Electrical Stimulation of the Amygdala Modifies the Negative Feedback Effect of Glucocorticoids on the Adrenocortical Responses to Stress

It is now well established that there is a bidirectional communication between the nervous system and the immune system. A large body of evidence has confirmed that reciprocal pathways of interaction between these two systems involve a neural pathway which includes direct contact between nerve terminals and lymphoid organs and humoral pathways via circulating glucocorticoids (GC) secreted following activation of the hypothalamo-pituitary-adrenal (HPA) axis. The activity of the HPA axis is regulated by extrahypothalamic structures including the amygdala (AMG), which plays a significant facilitatory role [1, 2]. Thus, acute electrical stimulation of the AMG in a variety of animals results in activation of the HPA axis [2]. This effect involves neural mechanisms mediated by amygdalar and hypothalamic norepinephrine and serotonin and by the secretion of hypothalamic corticotropin-releasing hormone (CRH-41) [3]. Bilateral lesions of the central and medial AMG have been found to inhibit HPA axis responses following a variety of stress modalities including olfactory, somatosensory, photic and acoustic stimulation, conditioned fear, laparotomy and immobilization stress [4, 5, 6]. In addition, several studies show that lesions of the central AMG inhibit the HPA axis responses to immune challenges such as herpes simplex virus infection and systemic injection of IL-1β [7]. We have previously reported that the AMG may facilitate the adrenocortical (AC) response to glutamate injected into the paraventricular nucleus [8].

The activity of the HPA axis is also regulated by the negative feedback exerted by corticosteroids which act predominantly at the hippocampus [9]. The effect of these hormones is mediated by 2 types of intracellular corticosteroid receptors [9, 10, 11, 12]. It was previously reported that repeated electrical stimulation of the AMG, resulting in kindling, caused a transient decrease in hippocampal GC receptor (GR) mRNA [13, 14, 15], and that this effect was associated with increased fearful behavior [15]. Many studies show that alterations in hippocampal GR activity may affect the responses of the HPA axis due to the impaired feedback action of the GC [16, 17, 18].

In view of these observations, we attempted to examine the effect of repeated electrical stimulation of the AMG on the responses to stress stimuli and on the function of the negative feedback exerted by GC.

Male rats (Hebrew University strain) weighing 180-200 g were purchased from Harlan (Hebrew University, Jerusalem) and housed in the animal care facility in compliance with the standard guidelines for animal care. All experiments were approved by the institutional committee for animal care. The rats were given free access to food and water. They were transferred to individual cages 24 h prior to stress exposure. Each experimental group consisted of 6 animals. All experiments were performed between 7 and 11 a.m.

Animals were anesthetized with ketamine-xylazine intraperitoneally (200 and 10 mg/kg body weight, respectively) and the stimulating electrodes were stereotaxically implanted into the central amygdalar nucleus. Stereotaxic coordinates [7], taking the bregma suture as the zero reference point, were the anterior-posterior plane (AP) = 0, lateral plane (L) = 4.0 and height (H) = -8.5. The electrodes consisting of concentric bipolar constructions were fixed to the skull with dental cement insulated to within 0.7 mm of the tip. One week after electrode implantation, electrical stimulation (0.5 mA for 1 ms and 100/s) was performed under anesthesia for 5 min once daily for 4 consecutive days. Sham stimulation consisted of complete anesthesia and connection of the electrodes to a stimulator but with no current being turned on. At 5 and 12 days after the onset of the electrical stimulation, the rats were exposed to stressful stimuli.

Acoustic Stimulus

Animals were exposed to a ringing bell with an intensity of 109 dB for 4 min.

Photic Stimulus

Animals were exposed, in a dark room, to a photo stimulator emitting flashes at a rate of 4/s for 4 min. All animals were sacrificed 15 min after the onset of the stimuli and trunk blood was collected for the determination of corticosterone (CS).

Animals were injected intraperitoneally with either vehicle or Dex (Sigma), 40 µg/kg of body weight, dissolved in saline containing 1% ethanol; 3.5 h later, the animals were exposed to either acoustic or photic stress as described above.

CS was determined by radioimmunoassay as previously described [19]. The sensitivity of the assay is 0.5 µg/100 ml. All data are presented as a mean ± SEM.

Binding Assay

For the corticosteroid-binding assay, the rats underwent bilateral adrenalectomy 24 h prior to the assay, in order to clear all binding sites from endogenous CS. The receptor-binding assay was performed as previously described [19]. In brief, aliquots of cytosol fraction of dorsal hippocampal tissue (300 µl, equivalent to 1.5 mg protein) were incubated with a saturating concentration of 20 nM³H-Dex (1,2,4-³H-Dex, 80 Ci/mmol; Amersham Life Science) for 20 h at 4°C. Nonspecific binding was determined in parallel incubations containing a 500-fold excess of unlabeled Dex. Following incubation, aliquots were run on Sephadex LH-20 (Sigma) to separate bound and free ligands. Specific binding was expressed as femtomoles of Dex/mg protein.

Analysis was performed with the Sigma-Stat software package (SPSS, USA). Data are presented as mean ± SEM. The results were analyzed with analysis of variance followed by the Student Newman-Keuls test.

Figure 1 shows that the acoustic and photic stress stimuli caused a marked 5-fold increase in serum CS in the sham-stimulated animals when compared to the nonstressed controls. A similar response to both stimuli was found in animals at 5 days following the onset of AMG stimulation. In this group, there was also a slight but significant increase of basal CS levels compared to in the sham-stimulated group. In sham-stimulated rats, the administration of Dex completely inhibited the AC responses to both photic and acoustic stimuli. In contrast, 5 days after AMG stimulation, the administration of Dex was significantly less effective in inhibiting the AC response to stress, i.e. Dex inhibited the CS response by only 50%. At 12 days after AMG stimulation, the AC responses to both stress modalities and the effect of Dex were similar to in sham-stimulated controls.

Next, we examined the effect of AMG stimulation on the ability of endogenous GC to terminate the stress-induced AC response. Figure 2 shows that 30 min after the onset of acoustic stress, CS serum levels markedly decreased by 60% (compared to the peak level at 10 min). However, at 30 min after stress stimulation in the AMG-stimulated group, serum levels of CS were significantly higher (2-fold) compared to the sham-stimulated control group at this time point.

Finally, we tested if the reduced feedback effect of GC following AMG stimulation is associated with changes in GR hippocampal binding activity. Figure 3 shows that at 5 days after the onset of AMG stimulation, the specific binding of ³H-Dex to corticosteroid receptors in hippocampal cytosol was significantly lower (40%) compared to in the sham-stimulated group. At 12 days after stimulation the binding of Dex returned to normal values, i.e. like in the sham-stimulated group.

The activation of the HPA axis depends on the secretion of CRH-41 from neurons located in the hypothalamic paraventricular nucleus [1, 20]. In response to a variety of stimuli including immune mediators such as IL-1, CRH-41 is released into the portal system, causing the secretion of pituitary adrenocorticotropic hormone and, consequently, AC GC. The activation of the HPA axis is regulated by extrahypothalamic limbic structures which play a significant facilitatory role [2, 6]. It is well known that the hippocampus plays a major role in the negative feedback action of GC upon the HPA axis [1, 9]. This effect is mediated by 2 types of corticosteroid receptors, the mineralocorticoid (MR) and the GR. These receptors differ in their localization and their affinities to GR and MR [10, 11, 21]. The relative involvement of each receptor type may depend on the type of stress modality [2, 10, 12]. Neuroendocrine studies show that the AMG projects to several hippocampal areas including the CA1 region [22, 23]. These neural pathways may potentially affect various aspects of hippocampal functions. However, the influence of the AMG on hippocampal functions related to the regulation of the HPA axis is not yet clear.

We found here that repeated electrical stimulations of the central AMG significantly attenuated the inhibitory action of Dex on the AC responses to both acoustic and photic stress stimuli. In addition, we tested the effect of AMG stimulation on the ability of endogenous GC to terminate the AC stress response. We found that AMG stimulation attenuated the decline in serum CS to basal levels. Also, AMG stimulation caused a slight increase in basal levels of CS; this may result from impaired feedback exerted by GC. It should be noted that the effect of AMG stimulation on the AC function is temporary; at day 12 after the stimulations, the AC responses to stress returned to normal values like those in the sham-stimulated groups.

Although we found that the AMG stimulation impaired the negative feedback action of GC, no change was found in the magnitude of the AC response to either acoustic or photic stimuli compared to the control groups. The reason for this apparent discrepancy is not clear.

Previous studies, however, showed that kindling caused by repeated electrical stimulations of the AMG increased the level of MR mRNA in the dentate gyrus of the hippocampus while the mRNA of GR decreased [14]. It is possible that the increase in hippocampal MR, known to modulate the AC responses to stress, may partially balance the feedback effect of circulating GC and thus blunt the effect of AMG stimulation on reducing the negative feedback action of GC.

To examine whether the impaired feedback caused by AMG stimulation may result from decreased hippocampal GR, we measured the binding of ³H-Dex by hippocampal tissue. It has been previously demonstrated that impaired GC feedback is associated with reduced hippocampal corticosteroid receptors. This reduction markedly attenuated the inhibitory effect of the hippocampus over the hypothalamus and impaired the negative feedback effect of GC on the AC responses to various stressful stimuli [17, 18, 19].

Our results showed that AMG stimulation caused a significant reduction in the maximal binding capacity of ³H-Dex by hippocampal cytosol. It may be assumed, therefore, that the impaired feedback of GC induced by AMG stimulation is mediated by the reduced level of hippocampal corticosteroid receptors.

In agreement with our results, previous studies showed that kindling in rats, induced by repeated AMG stimulations, decreased GC mRNA expression in hippocampal regions [13, 14, 15]. The mechanisms by which AMG stimulation caused the reduction of hippocampal GR is not yet clear. It was suggested that this effect is not due to the AMG-induced loss of hippocampal neurons because kindling induced by intense AMG stimulation increases benzodiazepine receptors in the hippocampus [15]. One mechanism which may be involved in reduced hippocampal GR is the downregulation caused by the hypersecretion of GC during AMG stimulations. It is well established that chronic stimulation of GR with GC results in decreased levels of GC binding and GR mRNA [14]. Another mechanism which may be involved in GR downregulation may be the stimulation of hippocampal N-methyl-D-aspartate receptors causing a rise in the intracellular calcium ion concentration and a downregulation in GR mRNA expression [13].

In conclusion, we showed here that impaired GC feedback induced by repeated AMG electrical stimulations may be involved in the regulatory role of this limbic structure on the HPA axis.

This work was supported by the Lena P. Harvey Endowment Fund for Neurological Research. This article is dedicated to the memory of Dr. Liora Yaar from whom I (J.W.) learned what stress is and how to cope with it.