Inescapable stress enhances extracellular acetylcholine in the rat hippocampus and prefrontal cortex but not the nucleus accumbens or amygdala

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Neuroscience Vol. 74, No. 3, pp. 767–774, 1996 Copyright ? 1996 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/96 $15.00+0.00 S0306-4522(96)00211-4

INESCAPABLE STRESS ENHANCES EXTRACELLULAR ACETYLCHOLINE IN THE RAT HIPPOCAMPUS AND PREFRONTAL CORTEX BUT NOT THE NUCLEUS ACCUMBENS OR AMYGDALA G. P. MARK,* P. V. RADA† and T. J. SHORS Department of Psychology, Princeton University, Princeton, NJ 08544-1010, U.S.A. Abstract––A number of experimental results has pointed to a cholinergic involvement in the stress response. Recently, analytical techniques have become available to measure acetylcholine release in vivo during exposure to various stressors. In these experiments, microdialysis was used to monitor acetylcholine output every 15 min in the dorsal hippocampus, amygdala, nucleus accumbens and prefrontal cortex before, during and after 1 h of restraint, including a 15-min session of intermittent tail-shock (1/min, 1 mA, 1-s duration) in rats. In response to the stressful event, acetylcholine release was significantly increased in the prefrontal cortex (186%; P < 0.01) and hippocampus (168%; P < 0.01) but not in the amygdala or nucleus accumbens. The sole effects observed in the amygdala and nucleus accumbens occurred upon release from the restrainer, at which point acetylcholine levels were significantly elevated in both areas (amygdala: 150%; P < 0.05; nucleus accumbens: 130%; P < 0.05). An enhanced acetylcholine release was also evident during this sample period in the hippocampus and prefrontal cortex. These data demonstrate an enhancement of cholinergic activity in response to stress in two acetylcholine projection systems (hippocampus and prefrontal cortex) but not in the intrinsic acetylcholine system of the nucleus accumbens or the extrinsic innervation of the amygdala. Moreover, the data showed that relief from stress was accompanied by a more ubiquitous acetylcholine response that extended to each site tested. Copyright ? 1996 IBRO. Published by Elsevier Science Ltd. Key words: restraint stress, tail-shock, acetylcholine release, microdialysis.

Stressful stimuli evoke a battery of central and peripheral neural reactions which are designed ostensibly to promote escape from or mitigate the deleterious effects of the stressor. Of these responses the involvement of the cholinergic system has received considerable attention since this transmitter system is thought to subserve a variety of functions which may be affected by stress, including learning and memory retrieval,3,4,59,66 locomotion11,18,43,54 and cortical activation.61,65 Several investigators have reported that the cholinergic pathways innervating the hypothalamus,16,26 ventral lateral medulla37 and the hippocampus15,23,46 are activated by exposure to stressors. With respect to the hippocampus, acetylcholine (ACh) activity in the dorsal hippocampus has been reported to be more responsive than in ventral regions.7,15 Despite differences in the relative amount of ACh released and the distinct anatomy of these regions, it appears that virtually every cholin*To whom correspondence should be addressed. Present address: Department of Behavioral Neuroscience, L470, Oregon Health Sciences University, School of Medicine, Portland, OR 97201, U.S.A. †Present address: Laboratorio de Fisiologia, Universidad de los Andes, Merida, Venezuela. Abbreviations: ACh, acetylcholine; DA, dopamine; NAc, nucleus accumbens; PFC, prefrontal cortex.

ergic system examined has revealed an excitatory reaction to stressful stimuli. One exception appears to be the ACh-containing interneurons of the caudate nucleus, which have been reported to be unresponsive to 120 min of restraint.29 In addition, several brain regions with prominent cholinergic innervation [namely, the amygdala, prefrontal cortex (PFC) and nucleus accumbens (NAc)] have not been studied. The preponderance of earlier studies evaluated the cholinergic response to stress based on poststress measurements of ACh tissue levels,15,16 receptors42,62 or ACh released from synaptosomes.19,22,23 While these in vitro or ex vivo techniques afford valuable insights into which systems respond to these stimuli, a more powerful method involves in vivo measurements of ACh release that are coincident with exposure to various stressors. In recent years microdialysis has afforded this opportunity to evaluate the effects of stressors on ACh release within discrete brain regions. Using this method, several groups have examined the relationship between stress and ACh output in the hippocampus,29,30,45,46 neostriatum29 and frontal cortex.51 In these experiments we have extended these findings to include the measurement of ACh release in several limbic forebrain regions before, during and after a period of restraint alone followed by restraint coupled with mild, intermittent tail-shock.

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G. P. Mark et al. EXPERIMENTAL PROCEDURES

Surgery and microdialysis procedure Twenty-five adult male Sprague–Dawley rats (obtained from the Department of Psychology, Princeton University vivarium) weighing 300–350 g were housed individually following surgery on a 15–9-h light–dark schedule (lights on 07.00–22.00) with food and water available ad libitum. For surgery, subjects were anesthetized with pentobarbital (20 mg/kg, i.p.) supplemented by ketamine (40 mg/kg, i.p.). Each animal was stereotaxically implanted with stainless steel guide shafts (21 ga.) at two of the following coordinates: posterior medial NAc (A 10.2 mm, L 1.2 mm, V 4.0 mm) and contralateral medial prefrontal cortex (A 12.2 mm, L 0.5 mm, V 2.0 mm); or dorsal hippocampus (A 5.2 mm, L 3 mm, V 1 mm) and amygdala (A 4 mm, L 5 mm, V 4 mm).49 Anterior, lateral and ventral coordinates were referenced to the interaural line, midsaggital sinus, and the surface of the level skull, respectively. Guide shafts were kept patent with 26-ga. stylets. After a one-week recovery period, microdialysis probes were inserted which extended 5 mm beyond the guide shafts, except for the probes placed in the dorsal hippocampus which extended 3 mm. Probes were constructed with silica glass tubing (37 ìm i.d.; Polymicro Tech.) inside a 26-ga. stainless steel tube with a microdialysis tip of cellulose tubing (0.2 mm o.d.; Spectrum Med. Co.) sealed at the end with epoxy cement. Tip lengths were 2 mm for NAc and hippocampus, 1.5 mm for amygdala and 3 mm for mPFC. The microdialysis fiber had a 6000 mol. wt cut-off. Detailed descriptions of this probe design are published elsewhere.27,40 Probes were perfused with a buffered Ringer solution (142 mM NaCl, 3.9 mM KCl, 1.2 mM CaCl2, 1.0 mM MgCl2, 1.35 mM Na2HPO4, 0.3 mM NaH2PO4; pH 7.3) at a flow rate of 1.0 ìl/min. Neostigmine (0.5 ìM; Sigma Co.) was added to the perfusion fluid to improve basal recovery of ACh by hindering its enzymatic degradation. The outlet branch of the probe led to a 400-ìl vial clipped to a flexible cable 25 cm above the head of the rat. Probes were implanted at least 24 h before each experiment to allow neurotransmitter recovery to stabilize. Restraint procedure One day prior to testing, rats were placed in Plexiglas chambers with ad libitum access to food and water. The following day food and water were removed and dialysates were collected every 15 min until a stable baseline was established of less than 15% variability in four consecutive samples. Subjects were then placed in a plastic restraining device designed to prevent body movement without disturbing the dialysis probes or perfusion lines. Sampling continued for two 15-min intervals of restraint. To facilitate future comparison of these neurochemical data with electrophysiological and behavioral data being collected in parallel investigations, rats were then exposed to 15, 1-mA, 1-s duration tail-shocks delivered at a rate of 1/min. Following a further 15 min of restraint alone, subjects were released and samples were obtained for an additional 1.5 h. Acetylcholine assay ACh was measured by reverse-phase, high-performance liquid chromatography with electrochemical detection using a single piston pump and pulse dampener (SSI Co.), a 50-ìl sample loop and an amperometric detector (EG & G Princeton Applied Research Corp.). The mobile phase contained 200 mM potassium phosphate at pH 8.0. ACh and Ch were separated on an 8-cm C18 analytical column (Chrompack) and then converted sequentially to betaine and hydrogen peroxide by an immobilized enzyme reactor (Chrompack; with acetylcholinesterase and choline oxidase from Sigma Co.). The resultant hydrogen peroxide was oxidized on a platinum electrode (BAS) set at 0.5 V with

Table 1. Average, absolute basal levels of acetylcholine (not corrected for probe recovery) obtained in the hippocampus, prefrontal cortex, nucleus accumbens and amygdala Site (N) Hippocampus (6) PFC (6) NAc (6) Amygdala (6)

pmol/15 ìl

S.E.M.

Range

0.199 0.521 0.519 0.291

0.033 0.022 0.021 0.022

0.08–0.79 0.12–0.91 0.17–1.12 0.15–0.63

respect to a Ag–AgCl reference electrode (Princeton Applied Res. Co.). Preliminary tests to confirm the identity of the putative ACh peak obtained from dialysates and its neuronal origin have been reported previously.39 The detection limit of this system, defined as the amount of ACh needed to achieve a 3 : 1 signal to noise ratio, was 20 fmol. Statistical analyses and histology Absolute basal recovery of ACh varied considerably between subjects. For this reason, peak heights were converted to a percentage of the mean of four baseline samples. Data were analysed by one-way analysis of variance followed by post hoc Newman–Keuls tests when justified. Histology was performed to verify probe placement. Subjects received an overdose of sodium pentobarbital and were perfused with 0.9% saline solution followed by formalin. Brains were removed and frozen for sectioning. Sections, 40 ìm thick, were taken from the anterior lobe caudally until probe tracks were identified. Data from any probe placement not entirely within a target nucleus were discarded.

RESULTS

Basal levels of acetylcholine Average absolute baseline levels of ACh obtained in dialysates from dorsal hippocampus, PFC and NAc and amygdala are listed in Table 1. Statistics were performed on the per cent of four baseline samples taken prior to stress exposure. An overall ANOVA with area and time as independent variables revealed a significant interaction between the areas tested and exposure to the stressors in terms of both absolute release (F36,240 = 4.20; P < 0.0001) and release relative to prestress baseline (F36,240 = 2.60; P < 0.0001). These results indicate that the effect of the stress depended on the area measured. The areas and the ways in which exposure to the stressor affected ACh release are presented sequentially. Effects of stress on acetylcholine release in the dorsal hippocampus The effect of restraint (stippled area) and intermittent tail-shock (hatched area) on ACh release in the hippocampus is illustrated in Fig. 1. Overall, exposure to the stressors was associated with a significant increase in ACh output (F12,60 = 14.5; P < 0.001). The release of ACh increased to 168% of baseline immediately after installing the animals in the restraining device, and then declined slightly to 149% of control in the next 15-min interval. The addition of

Stress and limbic ACh release

Fig. 1. Extracellular ACh in the dorsal hippocampus (n = 6) was increased to 168% of control levels in the first 15-min interval of restraint stress (stippled area). Thereafter, ACh release seemed to decline, becoming non-significant at the 1-h sample period. A 15-min interval of intermittent tailshock (hatched area) appeared to interrupt this decline but did not elevate ACh release above that observed during the first exposure to restraint. Finally, a marked increase in ACh output (193%) occurred in the 15-min interval after release from restraint. *P < 0.01 compared to baseline in post hoc analysis.

1 mA, 1/min tail-shock did not substantially alter ACh output, although exposure to this stressor may have offset a decline in ACh release in response to restraint. All samples taken during stress exposure were elevated significantly above baseline (P < 0.05). The level of ACh release returned to baseline within 1 h of release from the restraining apparatus. As with all areas tested, the most pronounced increase in release observed in this region of the hippocampus occurred in the 15-min interval that followed exposure to the stressors (193% above control). Effects of stress on acetylcholine release in the amygdala Exposure to the experimental conditions enhanced ACh release in the amygdala (F12,60 = 3.03; P < 0.01); (Fig. 2). However, post hoc analysis revealed that the only time-point in which the release of ACh was significantly enhanced relative to baseline levels was during the sample taken immediately after release from the restraining apparatus (150% of basal levels; P < 0.05). Thus, exposure to the stressor itself did not result in a statistically significant elevation of ACh levels, even though the means for these samples were consistently above baseline. Effects of stress on acetylcholine release in the prefrontal cortex The most robust increase in ACh release in response to the stress procedure occurred in the prefrontal cortex (F12,60 = 12.53; P < 0.001); (Fig. 3). Each of the manipulations resulted in a significant increase with respect to baseline. Thus, samples obtained during restraint alone (P < 0.05), shock and restraint (P < 0.05), and release from restraint

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Fig. 2. Exposure to restraint stress (stippled area) was accompanied by a transient, but non-significant increase (127%) in extracellular ACh in the amygdala (n = 6). This effect dissipated immediately and a second, non-significant increase (144%) occurred during 15 min of intermittent tail-shock (hatched area). Release from restraint was accompanied by a statistically reliable 150% increase in ACh output. *P < 0.05 compared to baseline in post hoc analysis.

Fig. 3. Extracellular ACh in the PFC (n = 6) increased substantially during exposure to restraint (stippled area) and a 15-min interval of tail-shock (hatched area). A maximal increase of 90% above prestress levels occurred in the 15-min sample immediately after relief from stress. * P < 0.01 compared to baseline in post hoc analysis.

(P < 0.05) were all elevated above prestress levels. The highest relative change from baseline also occurred in this area. ACh levels were comparable to the hippocampus during the restraint alone (161% of basal levels vs 168% in hippocampus). The addition of tail-shock caused an additional 25% increase (to 186% of prestress levels), although this was not a statistically significant enhancement compared to restraint alone. As with the other areas, the maximal level of ACh release (190%) was obtained during the 15-min interval that followed release from restraint. Thereafter ACh levels dropped substantially but remained slightly, albeit non-significantly, above prestress values (115–139%). Effects of stress on acetylcholine release in the nucleus accumbens As indicated for the amygdala, there was a significant impact of the experimental conditions on ACh release in the NAc (F12,60 = 3.03; P < 0.01).

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Fig. 4. Extracellular ACh in the NAc (n = 6) was unaffected by restraint (stippled area) or a 15-min interval of tailshock (hatched area) but there was a significant increase of 130% in the sample after release from restraint. *P < 0.05 compared to baseline in post hoc analysis.

However, upon post hoc analysis, it was determined that the only significant effect occurred upon release from restraint (130% of control levels; P < 0.05). Figure 4 illustrates these effects of restraint/shock on ACh release in the NAc. DISCUSSION

These results demonstrate that exposure to inescapable stressors is associated with an enhancement of ACh release in the dorsal hippocampus and prefrontal cortex, but not in the nucleus accumbens or amygdala. Relief from restraint, however, was associated with a more ubiquitous potentiation of ACh output such that, in addition to enhanced ACh release in the hippocampus and PFC, release in the NAc and amygdala was significantly elevated in the sample period which followed restraint stress. Increases in the activity of basal forebrain cholinergic cells (especially those innervating cortex) have been suggested to be associated with heightened arousal, and correlate with the behavioral state of the subject.31,65 The implication that such increases in cell firing result in augmented ACh release in frontal cortex is supported by our findings of substantially heightened ACh levels in PFC during and after exposure to stress. These results are consistent with those of previous studies demonstrating increases in cortical ACh release in vivo during various forms of behavioral arousal.11,30,32,51 ACh levels were also increased in the hippocampus during and immediately following restraint. The septohippocampal pathway consists of axons arising from somata in the medial septal nuclei coursing through the fimbria-fornix to innervate the hippocampus.2,17,53,60 This system is known to be responsive to a range of behaviorally relevant stimuli as well as general locomotor activity.11,13,14,43,46,63,65 Of particular interest here are the results from several experiments which provide evidence for an involvement in the stress response. These findings include stress-induced changes in hippocampal muscarinic

receptor binding,15,24,42,62 changes in the release of newly synthesized ACh from hippocampal synaptosomes after exposure to acute stress,19,23 and demonstrations of a cholinergic component in the behavioral responses of animals subjected to stress.36,55 For example, exposure to the stressor used in the present study is reported to enhance sensitization to external cues and facilitate associative learning.56,59 Interestingly, the sensitization but not the facilitated learning can be prevented by blocking muscarinic receptors during exposure to the stressor.58 It is also the case that the stressor used here enhances the occurrence of theta activity in the hippocampus,57 a rhythm which is dependent on cholinergic activation from the septum.5 These rhythms have been previously associated with movement20,64 but, owing to the restraint procedure employed, it is unlikely that the effects here are due to movement per se. Although it is widely known that ACh is excitatory to hippocampal pyramidal cells,34,44 the increase in ACh release was apparently not accompanied by an increase in cell excitability or synaptic efficacy in area CA1. A recent study using the same stress procedure employed here simultaneously recorded excitatory postsynaptic potentials in stratum radiatum. As reported here for the amygdala and nucleus accumbens, the only significant change in the synaptic response occurred upon release from restraint,57 where the response was depressed for several minutes. These results suggest that any increase in cell excitability that may occur in response to the release of ACh during the stressful event is either pre-empted or overcome by inhibitory influences in response to the stressor. In addition, they suggest that in contrast to the increase in ACh and theta activity during the stressor, these effects could be mediated via the increase in movement that occurred upon release from the restraining apparatus. Previous studies regarding the effects of various stressors on hippocampal ACh release in vivo have yielded results that are in general agreement with the microdialysis data reported here. Nilsson et al.46 demonstrated that handling stress was accompanied by a 94% increase in ACh release in the hippocampus, and this effect was nearly eliminated by severing the fimbria-fornix. Inglis and Fibiger30 confirmed this finding: a mild stressor (i.e. tactile stimulation) was accompanied by a 75–80% increase hippocampal ACh release. Using a more intense stress procedure (2-h restraint), Imperato et al.29 reported an ACh response in hippocampus that was remarkably similar to that observed in the present study. Moreover, consistent with our findings, these authors also observed a substantial increase in the release of ACh upon relief from restraint. This poststress effect was not reported by either Nilsson et al. or Inglis and Fibiger, although this may be due to differences in the duration or intensity of the stressors (handling and/or stroking the fur for 10–15 min) used by these

Stress and limbic ACh release

groups. It would thus appear that, at least under the conditions of restraint stress, an increase in hippocampal ACh output following release from stress is a consistent result, although the behavioral significance of this finding remains unclear. Both the nucleus accumbens and prefrontal cortex receive prominent dopamine (DA) inputs from the ventral tegmental area and, to some extent in the NAc, from the medial edge of the substantia nigra. The results of a variety of experiments have suggested that this mesolimbic DA system is more responsive to stressful stimuli than the nigrostriatal system which innervates the caudate nucleus.1,12,25 Dopamine neurons of the ventral tegmentum are also known to project to the septum as well as NAc and PFC,38,48 and these mesoseptal DA neurons have been reported to be activated by immobilization stress.52 These findings have led to the suggestion that dopamine may participate in the general modulation of the cholinergic response to stressors.8,21,47,50 Consistent with these suggestions, two groups have demonstrated increases in extracellular dopamine in the NAc and the PFC associated with restraint stress.1,29 While it is possible that the stress-induced increase in cortical or hippocampal ACh release observed in the present study was mediated in part by DA, this interaction appears to be neither ubiquitous nor obligatory since NAc ACh release was unaffected during restraint/shock. Moreover, Imperato et al.29 also reported an increase in accumbens DA following relief from restraint of nearly equal proportion to that observed during the stress. Thus, in one instance (during restraint), DA appears to be released in the NAc but ACh is not, whereas following release from restraint, levels of both transmitters increase. As a result of the difficulty in reconciling these two findings, we conclude that the release of ACh following restraint in the NAc coincides with, but is not contingent upon, the release of DA. Conversely, it would also appear that the increase in DA release in the accumbens during exposure to stress that has been reported by other groups is not caused by an activation of cholinergic interneurons. Based on our results, it would seem that while exposure to stress may activate ventral tegmental neurons and release DA into several projection sites (namely, NAc, PFC and hippocampus), the ability of DA to modulate ACh release may depend on the nature of the cholinergic system on which the monoamine impinges. In contrast to the hippocampus, PFC and amygdala, ACh in the NAc is derived from interneurons rather than projection neurons, an anatomical characteristic that it shares with the dorsal-lateral striatum.6,28,33 It is possible that a stress-responsive, and perhaps monoaminergic, input to cholinergic projection neurons is absent from these interneurons. Consistent with this hypothesis and our results in the NAc, Imperato et al.29 found no ACh release in response to restraint stress from cholinergic

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neurons in the striatum. Alternatively, the previously reported stress-induced increase in DA within the NAc1,29 may have multiple, and potentially opposing effects on NAc cholinergic interneurons. Damsma et al.9,10 have shown that the activation of D1 and D2 receptors has opposite effects on ACh release in striatum. As mentioned previously, the NAc maintains a very similar morphology to dorsal-lateral striatum and thus may also exibit this dualist interaction. If this is so, the possibility remains that DA released during stress may interact with ACh interneurons, but the opposition of stimulatory, D1-mediated, and inhibitory, D2-mediated effects resulted in little net observable response in ACh output. ACh release in the NAc and amygdala became significantly elevated in the 15-min sample period after subjects were released from restraint. These findings suggest that the activity of ACh neurons innervating the NAc and amygdala may be involved in locomotion, as previously suggested for cortical11,35 and hippocampal cholinergic systems.11,13,43 Alternatively, the NAc and amygdala may be responsive to the positive aspects associated with being released from restraint, or may be tuned to respond to situations from which escape is possible rather than impossible. In either event, cholinergic interneurons in the NAc and, to some extent, those that innervate the amygdala are relatively insensitive to stressful or arousing stimuli that readily activate PFC and hippocampal ACh systems. Unlike the NAc, the amygdala is innervated by cholinergic projection neurons arising in the C4 region of the nucleus basalis and horizontal limb of diagonal band.41 These cells appeared to be mildly responsive to any change in environmental conditions experienced by the animal; i.e. there were slight increases in ACh output during the initial restraint, after tail-shock and then again after subjects were released from restraint. In each intervening sample, however, ACh release decreased slightly, albeit not to baseline values. The fact that ACh levels were significantly elevated upon relief from stress may have been due to this cumulative effect of successive increases during stress. Alternatively, and as with other sites tested, the poststress response may have been primarily attributable to enhanced locomotion. Additional studies will be required to determine which of these or other possibilities accounts for the consistent increase in ACh output following relief from stress. CONCLUSIONS

The results presented here show differential effects of inescapable stress on ACh release in four limbic structures. Both the septohippocampal and basalocortical cholinergic systems were activated during stressor exposure. Moreover, relief from stress was associated with a strong increase in ACh release into

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the hippocampus. In contrast, ACh neurons in the nucleus accumbens and those innervating the amygdala were relatively non-responsive during stress, but both systems became activated immediately following release from restraint. These findings demonstrate a complex interaction between stress exposure and cholinergic activity consisting of sitespecific activation in response to stress and a more ubiquitous potentiation afterwards.

Acknowledgements—This research was supported by grants from the Whitehall Foundation, McDonnell-Pew Foundation, the Office of Naval Research and NSF to T.J.S. Procedures used in these studies conformed to the guidelines established by the National Institutes of Health for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used. Alternatives to in vivo techniques were explored. Experimental proceduers were approved by the Institutional Animal Care and Use Committee.

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