate released into the synaptic cleft (Duan et al., 1999; Liang et al., 2008). Further evidence of an MS-induced increase in glutamate release was shown also by the upregulation of glutamate transporter GLAST (SLC1A3) in MS animals (Pickering et al., 2006). Previous studies show that VGLUT1 is the major isoform in the hippocampus (Takamori et al., 2000), where it plays a key role in the vesicular uptake and synaptic transmission of glutamate (Wojcik et al., 2004). Decreased levels of this isoform would support the notion of a decreased uptake of glutamate, perhaps as a compensatory change in a situation of chronic stress. It is of interest that mice heterozygous for the vesicular glutamate transporter 1 (VGLUT1 þ/) showed increased depressive-like behavioural symptoms (Garcia-Garcia et al., 2009). A striking result is the upregulation of VGLUT2 found in MS rats, which is completely reversed in aged stressed animals. It could be suggested that the upregulation of VGLUT2 could contribute to the imbalance between excitatory/inhibitory ratio in the hippocampus, favouring the release of glutamate. However, the loss of VGLUT2 in aged rats could be consequence of the retraction of the dendritic arbor associated to chronic sustained corticosterone levels (see below). Because glutamate is required for the major neuronal GABA synthesis pathway (Mathews and Diamond, 2003), alterations in glutamate release, glial reuptake, or both would be expected to affect GABAergic neurotransmission, and therefore, stress-induced changes of excitatory synaptic composition could be counterbalanced by parallel changes of inhibitory synaptic networks. We suggest that the downregulation of VGAT and its coupled GABA synthesizing enzyme GAD65 in response to MS could lead to decreased GABA levels in the hippocampus. Recent studies addressing the effects of chronic mild stress on GABAergic neurotransmission showed results consistent with ours (Garcia-Garcia et al., 2009; Holm et al., 2011). Altogether, these changes could limit GABA synthesis and elevate levels of extracellular glutamate, which in turn could affect the efficiency of signalling. To further investigate this question, the expression of the different glutamate receptor subunits was studied. In a previous study, Pickering et al. (2006) observed a reduction in hippocampal mRNA expression of the NMDA NR2B and AMPA GluR1 and GluR2 subunits in MS, changes that have not been found presently, perhaps due to the fact that these changes in mRNA levels could be counteracted at the post-transcriptional level. Our results show that the subunit composition of glutamate receptors is altered, and it is especially sensitive to stress, hence affect the pharmacokinetic and physiological properties of the receptor. The present findings point to the importance of understanding the subunit composition of individual glutamate receptors
Fig. 6. Time course of glutamate and GABA release from rat hippocampal slices. Slices came form 3 months old male Wistar rats. Effects of 5, 10 or 30 min incubation with corticosterone on glutamate (left panel) or GABA release (right panel). One-way ANOVA, p < 0.05 vs control. CORT: corticosterone.
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Fig. 7. Glutamate and GABA release from hippocampal slices. Slices came from 3 months old male Wistar rats. In A, effects of different concentrations of corticosterone on glutamate (left panel) or GABA release (right panel). In B, effects of venlafaxine (1 mM) on corticosterone (10 mM)-induced changes. One-way ANOVA, *p < 0.05 vs control, yp < 0.05 vs corticosterone. CORT: corticosterone. Ven: venlafaxine.
in the stressed brain prior to the development of drugs targeted towards those receptors. 4.2. Could stress-induced corticosterone secretion be responsible for changes in glutamate/GABA release? The effects of stress are mediated by the activation of the hypothalamicepituitaryeadrenal (HPA) axis, culminating in increased levels of glucocorticoids. Aged rats, as well as elderly humans, show progressive loss of control of the HPA axis, resulting in hypersecretion of glucocorticoids (present results, Born et al., 1995; Sapolsky et al., 1983; Solas et al., 2010). A considerable body of work, carried out with in vivo microdialysis as well as other methodologies, has shown that both stress or corticosterone treatment induce enhancement of glutamate release, as further supported by the present in vitro results, and that the effects of acute stress on glutamate release are mediated by raised levels of corticosterone (Venero and Borrell, 1999; Stein-Behrens et al., 1994). Interestingly, when comparing results from rats with similar age (young 3 months old), chronic treatment with corticosterone was able to recapitulate the main key biochemical findings observed in MS rats. Regarding the differential results found concerning NR1, GluR1 and GluR2/3, there is no consensus in literature regarding the effects of stress/corticosterone treatment on expression of the different glutamate receptor subunits, which depend importantly on time of stress (neonatal vs corticosterone treatment on adulthood). In any case, and altogether, the present
results point to the idea that corticosterone secretion after stress could be responsible for the changes in glutamate/GABA cycling. In turn, GABA and glutamate play a major role in central integration of HPA stress responses (Herman et al., 2004). Even though we cannot exclude that non-specific membrane effect due to the high lipophilicity of corticosteroids could be associated to the effects of coticosterone on glutamate/GABA release, we propose that glucocorticoid receptors (GR or MR) could be involved, as either the GR antagonist mifepristone or the MR antagonist spironolactone were able to reverse the effects of corticosterone on glutamate/GABA release. Another interesting point is the early response to corticosterone (10 min): It has been shown that corticosteroids may stimulate very rapidly glutamate transmission in the hippocampus. The microdialysis technique has local administration of a high dose of corticosterone to rats increased extracellular glutamate levels in CA1 within 15 min (Venero and Borrell, 1999). Recent electrophysiological and live imaging experiments have however provided experimental data which reinforce the hypothesis that beside these delayed effects, corticosteroid hormones may also act rapidly through membrane receptors. Therefore, in addition to their predominant cytosolic location, corticosteroid receptors may also be located at the cell membrane where they mediate rapid (i.e. non-genomic) and transient effects on stimulation (Chaouloff and Groc, 2011). From the present results it could be suggested that the effects of corticosterone on glutamate release could be mediated, at least in part, by non-conventional MRs receptors.
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Fig. 8. Effect of blockade of glucocorticoid receptors on glutamate and GABA release from rat hippocampal slices. In A, effect of glucocorticoid receptor blockade with mifepristone (MIFE, 1 mM) on corticosterone (10 mM)-induced changes. In B, effect of mineralocorticoid receptor blockade with spironolactone (SPIRO, 1 mM) on corticosterone (10 mM)-induced changes. One-way ANOVA, *p < 0.05 vs control, yp < 0.05 vs corticosterone. CORT: corticosterone.
Results from preclinical studies suggest that stress- and glucocorticoid-induced enhancement of glutamate release and transmission plays a main role in the induction of maladaptive cellular effects, in turn responsible for dendritic remodelling. In a recent review, Musazzi et al. (2011) analysed the putative role that enhancement of glutamate release and transmission, induced by different stressors, leading to changes in neuroplasticity linked to pathophysiology of depression. The authors speculated that an inadequate or excessive stress response, through the action of glucocorticoids, may induce an excess of excitatory neurotransmission in select brain areas, including the hippocampus. This in turn could induce retraction of the dendritic arbor at various locations in these areas, with loss of dendritic spines/synapses which could be linked to volumetric changes consistently found in limbic and cortical areas of depressed subjects (Malykhin et al., 2010). In addition, it is worth mentioning that MS rats show decreased cell proliferation in the hippocampus and alteration in synaptic markers (decreased BDNF and synaptophysin mRNA levels, Aisa et al., 2007) which has been suggested to contribute to an increased vulnerability of the hippocampus to subsequent insults. Therefore, altered glutamate transmission in MS rats might be consequence of alterations in normal development in MS rats, but could also contribute to the maintenance of these alterations during ageing. 4.3. Is a pharmacological intervention with the antidepressant venlafaxine able to counteract the effects of stress/ageing? The combined serotoninenorepinephrine reuptake inhibitor, venlafaxine, has demonstrated better short-term efficacy than other antidepressants, including other selective serotonin reuptake inhibitors in epidemiological pooled analyses (Guelfi et al., 1995; Dierick et al., 1996). As venlafaxine treatment modulates corticosterone levels (present results on corticosterone levels and GR expression), the normalizing actions of venlafaxine on
corticosterone-evoked changes in glutamate/GABA release could be related to its actions on corticosterone signalling. However, as venlafaxine could not alter corticosterone levels in vitro, the dampening action of this drug on changes in glutamate/GABA release must be downstream of this process (Musazzi et al., 2010). Venlafaxine treatment not only modified corticosterone and corticosterone-associated changes in glutamate/GABA release, but also counteracted the changes in glutamate receptor subunits expression associated to MS. Therefore, it could be suggested that antidepressant treatment with venlafaxine in adulthood could prevent from changes associated to the interaction between stress and ageing. 4.4. Conclusions Corticosterone secretion associated to chronic stress could alter glutamate/GABA release which in turn, as compensatory mechanism, results in altered expression of proteins responsible for glutamate/GABA cycling. However, some of these changes could be observed only in aged stressed rats. The different neurochemical effects that chronic stress exerts in young and adult animals may explain the involvement of glucocorticoids in ageing-related diseases, including depression (Pariante and Lightman, 2008), memory impairments (Aisa et al., 2007; Lupien et al., 1998; Sapolsky et al., 1986) or AD (Elgh et al., 2006; Hartmann et al., 1997). An additional important issue is that modulation of glutamate release may be a relevant component of the therapeutic action of drugs such as venlafaxine (Musazzi et al., 2010; Reznikov et al., 2007). Indeed, a dampening of glutamate release could improve the signal to noise ratio, when it becomes compromised by excessive neuronal activation and release due to the action of stress. Understanding the action of antidepressant drugs on glutamate transmission could be of great help in developing drugs for the treatment of mood disorders.
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Fig. 9. Effect of a chronic treatment with corticosterone on proteins related to glutamate/GABA cycling in the hippocampus. Data is shown as % optical density of control saline rats. In each panel, a representative picture of western blot is shown. Statistical analysis by Student’s t-test, *p < 0.05. CORT: corticosterone; VGlut1: vesicular glutamate transporter 1; VGlut2: vesicular glutamate transporter 2; EAAT1: excitatory amino acid transporter 1; EAAT2: excitatory amino acid transporter 2; VGAT: GABA transporter; GAD65: glutamic acid decarboxylase 65.
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