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Effects of immobilization on in vivo release of norepinephrine in the bed nucleus of the stria terminalis in conscious rats
BRAIN RESEARCH ELSEVIER
Brain Research688 (1995) 242-246
Short communication
Effects of immobilization on in vivo release of norepinephrine in the bed nucleus of the stria terminalis in conscious rats Karel Pacak
a,
Richard McCarty b, ,, Miklos Palkovits c, Irwin J. Kopin a, David S. Goldstein
a
Clinical Neuroscience Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA b Department of Psychology, University of Virginia, 102 Gilmer Hall, Charlottesville, VA 22903-2477, USA c Laboratory ofCellBiology, Nationallnstitute ofMentalHealth, Bethesda, MD 20892, USA
Accepted9 May 1995
Abstract
Release of norepinepriine (NE) and its metabolites in the bed nucleus of the stria terminalis (BNST) was examined using in vivo microdialysis in conscious rats before, during and after 2 h of immobilization. Microdialysate levels of NE and of dihydroxyphenylglycol (DHPG) increased by 170-290% above basal levels during the 1st h of immobilization and decreased gradually thereafter. In contrast, levels of dihydroxyphenylacetic acid (DOPAC) increased gradually over the entire period of immobilization, peaking at 110% above baseline levels. These findings indicate that in rats a single immobilization is attended by increased synthesis, release and reuptake of NE within the BNST. The results are consistent with previous findings relating to stress-induced release of NE in the hypothalamic paraventricular nucleus, central nucleus of the amygdala and cerebral cortex and suggest concurrent noradrenergic activation in several brains centers during acute stress. Keywords: Microdialysis;Dihydroxyphenylglycol;Dihydroxyphenylaceticacid
The bed nucleus of the stria terminalis (BNST) serves as a relay center for conveying information from limbic areas, including the amygdala and the ventral hippocampus, to the hypothalamic paraventricular nucleus (PVN), lower brainstem nuclei and the spinal cord [3-5,9,24]. The BNST and PVN receive afferent input from several catecholamine-containing brainstem nuclei, including the A1 and A2 noradrenergic cell groups and the locus coeruleus [20,25]. In vivo studies have shown that NE release from nerve terminals within the PVN increases dramatically during exposure to acute stress [17]. In an in vitro study, electrical stimulation-induced NE release from nerve terminals in the BNST was demonstrated using fast cycle voltammetry [18]. In addition, the firing rates of a significant proportion of BNST neurons change in response to iontophoretically applied NE [2]. Cell bodies in the parvocellular PVN synthesize CRH and are stimulated by NE released from local noradrenergic terminals. CRH neurons in turn project to the median eminence and to autonomic centers in the brainstem and
* Corresponding author. Fax: (1X804) 982-4766. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0006-8993(95)00566-8
spinal cord, thereby affecting hypothalamic-pituitaryadrenocortical (HPA) and sympathetic-adrenal medullary responses to stressful stimuli [14]. Thus, BNST neurons are well-positioned to affect autonomic and endocrine responses to various stressors via their effects on midbrain, brainstem and spinal cord cardiovascular regulatory centers as well as the PVN [1,6,8,9,11,15]. In the present study, we assessed in vivo release of NE in the BNST of conscious rats during a single immobilization. Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) were housed in a vivarium room maintained at 22 ± I°C with a 12-h light-dark cycle (lights on from 06:00 to 18:00). Food and water were provided ad libitum. All procedures were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke, NIH. Rats (308 ± 9 g) were anesthetized with pentobarbital (50 m g / k g i.p.) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA), with the incisor bar fixed at 3.2 mm below the interaural line. The skull was exposed by a midline incision and a small hole was drilled over the bed nucleus of the stria terminalis to the right of the midline. A microdialysis probe (1 mm length, cutoff value of 20 kDa;
K. Pacak et al. / Brain Research 688 (1995) 242-246
CMA/Microdialysis, Acton, MA) was lowered into the right BNST with the following coordinates with respect to bregma: posterior 0.75 mm, lateral 1.4 mm and vertical - 7 . 8 mm, according to Paxinos and Watson [19]. The dialysis probe was anchored to the skull with stainless steel screws and acrylic dental cement. The inlet tube of each probe was attached to a swivel connector positioned above a cylindrical Plexiglas cage. Each cage contained laboratory chow and water. Postoperatively, rats were housed individually and allowed 20-24 h to recover prior to the acute stress experiment. Immediately after surgery, artificial cerebrospinal fluid (189 mM NaCI, 3.9 mM KCI and 3.37 mM CaC12; pH 6.3) was perfused continuously through the microdialysis probe at a rate of 1 /xl/min using a microinfusion pump (CMA/100; CMA/Microdialysis). After a baseline period of microdialysate collection (2 × 30 min), samples were collected every 30 min during a 2-h period of immobilization and for 90 min after the rats were returned to their home cages. Microdialysate samples were collected into vials that contained 20 /xl 0.2 N acetic acid. The vials were frozen immediately in dry ice and stored at - 7 0 ° C until assayed within 4 - 6 weeks. A t the end of each experiment, the probe was perfused for 5 min with a solution of toluidine blue. Brains were removed and stored in 10% formalin solution. Serial coronal sections (30 ~ m thickness) of the preoptic-anterior hypothalamic area were cut in a cryostat, the sections were stained with haemotoxylin-eosin and the position of the probe was localized using a stereomicroscope. Criteria for correct placement of the microdialysis probe included having the dialysis probe penetrate through the lateral ventricle without causing major damage or significant hemorrhage. The probe must have been situated in the center of
243
the BNST (Fig. 1) at the same level or just caudal to the cross-over fibers of the anterior commissure with the entire 1 mm length of the probe under the lateral ventricle. Also, probes that were found to have been placed either lateral or medial to the lateral ventricle were not accepted. Acceptable probe placements dialyzed the extracellular fluid of the dorsal, ventral and lateral subdivisions of the BNST equally. ~ 70% of probe placements were acceptable. The volume of the BNST is sufficient to accommodate a 1 × 0.5-mm microdialysis probe [12]. Microdialysate levels of NE, dihydroxyphenyglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC) were quantified using reverse-phase liquid chromatography with electrochemical detection [10]. The limits of detection for NE and DHPG were ~ 2 pg/sample and for DOPAC 8 pg/sample. The results are expressed as p g / m l of microdialysate and are presented as means _+ SEM for the indicated numbers of animals. Responses over time for NE, DHPG and DOPAC were analysed by one-way ANOVA for repeated measures, with posthoc Fisher PLSD tests. A P value of < 0.05 defined statistical significance. Baseline microdialysate levels of NE (93 +_ 12 pg/ml), DHPG (1970 + 320 p g / m l ) and DOPAC (12610 _+ 1870 p g / m l ) were similar during two prestress samples in rats with microdialysis probes properly positioned within the BNST (Fig. 2). The highest NE levels, 360 p g / m l ( ~ 4fold basal levels) were found in microdialysate samples collected during the first 30-rain interval of immobilization. Levels of NE during the remainder of the 2 h of immobilization were 250-290 p g / m l and progressively declined after immobilization was terminated. The highest levels of DHPG, 5200 p g / m l (2.7-fold basal levels), were obtained during the first two 30-rain intervals after the
Fig. 1. Coronal section through rat forebrain demonstrating placement of a microdialysis probe (see arrows) in BNST. A, anterior commissure; F, fornix; L, lateral ventricle; * third ventricle.
K. Pacak et al. / B r a i n Research 688 (1995) 242-246
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start of immobilization, declined to 4500 p g / m l during the 2nd h and fell towards basal levels when immobilization was terminated• DOPAC levels increased progressively during the 2 h of immobilization to peak at 25,400 p g / m l (just greater than twice basal levels) during the fourth 30-min collection interval during immobilization. Levels of DOPAC decreased gradually when animals were returned to their home cages. Levels of NE, DHPG and DOPAC remained significantly greater than basal levels 90 min after the termination of immobilization (Fig. 2). The present results provide evidence for substantial immobilization-induced activation of noradrenergic nerve terminals in the BNST. The increases in NE levels in microdialysate samples after immobilization stress were rapid in onset and significant in magnitude. Consistent with these findings, Saavedra [23] reported that immobilization decreased NE content in the BNST, with a return to control levels 2 - 4 h after termination of immobilization, whereas tissue dopamine levels were unchanged during and after immobilization. Microdialysate levels of DHPG, the main intraneuronal metabolite of NE, also increased rapidly after the onset of immobilization but peak values were attained slightly later than for NE. Increases in DOPAC levels, which at least partly reflect changes in catecholamine biosynthesis, were more gradual and proportionately smaller than those of NE and DHPG. Since the BNST receives input from both noradrenergic and dopaminergic neurons [13,21], immobilization-induced increases in DOPAC levels could reflect intraneuronal metabolism of both NE and dopamine [16]. However, we were unable to detect dopamine in microdialysate samples in the present study. Given the delay in DOPAC responses during a single immobilization, it appears that alterations in DOPAC levels in the extracellular fluid of the BNST at least partly reflect alterations in local catecholamine synthesis. Since in dopaminergic terminals DOPAC also would be formed rapidly from reuptake of released dopamine, the results tend to support the view that the delayed DOPAC response reflects, at least in part, augmented catecholamine synthesis in noradrenergic terminals [16]. The BNST receives input from several limbic areas, including the ventral hippocampus and the central nucleus of the amygdala and affects activity of the HPA axis [3,5]. Ibotenic acid lesions of the anterior portion of the BNST in unstressed rats decrease CRH mRNA expression in the PVN, without affecting circulating levels of ACTH or corticosterone. In contrast, ibotenic acid lesions of the lateral BNST attenuate plasma ACTH and corticosterone
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Fig. 2. Microdialysate levels of NE (panel A), DHPG (panel B) and D O P A C (panel C) in BNST of laboratory rats before, during and after acute immobilization stress. Results are presented as m e a n ± S . E . M . values for 9 rats. * P < 0.05, * * P < 0.01 vs. preimmobilization baseline values.
K. Pacak et al. / Brain Research 688 (1995) 242-246
responses of rats to a conditioned stressor [7]. Electrolytic lesions of the BNST blunt the increase in plasma corticosterone after electrical stimulation of the medial amygdala [5]. The well-described inhibitory influence of the hippocampus on the HPA axis may also be mediated in part by projections from the hippocampus to the BNST via the ventral subiculum [3]. Thus, the BNST appears to be a site of convergence of multiple pathways conveying stimulatory and inhibitory influences to the PVN. The inhibitory influences of the hippocampus on the PVN appear to be mediated in part by GABAergic neurons projecting from the BNST [3,22]. Subicular projections to the lateral BNST stimulate GABAergic neurons, which then inhibit the PVN. NE release in the BNST may inhibit PVN-projecting GABAergic neurons within the BNST, releasing PVN CRH-containing neurons from inhibition. Consistent with this suggestion, Casada and Dafny [1] reported that 70% of BNST neurons from which they recorded had decreased firing rates after iontophoretic application of NE. Such an effect of NE released within the BNST could work in concert with the direct stimulation of CRH-containing neurons in the PVN by noradrenergic projections from lower brainstem nuclei. NE release in the BNST may also affect autonomic function and behavior via descending peptidergic projections to midbrain, brainstem and spinal cord areas, including the central gray, parabrachial nucleus and the dorsal vagal complex [6,11,15]. In previous studies reported from this laboratory, NE release within the PVN was shown to increase markedly during single immobilization. The magnitude of immobilization-induced increases in levels of NE, DHPG and DOPAC in PVN microdialysate samples are similar to corresponding increases in levels of these substances in the BNST in the present study [16]. Woulfe and coworkers [25], using a double-labeling technique in combination with immunohistochemistry, demonstrated collateral projections from the same A1 cell bodies to the PVN and the BNST. These findings suggest that increases in firing rates of A1 noradrenergic neurons may simultaneously inhibit GABAergic neurons in the BNST and stimulate CRH-containing neurons in the parvocellular PVN. Each of these effects of NE could contribute to increased stimulation of the HPA axis during acute stress. Additional anatomical and physiological studies are required to clarify the nature and influences of GABAergic neurons within the BNST that project to the PVN.
References [1] Casada, J.H. and Dafny, N., Restraint and stimulation of bed nucleus of the stria terminalis produce similar stress-like behaviors, Brain Res. Bull., 27 (1991) 207-212. [2] Casada, J.H. and Dafny, N., Responses of neurons in bed nucleus of the stria terminalis to microiontophoretically applied morphine, n o r -
245
epinephrine and acetylcholine, Neuropharmacology, 32 (1993) 279284. [3] Cullinan, W.E., Herman, J.P. and Watson, S.J., Ventral subicular interaction with the hypothalamic paraventricular nucleus: evidence for a relay in the bed nucleus of the stria terminalis, J. Comp. Neurol., 332 (1993) 1-20. [4] Dunn, J.D., Plasma corticosterone responses to electrical stimulation of the bed nucleus of the stria terminalis, Brain Res., 407 (1987) 327-331. [5] Feldman, S., Conforti, N. and Saphier, D., The preoptic area and bed nucleus of the stria terminalis are involved in the effects of the amygdala on adrenocortical secretion, Neuroscience, 37 (1990) 775-779. [6] Gray, T.S. and Magnuson, D.J., Neuropeptide neuronal efferents from the bed nucleus of the stria terminalis and central amygdaloid nucleus to the dorsal vagal complex in the rat, J. Comp. Neurol., 262 (1987) 365-374. [7] Gray, T.S., Piechowski, R.A., Yracheta, J.M., Rittenhouse, P.A., Bethea, C.L. and Van de Kar, L.D., lbotenic acid lesions in the bed nucleus of the stria terminalis attenuate conditioned stress-induced increases in prolactin, ACTH and corticosterone, Neuroendocrinology, 57 (1993) 517-524. [8] Henke, P.G., The bed nucleus of the stria terminalis and immobilization-stress: unit activity, escape behaviour, and gastric pathology in rat, Behac. Brain Res., 11 (1984) 35-45. [9] Herman, J.P., Cullinan, W.E. and Watson, S.J., Involvement of the bed nucleus of the stria terminalis in tonic regulation of paraventricular hypothalamic CRH and AVP mRNA expression, J. Neuroendocrinol., 6 (1994) 433-442. [10] Holmes, C , Eisenhofer, G. and Goldstein, D.S., Improved assay for plasma dihydroxyphenylacetic acid and other catechols using highperformance liquid chromatography with electrochemical detection, J. Chromatogr. B, 653 (1994) 131-138. [11] Holstege, G., Meiners, L. and Tan, K., Projections of the bed nucleus of the stria terminalis to the mesencephalon, ports, and medulla oblongata in the cat, Exp. Brain Res., 58 (1985) 379-391. [12] Ju, G. and Swanson, L.W., Studies on the cellular architecture of the bed nuclei of the stria terminalis in the rat: I. cytoarchitecture, J. Comp. Neurol., 280 (1989) 587-602. [13] Ju, G., Swanson, L.W. and Simerly, R.B., Studies on the cellular architecture of the bed nuclei of the stria terminalis in the rat. II. Chemoarchitecture, J. Comp. Neurol., 280 (1989) 603-621. [14] Mezey, E. and Palkovits, M., CRF-containing neurons in the hypothalamic paraventricular nucleus: regulation, especially by catecholamines, Front. Neuroendocrinol., 12 (1991) 23-37. [15] Moga, M.M., Saper, C.B. and Gray, T.S., Bed nucleus of the stria terminalis: cytoarchitecture, immunohistochemistry, and projection to the parabrachial nucleus in the rat, .L Comp. Neurol.. 283 (1989) 315-332. [16] Pacak, K., Armando, I., Fukuhara, K., Kvetnansky, R., Palkovits, M., Kopin, I.J. and Goldstein, D.S., Noradrenergic activation in the paraventricular nucleus during acute and chronic immobilization stress in rats: an in vivo microdialysis study, Brain Res., 589 (1992) 91-96. [17] Pacak, K., Palkovits, M., Kopin, l.J. and Goldstein, D.S., Stress-induced norepinephrine release in the hypothalamic paraventricular nucleus and pituitary-adrenalocortical and sympathoadrenal activity: in vivo microdialysis studies, Front. Neuroendocrinol., 16 (1995) 89-150. [18] Palif, P. and Stamford, J.A., Real time monitoring of endogenous noradrenaline release in rat brain slices using fast cycle voltammetry. 2. Operational characteristics of the alpha 2 autoreceptor in the bed nucleus of the stria terminalis, pars ventralis, Brain Res., 607 (1993) 134-140. [19] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd Ed., New York, NY, 1986. [20] Phelix, C.F., Liposits, Z. and Paull, WK., Monoamine innervation
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of bed nucleus of stria terminalis: an electron microscopic investigation, Brain Res. Bull., 28 (1992) 949-965. [21] Phelix, C.F., Liposits, Z. and Paull, W.K., Catecholamine-CRF synaptic interaction in a septal bed nucleus: afferents of neurons in the bed nucleus of the stria terminalis, Brain Res. Bull., 33 (1994) 109-119. [22] Roland, B.L. and Sawchenko, P.E., Local origins of some GABAergic projections to the paraventricular and supraoptic nuclei of the hypothalamus in the rat, J. Comp. Neurol., 332 (1993) 123-143. [23] Saavedra, J.M., Changes in dopamine, noradrenaline and adrenaline
in specific septal and preoptic nuclei after acute immobilization stress, Neuroendocrinology, 35 (1982) 396-401. [24] Sun, N., Roberts, L. and Cassell, M.D., Rat central amygdaloid nucleus projections to the bed nucleus of the stria terminalis, Brain Res. Bull., 27 (1991) 651-662. [25] Woulfe, J.M., Hrycyshyn, A.W. and Flumerfelt, B.A., Collateral axonal projections from the A1 noradrenergic cell group to the paraventricular nucleus and bed nucleus of the stria terminalis in the rat, Exp. Neurol., 102 (1988) 121-124.
Brain Research688 (1995) 242-246
Short communication
Effects of immobilization on in vivo release of norepinephrine in the bed nucleus of the stria terminalis in conscious rats Karel Pacak
a,
Richard McCarty b, ,, Miklos Palkovits c, Irwin J. Kopin a, David S. Goldstein
a
Clinical Neuroscience Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA b Department of Psychology, University of Virginia, 102 Gilmer Hall, Charlottesville, VA 22903-2477, USA c Laboratory ofCellBiology, Nationallnstitute ofMentalHealth, Bethesda, MD 20892, USA
Accepted9 May 1995
Abstract
Release of norepinepriine (NE) and its metabolites in the bed nucleus of the stria terminalis (BNST) was examined using in vivo microdialysis in conscious rats before, during and after 2 h of immobilization. Microdialysate levels of NE and of dihydroxyphenylglycol (DHPG) increased by 170-290% above basal levels during the 1st h of immobilization and decreased gradually thereafter. In contrast, levels of dihydroxyphenylacetic acid (DOPAC) increased gradually over the entire period of immobilization, peaking at 110% above baseline levels. These findings indicate that in rats a single immobilization is attended by increased synthesis, release and reuptake of NE within the BNST. The results are consistent with previous findings relating to stress-induced release of NE in the hypothalamic paraventricular nucleus, central nucleus of the amygdala and cerebral cortex and suggest concurrent noradrenergic activation in several brains centers during acute stress. Keywords: Microdialysis;Dihydroxyphenylglycol;Dihydroxyphenylaceticacid
The bed nucleus of the stria terminalis (BNST) serves as a relay center for conveying information from limbic areas, including the amygdala and the ventral hippocampus, to the hypothalamic paraventricular nucleus (PVN), lower brainstem nuclei and the spinal cord [3-5,9,24]. The BNST and PVN receive afferent input from several catecholamine-containing brainstem nuclei, including the A1 and A2 noradrenergic cell groups and the locus coeruleus [20,25]. In vivo studies have shown that NE release from nerve terminals within the PVN increases dramatically during exposure to acute stress [17]. In an in vitro study, electrical stimulation-induced NE release from nerve terminals in the BNST was demonstrated using fast cycle voltammetry [18]. In addition, the firing rates of a significant proportion of BNST neurons change in response to iontophoretically applied NE [2]. Cell bodies in the parvocellular PVN synthesize CRH and are stimulated by NE released from local noradrenergic terminals. CRH neurons in turn project to the median eminence and to autonomic centers in the brainstem and
* Corresponding author. Fax: (1X804) 982-4766. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0006-8993(95)00566-8
spinal cord, thereby affecting hypothalamic-pituitaryadrenocortical (HPA) and sympathetic-adrenal medullary responses to stressful stimuli [14]. Thus, BNST neurons are well-positioned to affect autonomic and endocrine responses to various stressors via their effects on midbrain, brainstem and spinal cord cardiovascular regulatory centers as well as the PVN [1,6,8,9,11,15]. In the present study, we assessed in vivo release of NE in the BNST of conscious rats during a single immobilization. Male Sprague-Dawley rats (Taconic Farms, Germantown, NY) were housed in a vivarium room maintained at 22 ± I°C with a 12-h light-dark cycle (lights on from 06:00 to 18:00). Food and water were provided ad libitum. All procedures were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke, NIH. Rats (308 ± 9 g) were anesthetized with pentobarbital (50 m g / k g i.p.) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA), with the incisor bar fixed at 3.2 mm below the interaural line. The skull was exposed by a midline incision and a small hole was drilled over the bed nucleus of the stria terminalis to the right of the midline. A microdialysis probe (1 mm length, cutoff value of 20 kDa;
K. Pacak et al. / Brain Research 688 (1995) 242-246
CMA/Microdialysis, Acton, MA) was lowered into the right BNST with the following coordinates with respect to bregma: posterior 0.75 mm, lateral 1.4 mm and vertical - 7 . 8 mm, according to Paxinos and Watson [19]. The dialysis probe was anchored to the skull with stainless steel screws and acrylic dental cement. The inlet tube of each probe was attached to a swivel connector positioned above a cylindrical Plexiglas cage. Each cage contained laboratory chow and water. Postoperatively, rats were housed individually and allowed 20-24 h to recover prior to the acute stress experiment. Immediately after surgery, artificial cerebrospinal fluid (189 mM NaCI, 3.9 mM KCI and 3.37 mM CaC12; pH 6.3) was perfused continuously through the microdialysis probe at a rate of 1 /xl/min using a microinfusion pump (CMA/100; CMA/Microdialysis). After a baseline period of microdialysate collection (2 × 30 min), samples were collected every 30 min during a 2-h period of immobilization and for 90 min after the rats were returned to their home cages. Microdialysate samples were collected into vials that contained 20 /xl 0.2 N acetic acid. The vials were frozen immediately in dry ice and stored at - 7 0 ° C until assayed within 4 - 6 weeks. A t the end of each experiment, the probe was perfused for 5 min with a solution of toluidine blue. Brains were removed and stored in 10% formalin solution. Serial coronal sections (30 ~ m thickness) of the preoptic-anterior hypothalamic area were cut in a cryostat, the sections were stained with haemotoxylin-eosin and the position of the probe was localized using a stereomicroscope. Criteria for correct placement of the microdialysis probe included having the dialysis probe penetrate through the lateral ventricle without causing major damage or significant hemorrhage. The probe must have been situated in the center of
243
the BNST (Fig. 1) at the same level or just caudal to the cross-over fibers of the anterior commissure with the entire 1 mm length of the probe under the lateral ventricle. Also, probes that were found to have been placed either lateral or medial to the lateral ventricle were not accepted. Acceptable probe placements dialyzed the extracellular fluid of the dorsal, ventral and lateral subdivisions of the BNST equally. ~ 70% of probe placements were acceptable. The volume of the BNST is sufficient to accommodate a 1 × 0.5-mm microdialysis probe [12]. Microdialysate levels of NE, dihydroxyphenyglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC) were quantified using reverse-phase liquid chromatography with electrochemical detection [10]. The limits of detection for NE and DHPG were ~ 2 pg/sample and for DOPAC 8 pg/sample. The results are expressed as p g / m l of microdialysate and are presented as means _+ SEM for the indicated numbers of animals. Responses over time for NE, DHPG and DOPAC were analysed by one-way ANOVA for repeated measures, with posthoc Fisher PLSD tests. A P value of < 0.05 defined statistical significance. Baseline microdialysate levels of NE (93 +_ 12 pg/ml), DHPG (1970 + 320 p g / m l ) and DOPAC (12610 _+ 1870 p g / m l ) were similar during two prestress samples in rats with microdialysis probes properly positioned within the BNST (Fig. 2). The highest NE levels, 360 p g / m l ( ~ 4fold basal levels) were found in microdialysate samples collected during the first 30-rain interval of immobilization. Levels of NE during the remainder of the 2 h of immobilization were 250-290 p g / m l and progressively declined after immobilization was terminated. The highest levels of DHPG, 5200 p g / m l (2.7-fold basal levels), were obtained during the first two 30-rain intervals after the
Fig. 1. Coronal section through rat forebrain demonstrating placement of a microdialysis probe (see arrows) in BNST. A, anterior commissure; F, fornix; L, lateral ventricle; * third ventricle.
K. Pacak et al. / B r a i n Research 688 (1995) 242-246
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start of immobilization, declined to 4500 p g / m l during the 2nd h and fell towards basal levels when immobilization was terminated• DOPAC levels increased progressively during the 2 h of immobilization to peak at 25,400 p g / m l (just greater than twice basal levels) during the fourth 30-min collection interval during immobilization. Levels of DOPAC decreased gradually when animals were returned to their home cages. Levels of NE, DHPG and DOPAC remained significantly greater than basal levels 90 min after the termination of immobilization (Fig. 2). The present results provide evidence for substantial immobilization-induced activation of noradrenergic nerve terminals in the BNST. The increases in NE levels in microdialysate samples after immobilization stress were rapid in onset and significant in magnitude. Consistent with these findings, Saavedra [23] reported that immobilization decreased NE content in the BNST, with a return to control levels 2 - 4 h after termination of immobilization, whereas tissue dopamine levels were unchanged during and after immobilization. Microdialysate levels of DHPG, the main intraneuronal metabolite of NE, also increased rapidly after the onset of immobilization but peak values were attained slightly later than for NE. Increases in DOPAC levels, which at least partly reflect changes in catecholamine biosynthesis, were more gradual and proportionately smaller than those of NE and DHPG. Since the BNST receives input from both noradrenergic and dopaminergic neurons [13,21], immobilization-induced increases in DOPAC levels could reflect intraneuronal metabolism of both NE and dopamine [16]. However, we were unable to detect dopamine in microdialysate samples in the present study. Given the delay in DOPAC responses during a single immobilization, it appears that alterations in DOPAC levels in the extracellular fluid of the BNST at least partly reflect alterations in local catecholamine synthesis. Since in dopaminergic terminals DOPAC also would be formed rapidly from reuptake of released dopamine, the results tend to support the view that the delayed DOPAC response reflects, at least in part, augmented catecholamine synthesis in noradrenergic terminals [16]. The BNST receives input from several limbic areas, including the ventral hippocampus and the central nucleus of the amygdala and affects activity of the HPA axis [3,5]. Ibotenic acid lesions of the anterior portion of the BNST in unstressed rats decrease CRH mRNA expression in the PVN, without affecting circulating levels of ACTH or corticosterone. In contrast, ibotenic acid lesions of the lateral BNST attenuate plasma ACTH and corticosterone
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Fig. 2. Microdialysate levels of NE (panel A), DHPG (panel B) and D O P A C (panel C) in BNST of laboratory rats before, during and after acute immobilization stress. Results are presented as m e a n ± S . E . M . values for 9 rats. * P < 0.05, * * P < 0.01 vs. preimmobilization baseline values.
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responses of rats to a conditioned stressor [7]. Electrolytic lesions of the BNST blunt the increase in plasma corticosterone after electrical stimulation of the medial amygdala [5]. The well-described inhibitory influence of the hippocampus on the HPA axis may also be mediated in part by projections from the hippocampus to the BNST via the ventral subiculum [3]. Thus, the BNST appears to be a site of convergence of multiple pathways conveying stimulatory and inhibitory influences to the PVN. The inhibitory influences of the hippocampus on the PVN appear to be mediated in part by GABAergic neurons projecting from the BNST [3,22]. Subicular projections to the lateral BNST stimulate GABAergic neurons, which then inhibit the PVN. NE release in the BNST may inhibit PVN-projecting GABAergic neurons within the BNST, releasing PVN CRH-containing neurons from inhibition. Consistent with this suggestion, Casada and Dafny [1] reported that 70% of BNST neurons from which they recorded had decreased firing rates after iontophoretic application of NE. Such an effect of NE released within the BNST could work in concert with the direct stimulation of CRH-containing neurons in the PVN by noradrenergic projections from lower brainstem nuclei. NE release in the BNST may also affect autonomic function and behavior via descending peptidergic projections to midbrain, brainstem and spinal cord areas, including the central gray, parabrachial nucleus and the dorsal vagal complex [6,11,15]. In previous studies reported from this laboratory, NE release within the PVN was shown to increase markedly during single immobilization. The magnitude of immobilization-induced increases in levels of NE, DHPG and DOPAC in PVN microdialysate samples are similar to corresponding increases in levels of these substances in the BNST in the present study [16]. Woulfe and coworkers [25], using a double-labeling technique in combination with immunohistochemistry, demonstrated collateral projections from the same A1 cell bodies to the PVN and the BNST. These findings suggest that increases in firing rates of A1 noradrenergic neurons may simultaneously inhibit GABAergic neurons in the BNST and stimulate CRH-containing neurons in the parvocellular PVN. Each of these effects of NE could contribute to increased stimulation of the HPA axis during acute stress. Additional anatomical and physiological studies are required to clarify the nature and influences of GABAergic neurons within the BNST that project to the PVN.
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