European Journal of Pharmacology. 176 (1990) 229- 231 Elsevier
229
EJP 20558 Short communication
Stress increases noradrenaline release in the rat frontal cortex: prevention by di~epam Zvani L. Rossetti, Chiara Portas, Luca Pani, Susanna Carboni and Gian Luigi Gessa "B.B. Brodie' Department of Neuroscience, University of Cagliari, 09124 Cagliari, Italy
Received 1 December 1989, accepted 12 December 1989
Foot-shock produced a more than 2-fold increase in noradrenaline (NA) release from the frontal cortex of freely moving rats. The effect of acute stress was almost completely prevented by the administration of diazepam (5 mg/kg i.p.). Diazepam alone inhibited cortical NA release, the maximal inhibition (-57%) being observed 90 min after the injection. Cortical NA release therefore appears to be a reliable index of central noradrenergic activity in response to stressful conditions. Microdialysis; Noradrenaline; Foot-shock; Diazepam
1. Introduction
The frontal cortex is considered to play an important role in the mediation of arousal and stress (Glavin, 1985). After acute stress, noradrenaline (NA) neurons are activated and NA turnover is increased in this area (Korf et at., 1973). A major innervation to the frontal cortex is provided by the dense noradrenergic projections originating in the pontine nucleus locu coeruleus (LC). This nucleus has long been considered to be involved in the mediation of vigilance, anxiety and stress (Foote al., 1983). Indeed, stress increases the spontaneous electrical activity of LC neurons (Abercrombie and Jacobs, 1987), whereas, conversely, drugs known to possess anxiolytic and sedative effects inhibit LC neuronal activity (Grant and Redmond, 1980) and NA turnover in noradrenergic terminal areas (Ida et al., 1985). Recently, Abercrombie et al. (1988) have shown that stress stimulates NA release from the hippo-
Correspondence to: Z.L. Rossetti, 'B.B. Brodie' Department of Neuroscience. Via Porcell 4, 09124 Cagliari, Italy.
campus; however, the effect of acute stress on NA release in the rat frontal cortex is not known. Using brain microdialysis in freely moving animals, we provide evidence that foot-shock increases the release of NA from the rat frontal cortex, and show that this effect is prevented by diazepam, a classical anxiolytic drug.
2. Materials and methods 2.1. Dialysis and analytical procedures
Brain microdialysis was performed in freely moving male Sprague-Dawley CD rats (220-250 g; Charles River, Italy) essentially as described earlier (Rossetti et al., 1989). Acrylonitrile-sodium methallyl sulphonate fibers were used because of their high recovery in vitro (34% vs. 1 /.tM N A solution at 37°C). Dialysis membranes (220 ~m o.d., 40 kDa MW cut-off, Filtral 12, Hospal, France) were implanted transversely through the frontal cortex (A: +2.2; V: - 2 . 0 from bregma) 24 h before the experiment. The correct location of the dialysis membrane in the brain was verified
0014-2999/90/$03.50 ':'~1990 Elsevier Science Publishers B.V. (Biomedical Division)
230 by a n a t o m i c a l e x a m i n a t i o n after the a n i m a l s had been killed. T h e perfusates (50 t~l/30 min) were applied directly to the H P L C - E C e q u i p m e n t , using a c o m p u t e r - c o n t r o l l e d , fully a u t o m a t e d system which avoided m a n i p u l a t i o n of the animals. The mobile phase for the H P L C was c i t r a t e / a c e t a t e buffer 50 m M , p H 4.8, c o n t a i n i n g sodium octyl sulphate 70 m g / l , E D T A 1 m M a n d m e t h a n o l 3.5% v/v. T h e c o l u m n was 25 cm long a n d contained octadecyl silica, 5 ffm particle size (Supelco); the flow rate was 0.9 m l / m i n (Waters 510 pump). The potential applied was 0.7 V with respect to a n A g / A g C I reference electrode (Waters M o d 460 detector). A n integrator (Waters 710 D a t a Module) was used to record the signal. A stable baseline of N A release was observed within 120 m i n after the start of the perfusion a n d was m a i n t a i n e d for at least 6 h. Extracellular N A c o n c e n t r a t i o n s at baseline, arbitrarily a s s u m i n g equal diffusion kinetics in vivo a n d in vitro, corres p o n d e d to 11,6 4-_ 2.3 n M ( m e a n _+ S.E.M., n = 17). Statistical significance was evaluated with an A N O V A followed by S t u d e n t ' s t-test where appropriate.
2.2. Stressful stimulation After a stable baseline for the release of the a m i n e had been obtained, the a n i m a l s received saline or diazepam, 5 m g / k g i.p. (Valium, Roche), a n d 90 m i n later were exposed for 30 m i n to a series of electrical foot-shocks delivered via a brass grid placed on the cage floor. The a n i m a l s received c u r r e n t pulses of 2 m A intensity for 200 ms every 1 s.
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h Fig. I. Effect of electrical foot-shock on cortical NA release in the conscious, freely moving rats perfused transcortically by means of a dialysis fiber. The rats received saline (m) or diazepam, 5 mg/kg i.p, (e), and were exposed to foot-shock for 30 rain (hatched bar), or received diazepam alone (©). The arrow indicates the time of injection. Each point represents the average NA concentration in 30-min samples (mean_+S.E.M.) from four to six animals and is expressed as a percentage of the basal release. The baseline NA output was 6.6 fmol/min _+2.1, n = 17). * P < 0.01 vs. baseline levels. * * P < 0.05 with respect to the corresponding time point for the treated animals not exposed to foot-shock.
azepam (figs. 1 a n d 2). D i a z e p a m a d m i n i s t r a t i o n reduced the a m o u n t of N A released d u r i n g stimulation a n d 30 m i n thereafter to 23 +_ 3% of control, the values b e i n g 390 +__42 a n d 90 + 12 fmol ( m e a n _+ S.E.M.) in the saline- a n d d i a z e p a m - t r e a t e d rats, respectively (fig. 2). D i a z e p a m alone p r o d u c e d a 500-~o 400 .300--
3. Results A significant increase in cortical N A efflux was observed in response to foot-shock (fig. 1). Maximal increases in N A release were observed in the first 30-min period a n d reached a peak of 221 425.4% ( m e a n ± S.E.M., n = 5) of baseline. The N A levels progressively declined when the stress was terminated, returing to levels that were lower t h a n baseline. Stress had little effect o n the cortical N A outflow when the rats were pretreated with di-
~ < z
200.100.-
i
,
I
0 I
®
Fig. 2. Prevention by diazepam (hatched bar) of stress-induced NA release. The ordinates represent the amount of NA released over baseline as a consequence of foot-shock during 30 rain of electrical stimulation and 30 min thereafter, and are expressed as fmol/h. *P < 0.001 vs. saline-treated animals.
231 significant decrease in N A release, the m a x i m a l i n h i b i t i o n (56.5 _+ 14.8% of baseline) being observed 90 m i n after the a d m i n i s t r a t i o n of the drug. The i n h i b i t i o n of N A efflux lasted for more than 3 h (fig. 1).
Acknowledgements The generous supply of dialysis membranes from Hospal SpA, Bologna, Italy, is gratefully acknowledged. We thank Mr. Antonio Boi for this excellent technical assistance. This work was supported by CNR Grant No. 88.00626.04.
4. Discussion References The f i n d i n g that stress increases N A efflux in the frontal cortex m a y support the view that LC n e u r o n a l activity is increased by stressful stimuli (Foote et al., 1983). This view is consistent with the results of recent microdialysis studies (Aberc r o m b i e et al., 1988) showing that N A release from the n o r a d r e n e r g i c h i p p o c a m p a l projections, which originate in the LC, is also increased by repeated electrical shock. The almost complete p r e v e n t i o n by d i a z e p a m of stress-induced cortical N A release m a y be considered to be the result of the i n h i b i t i o n of LC n e u r o n a l activity ( G r a n t and R e d m o n d , 1980) a n d of N A t u r n o v e r (Ida et al., 1985) by benzodiazepines, thus giving further support to the hypothesis of the i n v o l v e m e n t of LC in the m e d i a t i o n of anxiety and fear. Our findings provide the first biochemical evidence in vivo for an i n h i b i t o r y action of b e n z o d i a z e p i n e s on cortical N A release a n d suggest that N A release, as studied by microdialysis, might represent a method for s t u d y i n g the effects of drugs with anxiolytic a n d sedative properties. Moreover, our results indicate that N A release might be a reliable index of central n o r a d r e n e r g i c activity in response to stressful conditions.
Abercrombie, E.D. and B.L. Jacobs, 1987. Single-unit response of noradrenergic neurons in the locus coeruleus of freelymoving cats. Acutely presented stressful and nonstressful stimuli, J. Neurosci. 7, 2837. Abercrombie, E.D., R.W. Keller, and M.J. Zigmond, 1988. Characterization of hippocampal norepinephrine release as measured by microdialysis perfusion: pharmacological and behavioural studies, Neuroscience 27, 897. Foote, S.L., F.E. Bloom and G. Aston-Jones, 1983, Nucleus locus coeruleus: new evidence of anatomical and physiological specificity, Physiol. Rev. 63, 844. Glavin, G.B., 1985, Stress and brain noradrenaline. A review, Behav. Neurosci. Rev. 9, 233. Grant, S.J. and D.E. Redmond, Jr., 1980, Benzodiazepines attenuate single unit activity in the locus coeruleus, Life Sci. 27, 223l. Ida, Y., M. Tanaka, A. Tsuda, S. Tsujimaru and N. Nagasaki, 1985, Attenuating effects of diazepam on stress-induced increase in noradrenaline turnover in specific brain regions of rats: Antagonism by Ro 15-1788, Life Sci. 37, 2491. Korf, J., G.K. Aghajanian and R.H. Roth, 1973. Increased turnover of norepinephrine in the rat cerebral cortex during stress: role of the locus coeruleus, Neuropharmacology 12, 933. Rossetti, Z.L., L. Pani, C. Portas and G.L. Gessa, 1989, Brain dialysis provides evidence for D 2 dopamine receptors modulating noradrenaline release in the rat frontal cortex, European J. Pharmacol. 163. 393.
229
EJP 20558 Short communication
Stress increases noradrenaline release in the rat frontal cortex: prevention by di~epam Zvani L. Rossetti, Chiara Portas, Luca Pani, Susanna Carboni and Gian Luigi Gessa "B.B. Brodie' Department of Neuroscience, University of Cagliari, 09124 Cagliari, Italy
Received 1 December 1989, accepted 12 December 1989
Foot-shock produced a more than 2-fold increase in noradrenaline (NA) release from the frontal cortex of freely moving rats. The effect of acute stress was almost completely prevented by the administration of diazepam (5 mg/kg i.p.). Diazepam alone inhibited cortical NA release, the maximal inhibition (-57%) being observed 90 min after the injection. Cortical NA release therefore appears to be a reliable index of central noradrenergic activity in response to stressful conditions. Microdialysis; Noradrenaline; Foot-shock; Diazepam
1. Introduction
The frontal cortex is considered to play an important role in the mediation of arousal and stress (Glavin, 1985). After acute stress, noradrenaline (NA) neurons are activated and NA turnover is increased in this area (Korf et at., 1973). A major innervation to the frontal cortex is provided by the dense noradrenergic projections originating in the pontine nucleus locu coeruleus (LC). This nucleus has long been considered to be involved in the mediation of vigilance, anxiety and stress (Foote al., 1983). Indeed, stress increases the spontaneous electrical activity of LC neurons (Abercrombie and Jacobs, 1987), whereas, conversely, drugs known to possess anxiolytic and sedative effects inhibit LC neuronal activity (Grant and Redmond, 1980) and NA turnover in noradrenergic terminal areas (Ida et al., 1985). Recently, Abercrombie et al. (1988) have shown that stress stimulates NA release from the hippo-
Correspondence to: Z.L. Rossetti, 'B.B. Brodie' Department of Neuroscience. Via Porcell 4, 09124 Cagliari, Italy.
campus; however, the effect of acute stress on NA release in the rat frontal cortex is not known. Using brain microdialysis in freely moving animals, we provide evidence that foot-shock increases the release of NA from the rat frontal cortex, and show that this effect is prevented by diazepam, a classical anxiolytic drug.
2. Materials and methods 2.1. Dialysis and analytical procedures
Brain microdialysis was performed in freely moving male Sprague-Dawley CD rats (220-250 g; Charles River, Italy) essentially as described earlier (Rossetti et al., 1989). Acrylonitrile-sodium methallyl sulphonate fibers were used because of their high recovery in vitro (34% vs. 1 /.tM N A solution at 37°C). Dialysis membranes (220 ~m o.d., 40 kDa MW cut-off, Filtral 12, Hospal, France) were implanted transversely through the frontal cortex (A: +2.2; V: - 2 . 0 from bregma) 24 h before the experiment. The correct location of the dialysis membrane in the brain was verified
0014-2999/90/$03.50 ':'~1990 Elsevier Science Publishers B.V. (Biomedical Division)
230 by a n a t o m i c a l e x a m i n a t i o n after the a n i m a l s had been killed. T h e perfusates (50 t~l/30 min) were applied directly to the H P L C - E C e q u i p m e n t , using a c o m p u t e r - c o n t r o l l e d , fully a u t o m a t e d system which avoided m a n i p u l a t i o n of the animals. The mobile phase for the H P L C was c i t r a t e / a c e t a t e buffer 50 m M , p H 4.8, c o n t a i n i n g sodium octyl sulphate 70 m g / l , E D T A 1 m M a n d m e t h a n o l 3.5% v/v. T h e c o l u m n was 25 cm long a n d contained octadecyl silica, 5 ffm particle size (Supelco); the flow rate was 0.9 m l / m i n (Waters 510 pump). The potential applied was 0.7 V with respect to a n A g / A g C I reference electrode (Waters M o d 460 detector). A n integrator (Waters 710 D a t a Module) was used to record the signal. A stable baseline of N A release was observed within 120 m i n after the start of the perfusion a n d was m a i n t a i n e d for at least 6 h. Extracellular N A c o n c e n t r a t i o n s at baseline, arbitrarily a s s u m i n g equal diffusion kinetics in vivo a n d in vitro, corres p o n d e d to 11,6 4-_ 2.3 n M ( m e a n _+ S.E.M., n = 17). Statistical significance was evaluated with an A N O V A followed by S t u d e n t ' s t-test where appropriate.
2.2. Stressful stimulation After a stable baseline for the release of the a m i n e had been obtained, the a n i m a l s received saline or diazepam, 5 m g / k g i.p. (Valium, Roche), a n d 90 m i n later were exposed for 30 m i n to a series of electrical foot-shocks delivered via a brass grid placed on the cage floor. The a n i m a l s received c u r r e n t pulses of 2 m A intensity for 200 ms every 1 s.
.._..250
*
(,9
2oo
~
150
7
50
tT..... I:......
o
:
0
,,
1
i
2
~
;ii~ii~:.
,,
.3fs
4
..
5
h Fig. I. Effect of electrical foot-shock on cortical NA release in the conscious, freely moving rats perfused transcortically by means of a dialysis fiber. The rats received saline (m) or diazepam, 5 mg/kg i.p, (e), and were exposed to foot-shock for 30 rain (hatched bar), or received diazepam alone (©). The arrow indicates the time of injection. Each point represents the average NA concentration in 30-min samples (mean_+S.E.M.) from four to six animals and is expressed as a percentage of the basal release. The baseline NA output was 6.6 fmol/min _+2.1, n = 17). * P < 0.01 vs. baseline levels. * * P < 0.05 with respect to the corresponding time point for the treated animals not exposed to foot-shock.
azepam (figs. 1 a n d 2). D i a z e p a m a d m i n i s t r a t i o n reduced the a m o u n t of N A released d u r i n g stimulation a n d 30 m i n thereafter to 23 +_ 3% of control, the values b e i n g 390 +__42 a n d 90 + 12 fmol ( m e a n _+ S.E.M.) in the saline- a n d d i a z e p a m - t r e a t e d rats, respectively (fig. 2). D i a z e p a m alone p r o d u c e d a 500-~o 400 .300--
3. Results A significant increase in cortical N A efflux was observed in response to foot-shock (fig. 1). Maximal increases in N A release were observed in the first 30-min period a n d reached a peak of 221 425.4% ( m e a n ± S.E.M., n = 5) of baseline. The N A levels progressively declined when the stress was terminated, returing to levels that were lower t h a n baseline. Stress had little effect o n the cortical N A outflow when the rats were pretreated with di-
~ < z
200.100.-
i
,
I
0 I
®
Fig. 2. Prevention by diazepam (hatched bar) of stress-induced NA release. The ordinates represent the amount of NA released over baseline as a consequence of foot-shock during 30 rain of electrical stimulation and 30 min thereafter, and are expressed as fmol/h. *P < 0.001 vs. saline-treated animals.
231 significant decrease in N A release, the m a x i m a l i n h i b i t i o n (56.5 _+ 14.8% of baseline) being observed 90 m i n after the a d m i n i s t r a t i o n of the drug. The i n h i b i t i o n of N A efflux lasted for more than 3 h (fig. 1).
Acknowledgements The generous supply of dialysis membranes from Hospal SpA, Bologna, Italy, is gratefully acknowledged. We thank Mr. Antonio Boi for this excellent technical assistance. This work was supported by CNR Grant No. 88.00626.04.
4. Discussion References The f i n d i n g that stress increases N A efflux in the frontal cortex m a y support the view that LC n e u r o n a l activity is increased by stressful stimuli (Foote et al., 1983). This view is consistent with the results of recent microdialysis studies (Aberc r o m b i e et al., 1988) showing that N A release from the n o r a d r e n e r g i c h i p p o c a m p a l projections, which originate in the LC, is also increased by repeated electrical shock. The almost complete p r e v e n t i o n by d i a z e p a m of stress-induced cortical N A release m a y be considered to be the result of the i n h i b i t i o n of LC n e u r o n a l activity ( G r a n t and R e d m o n d , 1980) a n d of N A t u r n o v e r (Ida et al., 1985) by benzodiazepines, thus giving further support to the hypothesis of the i n v o l v e m e n t of LC in the m e d i a t i o n of anxiety and fear. Our findings provide the first biochemical evidence in vivo for an i n h i b i t o r y action of b e n z o d i a z e p i n e s on cortical N A release a n d suggest that N A release, as studied by microdialysis, might represent a method for s t u d y i n g the effects of drugs with anxiolytic a n d sedative properties. Moreover, our results indicate that N A release might be a reliable index of central n o r a d r e n e r g i c activity in response to stressful conditions.
Abercrombie, E.D. and B.L. Jacobs, 1987. Single-unit response of noradrenergic neurons in the locus coeruleus of freelymoving cats. Acutely presented stressful and nonstressful stimuli, J. Neurosci. 7, 2837. Abercrombie, E.D., R.W. Keller, and M.J. Zigmond, 1988. Characterization of hippocampal norepinephrine release as measured by microdialysis perfusion: pharmacological and behavioural studies, Neuroscience 27, 897. Foote, S.L., F.E. Bloom and G. Aston-Jones, 1983, Nucleus locus coeruleus: new evidence of anatomical and physiological specificity, Physiol. Rev. 63, 844. Glavin, G.B., 1985, Stress and brain noradrenaline. A review, Behav. Neurosci. Rev. 9, 233. Grant, S.J. and D.E. Redmond, Jr., 1980, Benzodiazepines attenuate single unit activity in the locus coeruleus, Life Sci. 27, 223l. Ida, Y., M. Tanaka, A. Tsuda, S. Tsujimaru and N. Nagasaki, 1985, Attenuating effects of diazepam on stress-induced increase in noradrenaline turnover in specific brain regions of rats: Antagonism by Ro 15-1788, Life Sci. 37, 2491. Korf, J., G.K. Aghajanian and R.H. Roth, 1973. Increased turnover of norepinephrine in the rat cerebral cortex during stress: role of the locus coeruleus, Neuropharmacology 12, 933. Rossetti, Z.L., L. Pani, C. Portas and G.L. Gessa, 1989, Brain dialysis provides evidence for D 2 dopamine receptors modulating noradrenaline release in the rat frontal cortex, European J. Pharmacol. 163. 393.
