G. Ixart

A single brain-derived neurotrophic factor injection modifies hypothalamo–pituitary–adrenocortical axis activity in adult male rats

Immobilization stress induces in adult male rats rapid activation of brain derived neurotrophic factor (BDNF) expression in the hypothalamic paraventricular nucleus (PVN) preceding the increases in corticotropin releasing hormone (CRH) and arginin-vasopressin (AVP) expression. The BDNF mRNA signal belatedly co-localizes with CRH and AVP mRNA signals in the PVN, as determined by in situ hybridization. Intracerebroventricular BDNF injections (5 microg/rat) in non-anesthetized adult male rats induce a gradual increase in the CRH mRNA signal whereas AVP mRNA signal progressively decreases in the parvocellular and magnocellular PVN portions. At the same time, the CRH hypothalamic content decreases while the AVP content increases. These variations are accompanied by increases in ACTH and corticosterone plasma concentrations. These results strongly suggest that BDNF could be a stress-responsive intercellular messenger since when it is exogenously administered acts as an important and early component in the activation and recruitment of hypothalamic CRH and AVP neurons.

Intrahypothalamic infusion of interleukin-1β increases the release of corticotropin-releasing hormone (CRH 41) and adrenocorticotropic hormone (ACTH) in free-moving rats bearing a push-pull cannula in the median eminence

In two simultaneous studies on unanesthetized rats implanted 1 week earlier with either an intracerebral (i.c.) cannula adjacent to the paraventricular nucleus of the hypothalamus and an intracarotid cannula, or the same i.c. cannula together with a push-pull cannula in the median eminence (ME), we explored the effect of i.c. infused interleukin-1 beta (IL 1 beta, 5 ng in 0.25 microliter of vehicle within 2 min) on the release of corticotropin-releasing hormone (CRH) 41 and adrenocorticotropic hormone (ACTH). Intracerebral infusion of the vehicle alone had no significant effect on either the pulsatility or the level of CRH 41 release and only a short-lived minor effect on plasma ACTH, whereas i.c. IL 1 beta injection led to a significant and long lasting (1-2 h) rise in CRH 41 release peaking 3 times higher than the mean peaks of basal pulsatility (26.1 +/- 3.5 pg/5 min vs 9.5 +/- 0.7 pg/5 min), and in plasma ACTH culminating 15-20 times higher than basal levels. Simultaneously, body temperature was increased by 2.3 +/- 0.3 degrees C. In another experiment, i.c.v. infusion of IL 1 beta produced a similar increase in plasma ACTH in rats whose catecholaminergic innervation to the hypothalamus had been obliterated by a bilateral injection of 6-hydroxydopamine into the ventral noradrenergic bundle, which appears to rule out modulation of this innervation in the stimulatory effect of IL 1 beta. The precise cellular site of action of IL 1 beta on CRH 41 secreting neurons and the physiological relevance of the study are discussed within the framework of functional interactions between the neuroendocrine and immune systems.

Time-course and regional analyses of the physiopathological changes induced after cerebral injection of an amyloid β fragment in rats.

Alzheimers disease (AD) is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is an amyloid β protein (Aβ) formed by pathological processing of the Aβ precursor protein. We assessed the time-course and regional effects of a single intracerebroventricular injection of aggregated Aβ fragment 25-35 (Aβ(25-35)) in rats. Using a combined biochemical, behavioral, and morphological approach, we analyzed the peptide effects after 1, 2, and 3 weeks in the hippocampus, cortex, amygdala, and hypothalamus. The scrambled Aβ(25-35) peptide was used as negative control. The aggregated forms of Aβ peptides were first characterized using electron microscopy, infrared spectroscopy, and Congo Red staining. Intracerebroventricular injection of Aβ(25-35) decreased body weight, induced short- and long-term memory impairments, increased endocrine stress, cerebral oxidative and cellular stress, neuroinflammation, and neuroprotective reactions, and modified endogenous amyloid processing, with specific time-course and regional responses. Moreover, Aβ(25-35), the presence of which was shown in the different brain structures and over 3 weeks, provoked a rapid glial activation, acetylcholine homeostasis perturbation, and hippocampal morphological alterations. In conclusion, the acute intracerebroventricular Aβ(25-35) injection induced substantial central modifications in rats, highly reminiscent of the human physiopathology, that could contribute to physiological and cognitive deficits observed in AD.

Immobilization Stress Rapidly and Differentially Modulates BDNF and TrkB mRNA Expression in the Pituitary Gland of Adult Male Rats

Brain-derived neurotrophic factor (BDNF) is a neurotrophin involved in neuronal survival and plasticity that binds to high-affinity receptors named TrkB. In the central nervous system, brain insults, including stress, induce modifications in BDNF messenger RNA (mRNA) expression. The present study attempted to determine in the adult rat pituitary, a peripheral structure relevant for the stress response: (1) whether BDNF and TrkB mRNA expression is influenced by different durations (15, 30, 60, 180 and 300 min) of single immobilization stress; (2) the expression of BDNF transcripts containing the different exons and their possible variations after stress exposure. Plasma corticotropin (ACTH) and corticosterone concentrations were strongly and significantly increased as early as 5 min after the stress stimulus. Using RNAse protection assay and in situ hybridization, a rapid increase in BDNF mRNA occurred at 15 min. This was accompanied by an increase in BDNF protein at 60 min, and by a rapid and significant decrease in TrkB mRNA expression observed at 15 and 30 min after stress application. RT-PCR analysis of BNDF transcripts showed strong basal expression of exons III and IV, whereas transcripts containing exons I and II seemed weakly expressed. After stress application, transcripts containing exons III and IV were rapidly and significantly increased at 30 min, whereas transcripts containing exons I and II remained unchanged. These results show that pituitary BDNF transcripts expression is differentially affected by immobilization stress.

Continuous i.c.v. infusion of brain-derived neurotrophic factor modifies hypothalamic–pituitary–adrenal axis activity, locomotor activity and body temperature rhythms in adult male rats

Brain-derived neurotrophic factor is a neurotrophin belonging to the nerve growth factor family, which is involved in the differentiation and survival of many types of neurons. It also participates in neuroprotection and neuronal plasticity in adult rats. Our previous studies showed that a single brain-derived neurotrophic factor injection modifies hypothalamic-pituitary-adrenal axis activity in adult male rats. To investigate the effect of chronic brain-derived neurotrophic factor administration on some physiological parameters, adult rats were implanted with osmotic micro-pumps to deliver brain-derived neurotrophic factor continuously for 14 days in the lateral ventricle (12 microg/day/rat). mRNA levels were evaluated by in situ hybridization analysis, peptide contents and plasma hormone concentrations by radioimmunoassay. Animals were also equipped with telemetric transmitters to study locomotor activity and temperature rhythms modifications, since hypothalamic-pituitary-adrenal axis is known to modulate these two parameters. Decreased body weight was used as a control of brain-derived neurotrophic factor access to hypothalamic areas as already documented. In the hypothalamus the continuous brain-derived neurotrophic factor treatment increases: (i) the mRNA steady state levels of corticotropin releasing hormone and arginin-vasopressin in the paraventricular nucleus, the supraoptic nucleus, and the suprachiasmatic nucleus; (ii) the surface of corticotropin releasing hormone and arginin-vasopressin mRNA signals in these nuclei as detected by in situ hybridization, and (iii) the corticotropin releasing hormone and arginin-vasopressin contents. The plasma concentrations of adrenocorticotropic hormone and corticosterone were decreased and increased, respectively. Finally, this treatment increased daily locomotor activity and temperature, and provoked some circadian perturbations. These results obtained after chronic brain-derived neurotrophic factor administration extend data on the brain-derived neurotrophic factor involvement in the hypothalamic-pituitary-adrenal axis regulation and illustrate its effects on the locomotor and temperature rhythms. They also allow demonstrating that the regulation of the hypothalamic-pituitary-adrenal axis by brain-derived neurotrophic factor differs according to the brain-derived neurotrophic factor administration mode, i.e. acute injection or chronic administration.

Evidence for basal and stress-induced release of corticotropin releasing factor in the push-pull cannulated median eminence of conscious free-moving rats

Fourteen adult male rats were successfully implanted in their median eminence with a push-pull cannula perfusing an artificial fluid at a rate of 13 microliters/min, to measure the release of corticotropin releasing factor (rCRF-41) under physiological conditions assessed by baseline plasma adrenocorticotropic hormone (ACTH) levels. In the basal conscious free-moving state, rCRF-41 release displayed a fluctuating pattern, with peaks about every 45 min at a mean value of 9.0 +/- 0.7 pg/15 min sample (n = 42) vs a mean trough value of 4.1 +/- 0.3 pg/sample (n = 44). Ether stress was followed by a striking rise in rCRF-41 release which generally lasted about 45 min and reached mean peak values of 54.3 +/- 3.2 pg/15 min sample (n = 7). These data constitute the first direct measurements of basal and stress-induced CRF releases in conscious unrestrained rats.

Alzheimer's Disease Related Markers, Cellular Toxicity and Behavioral Deficits Induced Six Weeks after Oligomeric Amyloid-β Peptide Injection in Rats

Alzheimer’s disease (AD) is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-β protein (Aβ). Recently, we characterized the effects of a single intracerebroventricular (icv) injection of Aβ fragment (25–35) oligomers (oAβ25–35) for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAβ25–35 and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAβ25–35 was still present in the brain after 6 weeks. oAβ25–35 injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation), mitochondrial (caspase-9 levels) and reticulum stress (caspase-12 levels), astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAβ25–35 injection resulted in increased APP expression, Aβ1–42 generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aβ, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.

A subpopulation of corticotopin-releasing hormone neurosecretory cells in the paraventricular nucleus of the hypothalamus also contain NADPH-diaphorase

The coexistence of ND with CRH 41 was explored in the parvicellular neurons of the PVN, using dual histochemical and radioimmunocytochemical labelling with the light microscope, in rats treated with colchicine. Even though the ND staining was scarce, a clear colocalization was evidenced in the parvicellular part of the PVN. Under these conditions, the ratio of neurons expressing both markers, ND and CRH, amounted about 15% of the CRH-containing neuron population. This result provides a useful tool to study morphological plastic changes in the PVN in response to environmental variations.

Adrenocorticotropic Regulations after Bilateral Lesions of the Paraventricular or Supraoptic Nuclei and in Brattleboro Rats

The circadian rhythm and ether stress responsiveness of plasma ACTH and corticosterone were explored in chronically cannulated female Sprague-Dawley rats with bilateral lesions of either the paraventricular nuclei (PVN) or supraoptic nuclei and also in female Brattleboro rats. 3 weeks after PVN lesions, the circadian ACTH rhythm had dropped by at least half. Less drastic alterations were measured for the corticosterone rhythm, and only its diurnal maximum and amplitude diminished by one third. Although the kinetics of plasma ACTH and corticosterone stimulation after ether stress were essentially normal, the peaks for both hormones, 5–15 min after stress, were only half as high as the control peaks. The effects of bilateral abolition of supraoptic nuclei on the circadian rhythm of the adrenocorticotropic system were slight, and only the diurnal minimum and mean level of ACTH dropped significantly. Intriguingly, both ACTH and corticosterone levels in rats with supraoptic lesions rose to twice the control levels after ether stress. In Brattleboro rats, neither the circadian rhythm nor stress-induced stimulation of ACTH and corticosterone differed from those of the controls. These data indicate that vasopressin is not essential for either of the regulations of the adrenocorticotropic system. The production of corticotropin-releasing factor (CRF) involved in both types of regulation clearly originates, at least partly, from the PVN area and no significant contribution is made by the region of the supraoptic nuclei, a finding which conforms to neuroanatomical data. However, it is not yet clear whether the PVN area is the site of origin of the recently characterized 41 amino-acid CRF or of other CRF fractions distinct from vasopressin.

Deregulation of hypothalamic-pituitary-adrenal axis functions in an Alzheimer's disease rat model

Elevated cortisol evidence in Alzheimers disease (AD) patients prompted the hypothesis that stress and glucocorticoids are involved in the development and/or maintenance of AD. We investigated the hypothalamic-pituitary-adrenal (HPA) axis activity, functionality, and reactivity for up to 6 weeks after an intracerebroventricular injection of amyloid-β(25-35) peptide (Aβ(25-35)) in rat, a validated acute model of AD. Aβ(25-35) induces memory impairment, alteration of anxiety responses, HPA axis hyperactivity, and glucocorticoid (GR) and mineralocorticoid (MR) receptor increases in brain regions related to HPA axis functions. GR are progressively translocated in neurons nucleus, while membrane version of MR is evidenced in all structures considered. The MR/GR ratio was modified in all structures considered. Aβ(25-35) induces a subtle disturbance in the feedback of the HPA axis, without modifying its functionality. The reactivity alteration is long-lasting, suggesting that amyloid toxicity affects the HPA axis adaptive response to stress. These findings are evidence of progressive HPA axis deregulation after Aβ(25-35), which is associated with an imbalance of MR/GR ratio and a disruption of the glucocorticoid receptors nucleocytoplasmic shuttling, and suggest that elevated glucocorticoids observed in AD could be first a consequence of amyloid toxicity.