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Vasopressin selectively modulates the release of taurine within the septum of the rat brain
Neuroscience Letters 277 (1999) 68±70 www.elsevier.com/locate/neulet
Vasopressin selectively modulates the release of taurine within the septum of the rat brain Nicolas Singewald a, Karl Ebner b, Rainer Landgraf b, Carsten T. Wotjak a, c, Mario Engelmann b, d,* a
UniversitaÈt Innsbruck, Institut fuÈr Pharmakologie and Toxikologie, A-6020 Innsbruck, Austria b Max-Planck-Institut fuÈr Psychiatrie, Kraepelinstrasse 2, D-80804 MuÈnchen, Germany c Zentrum fuÈr Molekulare Neurobiologie, Martinistrasse 52, D-20246 Hamburg, Germany d Otto-von-Guericke-UniversitaÈt, Inst fuÈr Medizinische Neurobiologie, Leipziger Strasse 44, D-39120 Magdeburg, Germany Received 3 September 1999; received in revised form 15 October 1999; accepted 15 October 1999
Abstract Previous experiments have shown that arginine vasopressin (AVP) released within the septal brain area of adult male rats in response to de®ned stressor exposure is involved in emotionality-related behavior. We report here that a 10-min forced swimming session stimulated the release of glutamate, aspartate, arginine, g-aminobutyric acid (GABA) and taurine but not alanine and serine in the medio-lateral part of this brain structure. Local administration of the AVP V1 receptor antagonist d(CH2)5Tyr(Me)AVP by inverse microdialysis caused a signi®cant increase in the concentration of taurine in microdialysates under resting conditions that was further potentiated during forced swimming. In contrast, the release of alanine, arginine, GABA and serine was unaffected by antagonist treatment. Taken together with previous data, our results suggest that the effects of intraseptally released AVP on stress-coping strategies might be mediated at least in part via its in¯uence on the local release of taurine. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Emotionality; Septum; Amino acids; g-Aminobutyric acid; Glutamate; Swim stress; Microdialysis
Arginine vasopressin (AVP) has been postulated to act as a neurotransmitter/neuromodulator in the septal brain area of rodents, where it signi®cantly in¯uences learning and memory [5±7]. Recent ®ndings suggest that intraseptally released AVP plays also a crucial role for animals' emotionality in particular under stressful conditions ([13], see also Ref. [11] for review). Indeed, we reported recently that: (1) during a 10-min forced swimming session AVP release is triggered within the septum and (2) intraseptally released AVP affects the behavior in the forced swimming task [3]. Since AVP seems to act not only as a neurotransmitter but also as a neuromodulator within the septum (see Refs. [4,12] for review), it is likely that additional neurotransmitter systems are modulated by locally released AVP. To test this hypothesis, we monitored the extracellular concentration of various amino acids with established local neurotransmitter function including glutamate, aspartate, gaminobutyric acid (GABA) and taurine [9] in response to * Corresponding author. Tel.: 149-391-671-4363; fax: 149-391671-4365. E-mail address: [email protected] (M. Engelmann)
forced with or without local administration of an AVP V1 receptor antagonist. Adult male Wistar rats (300±400 g, n 30) were purchased from a Charles River (Sulzfeld, Germany) and kept under controlled laboratory conditions. Animals were implanted with microdialysis probes aimed at the mediolateral part of the septum under halothane anesthesia as described in detail by Ebner et al. [3] (implantation coordinates: 0.8 mm rostral to bregma, 2.3 mm lateral to midline, 6.3 mm below the surface of the skull with an angle of 228 to avoid damage to the sagittal sinus). Immediately after surgery rats were injected with antibiotics and individually housed in polycarbon cages (23 £ 29 £ 36 cm). The day after surgery animals were familiarized with the microdialysis procedure and handled for 5±10 min. On the following day animals were connected at 08.00 h (approx. 48 h after surgery) to the microdialysis equipment and the probes were perfused with sterile Ringer's solution (3.3 ml/ min). Initially probes were perfused for 2 h to allow to establish an equilibrium between inside and outside of the dialysis membrane. During this period, sample collection was simulated every 30 min.
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 99) 0 08 41- 1
N. Singewald et al. / Neuroscience Letters 277 (1999) 68±70
Fig. 1. Concentration of amino acids in 30-min microdialysates obtained from the medio-lateral septum of freely moving rats (means ^ SEM) and expressed as percentage of the averaged ®rst two samples (100%; dotted line). Animals remained undisturbed in their home cages throughout the experiment except for the beginning of the collection of sample number 4, when rats were forced to swim for 10 min.**P , 0:01 and *P , 0:05 vs. samples 1±3.
After this initial period six consecutive 30-min dialysates were collected directly in 0.5-ml Eppendorf tubes and immediately stored on dry ice. At the beginning of the collection of sample four animals were transferred to forced swimming tanks (30 cm in diameter, ®lled to a depth of 40 cm with water of a temperature of 20 ^ 18C) and forced to swim for 10 min. Immediately before swimming, rats were equipped with a small piece of styrofoam on the back of the neck, to aid ¯oating without the necessity of additional leg movements. The behavior of the animals was monitored during the entire swimming session. Behavioral data obtained during the experiment had already been reported in a previous paper [3]. In a second set of experiments, the dialysis medium was switched to Ringer's solution containing the AVP V1 receptor antagonist d(CH2)5Tyr(Me)AVP (Dr. M. Manning, Toledo, USA) in 15 rats during the dialysis periods 3 and 4. The total amount of antagonist administered was calculated to be approximately 10 ng [6]. Controls (n 15) received Ringers' solution only.
69
Amino acids were determined in microdialysates by high performance liquid chromatography (HPLC) and ¯uorimetric detection (Merck-Hitachi, Tokyo, Japan) after derivatization with o -phtahldialdehyd (OPA) as previously described [14]. In the present study 75 ml of a mixture of 80 ml of the dialysate and 20 ml of OPA reaction mixture were injected. The reproducibility of the derivatization was controlled by addition of S-carboxymethyl-l-cysteine as an internal standard. Concentration of amino acids in microdialysates were not corrected for recovery. Data (means and SEM) are presented as percentage of the two averaged basal values (100%). For statistical analysis, they were transformed by arc-tan to ®t to Gaussian distribution and submitted to a oneway (effect of forced swimming on controls) or two-way (treatment and forced swimming) ANOVA with repeated measures. Fisher's least signi®cant difference was applied if appropriate. P , 0:05 was considered to be statistically signi®cant. Histological veri®cation of the implantation site and behavioral performance of the animals had already been reported in the study of Ebner et al. [3]. During HPLC analysis samples of four controls and two AVP V1 receptor antagonist treated animals had missing values. As a consequence, these animals were not included in the statistical analysis which reduced the number of animals per group to controls: n 11 and AVP V1 receptor antagonist: n 13. As shown in Fig. 1, forced swimming caused a signi®cant rise in microdialysate concentration of aspartate, glutamate, GABA, taurine, and arginine, whereas the release of serine and alanine was largely unaffected (Fig. 1). Administration of the V1 antagonist had no effects on the release of alanine, arginine, serine (data and statistics not shown) and GABA (Fig. 2) either before (P . 0:05) or during forced swimming
Fig. 2. Concentration of amino acids in 30-min microdialysates obtained from the medio-lateral septum of freely moving rats. Data are expressed as percentage of the averaged ®rst two samples. During collection of sample numbers 3 and 4, the dialysis medium was either Ringer's solution containing the AVP V1 receptor antagonist d(CH2)5Tyr(Me)AVP (solid line with black circles) or Ringer's solution alone (dotted line with open circle; data obtained from Fig. 1). At the beginning of the collection of sample number 4 the animals were forced to swim for 10 min in 208C warm water. *P , 0:05 vs. the same sample of the Ringer's group and P , 0:01 vs. samples 1 and 2.
70
N. Singewald et al. / Neuroscience Letters 277 (1999) 68±70
(P . 0:05). In contrast, administration of the antagonist caused a signi®cant increase in the release of taurine already under pre-stress conditions (P , 0:05). This increase was further potentiated when the animals were forced to swim (Fig. 2; P , 0:01). Aspartate and glutamate had to be excluded from further analyses because of a priori contamination of the antagonist-containing dialysis medium with these two amino acids. Our data demonstrate that a 10-min forced swimming session triggers intraseptal release of glutamate, aspartate, arginine, GABA and taurine but not other amino acids investigated (Fig. 1). Although extensive histological investigations imply GABA as the primary candidate for the interaction with AVP at the level of the septum [8±10], we failed to detect such an effect by local administration of a AVP V1 antagonist (Fig. 2). In contrast the release of taurine as a different inhibitory amino acid was signi®cantly increased by blockade of the endogenously released AVP (Fig. 2). This ®nding adds to the growing body of evidence for a close interaction between AVP and taurine within the rat brain [1,2]. As reported previously, behavioral consequences of the antagonist treatment resulted in higher ¯oating and shorter swimming duration as compared with controls [3]. It is tempting to speculate that the interplay between AVP and taurine contributes to these behavioral changes. However, the behavioral alterations observed do not correlate directly with extracellular concentration of taurine measured in the present study (unpublished data). This argues against taurine as a key player for the behavioral effects of intraseptally released AVP. Further studies will have to investigate explicitly the role of taurine in the physiological signi®cance of the stress-induced taurine release by administration of speci®c taurine antagonists. [1] Brust, P., Christensen, T. and Diemer, N.H., Decrease of extracellular taurine in the rat dorsal hippocampus after central nervous administration of vasopressin. J. Neurochem., 58 (1992) 1427±1431. [2] Campistron, G., Geffard, M. and Buijs, R.M., Immunological
[3]
[4] [5] [6]
[7]
[8]
[9] [10]
[11]
[12] [13] [14]
approach to the detection of taurine and immunocytochemical results. J. Neurochem., 46 (1986) 862±868. Ebner, K., Wotjak, C.T., Holsboer, F., Landgraf, R. and Engelmann, M., Vasopressin released within the septal brain area during swim stress modulates the behavioral stress response in male rats. Eur. J. Neurosci., 11 (1999) 997±1002. Fuxe, K. and Agnati, L.F., Volume Transmission in the Brain: Novel Mechanisms for Neural Transmission, Raven Press, New York, 1991. Dantzer, R., Koob, G.F., BlutheÂ, R.-M. and Le Moal, M., Septal vasopressin modulates social memory in male rats. Brain Res., 457 (1988) 143±147. Engelmann, M. and Landgraf, R., Microdialysis administration of vasopressin into the septum improves social recognition in Brattleboro rats. Physiol. Behav., 55 (1994) 145± 149. Engelmann, M., Ludwig, M. and Landgraf, R., Microdialysis administration of vasopressin and vasopressin antagonists into the septum during pole-jumping behavior in rats. Behav. Neural Biol., 58 (1992) 51±57. Jakab, R.L. and Leranth, C., Catecholaminergic, GABAergic, and hippocamposeptal innervation of GABAergic `somatospiny' neurons in the rat lateral septal area. J. Comp. Neurol., 302 (1990) 305±321. Jakab, R.L. and Leranth, C., Septum. In G. Paxinos and C. Watson (Eds.), The Rat Nervous System, Academic Press, New York, 1995, pp. 405±442. Jakab, R.L., Naftolin, F. and Leranth, C., Convergent vasopressinergic and hippocampal input onto somatospiny neurons of the rat lateral septal area. Neuroscience, 40 (1991) 413±421. Koolhaas, J.M., Everts, H., de Ruiter, A.J., de Boer, S.F. and Bohus, B., Coping with stress in rats and mice: differential peptidergic modulation of the amygdala-lateral septum complex. Prog. Brain Res., 119 (1998) 437±448. Landgraf, R., Intracerebrally released vasopressin and oxytocin: measurement, mechanisms and behavioural concequences. J. Neuroendocrinol., 7 (1995) 243±253. Liebsch, G., Wotjak, C.T., Landgraf, R. and Engelmann, M., Septal vasopressin modulates anxiety-related behavior of adult male rats. Neurosci. Lett., 217 (1996) 101±104. Singewald, N., Guo, L. and Philippu, A., Release of endogenous GABA in the posterior hypothalamus of the conscious rat; effects of drugs and experimentally induced blood pressure changes. Naunyn-Schmiedeberg's Arch. Pharmacol., 347 (1993) 402±406.
Vasopressin selectively modulates the release of taurine within the septum of the rat brain Nicolas Singewald a, Karl Ebner b, Rainer Landgraf b, Carsten T. Wotjak a, c, Mario Engelmann b, d,* a
UniversitaÈt Innsbruck, Institut fuÈr Pharmakologie and Toxikologie, A-6020 Innsbruck, Austria b Max-Planck-Institut fuÈr Psychiatrie, Kraepelinstrasse 2, D-80804 MuÈnchen, Germany c Zentrum fuÈr Molekulare Neurobiologie, Martinistrasse 52, D-20246 Hamburg, Germany d Otto-von-Guericke-UniversitaÈt, Inst fuÈr Medizinische Neurobiologie, Leipziger Strasse 44, D-39120 Magdeburg, Germany Received 3 September 1999; received in revised form 15 October 1999; accepted 15 October 1999
Abstract Previous experiments have shown that arginine vasopressin (AVP) released within the septal brain area of adult male rats in response to de®ned stressor exposure is involved in emotionality-related behavior. We report here that a 10-min forced swimming session stimulated the release of glutamate, aspartate, arginine, g-aminobutyric acid (GABA) and taurine but not alanine and serine in the medio-lateral part of this brain structure. Local administration of the AVP V1 receptor antagonist d(CH2)5Tyr(Me)AVP by inverse microdialysis caused a signi®cant increase in the concentration of taurine in microdialysates under resting conditions that was further potentiated during forced swimming. In contrast, the release of alanine, arginine, GABA and serine was unaffected by antagonist treatment. Taken together with previous data, our results suggest that the effects of intraseptally released AVP on stress-coping strategies might be mediated at least in part via its in¯uence on the local release of taurine. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Emotionality; Septum; Amino acids; g-Aminobutyric acid; Glutamate; Swim stress; Microdialysis
Arginine vasopressin (AVP) has been postulated to act as a neurotransmitter/neuromodulator in the septal brain area of rodents, where it signi®cantly in¯uences learning and memory [5±7]. Recent ®ndings suggest that intraseptally released AVP plays also a crucial role for animals' emotionality in particular under stressful conditions ([13], see also Ref. [11] for review). Indeed, we reported recently that: (1) during a 10-min forced swimming session AVP release is triggered within the septum and (2) intraseptally released AVP affects the behavior in the forced swimming task [3]. Since AVP seems to act not only as a neurotransmitter but also as a neuromodulator within the septum (see Refs. [4,12] for review), it is likely that additional neurotransmitter systems are modulated by locally released AVP. To test this hypothesis, we monitored the extracellular concentration of various amino acids with established local neurotransmitter function including glutamate, aspartate, gaminobutyric acid (GABA) and taurine [9] in response to * Corresponding author. Tel.: 149-391-671-4363; fax: 149-391671-4365. E-mail address: [email protected] (M. Engelmann)
forced with or without local administration of an AVP V1 receptor antagonist. Adult male Wistar rats (300±400 g, n 30) were purchased from a Charles River (Sulzfeld, Germany) and kept under controlled laboratory conditions. Animals were implanted with microdialysis probes aimed at the mediolateral part of the septum under halothane anesthesia as described in detail by Ebner et al. [3] (implantation coordinates: 0.8 mm rostral to bregma, 2.3 mm lateral to midline, 6.3 mm below the surface of the skull with an angle of 228 to avoid damage to the sagittal sinus). Immediately after surgery rats were injected with antibiotics and individually housed in polycarbon cages (23 £ 29 £ 36 cm). The day after surgery animals were familiarized with the microdialysis procedure and handled for 5±10 min. On the following day animals were connected at 08.00 h (approx. 48 h after surgery) to the microdialysis equipment and the probes were perfused with sterile Ringer's solution (3.3 ml/ min). Initially probes were perfused for 2 h to allow to establish an equilibrium between inside and outside of the dialysis membrane. During this period, sample collection was simulated every 30 min.
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0 30 4- 39 40 ( 99) 0 08 41- 1
N. Singewald et al. / Neuroscience Letters 277 (1999) 68±70
Fig. 1. Concentration of amino acids in 30-min microdialysates obtained from the medio-lateral septum of freely moving rats (means ^ SEM) and expressed as percentage of the averaged ®rst two samples (100%; dotted line). Animals remained undisturbed in their home cages throughout the experiment except for the beginning of the collection of sample number 4, when rats were forced to swim for 10 min.**P , 0:01 and *P , 0:05 vs. samples 1±3.
After this initial period six consecutive 30-min dialysates were collected directly in 0.5-ml Eppendorf tubes and immediately stored on dry ice. At the beginning of the collection of sample four animals were transferred to forced swimming tanks (30 cm in diameter, ®lled to a depth of 40 cm with water of a temperature of 20 ^ 18C) and forced to swim for 10 min. Immediately before swimming, rats were equipped with a small piece of styrofoam on the back of the neck, to aid ¯oating without the necessity of additional leg movements. The behavior of the animals was monitored during the entire swimming session. Behavioral data obtained during the experiment had already been reported in a previous paper [3]. In a second set of experiments, the dialysis medium was switched to Ringer's solution containing the AVP V1 receptor antagonist d(CH2)5Tyr(Me)AVP (Dr. M. Manning, Toledo, USA) in 15 rats during the dialysis periods 3 and 4. The total amount of antagonist administered was calculated to be approximately 10 ng [6]. Controls (n 15) received Ringers' solution only.
69
Amino acids were determined in microdialysates by high performance liquid chromatography (HPLC) and ¯uorimetric detection (Merck-Hitachi, Tokyo, Japan) after derivatization with o -phtahldialdehyd (OPA) as previously described [14]. In the present study 75 ml of a mixture of 80 ml of the dialysate and 20 ml of OPA reaction mixture were injected. The reproducibility of the derivatization was controlled by addition of S-carboxymethyl-l-cysteine as an internal standard. Concentration of amino acids in microdialysates were not corrected for recovery. Data (means and SEM) are presented as percentage of the two averaged basal values (100%). For statistical analysis, they were transformed by arc-tan to ®t to Gaussian distribution and submitted to a oneway (effect of forced swimming on controls) or two-way (treatment and forced swimming) ANOVA with repeated measures. Fisher's least signi®cant difference was applied if appropriate. P , 0:05 was considered to be statistically signi®cant. Histological veri®cation of the implantation site and behavioral performance of the animals had already been reported in the study of Ebner et al. [3]. During HPLC analysis samples of four controls and two AVP V1 receptor antagonist treated animals had missing values. As a consequence, these animals were not included in the statistical analysis which reduced the number of animals per group to controls: n 11 and AVP V1 receptor antagonist: n 13. As shown in Fig. 1, forced swimming caused a signi®cant rise in microdialysate concentration of aspartate, glutamate, GABA, taurine, and arginine, whereas the release of serine and alanine was largely unaffected (Fig. 1). Administration of the V1 antagonist had no effects on the release of alanine, arginine, serine (data and statistics not shown) and GABA (Fig. 2) either before (P . 0:05) or during forced swimming
Fig. 2. Concentration of amino acids in 30-min microdialysates obtained from the medio-lateral septum of freely moving rats. Data are expressed as percentage of the averaged ®rst two samples. During collection of sample numbers 3 and 4, the dialysis medium was either Ringer's solution containing the AVP V1 receptor antagonist d(CH2)5Tyr(Me)AVP (solid line with black circles) or Ringer's solution alone (dotted line with open circle; data obtained from Fig. 1). At the beginning of the collection of sample number 4 the animals were forced to swim for 10 min in 208C warm water. *P , 0:05 vs. the same sample of the Ringer's group and P , 0:01 vs. samples 1 and 2.
70
N. Singewald et al. / Neuroscience Letters 277 (1999) 68±70
(P . 0:05). In contrast, administration of the antagonist caused a signi®cant increase in the release of taurine already under pre-stress conditions (P , 0:05). This increase was further potentiated when the animals were forced to swim (Fig. 2; P , 0:01). Aspartate and glutamate had to be excluded from further analyses because of a priori contamination of the antagonist-containing dialysis medium with these two amino acids. Our data demonstrate that a 10-min forced swimming session triggers intraseptal release of glutamate, aspartate, arginine, GABA and taurine but not other amino acids investigated (Fig. 1). Although extensive histological investigations imply GABA as the primary candidate for the interaction with AVP at the level of the septum [8±10], we failed to detect such an effect by local administration of a AVP V1 antagonist (Fig. 2). In contrast the release of taurine as a different inhibitory amino acid was signi®cantly increased by blockade of the endogenously released AVP (Fig. 2). This ®nding adds to the growing body of evidence for a close interaction between AVP and taurine within the rat brain [1,2]. As reported previously, behavioral consequences of the antagonist treatment resulted in higher ¯oating and shorter swimming duration as compared with controls [3]. It is tempting to speculate that the interplay between AVP and taurine contributes to these behavioral changes. However, the behavioral alterations observed do not correlate directly with extracellular concentration of taurine measured in the present study (unpublished data). This argues against taurine as a key player for the behavioral effects of intraseptally released AVP. Further studies will have to investigate explicitly the role of taurine in the physiological signi®cance of the stress-induced taurine release by administration of speci®c taurine antagonists. [1] Brust, P., Christensen, T. and Diemer, N.H., Decrease of extracellular taurine in the rat dorsal hippocampus after central nervous administration of vasopressin. J. Neurochem., 58 (1992) 1427±1431. [2] Campistron, G., Geffard, M. and Buijs, R.M., Immunological
[3]
[4] [5] [6]
[7]
[8]
[9] [10]
[11]
[12] [13] [14]
approach to the detection of taurine and immunocytochemical results. J. Neurochem., 46 (1986) 862±868. Ebner, K., Wotjak, C.T., Holsboer, F., Landgraf, R. and Engelmann, M., Vasopressin released within the septal brain area during swim stress modulates the behavioral stress response in male rats. Eur. J. Neurosci., 11 (1999) 997±1002. Fuxe, K. and Agnati, L.F., Volume Transmission in the Brain: Novel Mechanisms for Neural Transmission, Raven Press, New York, 1991. Dantzer, R., Koob, G.F., BlutheÂ, R.-M. and Le Moal, M., Septal vasopressin modulates social memory in male rats. Brain Res., 457 (1988) 143±147. Engelmann, M. and Landgraf, R., Microdialysis administration of vasopressin into the septum improves social recognition in Brattleboro rats. Physiol. Behav., 55 (1994) 145± 149. Engelmann, M., Ludwig, M. and Landgraf, R., Microdialysis administration of vasopressin and vasopressin antagonists into the septum during pole-jumping behavior in rats. Behav. Neural Biol., 58 (1992) 51±57. Jakab, R.L. and Leranth, C., Catecholaminergic, GABAergic, and hippocamposeptal innervation of GABAergic `somatospiny' neurons in the rat lateral septal area. J. Comp. Neurol., 302 (1990) 305±321. Jakab, R.L. and Leranth, C., Septum. In G. Paxinos and C. Watson (Eds.), The Rat Nervous System, Academic Press, New York, 1995, pp. 405±442. Jakab, R.L., Naftolin, F. and Leranth, C., Convergent vasopressinergic and hippocampal input onto somatospiny neurons of the rat lateral septal area. Neuroscience, 40 (1991) 413±421. Koolhaas, J.M., Everts, H., de Ruiter, A.J., de Boer, S.F. and Bohus, B., Coping with stress in rats and mice: differential peptidergic modulation of the amygdala-lateral septum complex. Prog. Brain Res., 119 (1998) 437±448. Landgraf, R., Intracerebrally released vasopressin and oxytocin: measurement, mechanisms and behavioural concequences. J. Neuroendocrinol., 7 (1995) 243±253. Liebsch, G., Wotjak, C.T., Landgraf, R. and Engelmann, M., Septal vasopressin modulates anxiety-related behavior of adult male rats. Neurosci. Lett., 217 (1996) 101±104. Singewald, N., Guo, L. and Philippu, A., Release of endogenous GABA in the posterior hypothalamus of the conscious rat; effects of drugs and experimentally induced blood pressure changes. Naunyn-Schmiedeberg's Arch. Pharmacol., 347 (1993) 402±406.