Psychopharmacology
Psychopharmacology (1990) 100: 60-65
© Springer-Verlag 1990
Regional neurotransmitter responses after acute and chronic electroconvulsive shock Paul Glue 1, Michael J. CosteIlo:, Agu Pert 2, Andrea Mele z, and David J. Nutt I 1 National Institute on Alcohol Abuse and Alcoholism, and 2 National Institute of Mental Health, Bethesda, MD 20892, USA
Abstract. Regional neurotransmitter changes after acute and chronic electroconvulsive shock (ECS) were studied using the technique of repeated microdiatysis. Microdialysis was carried out on alternate sides of the brains of anaesthetised rats before and during the first and the eighth ECS or sham (control) treatments. Extracellular fluid release of monoamines and their metabolites was measured in the frontal cortex, striatum and nucleus accumbens using HPLC with electrochemical detection. The first ECS produced selective regional responses, shown by increased concentrations of noradrenaline (NA) and dopamine (DA) in frontal cortex, by unchanged DA content in striatum, and by a small rise in NA and a fall in DA concentrations in nucleus accumbens. Concentrations of metabolites increased after ECS in all regions studied, and for homovanillic acid and dihydroxyphenylacetic acid, the temporal pattern of these changes did not resemble that of DA. Comparison of neurotransmitter responses as per cent of baseline release after the first and eighth ECS treatments showed they were identical. Basal release of monoamines and metabolites before the first ECS or sham treatment was similar in all regions studied. Prior to the eighth treatment, basal release of NA in the frontal cortex and DA in the striatum was elevated in the ECS-treated animals, while basal release of N A in the nucleus accumbens was reduced in both ECSand sham-treated animals. These data suggest that acute and chronic ECS have different and region-specific effects on neurotransmitter release, although the overall pattern of these responses is not changed by chronic treatment. The catecholamine-releasing actions of ECS, and the changes in basal release of neurotransmitters seen after chronic treatment may contribute to its therapeutic effects. Key words: Microdialysis- Electroconvulsive shock - N o r adrenaline - Dopamine Regional responses - Rat
Etectroconvulsive therapy is an effective and safe method of treating severe depression (Kendell 1981). It mechanism of action is unclear, although it probably includes some effects on monoamine neurotransmitters (for reviews, see Green and Nutt 1987; Gleiter and Nutt 1989). Most investigations into these changes have been carried out using elecOffprint requests to: P. Glue, Reckitt and Colman Psychopharmacotogy Unit, Department of Pharmacology, The Medical School, University Walk, Bristol BS8 ITD, UK
troconvulsive shock (ECS) in animals, and have looked at either changes in biochemistry, or functional or behaviourat changes of a number of receptor systems (see reviews by Lerer and Belmaker 1982; Green and Nutt 1987; Gleiter and Nutt 1989). The findings from studies of biochemical changes after ECS have been inconsistent (Schildkraut and Draskoczy 1974; Arato et al. t980; Lerer and Belmaker 1982), although more consistent alterations in receptor binding have been demonstrated with reduced beta- and alpha-2-adrenoceptor (for reviews see Lerer 1984; Gleiter and Nutt 1989), and increased 5-HTz receptor number (Vetulani et al. ]981, 1983; Goodwin et al. 1985). These are supported by studies using behavioural models of receptor function (Grahame-Smith et al. 1978; Green and Deakin 1980; Lerer and Belmaker 1982; Green et al. 1986). Overall, these studies indicate that ECS produces alterations in NA and DA function, but as yet there are no studies of the actual effects of ECS upon monoamine release, especially with regard to regional differences. To this end, we have used the technique of repeated microdialysis to examine the effects of acute and chronic ECS on extracellular fluid release of monoamines and metabolites in three rat brain regions, that have been implicated in the actions of ECS.
Materials and methods Animals and surgery. Male Sprague-Dawley rats (30(~350 g,
Taconic Farms) were used in this study. They were housed in groups of six in wire cages, with a 12 h light/dark cycle, and had free access to food and water. Animals were anaesthetised with chloral hydrate (400 mg/kg IP), and placed in a stereotaxic frame. Anaesthesia was maintained at a level whereby corneal reflexes were abolished, by further administration of chloral hydrate. After exposing the skull, a microdialysis probe (Carnegie Medicin, Stockholm) was inserted through a small hole drilled in the skull above one of three brain regions, the frontal cortex (AP + 13.2; ML_+0.8; DV+4.2), striatum (AP+9.7; ML_+3.2; D V + 3.0), or nucleus accumbens (AP + 10.5; ML_+ 1.5; DV + 0.9) (earbar zero; based on the atlas of Paxinos and Watson 1982). Two medio-lateral co-ordinates are given, as microdialysis was carried out "twice in each rat, at the first and eighth ECS or sham treatment. On the first occasion, the side of the brain to be dialysed was chosen randomly, and on the second occasion the other side was used. After the first episode of microdialysis, the probe was removed and
6I the head incision closed using wound clips. After the second microdialysis, rats were sacrificed and coronal sections obtained from frozen brain tissue to allow microscopic confirmation of probe placements.
Microdialysis, ECS and sham treatment procedures. Artificial CSF (NaC1 8.6 g, CaC12.2HzO 0.335 g, and KC1 0.3 g in I 1 distilled water, pH 6.6) was pumped through the microdialysis probes at 1.33 gl/min by a microinfusion pump (Carnegie Medicin, Stockholm). No samples were collected until 45 min after the probes were inserted into brain tissue to avoid abnormally high neurotransmitter levels caused by tissue damage. Subsequent samples were collected every 15 rain into polyethylene tubes containing 10 gl 0.15 M perchloric acid, and injected immediately into an HPLC system. Four baseline samples were obtained, with the mean neurotransmitter concentrations of the last two baselines presented in this study as basal release. After the fourth baseline sample was obtained, earclips were attached, and the rats were administered either ECS (ECS 1) (100 mA for 10 s; Annett's Industries, Oxford) ( n = 6 for each region), or the clips alone (n = 6 for each region). A full tonic/ clonic convulsion was observed in each rat receiving ECS. Firm restraint by the stereotaxic frame prevented any head movement during the ECS or subsequent convulsion. Four further samples were collected before removal of the probe. ECS or sham treatments were continued on a once-daily basis for the next 6 days, with unanaesthetised animals receiving either further ECS (80 mA for 0.5 s) or earclipping, according to their first treatment. Again, full tonic-clonic convulsions were observed in all ECS-treated rats. On the 8th day, microdialysis was performed under chloral hydrate anaesthesia on the opposite brain region as described above, and the last ECS (ECS 8) or clipping performed. For the striatum and frontal cortex, 3 mm probes were used; for nucleus accumbens, 2 mm probes were used.
Biochemical analysis. Microdialysis samples were injected immediately after collection into an HPLC system. This comprised an LKB pump, and a coulometric detector (ESA), with detector 1 set at --0.4 V, and detector 2 set at +0.35 V. Mobile phase was made up of sodium acetate 2.6 g, citric acid 4.0 g, sodium octyl sulphate 350rag, EDTA 100 mg, and methanol 180 ml in 1.3 1 distilled water (Sigma Chemical Co., St. Louis, Mo.; all chemicals of HPLC grade). This was pumped through a 10 cm C-18 5 gm reversed phase column (Axxiom) at 0.9 ml/min. Concentrations of monoamines and metabolites were calculated by comparing peak heights from the microdialysis samples with those of external standards of known concentration, of NA, DA, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) (Sigma Chemical Co., St. Louis, Mo.). Responses of monoamines and metabolites after ECS were expressed as percent of basal release. Statistical analysis. The BMDP statistical package Dixon 1981) was used for all analyses. Basal release of neurotransmitters or metabolites was calculated by taking the mean of the last two samples collected before administration of ECS. Comparison of basal release in each treatment group, before the first and eighth treatments, was made by paired t-test. Basal release before 8th ECS or sham treatment was compared by independent t-test. Responses after ECS were
expressed as percentage of baseline release, and comparisons of these %-change responses after the first and eighth treatments were made by two-way analysis of variance with repeated measures (ANOVAR). Results
Basal release. treatment 1 compared with treatment 8 Noradrenaline (NA). Basal release of NA before the first treatment was similar in both the ECS- and sham-treated groups, in all three brain regions (Table 1). Before the eighth treatment, release of N A in frontal cortex was significantly higher compared with both the first ECS treatment and the chronic sham controls, whilst release in the shamtreated rats was unaltered (Table 1). Basal N A release in nucleus accumbens was significantly reduced in both groups before the eighth treatment, compared with release before treatment, and was also not different between treatments (Table 1). Basal release of N A was not detectable in striaturn.
Dopamine (DA). Basal release of D A before the first treatments was similar in both the ECS- and sham-treated groups, in all three brain regions (Table 1). In frontal cortex, release of DA before the eighth treatment was not significantly different from release before the first treatment in either group, nor between groups before the eighth treatments (Table 1). In nucleus accumbens, baseline DA concentrations before ECS 8 were not significantly greater than before ECS 1. However, baseline DA before ECS 8 was significantly greater than before sham 8, although as can be seen from Table l, this reflects a fall in the sham 8 concentrations rather than a rise in the ECS 8 values, tn striatuna, basal DA release was increased before the eighth treatment compared with the first treatment in rats given ECS, whilst in the sham-treated group, basal DA release was significantly reduced (Table 1). Comparison of sham 8 and ECS 8 concentrations in striatum also showed significant differences. Table 1. Basal regional noradrenaline and dopamine release before ist and 8th ECS or sham treatments (mean_+SEM; n =6) Region
Treatment
NA (fmole)
DA (fmole)
Frontal cortex
ECS 1 ECS 8
9.9_+ 1.4 18.3 +2.7**
4.9 ± 1.3 4.6+ 1.4
Sham 1 Sham 8
11.0_+1.2 8.1 - + 0 . 4 b
4.1_0.4 3.1 + 0.5
Nucleus accumbens
ECS 1 ECS 8 Sham I Sham8
26.8 _+3.2 I7.9_+2.9" 22.7-t-3.8 12.3_+1.0"
14,8 ± 1.5 17.9 ~-2.3 14.0+__2.0 9.1___0.5b
Striatum
ECS 1 ECS 8 Sham 1 Sham 8
not detected
43.8 + 5.8 75.5± 14.8" 41.2 ± 3.5 23.3 ± 1.9"*'"
not detected
Comparison of treatments 1 vs 8: * P
Psychopharmacology (1990) 100: 60-65
© Springer-Verlag 1990
Regional neurotransmitter responses after acute and chronic electroconvulsive shock Paul Glue 1, Michael J. CosteIlo:, Agu Pert 2, Andrea Mele z, and David J. Nutt I 1 National Institute on Alcohol Abuse and Alcoholism, and 2 National Institute of Mental Health, Bethesda, MD 20892, USA
Abstract. Regional neurotransmitter changes after acute and chronic electroconvulsive shock (ECS) were studied using the technique of repeated microdiatysis. Microdialysis was carried out on alternate sides of the brains of anaesthetised rats before and during the first and the eighth ECS or sham (control) treatments. Extracellular fluid release of monoamines and their metabolites was measured in the frontal cortex, striatum and nucleus accumbens using HPLC with electrochemical detection. The first ECS produced selective regional responses, shown by increased concentrations of noradrenaline (NA) and dopamine (DA) in frontal cortex, by unchanged DA content in striatum, and by a small rise in NA and a fall in DA concentrations in nucleus accumbens. Concentrations of metabolites increased after ECS in all regions studied, and for homovanillic acid and dihydroxyphenylacetic acid, the temporal pattern of these changes did not resemble that of DA. Comparison of neurotransmitter responses as per cent of baseline release after the first and eighth ECS treatments showed they were identical. Basal release of monoamines and metabolites before the first ECS or sham treatment was similar in all regions studied. Prior to the eighth treatment, basal release of NA in the frontal cortex and DA in the striatum was elevated in the ECS-treated animals, while basal release of N A in the nucleus accumbens was reduced in both ECSand sham-treated animals. These data suggest that acute and chronic ECS have different and region-specific effects on neurotransmitter release, although the overall pattern of these responses is not changed by chronic treatment. The catecholamine-releasing actions of ECS, and the changes in basal release of neurotransmitters seen after chronic treatment may contribute to its therapeutic effects. Key words: Microdialysis- Electroconvulsive shock - N o r adrenaline - Dopamine Regional responses - Rat
Etectroconvulsive therapy is an effective and safe method of treating severe depression (Kendell 1981). It mechanism of action is unclear, although it probably includes some effects on monoamine neurotransmitters (for reviews, see Green and Nutt 1987; Gleiter and Nutt 1989). Most investigations into these changes have been carried out using elecOffprint requests to: P. Glue, Reckitt and Colman Psychopharmacotogy Unit, Department of Pharmacology, The Medical School, University Walk, Bristol BS8 ITD, UK
troconvulsive shock (ECS) in animals, and have looked at either changes in biochemistry, or functional or behaviourat changes of a number of receptor systems (see reviews by Lerer and Belmaker 1982; Green and Nutt 1987; Gleiter and Nutt 1989). The findings from studies of biochemical changes after ECS have been inconsistent (Schildkraut and Draskoczy 1974; Arato et al. t980; Lerer and Belmaker 1982), although more consistent alterations in receptor binding have been demonstrated with reduced beta- and alpha-2-adrenoceptor (for reviews see Lerer 1984; Gleiter and Nutt 1989), and increased 5-HTz receptor number (Vetulani et al. ]981, 1983; Goodwin et al. 1985). These are supported by studies using behavioural models of receptor function (Grahame-Smith et al. 1978; Green and Deakin 1980; Lerer and Belmaker 1982; Green et al. 1986). Overall, these studies indicate that ECS produces alterations in NA and DA function, but as yet there are no studies of the actual effects of ECS upon monoamine release, especially with regard to regional differences. To this end, we have used the technique of repeated microdialysis to examine the effects of acute and chronic ECS on extracellular fluid release of monoamines and metabolites in three rat brain regions, that have been implicated in the actions of ECS.
Materials and methods Animals and surgery. Male Sprague-Dawley rats (30(~350 g,
Taconic Farms) were used in this study. They were housed in groups of six in wire cages, with a 12 h light/dark cycle, and had free access to food and water. Animals were anaesthetised with chloral hydrate (400 mg/kg IP), and placed in a stereotaxic frame. Anaesthesia was maintained at a level whereby corneal reflexes were abolished, by further administration of chloral hydrate. After exposing the skull, a microdialysis probe (Carnegie Medicin, Stockholm) was inserted through a small hole drilled in the skull above one of three brain regions, the frontal cortex (AP + 13.2; ML_+0.8; DV+4.2), striatum (AP+9.7; ML_+3.2; D V + 3.0), or nucleus accumbens (AP + 10.5; ML_+ 1.5; DV + 0.9) (earbar zero; based on the atlas of Paxinos and Watson 1982). Two medio-lateral co-ordinates are given, as microdialysis was carried out "twice in each rat, at the first and eighth ECS or sham treatment. On the first occasion, the side of the brain to be dialysed was chosen randomly, and on the second occasion the other side was used. After the first episode of microdialysis, the probe was removed and
6I the head incision closed using wound clips. After the second microdialysis, rats were sacrificed and coronal sections obtained from frozen brain tissue to allow microscopic confirmation of probe placements.
Microdialysis, ECS and sham treatment procedures. Artificial CSF (NaC1 8.6 g, CaC12.2HzO 0.335 g, and KC1 0.3 g in I 1 distilled water, pH 6.6) was pumped through the microdialysis probes at 1.33 gl/min by a microinfusion pump (Carnegie Medicin, Stockholm). No samples were collected until 45 min after the probes were inserted into brain tissue to avoid abnormally high neurotransmitter levels caused by tissue damage. Subsequent samples were collected every 15 rain into polyethylene tubes containing 10 gl 0.15 M perchloric acid, and injected immediately into an HPLC system. Four baseline samples were obtained, with the mean neurotransmitter concentrations of the last two baselines presented in this study as basal release. After the fourth baseline sample was obtained, earclips were attached, and the rats were administered either ECS (ECS 1) (100 mA for 10 s; Annett's Industries, Oxford) ( n = 6 for each region), or the clips alone (n = 6 for each region). A full tonic/ clonic convulsion was observed in each rat receiving ECS. Firm restraint by the stereotaxic frame prevented any head movement during the ECS or subsequent convulsion. Four further samples were collected before removal of the probe. ECS or sham treatments were continued on a once-daily basis for the next 6 days, with unanaesthetised animals receiving either further ECS (80 mA for 0.5 s) or earclipping, according to their first treatment. Again, full tonic-clonic convulsions were observed in all ECS-treated rats. On the 8th day, microdialysis was performed under chloral hydrate anaesthesia on the opposite brain region as described above, and the last ECS (ECS 8) or clipping performed. For the striatum and frontal cortex, 3 mm probes were used; for nucleus accumbens, 2 mm probes were used.
Biochemical analysis. Microdialysis samples were injected immediately after collection into an HPLC system. This comprised an LKB pump, and a coulometric detector (ESA), with detector 1 set at --0.4 V, and detector 2 set at +0.35 V. Mobile phase was made up of sodium acetate 2.6 g, citric acid 4.0 g, sodium octyl sulphate 350rag, EDTA 100 mg, and methanol 180 ml in 1.3 1 distilled water (Sigma Chemical Co., St. Louis, Mo.; all chemicals of HPLC grade). This was pumped through a 10 cm C-18 5 gm reversed phase column (Axxiom) at 0.9 ml/min. Concentrations of monoamines and metabolites were calculated by comparing peak heights from the microdialysis samples with those of external standards of known concentration, of NA, DA, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) (Sigma Chemical Co., St. Louis, Mo.). Responses of monoamines and metabolites after ECS were expressed as percent of basal release. Statistical analysis. The BMDP statistical package Dixon 1981) was used for all analyses. Basal release of neurotransmitters or metabolites was calculated by taking the mean of the last two samples collected before administration of ECS. Comparison of basal release in each treatment group, before the first and eighth treatments, was made by paired t-test. Basal release before 8th ECS or sham treatment was compared by independent t-test. Responses after ECS were
expressed as percentage of baseline release, and comparisons of these %-change responses after the first and eighth treatments were made by two-way analysis of variance with repeated measures (ANOVAR). Results
Basal release. treatment 1 compared with treatment 8 Noradrenaline (NA). Basal release of NA before the first treatment was similar in both the ECS- and sham-treated groups, in all three brain regions (Table 1). Before the eighth treatment, release of N A in frontal cortex was significantly higher compared with both the first ECS treatment and the chronic sham controls, whilst release in the shamtreated rats was unaltered (Table 1). Basal N A release in nucleus accumbens was significantly reduced in both groups before the eighth treatment, compared with release before treatment, and was also not different between treatments (Table 1). Basal release of N A was not detectable in striaturn.
Dopamine (DA). Basal release of D A before the first treatments was similar in both the ECS- and sham-treated groups, in all three brain regions (Table 1). In frontal cortex, release of DA before the eighth treatment was not significantly different from release before the first treatment in either group, nor between groups before the eighth treatments (Table 1). In nucleus accumbens, baseline DA concentrations before ECS 8 were not significantly greater than before ECS 1. However, baseline DA before ECS 8 was significantly greater than before sham 8, although as can be seen from Table l, this reflects a fall in the sham 8 concentrations rather than a rise in the ECS 8 values, tn striatuna, basal DA release was increased before the eighth treatment compared with the first treatment in rats given ECS, whilst in the sham-treated group, basal DA release was significantly reduced (Table 1). Comparison of sham 8 and ECS 8 concentrations in striatum also showed significant differences. Table 1. Basal regional noradrenaline and dopamine release before ist and 8th ECS or sham treatments (mean_+SEM; n =6) Region
Treatment
NA (fmole)
DA (fmole)
Frontal cortex
ECS 1 ECS 8
9.9_+ 1.4 18.3 +2.7**
4.9 ± 1.3 4.6+ 1.4
Sham 1 Sham 8
11.0_+1.2 8.1 - + 0 . 4 b
4.1_0.4 3.1 + 0.5
Nucleus accumbens
ECS 1 ECS 8 Sham I Sham8
26.8 _+3.2 I7.9_+2.9" 22.7-t-3.8 12.3_+1.0"
14,8 ± 1.5 17.9 ~-2.3 14.0+__2.0 9.1___0.5b
Striatum
ECS 1 ECS 8 Sham 1 Sham 8
not detected
43.8 + 5.8 75.5± 14.8" 41.2 ± 3.5 23.3 ± 1.9"*'"
not detected
Comparison of treatments 1 vs 8: * P
