Fred J.H. Tilders

Activation of the hypothalamus-pituitary-adrenal axis by bacterial endotoxins: Routes and intermediate signals

Peripheral administration of endotoxin induces brain-mediated responses, including activation of the hypothalamus-pituitary-adrenal (HPA) axis and changes in thermoregulation. This paper reviews the mechanisms by which endotoxin affects these responses. The effects on thermoregulation are complex and include macrophage-dependent hyperthermic and hypothermic responses. Low doses of endotoxin, given IP, activate peripheral macrophages to produce interleukin (IL)-1 beta, which enters the circulation and acts as a hormonal signal. IL-1 may pass fenestrated endothelium in the median eminence to stimulate corticotropin-releasing hormone (CRH) secretion from the CRH nerve-terminals. In addition, IL-1 may activate brain endothelial cells to produce IL-1, IL-6, prostaglandins, etc., and secrete these substances into the brain. By paracrine actions, these substances may affect neurons (e.g., CRH neurons) or act on microglial cells, which show IL-1-induced IL-1 production and therefore amplify and prolong the intracerebral IL-1 signal. In contrast, high doses of endotoxin given i.v. may directly stimulate endothelial cells to produce IL-1, IL-6, and prostaglandin-E2 (PGE2) and thereby activate the HPA axis in a macrophage-independent manner.

A Single Exposure to Amphetamine Is Sufficient to Induce Long-Term Behavioral, Neuroendocrine, and Neurochemical Sensitization in Rats

Repeated treatment with psychostimulant drugs causes long-lasting behavioral sensitization and associated neuroadaptations. Although sensitization induced by a single psychostimulant exposure has also been reported, information on the behavioral and neurochemical consequences of a single psychostimulant exposure is sparse. Therefore, to evaluate whether behavioral sensitization evoked by single and repeated psychostimulant pretreatment regimens represent the same neurobiological phenomenon, the time-dependent expression of behavioral, neurochemical, and neuroendocrine sensitization after a single exposure to amphetamine was investigated in rats. A single exposure to amphetamine (5 mg/kg, i.p.) caused context-independent sensitization of the locomotor effects of amphetamine, which intensified over time. Thus, sensitization to amphetamine was marginal at 3 d after treatment and more evident after 1 week, whereas 3 weeks after treatment, profound sensitization, as well as cross-sensitization, to cocaine was observed. Amphetamine pretreatment caused an increase in the electrically evoked release of [3H]dopamine from nucleus accumbens, caudate putamen, and medial prefrontal cortex slices and of [14C]acetylcholine from accumbens and caudate slices. The hyperreactivity of dopaminergic nerve terminals appeared to parallel the development of locomotor sensitization, i.e., whereas hyperreactivity of accumbens dopaminergic terminals increased between 3 d and 3 weeks after treatment, the hyperreactivity of medial prefrontal dopaminergic terminals decreased. Pre-exposure to amphetamine also sensitized the hypothalamus–pituitary–adrenal axis response to amphetamine at 1 and 3 weeks, but not at 3 d after treatment. Because these data closely resemble those reported previously for repeated amphetamine pretreatment, it is concluded that a single exposure to amphetamine is sufficient to induce long-term behavioral, neurochemical, and neuroendocrine sensitization in rats.

Repeated stress-induced activation of corticotropin-releasing factor neurons enhances vasopressin stores and colocalization with corticotropin-releasing factor in the median eminence of rats.

Stress-induced release of corticotropin-releasing factor (CRF) and vasopressin (AVP) was studied in rats by measuring the decline of CRF and AVP stores in the median eminence after blockade of fast axonal transport with colchicine (5 micrograms per rat intracisternally). Quantitative immunocytochemistry was used to detect changes in CRFi and AVPi in the external zone of the median eminence (ZEME) selectively. Immobilization stress induced a fast ACTH response to 1,000-2,000 pg/ml which was associated with a fall in both CRFi and AVPi of 34% during the first 30 min. This is followed by different time courses of further AVPi and CRFi depletion. In addition, we investigated the effect of repeated daily stress exposure on CRFi and AVPi in the ZEM 1 day after stress exposure. Repeated daily immobilization for 9 or 16 subsequent days did not affect the CRFi stores in the ZEME, but increased the AVPi stores to 161 +/- 13% and 218 +/- 11% respectively. Quantitative analysis of electron microphotographs of repeatedly handled rats showed a mean density of CRF positive profiles in the ZEME of 45.5 +/- 2.5 per 500 microns 2 of which 25% also stained for pro-AVP-derived peptides. After 9 subsequent days of immobilization the total density of CRF-positive profiles remained unchanged, but the fraction of CRF swellings that also stained for pro-AVP-derived peptides increased approximately 2-fold. We conclude that (1) the secretion of AVPi and CRFi from the ZEME are independently controlled, indicating differential activation of AVP containing and AVP deficient CRF neurons during acute immobilization, and (2) repeated stress leads to plastic changes in hypothalamic CRF neurons resulting in increased AVP stores and colocalization in CRF nerve terminals.

Interleukin-10, interleukin-4, and transforming growth factor-β differentially regulate lipopolysaccharide-induced production of pro-inflammatory cytokines and nitric oxide in co-cultures of rat astroglial and microglial cells

The pro‐inflammatory cytokines interleukin‐1β (IL‐1β), IL‐6, tumor necrosis factor‐α (TNF‐α), and nitric oxide (NO) can be produced by activated glial cells and play a critical role in various neurological diseases. Using primary co‐cultures of rat microglial and astroglial cells, we investigated the effects of the anti‐inflammatory cytokines transforming growth factor‐β1 (TGF‐β1)/β2, IL‐4, and IL‐10 on the production of (pro‐) inflammatory mediators after stimulation of the cells with lipopolysaccharide (LPS; 0.1 μg/ml, 24 h). IL‐10 (10 and 100 ng/ml) and IL‐4 (5 and 50 U/ml) suppressed the LPS‐induced production of NO, IL‐6, and TNF‐α in a dose‐dependent manner, whereas TGF‐β1/β2 (2 and 20 ng/ml) only suppressed NO production. LPS‐induced levels of IL‐1β were suppressed by IL‐10, but not by IL‐4 and TGF‐β1/β2. Conversely, co‐incubation of the glial cells with LPS and antibodies to TGF‐β1/β2 selectively enhanced LPS‐induced NO production, whereas co‐incubation with antibody to IL‐10 enhanced LPS‐induced production of all pro‐inflammatory cytokines and NO. This finding strongly suggests that effective concentrations of TGF‐β1/β2 and IL‐10 are produced by LPS‐stimulated glial cell co‐cultures. Production of IL‐10 in these co‐cultures was confirmed by measurement of rat IL‐10 by radioimmunoassay. We conclude that anti‐inflammatory cytokines affect the production of inflammatory mediators in LPS‐activated co‐cultures of microglial and astroglial cells differentially. GLIA 30:134–142, 2000.

Short Inescapable Stress Produces Long-Lasting Changes in the Brain-Pituitary-Adrenal Axis of Adult Male Rats

Recently, we reported that rats exposed to a single and short session of inescapable footshocks showed alterations in behavioural response to environmental stimuli which developed progressively over a week and remained present for at least 28 days. The aim of the present study was to investigate whether these behavioural changes were accompanied by alterations in the brain-pituitary-adrenal axis. Male Wistar rats were subjected to 10 inescapable footshocks (S) of 6 s duration and 1 mA intensity during a period of 15 min. Control rats (C) were placed in the shock apparatus for 15 min without receiving shocks. The effects of these experimental procedures were studied 14 days later. Exposure to shocks did not affect basal plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT). However, the novelty-induced ACTH response was increased in S rats as compared to C rats whereas the CORT response did not differ between C and S rats. The ACTH content of the anterior pituitary gland and adrenal weight were not affected by exposure to inescapable footshocks 14 days earlier. Quantitative immunocytochemistry of vasopressin (AVP) and corticotropin-releasing factor (CRF) in the external zone of the median eminence showed that prior footshock exposure increased the AVPi stores to 167% as compared to C rats, whereas CRFi content was not changed. In addition, S rats showed increased mineralocorticoid (MR) and glucocorticoid (GR) receptor binding capacity in the hippocampus as compared to C rats, whereas affinities were not affected. We conclude that a single and short session of inescapable footshocks has long-lasting effects on brain-pituitary-adrenal functioning concomitant with behavioural alterations.

Inescapable footshocks induce progressive and long-lasting behavioural changes in male rats.

Long-term behavioural consequences of exposure to a brief (15 min) session of inescapable footshocks (10 x 6 s, 1 mA) were investigated in male rats. The time course of the effects of inescapable footshocks was assessed by studying the behaviour of groups of rats at different post-stress intervals. Footshocked rats (S) did not differ from control (C) rats (exposed to the shock box for 15 min) in their behavioural response to an open field whether tested 1 h or 4 h post-stress. However, one day after shocks, S rats showed less locomotion and rearing, and more immobility and attention as compared to C rats. At 7 days or 14 days post-stress, S rats exhibited decreased locomotion, rearing, sniffing, and grooming, and increased immobility, attention, and defecation relative to C rats. In a second experiment, we investigated whether footshocks affect the behavioural response to a sudden drop in background noise during exposure to a novel environment. At 21 days post-stress, S rats showed a markedly enhanced immobility response to this stimulus as compared to C rats. In order to investigate whether rats could be exposed repeatedly to the open field without affecting the differences in behaviour between the two treatment groups, C and S rats were tested in an open field for the first time at 7 days post-stress, which yielded the typical effects of footshocks. When these rats were exposed to a second open-field test one week later, the behavioural responses of C and S rats were not different.(ABSTRACT TRUNCATED AT 250 WORDS)

Repeated stress enhances vasopressin synthesis in corticotropin releasing factor neurons in the paraventricular nucleus

The effect of repeated stress on corticotropin-releasing factor (CRF) and vasopressin (AVP) synthesis in parvocellular neurons of the paraventricular nucleus (PVN) was studied by means of double label immunocytochemistry. Once daily immobilization of male rats for 16 days leads to a 75% increase in the number of CRF immunoreactive neurons, but a 5-fold elevation in the number of AVP containing CRF cell bodies. These results demonstrate that repeated stress activates AVP synthesis in CRF neurons.

Characterization of stress-induced long-term behavioural changes in rats: Evidence in favor of anxiety

Recently, we reported that rats exposed to one brief session of inescapable footshocks showed a gradually developing and long-lasting decrease in behavioural activity and an increase in defecation in an open field. The aim of the present study was to further characterize the long-lasting changes in behavioural responsiveness to environmental stimuli. For this purpose, behavioural paradigms validated as tools in the preclinical study of the psychobiology of depression were used. Footshocked rats (S) showed a decreased response latency in an one-way avoidance-escape task and decreased immobility in a forced swim test as compared to nonshocked control rats (C) 14 days after shock exposure. These S rats showed decreased behavioural activity and increased defecation as compared to the C rats in an open field test carried out 28 days after footshock exposure. In addition, footshock exposure did not affect the preference for or consumption of a 0.05% saccharin solution on a long-term basis, although a decreased consumption of this solution was evident in S rats on day 1 postshock. These S rats showed an exaggerated immobility response to a sudden reduction in background noise level compared to C rats while placed in a novel environment on day 11 postshock. We conclude that the long-term effects of one short session of inescapable footshocks are not compatible with what is supposed to represent behavioural manifestations of depression in animals. It is argued that the common denominator of shock-induced long-lasting changes is increased behavioural defensiveness, which is more likely related to increased fear and/or anxiety.

Induction of plasma interleukin-6 by circulating adrenaline in the rat

Adrenaline, which is secreted from the adrenal medulla during stress, is considered to be involved in the control of inflammation and immune responses. Therefore, we studied the effects of adrenaline on the plasma levels of one of the major pro-inflammatory cytokines, interleukin-6 (IL-6). Here we describe that in rats, SC administration of adrenaline induces a dose-dependent increase in plasma IL-6 concentrations, reaching its maximum after 2 h. In addition, intravenous (IV) infusion of adrenaline in a dose resulting in circulating adrenaline concentrations similar to those observed during stress, enhanced heart rate and increased plasma IL-6 concentrations. The increase in plasma IL-6 in response to adrenaline given by subcutaneous (SC) route and by IV infusion could be blocked by the beta-adrenergic receptor antagonist l-propranolol but not by d-propranolol. Based on these data we conclude that under physiological conditions circulating adrenaline may be involved in the control of IL-6 production, and thereby may modulate inflammatory responses.

Long-lasting deficient dexamethasone suppression of hypothalamic-pituitary-adrenocortical activation following peripheral CRF challenge in socially defeated rats

The present study focuses on the long‐term changes in the regulation of the hypothalamic‐pituitary‐adrenocortical (HPA) axis following two short‐lasting episodes of intensive stress in the rat stress model of social defeat and the possible similarities with HPA functioning in human affective disorders. Male Wistar rats experienced social defeats on 2 consecutive days by an aggressive male conspecific. The long‐term effect of these defeats on resting and ovine corticotropin‐releasing factor (oCRF; intravenous (i.v.) 0.5 μg/kg) induced levels of plasma ACTH and corticosterone (CORT) were measured 1 and 3 weeks later. In a second experiment the glucocorticoid feedback regulation of HPA function was tested in a combined dexamethasone (DEX)/CRF test (DEX; 25 μg/kg s.c., 90 min before oCRF injection, 0.5 μg/kg). The oCRF challenges were performed between 11.00 and 13.00 h (about three hours after start of the light phase). One week after defeat the ACTH response to CRF was significantly enhanced in defeated rats as compared to controls. Three weeks after defeat the ACTH response was back to control levels. The increased ACTH response 1 week after the stressor was not reflected in higher CORT levels. Neither were baseline ACTH and CORT levels affected by the prior stress exposure. DEX pretreatment inhibited pituitary adrenocortical activity, reflected both in reduced baseline and response values of ACTH and CORT. The ACTH response to CRF following DEX administration was significantly higher in defeated rats as compared to controls both at one and three weeks after defeat. A reduced DEX suppression of baseline secretion of ACTH appeared 3 weeks after defeat. The same tendency was apparent in response and baseline values of CORT. The differences in CORT between socially stressed and control treated rats, however, did not reach significance. The possible role of changes in glucocorticoid‐(GR) and mineralocorticoid receptor (MR) binding in the altered regulation of HPA activity following defeat were studied in brain and pituitary of male Wistar rats 1 and 3 weeks after defeat. One week after defeat GR‐binding decreased in hippocampus and hypothalamus. No changes were observed in GR‐binding in the pituitary nor in MR‐binding in any of the regions analysed. Three weeks after defeat GR‐binding recovered in hippocampus and hypothalamus but at this time MR‐binding in hippocampal tissue was seriously decreased. In a fourth experiment vasopressin (AVP) and CRF stores in the external zone of the median eminence (ZEME) were measured by quantitative immunocytochemistry one and three weeks after defeat and compared with controls. Social defeat failed to induce a change in the immunocytochemical stores of AVP or CRF. The present findings show that in rats short‐lasting stressors like defeat induce long‐lasting, temporal dynamic changes in the regulation of the HPA axis. Since these changes in time are reflected in GRs and MRs in different brain areas an altered corticosteroid receptor binding might play an important role in the affected HPA activity following defeat.