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CRF Receptor Antagonist Attenuates Immobilization Stress-Induced Norepinephrine Release in the Prefrontal Cortex in Rats
Brain Research Bulletin, Vol. 42, No. 6, pp. 431–434, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(96)00368-1
CRF Receptor Antagonist Attenuates Immobilization Stress-Induced Norepinephrine Release in the Prefrontal Cortex in Rats GENNADY N. SMAGIN, 1 JUN ZHOU, RUTH B. S. HARRIS AND DONNA H. RYAN Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Rd., Baton Rouge, Louisiana 70808 [Received 1 July 1996; Accepted 7 October 1996] ABSTRACT: Neuroanatomical, neurophysiological, and behavioral studies suggest that brain stem nucleus locus coeruleus (LC) plays an important role in stress response. The present study was designed to clarify, whether infusion of CRF antagonist, ahCRF, into LC could attenuate or block stress-induced changes in norepinephrine (NE) concentrations in microdialysates collected from the medial prefrontal cortex (PFM). Rats were implanted with a bilateral cannulae assembly aimed in the LC and a microdialysis probe (4 mm active membrane length) into the LC. Immobilization of animals significantly increased the concentration of NE in microdialysates from PFM to a maximum of 170.8 { 12.8% of the baseline ten minutes after the onset of stressor. Concentration of NE in dialysates remained significantly elevated for the next 40 min. Infusion of ahCRF into the LC significantly attenuated stress-induced increase in PFM NE concentration in samples collected at 10, 20, 30, and 50 min after the onset of immobilization. Infusion of ahCRF alone (no immobilization) did not change concentrations at any time during sample collection. These results are consistent with other studies and suggest that stress can facilitate NE release in the PFM through the activation of the CRF system in the brain. Q 1997 Elsevier Science Inc.
[13]. Because PFM receives dense noradrenergic innervation from the LC [21] and is important in the mediation of stress, the changes in NE metabolism in the PFM likely result from increased activity of LC neurons. Although such activation occurs after ICV administration of CRF [11] and Shimizu et al. [15] have demonstrated that CRF antagonist infused ICV attenuates the immobilization stress induced NE release in the PFM of rats, the specific involvement of the LC in the stress-related response has not been established. The present study was designed to clarify whether a CRF antagonist, ahCRF, infused locally in the region of the LC could attenuate, or block, immobilization stress-induced changes in NE concentrations in microdialysates collected from the PFM of rats. METHOD Animals Male Sprague–Dawley rats were obtained from Harlan– Spague–Dawley (Indianapolis, IN) and weighed 250–350 g at the time of the experiment. They were housed singly in plastic cages for at least 1 week prior to the experiment with ad lib access to rat chow (Purina 5001; Purina Mills, St. Louis, MO) and tap water. A 12–12 h light–dark cycle was maintained with lights on at 0700 h. All animal procedures were approved by the Pennington Biomedical Research Center IACUC.
KEY WORDS: Corticotropin-releasing factor, Immobilization stress, Noradrenergic system, Microdialysis, Locus coeruleus.
INTRODUCTION Surgery
Overwhelming evidence indicates that corticotropin releasing factor (CRF) serves to integrate autonomic, endocrine, and behavioral responses to stress [4,7,9,12]. Wide distribution of CRF-binding sites in the brain suggest that CRF is a possible mediator of stress-induced physiological and behavioral responses. It is also well established that stress activates the noradrenergic system [10,18]. Many reports indicate that there is an interaction between CRF and noradrenergic systems. One site of interaction is the brain stem LC. It has been shown that stress increases neuronal activity of the LC [1] elevates the concentration of CRF-like immunoreactivity in the LC and induces expression of CRF mRNA [7]. CRF injected into the LC increases the rate of firing of LC neurons [22] and increases NE concentration in dialysates collected from PFM [17] and hippocampus 1
Rats were anesthetized by IP injection of Ketamine hydrochloride (90 mg/kg) and Xylazine (10 mg/kg). They were implanted with a double barreled cannulae assembly, consisted of two 22 gauge guide cannulae spaced 2.4 mm apart and two dummy inserts (Plastics One, Roanake, VA) aimed at the LC and a CMA/12 guide cannula with a dummy insert (CMA/Microdialysis AB, Stockholm, Sweden) aimed at the PFM. For positioning in the LC the following coordinates were applied to a skull leveled between bregma and lambda: AP 01.2 from the interaural line; ML 1.2 mm and V 6.8 mm from the skull surface. For the medial prefrontal cortex: AP /3.3 mm from the bregma; L 00.6 mm; V 0.6 mm. The cannulae were secured to the skull with three anchor screws and dental cement.
To whom requests for reprints should be addressed.
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FIG. 1. A thionin-stained coronal section of the brain at the level of the locus coeruleus.
Microdialysis Procedure Concentric design microdialysis probes CMA/12, (CMA/ Microdialysis AB) were used for microdialysis. The active length of the dialysis membrane was 4 mm. The probes were perfused with artificial cerebrospinal fluid (aCSF: 1.2 mM CaCl2, 1.2 mM Na2HPO4 , 0.3 mM NaH2PO4 , 3.4 mM KCl, 140 mM NaCl, adjusted to pH 7.2) at a flow rate of 2.5 ml/min using a CMA/100 Microinjection pump. Microdialysis samples were collected by a CMA/140 Microfraction collector every 10 min directly into microvials containing 20 ml of 0.1 M HClO4 . The samples were analyzed for NE and other metabolites using an HPLC system (ESA, Inc., Chelmsford, MA) consisting of a pump model 420, autosampler model 460, Coulochem detector model 5100A, model 5014 High-Performance analytical cell, and MD-150 column. The mobile phase was 75 mM NaH2PO4 , 1.4 mM OSA, 10 mM EDTA, 10% Acetonitrile adjusted to pH 3.1 with H3PO4 . Flow rate was maintained at 0.8 ml/min. Minor changes of pH and OSA concentration were made to obtain optimal separation. Detector potentials were set: electrode 1 at 00.04 V, electrode 2 at /0.35 V; guard cell at /0.4 V. Experimental Protocol Following surgery, rats were allowed to recover for 6–7 days in their home cages. The evening before the experiment microdialysis probes were inserted through the guide cannula and infusion fluid was continuously pumped overnight at a flow rate of 0.5 ml/min. On the day of the experiment flow rate was switched to 2.5 ml/min. After a stabilization period of 2 h, baseline samples were collected for 1 h. The CRF receptor antagonist ahCRF was dissolved in aCSF and 1 mg of ahCRF in 300 nl was infused bilaterally into the LC over a 2-min period using an infusion pump. Injectors were made from fused silica tubing (o.d. 0.175 mm, Polymicro Technologies, Phoenix, AZ) inserted into 28 gauge stainless steel tubing and projected 1.0 mm beyond the tips of guide cannulae. Injectors were left in place for an additional 1 min and then replaced with dummy inserts. Ten minutes after infusion animals were immobilized for 30 min by strapping them with VELCROt tape to a plastic board. Control animals (vehicle / IMB) received an infusion of aCSF. A second control
group of animals ( ahCRF alone) was not immobilized following infusion of ahCRF. The amount of ahCRF chosen was based on published data that a 10-fold excess of ahCRF is required to prevent the pituitary responses to CRF and that 100–200 ng of the peptide injected into the LC was effective in blocking the shock-induced freezing behavior [20]. After completion of procedures rats were overdosed with sodium pentobarbital (120 mg/kg) and perfused intracardially through the left ventricle with 0.9% saline followed by 4% formalin. Brains were removed, stored at least 48 h in 4% formalin, and coronal sections, cut at 50 mm, were stained with thionin. Placement of the injector tips in the brain stem were determined with reference to the stereotaxic atlas of Paxinos and Watson [14]. Only the results from animals in which the injection sites were within the LC were included in data analysis. Data Analysis Values of NE concentration in six baseline samples were averaged and then all data were expressed as percent of baseline. Statistical analyses were performed using two-way analysis of variance (ANOVA). Dunnett’s multiple range test was used for post hoc analysis. A p-value of less than 0.05 was considered to indicate statistical significance. RESULTS Histological analysis indicated that the injector tips were located within the LC in six rats that received infusions of ahCRF without immobilization, in seven rats that received infusion of ahCRF and immobilized, and in six infused with aCSF and immobilized. A typical example of histological verification of cannulae tip placement is shown in Fig. 1. Changes in NE concentrations in microdialysates collected from the PFM before, during and after immobilization of animals are shown in Fig. 2. The basal level of NE was 1.03 { 0.06, 1.07 { 0.11, and 0.9 { 0.14 ng/ml of dialysate for ahCRF, vehicle / IMB, and ahCRF/ IMB groups, respectively, and was stable before immobilization. Immobilization of animals significantly increased the concentration of NE in microdialysates collected from the PFM reaching a maximum of 170.8 { 12.8% of baseline
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FIG. 2. The effect of immobilization stress and infusion of ahCRF/aCSF into the LC on concentration of NE in microdialysates collected from the medial prefrontal cortex. Injection of ahCRF/aCSF was made 10 min before immobilization (indicated by an arrow). The duration of immobilization (IMB) is indicated by the horizontal bar. *Significant difference from the basal level (Dunnett’s multiple range test, p õ 0.05), /difference from corresponding values of the control group (veh / IMB, Dunnett’s t-test). Two-way ANOVA indicated significant difference between the groups ( p õ 0.001).
by 10 min after onset of immobilization. The concentration of NE remained elevated during 30 min of stress and was 151.8 { 9.6% of baseline at the end of immobilization. Analysis of variance indicated a significant effect of time, F (19, 135) Å 2.7, p Å 0.002, on NE concentration. Post hoc determination (Dunnett’s test) of differences over the baseline level indicated that the immobilization stress-induced increase in NE concentrations in microdialysates was significant for the first 50 min of immobilization. Infusion of ahCRF significantly attenuated the immobilization stress-induced NE release in the PFM. A two-way ANOVA with repeated measures indicated a significant difference between groups, F(2, 364) Å 30.06, p õ 0.001. Dunnet’s was used to compare corresponding values of the vehicle / IMB group and ahCRF / IMB group and revealed significant differences at 10-, 20-, 30-, and 50-min time points (Fig. 2.). Infusion of ahCRF alone (no immobilization) did not significantly change NE concentrations from baseline level during the 130-min period of samples collection. Because the HPLC system was optimized for detection of NE, other catecholamines and metabolites were not reliably detected in microdialysis samples and data not shown. DISCUSSION In vivo microdialysis studies permit measurements of extracellular concentrations of neurotransmitters, and reflect the ‘‘overflow’’ of released neurotransmitters. Although such extracellular concentrations of NE are an indirect estimate of release, several studies with drugs that inhibit action potentials (e.g., tetrodotoxin) have demonstrated that dialysate concentrations of catecholamines generally reflect synaptic release [2,6]. Changes in extracellular concentrations are likely to reflect changes in synaptic release.
A number of studies have demonstrated that CRF, the hypothalamic hormone, may also serve as neurotransmitter in the LC and may mediate various aspects of the stress response. There are CRF-containing nerve terminals and CRF binding sites in the LC [19,26], and there is ultrastructural evidence that axon terminals containing CRF directly contact catecholamine-containing dendrites in the LC [25]. Local infusion of CRF into the LC increases discharge rate of neurons [22] and increases norepinephrine release in PFM, a region whose main source of NE derives from LC [17]. Behavioral results also suggest a role of CRF in stress-induced behavioral changes. CRF administered into the LC induced defensive withdrawal in rats and was more potent than injected ICV [5]. The rationale to perform bilateral infusions of CRF antagonist in our experiments appears from the observed laterality of the noradrenergic response to CRF injection into the LC [17]. The existing data suggest that unilateral stimulation of the LC is necessary to produce an activation, but bilateral infusions are likely to be necessary to block the response. In our microdialysis study [17] we found that there was an apparent increase in dialysate NE on the contralateral side (although it was not statistically significant). This could have occurred because of nonspecific activation of the two loci. If both loci coertulei were activated, then bilateral blockage or lesion would be necessary to prevent the activating effect. It has been reported that ahCRF injected into the LC bilaterally attenuated shock-induced freezing [20] and immobilization stress-induced defensive withdrawal [16]. Exposure to stressful stimuli activates release of NE in the cortex [1,3] and immobilization stress elevates the concentration of CRF-like immunoreactivity in the LC [7]. Microdialysis experiments showed that infusion of ahCRF ICV attenuated immobilization stress-induced NE release in PFM [15]. Addition-
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ally, activation of LC neurons by hypotensive stress can be prevented by microinfusion of ahCRF into the LC [8,23]. The present study demonstrated that immobilization stress caused a significant increase in NE concentrations in microdialysates collected from PFM, and that the release was significantly attenuated by infusion of CRF antagonist into the region of the LC. It is important to note that in our experiments we observe total elimination of stress-induced increase of NE concentrations in microdialysates collected from PFM, while other authors reported just attenuation [17]. In our experiments we were using 30-min immobilization stress, and bilateral infusion of a large dose of ahCRF bilaterally into the region of the LC, comparing to 1-h immobilization stress and ICV infusion of ahCRF as reported by Shimizu et al. [15] . All these factors may explain the difference in the results. These results suggest that immobilization stress-induced activation of CRF receptors in the LC leads to the release of NE in the PFM and the increased concentrations of NE in microdialysates collected from the PFM. It has been suggested that CRF may affect LC neuronal activity at several sites: CRF could directly affect LC discharge via synaptic contacts; indirectly affect LC discharge via presynaptic actions on terminals; modulate the effect of other neurotransmitters with which it is colocalized [25]. Whether or not CRF is mediating the noradrenergic response may depend on the form of stress. Valentino et al. [24] has shown that the local infusion of ahCRF into LC can prevent activation of LC neurons in response to sodium nitroprusside infusion, but this did not occur when LC neurons were activated by sciatic nerve stimulation. In summary, our results suggest that local infusion of ahCRF in the region of the brain stem LC attenuates the immobilization stress-induced synaptic release of NE in the PFM. This is consistent with anatomical and physiological data that show that stress can facilitate NE release in the PFM through the activation of the CRF system in the brain. ACKNOWLEDGEMENTS
We thank Dr. I. I. Rybkin for the help with histology. This research was supported by the U.S. Army Research and Development Command Grant DAMD 17-92-V-2009. Opinions, interpretations, conclusions are those of the authors and are not necessarily endorsed by U.S. Army.
REFERENCES 1. Abercombie, E. D.; Jacobs, B. L. Single-unit response of noradrenergic neurons in the locus coeruleus of freely moving cats. I. Acutely presented stressful and nonstressful stimuli. J. Neurosci. 7:2837– 2843; 1987. 2. Benveniste, H.; Huttemeier, P. C. Microdialysis—Theory and application. Prog. Neorobiol. 35:195–215; 1990. 3. Britton, K. T.; Segal, D. S.; Kuczenski, R.; Hauger, R. Dissociation between in vivo hippocampal norepinephrine response and behavioral/neuroendocrine responses to noise stress in rats. Brain Res. 574:125–130; 1992. 4. Brown, M. R.; Fisher, L. A. Corticotropin-releasing factor: Effects on the autonomic nervous system and visceral systems. Fed. Proc. 44:243–248; 1985. 5. Butler, P. D.; Weiss, J. M.; Stout, J. C.; Nemeroff, C. B. Corticotropin-releasing factor produces fear-enhancing and behavioral activating effects following infusion into the locus coeruleus. J. Neurosci. 10:176–183; 1990. 6. Cenci, M. A.; Kalen, P.; Mandel, R. J.; Bjorklund, A. Regional differences in the regulation of dopamine and noradrenaline release in medial frontal cortex, nucleus accumbens and caudate-putamen: A microdialysis study in the rat. Brain Res. 581:217–228; 1992.
7. Chappell, P. B.; Smith, M. A.; Kilts, C. D.; Bissette, G.; Ritchie, J.; Anderson, C.; Nemeroff, C. B. Alterations in corticotropin-releasing factor-like immunoreactivity in discrete rat brain regions after acute and chronic stress. J. Neurosci. 6:2908–2914; 1986. 8. Curtis, A. L.; Drolet, G.; Valentino, R. J. Hemodynamic stress activates locus coeruleus neurons of unanesthetized rats. Brain Res. Bull. 31:737–744; 1993. 9. Dunn, A. J.; Berridge, C. W. Corticotropin-releasing factor administration elicits a stress-like activation of cerebral catecholaminergic systems. Pharmacol. Biochem. Behav. 27:685–691; 1987. 10. Dunn, A. J.; Kramarcy, N. R. Neurochemical responses in stress: Relationships between the hypothalamic–pituitary–adrenal and catecholamine systems. Handbook Psychopharmacol. 18:455–515; 1984. 11. Lavicky, J.; Dunn, A. J. Corticotropin-releasing factor stimulates catecholamine release in hypothalamus and prefrontal cortex in freely moving rats as assessed by microdialysis. J. Neurochem. 60:602–612; 1993. 12. Owens, M. J.; Nemeroff, C. B. Physiology and pharmacology of corticotropin-releasing factor. Pharmacol. Rev. 43:425–474; 1991. 13. Page, M. E.; Abercombie, E. D. Microinfusion of corticotropin-releasing factor in the locus coeruleus increases extracellular norepinephrine in the rat hippocampus. Soc. Neurosci. Abstr. 21:634; 1995. 14. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. San Diego: Academic Press; 1986. 15. Shimizu, N.; Nakane, H.; Hori, T.; Hayashi, Y. CRF receptor antagonist attenuates stress-induced noradrenaline release in the medial prefrontal cortex of rats. Brain Res. 654:145–148; 1994. 16. Smagin, G. N. Corticotropin-releasing factor receptor antagonist infused into the locus coeruleus attenuates immobilization stress-induced defensive withdrawal in rats. Soc. Neurosci. Abstr. 21:500; 1995. 17. Smagin, G. N.; Swiergiel, A. H.; Dunn, A. J. Corticotropin-releasing factor administered into the locus coeruleus, but not the parabrachial nucleus, stimulates norepinephrine release in the prefrontal cortex. Brain Res. Bull. 19:71–76; 1995. 18. Stone, E. A. Stress and catecholamines. In: Friedhoff, A. J., eds. Catecholamines and behavior, vol. 2. Neuropsychopharmacology. New York: Plenum Press; 1975:31–72. 19. Swanson, L. W.; Sawchenko, P. E.; Rivier, J.; Vale, W. W. Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: An immunohistochemical study. Neuroendocrinology 36:165–186; 1983. 20. Swiergiel, A. H.; Takahashi, L. K.; Rubin, W. W.; Kalin, N. H. Antagonism of corticotropin-releasing factor receptors in the locus coeruleus attenuates shock-induced freezing in rats. Brain Res. 587:263–268; 1992. 21. Ungerstedt, U. Mapping of the central doamine, noradrenaline and 5-hydroxytryptamine pathways. Acta Physiol. Scand. 367:1–43; 1971. 22. Valentino, R. J.; Foote, S. L.; Aston–Jones, G. Corticotropin-releasing factor activates noradrenergic neurons of the locus coeruleus. Brain Res. 270:363–367; 1983. 23. Valentino, R. J.; Page, M. E.; Curtis, A. L. Activation of noradrenergic locus coeruleus neurons by hemodynamic stress is due to local release of corticotropin-releasing factor. Brain Res. 555:25–34; 1991. 24. Valentino, R. J.; Wehby, R. G. Corticotropin-releasing factor: Evidence for a neurotransmitter role in the locus ceruleus during hemodynamic stress. Neuroendocrinology 48:674–677; 1988. 25. Van Bockstaele, E. J.; Colago, E. E. O.; Valentino, R. J. Corticotropin-releasing factor-containing axon terminals synapse onto catecholamine dendrites and may presynaptically modulate other afferents in the rostral pole of the nucleus locus coeruleus in the rat brain. J. Comp. Neurol. 364:523–534; 1996. 26. Wynn, P. C.; Hauger, R. L.; Holmes, M. C.; Millan, M. A.; Catt, K. J.; Aguilera, G. Brain and pituitary receptors for corticotropin releasing factor: Localization and differential regulation after adrenalectomy. Peptides 5:1077–1084; 1984.
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CRF Receptor Antagonist Attenuates Immobilization Stress-Induced Norepinephrine Release in the Prefrontal Cortex in Rats GENNADY N. SMAGIN, 1 JUN ZHOU, RUTH B. S. HARRIS AND DONNA H. RYAN Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Rd., Baton Rouge, Louisiana 70808 [Received 1 July 1996; Accepted 7 October 1996] ABSTRACT: Neuroanatomical, neurophysiological, and behavioral studies suggest that brain stem nucleus locus coeruleus (LC) plays an important role in stress response. The present study was designed to clarify, whether infusion of CRF antagonist, ahCRF, into LC could attenuate or block stress-induced changes in norepinephrine (NE) concentrations in microdialysates collected from the medial prefrontal cortex (PFM). Rats were implanted with a bilateral cannulae assembly aimed in the LC and a microdialysis probe (4 mm active membrane length) into the LC. Immobilization of animals significantly increased the concentration of NE in microdialysates from PFM to a maximum of 170.8 { 12.8% of the baseline ten minutes after the onset of stressor. Concentration of NE in dialysates remained significantly elevated for the next 40 min. Infusion of ahCRF into the LC significantly attenuated stress-induced increase in PFM NE concentration in samples collected at 10, 20, 30, and 50 min after the onset of immobilization. Infusion of ahCRF alone (no immobilization) did not change concentrations at any time during sample collection. These results are consistent with other studies and suggest that stress can facilitate NE release in the PFM through the activation of the CRF system in the brain. Q 1997 Elsevier Science Inc.
[13]. Because PFM receives dense noradrenergic innervation from the LC [21] and is important in the mediation of stress, the changes in NE metabolism in the PFM likely result from increased activity of LC neurons. Although such activation occurs after ICV administration of CRF [11] and Shimizu et al. [15] have demonstrated that CRF antagonist infused ICV attenuates the immobilization stress induced NE release in the PFM of rats, the specific involvement of the LC in the stress-related response has not been established. The present study was designed to clarify whether a CRF antagonist, ahCRF, infused locally in the region of the LC could attenuate, or block, immobilization stress-induced changes in NE concentrations in microdialysates collected from the PFM of rats. METHOD Animals Male Sprague–Dawley rats were obtained from Harlan– Spague–Dawley (Indianapolis, IN) and weighed 250–350 g at the time of the experiment. They were housed singly in plastic cages for at least 1 week prior to the experiment with ad lib access to rat chow (Purina 5001; Purina Mills, St. Louis, MO) and tap water. A 12–12 h light–dark cycle was maintained with lights on at 0700 h. All animal procedures were approved by the Pennington Biomedical Research Center IACUC.
KEY WORDS: Corticotropin-releasing factor, Immobilization stress, Noradrenergic system, Microdialysis, Locus coeruleus.
INTRODUCTION Surgery
Overwhelming evidence indicates that corticotropin releasing factor (CRF) serves to integrate autonomic, endocrine, and behavioral responses to stress [4,7,9,12]. Wide distribution of CRF-binding sites in the brain suggest that CRF is a possible mediator of stress-induced physiological and behavioral responses. It is also well established that stress activates the noradrenergic system [10,18]. Many reports indicate that there is an interaction between CRF and noradrenergic systems. One site of interaction is the brain stem LC. It has been shown that stress increases neuronal activity of the LC [1] elevates the concentration of CRF-like immunoreactivity in the LC and induces expression of CRF mRNA [7]. CRF injected into the LC increases the rate of firing of LC neurons [22] and increases NE concentration in dialysates collected from PFM [17] and hippocampus 1
Rats were anesthetized by IP injection of Ketamine hydrochloride (90 mg/kg) and Xylazine (10 mg/kg). They were implanted with a double barreled cannulae assembly, consisted of two 22 gauge guide cannulae spaced 2.4 mm apart and two dummy inserts (Plastics One, Roanake, VA) aimed at the LC and a CMA/12 guide cannula with a dummy insert (CMA/Microdialysis AB, Stockholm, Sweden) aimed at the PFM. For positioning in the LC the following coordinates were applied to a skull leveled between bregma and lambda: AP 01.2 from the interaural line; ML 1.2 mm and V 6.8 mm from the skull surface. For the medial prefrontal cortex: AP /3.3 mm from the bregma; L 00.6 mm; V 0.6 mm. The cannulae were secured to the skull with three anchor screws and dental cement.
To whom requests for reprints should be addressed.
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FIG. 1. A thionin-stained coronal section of the brain at the level of the locus coeruleus.
Microdialysis Procedure Concentric design microdialysis probes CMA/12, (CMA/ Microdialysis AB) were used for microdialysis. The active length of the dialysis membrane was 4 mm. The probes were perfused with artificial cerebrospinal fluid (aCSF: 1.2 mM CaCl2, 1.2 mM Na2HPO4 , 0.3 mM NaH2PO4 , 3.4 mM KCl, 140 mM NaCl, adjusted to pH 7.2) at a flow rate of 2.5 ml/min using a CMA/100 Microinjection pump. Microdialysis samples were collected by a CMA/140 Microfraction collector every 10 min directly into microvials containing 20 ml of 0.1 M HClO4 . The samples were analyzed for NE and other metabolites using an HPLC system (ESA, Inc., Chelmsford, MA) consisting of a pump model 420, autosampler model 460, Coulochem detector model 5100A, model 5014 High-Performance analytical cell, and MD-150 column. The mobile phase was 75 mM NaH2PO4 , 1.4 mM OSA, 10 mM EDTA, 10% Acetonitrile adjusted to pH 3.1 with H3PO4 . Flow rate was maintained at 0.8 ml/min. Minor changes of pH and OSA concentration were made to obtain optimal separation. Detector potentials were set: electrode 1 at 00.04 V, electrode 2 at /0.35 V; guard cell at /0.4 V. Experimental Protocol Following surgery, rats were allowed to recover for 6–7 days in their home cages. The evening before the experiment microdialysis probes were inserted through the guide cannula and infusion fluid was continuously pumped overnight at a flow rate of 0.5 ml/min. On the day of the experiment flow rate was switched to 2.5 ml/min. After a stabilization period of 2 h, baseline samples were collected for 1 h. The CRF receptor antagonist ahCRF was dissolved in aCSF and 1 mg of ahCRF in 300 nl was infused bilaterally into the LC over a 2-min period using an infusion pump. Injectors were made from fused silica tubing (o.d. 0.175 mm, Polymicro Technologies, Phoenix, AZ) inserted into 28 gauge stainless steel tubing and projected 1.0 mm beyond the tips of guide cannulae. Injectors were left in place for an additional 1 min and then replaced with dummy inserts. Ten minutes after infusion animals were immobilized for 30 min by strapping them with VELCROt tape to a plastic board. Control animals (vehicle / IMB) received an infusion of aCSF. A second control
group of animals ( ahCRF alone) was not immobilized following infusion of ahCRF. The amount of ahCRF chosen was based on published data that a 10-fold excess of ahCRF is required to prevent the pituitary responses to CRF and that 100–200 ng of the peptide injected into the LC was effective in blocking the shock-induced freezing behavior [20]. After completion of procedures rats were overdosed with sodium pentobarbital (120 mg/kg) and perfused intracardially through the left ventricle with 0.9% saline followed by 4% formalin. Brains were removed, stored at least 48 h in 4% formalin, and coronal sections, cut at 50 mm, were stained with thionin. Placement of the injector tips in the brain stem were determined with reference to the stereotaxic atlas of Paxinos and Watson [14]. Only the results from animals in which the injection sites were within the LC were included in data analysis. Data Analysis Values of NE concentration in six baseline samples were averaged and then all data were expressed as percent of baseline. Statistical analyses were performed using two-way analysis of variance (ANOVA). Dunnett’s multiple range test was used for post hoc analysis. A p-value of less than 0.05 was considered to indicate statistical significance. RESULTS Histological analysis indicated that the injector tips were located within the LC in six rats that received infusions of ahCRF without immobilization, in seven rats that received infusion of ahCRF and immobilized, and in six infused with aCSF and immobilized. A typical example of histological verification of cannulae tip placement is shown in Fig. 1. Changes in NE concentrations in microdialysates collected from the PFM before, during and after immobilization of animals are shown in Fig. 2. The basal level of NE was 1.03 { 0.06, 1.07 { 0.11, and 0.9 { 0.14 ng/ml of dialysate for ahCRF, vehicle / IMB, and ahCRF/ IMB groups, respectively, and was stable before immobilization. Immobilization of animals significantly increased the concentration of NE in microdialysates collected from the PFM reaching a maximum of 170.8 { 12.8% of baseline
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FIG. 2. The effect of immobilization stress and infusion of ahCRF/aCSF into the LC on concentration of NE in microdialysates collected from the medial prefrontal cortex. Injection of ahCRF/aCSF was made 10 min before immobilization (indicated by an arrow). The duration of immobilization (IMB) is indicated by the horizontal bar. *Significant difference from the basal level (Dunnett’s multiple range test, p õ 0.05), /difference from corresponding values of the control group (veh / IMB, Dunnett’s t-test). Two-way ANOVA indicated significant difference between the groups ( p õ 0.001).
by 10 min after onset of immobilization. The concentration of NE remained elevated during 30 min of stress and was 151.8 { 9.6% of baseline at the end of immobilization. Analysis of variance indicated a significant effect of time, F (19, 135) Å 2.7, p Å 0.002, on NE concentration. Post hoc determination (Dunnett’s test) of differences over the baseline level indicated that the immobilization stress-induced increase in NE concentrations in microdialysates was significant for the first 50 min of immobilization. Infusion of ahCRF significantly attenuated the immobilization stress-induced NE release in the PFM. A two-way ANOVA with repeated measures indicated a significant difference between groups, F(2, 364) Å 30.06, p õ 0.001. Dunnet’s was used to compare corresponding values of the vehicle / IMB group and ahCRF / IMB group and revealed significant differences at 10-, 20-, 30-, and 50-min time points (Fig. 2.). Infusion of ahCRF alone (no immobilization) did not significantly change NE concentrations from baseline level during the 130-min period of samples collection. Because the HPLC system was optimized for detection of NE, other catecholamines and metabolites were not reliably detected in microdialysis samples and data not shown. DISCUSSION In vivo microdialysis studies permit measurements of extracellular concentrations of neurotransmitters, and reflect the ‘‘overflow’’ of released neurotransmitters. Although such extracellular concentrations of NE are an indirect estimate of release, several studies with drugs that inhibit action potentials (e.g., tetrodotoxin) have demonstrated that dialysate concentrations of catecholamines generally reflect synaptic release [2,6]. Changes in extracellular concentrations are likely to reflect changes in synaptic release.
A number of studies have demonstrated that CRF, the hypothalamic hormone, may also serve as neurotransmitter in the LC and may mediate various aspects of the stress response. There are CRF-containing nerve terminals and CRF binding sites in the LC [19,26], and there is ultrastructural evidence that axon terminals containing CRF directly contact catecholamine-containing dendrites in the LC [25]. Local infusion of CRF into the LC increases discharge rate of neurons [22] and increases norepinephrine release in PFM, a region whose main source of NE derives from LC [17]. Behavioral results also suggest a role of CRF in stress-induced behavioral changes. CRF administered into the LC induced defensive withdrawal in rats and was more potent than injected ICV [5]. The rationale to perform bilateral infusions of CRF antagonist in our experiments appears from the observed laterality of the noradrenergic response to CRF injection into the LC [17]. The existing data suggest that unilateral stimulation of the LC is necessary to produce an activation, but bilateral infusions are likely to be necessary to block the response. In our microdialysis study [17] we found that there was an apparent increase in dialysate NE on the contralateral side (although it was not statistically significant). This could have occurred because of nonspecific activation of the two loci. If both loci coertulei were activated, then bilateral blockage or lesion would be necessary to prevent the activating effect. It has been reported that ahCRF injected into the LC bilaterally attenuated shock-induced freezing [20] and immobilization stress-induced defensive withdrawal [16]. Exposure to stressful stimuli activates release of NE in the cortex [1,3] and immobilization stress elevates the concentration of CRF-like immunoreactivity in the LC [7]. Microdialysis experiments showed that infusion of ahCRF ICV attenuated immobilization stress-induced NE release in PFM [15]. Addition-
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ally, activation of LC neurons by hypotensive stress can be prevented by microinfusion of ahCRF into the LC [8,23]. The present study demonstrated that immobilization stress caused a significant increase in NE concentrations in microdialysates collected from PFM, and that the release was significantly attenuated by infusion of CRF antagonist into the region of the LC. It is important to note that in our experiments we observe total elimination of stress-induced increase of NE concentrations in microdialysates collected from PFM, while other authors reported just attenuation [17]. In our experiments we were using 30-min immobilization stress, and bilateral infusion of a large dose of ahCRF bilaterally into the region of the LC, comparing to 1-h immobilization stress and ICV infusion of ahCRF as reported by Shimizu et al. [15] . All these factors may explain the difference in the results. These results suggest that immobilization stress-induced activation of CRF receptors in the LC leads to the release of NE in the PFM and the increased concentrations of NE in microdialysates collected from the PFM. It has been suggested that CRF may affect LC neuronal activity at several sites: CRF could directly affect LC discharge via synaptic contacts; indirectly affect LC discharge via presynaptic actions on terminals; modulate the effect of other neurotransmitters with which it is colocalized [25]. Whether or not CRF is mediating the noradrenergic response may depend on the form of stress. Valentino et al. [24] has shown that the local infusion of ahCRF into LC can prevent activation of LC neurons in response to sodium nitroprusside infusion, but this did not occur when LC neurons were activated by sciatic nerve stimulation. In summary, our results suggest that local infusion of ahCRF in the region of the brain stem LC attenuates the immobilization stress-induced synaptic release of NE in the PFM. This is consistent with anatomical and physiological data that show that stress can facilitate NE release in the PFM through the activation of the CRF system in the brain. ACKNOWLEDGEMENTS
We thank Dr. I. I. Rybkin for the help with histology. This research was supported by the U.S. Army Research and Development Command Grant DAMD 17-92-V-2009. Opinions, interpretations, conclusions are those of the authors and are not necessarily endorsed by U.S. Army.
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