189
BrainResearch, 583 (1992) 189-193 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05 .00
BRES 17892
Social isolation increases the density of [l25 I]w-conotoxin GVIA binding sites in the rat frontal cortex and caudate nucleus Anna Czyrak
a,
David J. Dooley
b,
Graham H. Jones
c
and Trevor W. Robbins
d
a Instituteof Pharmacology, Polish Academy of Sciences, Cracow (Poland), b Department of Neuroscience, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, MI48106-1047 (USA), C Department of Neuropsychopharmacology, Schering AG, Berlin (FRG) and d Department of Experimental Psychology, Cambridge (uK)
(Accepted 18 February 1992)
Key words: Isolation-rearing; Voltage-s ensitive calcium channel ; [3HlIsradipine; [l 25 Ilw-Co notoxin GVIA; Rat brain
Rats were reared from wean ing either in isolation or in social group s for 12 weeks . Potential isolation-related changes in L- and N-type voltage-sensitive calcium channels (VSCCS) were assessed by the in vitro binding of [3Hlisradipine (100 pM) and [l 25Ilw-conotoxin GVIA (4 pM) to membranes prepared from three discrete central nervous system regions : frontal cortex , caudate nucleus and hippocampus. The [3Hlisradipine bind ing was generally not affected by isolation. However, [l25I)w-conotoxin GVIA binding was significantly higher in frontal cortex (52%) and caudate nucleu s (75%) of isolated rat s when compared with socially reared controls. The increased [l 25Ilw-conotoxin GVIA binding reflected an elevated density of binding sites without an alteration of receptor affinity. The possible contribution of an increased density of neuronal N-VSCCs (as labeled with [ l25Ilw-conotoxin GVIA) to the behavioral and neurochemical changes observed in ' isolation syndrome ' is discussed .
INTRODUCTION
Isolated rats display many behavioral disturbances such as increased locomotor activity in novel environments12,33,39, altered exploratory behavior 27,32, and deficits in both learning 18,23 and spatial memory tasks 20. Isolates are also more responsive to tail-pinch stimulatiorr" ; they show increased sensitivity to reward-related stimuli 17 and increased stereotyped responses to psychomotor stimulant drugs , including o-amphetamine and apomorphine'v'Pr" . There also has been much research into the neurochemical effects of social isolation. Blanc et a1. 2 suggested that long-term isolation reduces dopamine turnover in mesocortical projections mea sured in nonstress conditions, but footshock causes much greater increases in frontal cortex dopamine turnover in isolates than in group-reared animals . According to Weinstock et a1. 4O, rats reared in isolation show significantly lower noradrenaline turnover in the home cage, but apparently greater noradrenaline turnover than that of
group-reared rats when exposed to an open field. More recent work using in vivo dialysis suggests that isolates have increased extracellular levels of dopamine in response to o-amphetamine". The activity of brain tyrosine hydroxylase is elevated in the isolated rats when compared to grouped controls". One brief communication also reports changes in striatal 02 receptors in rats reared in isolation':', It has also been suggested that neurochemical changes in 5-hydroxytryptam ine (5HT) 24,41,42, opiate 35 and benzodiazepine" systems are important in the effects of isolation although the pre cise mechanisms underlying the apparent isolation-induced dysregulation of central neurotransmitter systems still remain to be elucidated. Transmembrane calcium movement from the extracellular fluid into the cytosol serves as a signal for initiation of many cellular and synaptic processes in neurons. Most of the influx of calcium into the neuron during cell depolarization occurs through voltage-sensitive calcium channels (VSCCs) located in neuronal cell membranes'r". The existence of multiple types of
Correspondence: D.l. Dooley, Department of Neuroscience, Parke-Davis Pharm aceutical Research Division, Warner-Lambert Co., Ann Arbor, MI 48106-1047, USA.
190 VSCCs has been established in neurons 22,25. Two of these neuronal VSCCs, designated as L-type (L-VSCC) and N-type (N-VSCC), have received increasing attention owing to the availability of appropriate pharmacological tools and radioligands. For example, the dihydropyridines, such as [3H]isradipine ([3H]ISR), selectively label the L-VSCC 9,37, which has a significant somadendritic localization and potential role in longterm potentiation at central synapses 4,19,22. The peptide [125I]w-conotoxin GVIA ([125I ]w-CT) selectively binds to the N_VSCC 7,26,28; this VSCC appears to be present on axon terminals and is important in the regulation of neurotransmitter release 8,22,29 and synthesis30. Depolarization-induced calcium influx through Land N-VSCCs is undoubtedly necessary for the occurrence of both intraneuronal and multisynaptic events, and changes in the physical state of these channels might precede or subserve isolation-induced dysregulation of brain neurotransmitter systems. We have therefore investigated possible isolation-related alterations of central L- and N-VSCCs by preparing crude membrane suspensions from discrete central nervous system regions of rats reared either in isolation or in social groups, and assessing the binding of [3H]ISR and [125I]w_Cf to these membranes. MATERIALS AND METHODS The female Lister hooded rats used in these experiments were obtained at 21 days of age (Olac, Bicester UK) and were divided into the two rearing condition groups counterbalanced by body weight. Rats were housed in isolation or in social groups of six rats per cage. All cages were constructed of plastic with grid floors and an underhanging sawdust tray. Isolation reared rats were housed singly in cages 45 X 28 x 20 em high. The social-group cages were 56 x 38 x 18 ern high. Both groups had continuous access to food and water. All rats were housed in a colony room maintained at 21°C, on a 12-h light/dark cycle (lights-on, 07.00-19.00 h) and could see, hear, and smell other rats. After 12 weeks, isolated and group-reared rats were killed by decapitation. Each brain was rapidly removed and placed in a dissecting block on an ice-cold plate. Following removal of the olfactory bulbs, the brain was dissected as follows: a 3-mm thick coronal section was made from the frontal pole, and non-cortical tissue ventral to the genu of the corpus callosum and lateral to the anterior commissure was removed. The remaining tissue was defined as frontal cortex. The anterior part of the caudate nucleus was obtained from the next 1.5 mm coronal section. The olfactory tubercles were removed, and the corpus callosum, septum, and anterior commissure used as landmarks. The posterior part of the caudate nucleus was dissected from the next 2 mm slice. The two parts of the caudate nucleus were combined. The whole hippocampus was also dissected and combined. After dissection, all brain regions were frozen in liquid nitrogen. Crude membrane fractions were subsequently prepared for use in the radioligand binding assays. Brain regions were placed in a minimum of 10 vols. (wt.yvol.) of ice-cold 50 mM HEPES-NaOH buffer (pH 7.4 at 22°C). Hornogenates, prepared using a glass/Teflon homogenizer (800 r.p.m., eight strokes) were washed twice by centrifugation (48,000 g for 15 min at 4°C); the final pellets were rehomogenized (800 r.p.m., four strokes) in fresh buffer, and the
resultant suspensions were pipetted into plastic ampules, which were rapidly frozen in liquid nitrogen. For subsequent experiments, membrane suspensions were thawed and diluted with appropriate volumes of either ice-cold 50 mM Tris-HCI buffer (pH 7.4 at 22°C) containing 1 mM CaC!z ([3HIISR binding) or 50 mM HEPES-NaOH buffer ([l25I)w-CT binding). Protein concentration was measured by the method of Bradford? using bovine serum albumin (BSA) as standard.
[3Hl/sradipine bindingassay The [3HlISR (3.15 TBq/mmol, Amersham) binding assay was a modification of that described by Supavilai and Karobath". Incubation mixtures (2 ml) consisted of 1.8 ml of assay buffer (50 mM Tris-HCI buffer (pH 7.4) containing 1 mM CaCl z), 100 Jd of membrane suspension (approximately 50 JLg of protein), 5 JLI of either assay buffer (total binding) or a solution containing unlabeled ISR (final concentration of 100 nM for non-specific binding) and 100 JLI of [3H]ISR (final concentration of 100 plvl), Incubations, performed in duplicate at 22°C under a sodium vapor lamp, were terminated after 60 min by rapid filtration through glass-fiber filters (GF/C, Whatman), The filters were immediately rinsed with three 4-ml volumes of ice cold assay buffer and transferred to scintillation vials containing 10 ml of scintillator (Aqualuma Plus 8594; J.T. Baker Chemicals). Radioactivity in the filters was measured by liquid scintillation counting at an efficiency of 50%.
{125/1 w-Conotoxin GVIA binding assay
The [l 25Ilw-CT (76 TBq/mmol, Amersham) binding assay was carried out as described by Dooley et a1. 7 • Incubation mixtures (0.5 ml) consisted of 400 JLI of assay buffer (50 mM HEPES-NaOH buffer (pH 7.4) containing 0.2% BSA), 50 JLI of membrane suspension (approximately 0.5 JLg of protein), 5 JLI of either assay buffer (total binding) or a solution containing unlabeled w-CT (final concentration of 10 nM for non-specific binding; Peninsula Labs.), and 50 JLI of [lZ5I)w_CT (final concentration of 4 or 0.1-10 pM). Membranes were preincubated in duplicate in the presence or absence of unlabeled w-CT for 30 min at 22°C; the final incubations were initiated by the addition of [l 25Ilw-CT and terminated after 15 min at 22°C by rapid filtration through presoaked glass-fiber filters (GF/C, Whatman). The filters were immediately rinsed with three 4-ml volumes of ice cold assay buffer containing 0.2 M NaC!. Radioactivity was measured by gamma counting at an efficiency of 50%.
Data analysis
Specific binding of [3H]ISR and [l z5I]w-CT was defined as the difference between total and non-specific binding. For saturation experiments, the binding of increasing concentrations of [l25I)w-CT was analyzed as described by Scatchard 34, and the K D (apparent dissociation constant), B m ax (maximal binding capacity) and r (correlation coefficient) were determined for each individual rat brain region. The results were analyzed using one-way analysis of variance (ANOVA).
RESULTS
The specific binding of both radioligands was generally high in all the brain regions studied in both groups of rats (Figs. 1 and 2). There were no differences in [3H]ISR binding (100 pM) in brain regions from rats reared in isolation compared with those from social groups (Fig. 1). The binding of [l25I]w-CT (4 pM) was, however, significantly higher in the frontal cortex (52%) and caudate nucleus (75%) of isolated rats when compared with that of control animals reared in social groups; hippocampal binding of [125I]w_CT was not significantly different for the two groups of rats (Fig.
191 TABLE I
300
Parameters of [l 25I]w -CT binding to membranes of frontal cortex and caudate nucleus from socially-reared and isolated rats.
c
.~
ll.l
~
Data represent means ± S.E.M., n = 4. One-way ANOVA indicated statistically significant differences in B max values in frontal cortex (F1,6 = 6.89, * P < 0.05) and caudate nucleus (F1,6 = 31.78, * * P
BrainResearch, 583 (1992) 189-193 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05 .00
BRES 17892
Social isolation increases the density of [l25 I]w-conotoxin GVIA binding sites in the rat frontal cortex and caudate nucleus Anna Czyrak
a,
David J. Dooley
b,
Graham H. Jones
c
and Trevor W. Robbins
d
a Instituteof Pharmacology, Polish Academy of Sciences, Cracow (Poland), b Department of Neuroscience, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, MI48106-1047 (USA), C Department of Neuropsychopharmacology, Schering AG, Berlin (FRG) and d Department of Experimental Psychology, Cambridge (uK)
(Accepted 18 February 1992)
Key words: Isolation-rearing; Voltage-s ensitive calcium channel ; [3HlIsradipine; [l 25 Ilw-Co notoxin GVIA; Rat brain
Rats were reared from wean ing either in isolation or in social group s for 12 weeks . Potential isolation-related changes in L- and N-type voltage-sensitive calcium channels (VSCCS) were assessed by the in vitro binding of [3Hlisradipine (100 pM) and [l 25Ilw-conotoxin GVIA (4 pM) to membranes prepared from three discrete central nervous system regions : frontal cortex , caudate nucleus and hippocampus. The [3Hlisradipine bind ing was generally not affected by isolation. However, [l25I)w-conotoxin GVIA binding was significantly higher in frontal cortex (52%) and caudate nucleu s (75%) of isolated rat s when compared with socially reared controls. The increased [l 25Ilw-conotoxin GVIA binding reflected an elevated density of binding sites without an alteration of receptor affinity. The possible contribution of an increased density of neuronal N-VSCCs (as labeled with [ l25Ilw-conotoxin GVIA) to the behavioral and neurochemical changes observed in ' isolation syndrome ' is discussed .
INTRODUCTION
Isolated rats display many behavioral disturbances such as increased locomotor activity in novel environments12,33,39, altered exploratory behavior 27,32, and deficits in both learning 18,23 and spatial memory tasks 20. Isolates are also more responsive to tail-pinch stimulatiorr" ; they show increased sensitivity to reward-related stimuli 17 and increased stereotyped responses to psychomotor stimulant drugs , including o-amphetamine and apomorphine'v'Pr" . There also has been much research into the neurochemical effects of social isolation. Blanc et a1. 2 suggested that long-term isolation reduces dopamine turnover in mesocortical projections mea sured in nonstress conditions, but footshock causes much greater increases in frontal cortex dopamine turnover in isolates than in group-reared animals . According to Weinstock et a1. 4O, rats reared in isolation show significantly lower noradrenaline turnover in the home cage, but apparently greater noradrenaline turnover than that of
group-reared rats when exposed to an open field. More recent work using in vivo dialysis suggests that isolates have increased extracellular levels of dopamine in response to o-amphetamine". The activity of brain tyrosine hydroxylase is elevated in the isolated rats when compared to grouped controls". One brief communication also reports changes in striatal 02 receptors in rats reared in isolation':', It has also been suggested that neurochemical changes in 5-hydroxytryptam ine (5HT) 24,41,42, opiate 35 and benzodiazepine" systems are important in the effects of isolation although the pre cise mechanisms underlying the apparent isolation-induced dysregulation of central neurotransmitter systems still remain to be elucidated. Transmembrane calcium movement from the extracellular fluid into the cytosol serves as a signal for initiation of many cellular and synaptic processes in neurons. Most of the influx of calcium into the neuron during cell depolarization occurs through voltage-sensitive calcium channels (VSCCs) located in neuronal cell membranes'r". The existence of multiple types of
Correspondence: D.l. Dooley, Department of Neuroscience, Parke-Davis Pharm aceutical Research Division, Warner-Lambert Co., Ann Arbor, MI 48106-1047, USA.
190 VSCCs has been established in neurons 22,25. Two of these neuronal VSCCs, designated as L-type (L-VSCC) and N-type (N-VSCC), have received increasing attention owing to the availability of appropriate pharmacological tools and radioligands. For example, the dihydropyridines, such as [3H]isradipine ([3H]ISR), selectively label the L-VSCC 9,37, which has a significant somadendritic localization and potential role in longterm potentiation at central synapses 4,19,22. The peptide [125I]w-conotoxin GVIA ([125I ]w-CT) selectively binds to the N_VSCC 7,26,28; this VSCC appears to be present on axon terminals and is important in the regulation of neurotransmitter release 8,22,29 and synthesis30. Depolarization-induced calcium influx through Land N-VSCCs is undoubtedly necessary for the occurrence of both intraneuronal and multisynaptic events, and changes in the physical state of these channels might precede or subserve isolation-induced dysregulation of brain neurotransmitter systems. We have therefore investigated possible isolation-related alterations of central L- and N-VSCCs by preparing crude membrane suspensions from discrete central nervous system regions of rats reared either in isolation or in social groups, and assessing the binding of [3H]ISR and [125I]w_Cf to these membranes. MATERIALS AND METHODS The female Lister hooded rats used in these experiments were obtained at 21 days of age (Olac, Bicester UK) and were divided into the two rearing condition groups counterbalanced by body weight. Rats were housed in isolation or in social groups of six rats per cage. All cages were constructed of plastic with grid floors and an underhanging sawdust tray. Isolation reared rats were housed singly in cages 45 X 28 x 20 em high. The social-group cages were 56 x 38 x 18 ern high. Both groups had continuous access to food and water. All rats were housed in a colony room maintained at 21°C, on a 12-h light/dark cycle (lights-on, 07.00-19.00 h) and could see, hear, and smell other rats. After 12 weeks, isolated and group-reared rats were killed by decapitation. Each brain was rapidly removed and placed in a dissecting block on an ice-cold plate. Following removal of the olfactory bulbs, the brain was dissected as follows: a 3-mm thick coronal section was made from the frontal pole, and non-cortical tissue ventral to the genu of the corpus callosum and lateral to the anterior commissure was removed. The remaining tissue was defined as frontal cortex. The anterior part of the caudate nucleus was obtained from the next 1.5 mm coronal section. The olfactory tubercles were removed, and the corpus callosum, septum, and anterior commissure used as landmarks. The posterior part of the caudate nucleus was dissected from the next 2 mm slice. The two parts of the caudate nucleus were combined. The whole hippocampus was also dissected and combined. After dissection, all brain regions were frozen in liquid nitrogen. Crude membrane fractions were subsequently prepared for use in the radioligand binding assays. Brain regions were placed in a minimum of 10 vols. (wt.yvol.) of ice-cold 50 mM HEPES-NaOH buffer (pH 7.4 at 22°C). Hornogenates, prepared using a glass/Teflon homogenizer (800 r.p.m., eight strokes) were washed twice by centrifugation (48,000 g for 15 min at 4°C); the final pellets were rehomogenized (800 r.p.m., four strokes) in fresh buffer, and the
resultant suspensions were pipetted into plastic ampules, which were rapidly frozen in liquid nitrogen. For subsequent experiments, membrane suspensions were thawed and diluted with appropriate volumes of either ice-cold 50 mM Tris-HCI buffer (pH 7.4 at 22°C) containing 1 mM CaC!z ([3HIISR binding) or 50 mM HEPES-NaOH buffer ([l25I)w-CT binding). Protein concentration was measured by the method of Bradford? using bovine serum albumin (BSA) as standard.
[3Hl/sradipine bindingassay The [3HlISR (3.15 TBq/mmol, Amersham) binding assay was a modification of that described by Supavilai and Karobath". Incubation mixtures (2 ml) consisted of 1.8 ml of assay buffer (50 mM Tris-HCI buffer (pH 7.4) containing 1 mM CaCl z), 100 Jd of membrane suspension (approximately 50 JLg of protein), 5 JLI of either assay buffer (total binding) or a solution containing unlabeled ISR (final concentration of 100 nM for non-specific binding) and 100 JLI of [3H]ISR (final concentration of 100 plvl), Incubations, performed in duplicate at 22°C under a sodium vapor lamp, were terminated after 60 min by rapid filtration through glass-fiber filters (GF/C, Whatman), The filters were immediately rinsed with three 4-ml volumes of ice cold assay buffer and transferred to scintillation vials containing 10 ml of scintillator (Aqualuma Plus 8594; J.T. Baker Chemicals). Radioactivity in the filters was measured by liquid scintillation counting at an efficiency of 50%.
{125/1 w-Conotoxin GVIA binding assay
The [l 25Ilw-CT (76 TBq/mmol, Amersham) binding assay was carried out as described by Dooley et a1. 7 • Incubation mixtures (0.5 ml) consisted of 400 JLI of assay buffer (50 mM HEPES-NaOH buffer (pH 7.4) containing 0.2% BSA), 50 JLI of membrane suspension (approximately 0.5 JLg of protein), 5 JLI of either assay buffer (total binding) or a solution containing unlabeled w-CT (final concentration of 10 nM for non-specific binding; Peninsula Labs.), and 50 JLI of [lZ5I)w_CT (final concentration of 4 or 0.1-10 pM). Membranes were preincubated in duplicate in the presence or absence of unlabeled w-CT for 30 min at 22°C; the final incubations were initiated by the addition of [l 25Ilw-CT and terminated after 15 min at 22°C by rapid filtration through presoaked glass-fiber filters (GF/C, Whatman). The filters were immediately rinsed with three 4-ml volumes of ice cold assay buffer containing 0.2 M NaC!. Radioactivity was measured by gamma counting at an efficiency of 50%.
Data analysis
Specific binding of [3H]ISR and [l z5I]w-CT was defined as the difference between total and non-specific binding. For saturation experiments, the binding of increasing concentrations of [l25I)w-CT was analyzed as described by Scatchard 34, and the K D (apparent dissociation constant), B m ax (maximal binding capacity) and r (correlation coefficient) were determined for each individual rat brain region. The results were analyzed using one-way analysis of variance (ANOVA).
RESULTS
The specific binding of both radioligands was generally high in all the brain regions studied in both groups of rats (Figs. 1 and 2). There were no differences in [3H]ISR binding (100 pM) in brain regions from rats reared in isolation compared with those from social groups (Fig. 1). The binding of [l25I]w-CT (4 pM) was, however, significantly higher in the frontal cortex (52%) and caudate nucleus (75%) of isolated rats when compared with that of control animals reared in social groups; hippocampal binding of [125I]w_CT was not significantly different for the two groups of rats (Fig.
191 TABLE I
300
Parameters of [l 25I]w -CT binding to membranes of frontal cortex and caudate nucleus from socially-reared and isolated rats.
c
.~
ll.l
~
Data represent means ± S.E.M., n = 4. One-way ANOVA indicated statistically significant differences in B max values in frontal cortex (F1,6 = 6.89, * P < 0.05) and caudate nucleus (F1,6 = 31.78, * * P
