Research reportElectrophysiological effects of sustained delivery of CRF and its receptor agonists in hippocampal slices
Introduction
The corticotropin-releasing factor (CRF) is a hypothalamic hormone which stimulates the release and secretion, from the anterior pituitary gland, of adrenocorticotropic hormone (ACTH) and β-endorphin. In 1981 Vale et al. [18] determined the primary structure of ovine CRF and found that its sequence is very close to that of rats, pigs, goats, cows and humans.
Autoradiographic findings provide evidence that each subtype of CRF receptors has a different regional distribution in the brain, strengthening the suggestion that CRF-R1 and CRF-R2 receptors have different roles in mediating CRF function [12]. CRF binding sites have been discovered in many different brain regions: brainstem, striatum, median eminence, cerebral cortex, septum, hippocampus, thalamus, cerebellum and hypothalamus [4]. CRF seems to mediate stress responses and both receptors seem to be involved in mediation of anxiety-like behavior [13], [14], [17]. CRF is largely released from the paraventricular nucleus of the hypothalamus, although it was found in various brain regions as the limbic system, the telencephalon, the neocortex and the cerebellar nuclei [5].
Localization of the CRF in the limbic system suggests that it can play an important role in activating the sympathetic nervous system for adaptive, autonomic, behavioral and immune responses to stress in mammals [1], [15] and in modulating learning memories performance in rats [7], [10], [20]. Systemic CRF administration causes a decrease in peripheral vascular resistance with consequent hypotension and tachycardia, a decrease of appetite, sleep, libido and symptoms of depression. Intra-amygdala injection improves memory retention and intra-hippocampal administration improves performance of a passive avoidance learning [8]. The electrophysiological effects of CRF in hippocampal neurons are largely uncharacterized. In 1983 Aldenhoff and colleagues [1] showed that in in vitro hippocampal slices CRF in nanomolar concentrations (15–250 nM) increases the spontaneous discharge of pyramidal neurons and decrease the afterhyperpolarization that follows bursts or trains of action potentials. Higher concentrations (more than 250 nM) have a similar effect, depolarizing CA1 and CA3 pyramidal neurons and increasing their spontaneous firing rate. Lee and colleagues [8] demonstrated that CRF activates NMDA receptors in the dentate gyrus of the hippocampus. In their experiments this was associated with enhanced memory consolidation in the CRF-treated rats. In the serotonergic dorsal raphe nucleus low-dose CRF has an inhibitory action on neuronal activity, while paradoxically higher doses cause less inhibition and even excitation [6]. We studied the effects of CRF and of its two receptor agonists d-Pro-5-CRF and d-Glu-20-CRF [19] on hippocampal LTP, a process which is considered to be a mechanism of learning and memory processes [3]. The two receptor agonists are selective for the CRF-R1 and the CRF-R2 receptors, respectively [19].
Section snippets
Slices preparation and incubation
Sprague–Dawley female rats (120–160 g) were anesthetized with chloroform and decapitated. Their hippocampi were dissected and cut into 600-μm thick transversal slices under ice-cold artificial cerebrospinal fluid (aCSF) containing in mM: NaCl 130, KCl 3.5, NaH2PO4 1.25, NaHCO3 24, CaCl2 2.4, MgSO4 1.2, glucose 10, pH of 7.35–7.40, and previously bubbled with a gas mixture of 95% O2/5% CO2 for about 5 min. Slices were incubated, for not less than 1 h, in a beaker with aCSF bubbled with the same
Results
In Fig. 1, it can be observed that sustained administration of CRF, d-Glu-20-CRF and d-Pro-5-CRF has in all cases a depressing action on population spike (PS) amplitude. This effect is obvious for CRF, which causes the deepest and fastest decrease, beginning already 10 min after bath application and reaching 20±22% (mean±S.D., n=8) of baseline after 1 h. The depressing effect of either one the two receptor agonists alone is slower and less pronounced. With d-Glu-20-CRF, the decrease is observed
Discussion
Previous electrophysiological studies on CRF found an excitatory effect on hippocampal neurons at both 1 nM and 1 μM concentration shortly after its administration [1]. More recently, Kirby et al. [6] found in a different neuronal population (neurons of the dorsal raphe nucleus) that low concentrations of CRF have an inhibitory effect which is abolished or even reversed at higher concentrations.
We did not investigate short-term effects of CRF, because these have already been studied in this
Acknowledgements
This work was funded by grant BIO 04-CT97-2307 of the European Commission. N.I. was partially funded by the Italian Ministry of Foreign Affairs within the framework of the First Protocol of Scientific and Technological Cooperation between Italy and the Russian Federation. The financial support of Telethon — Italy (Grant no. E.1237) and of INTAS (Grant no. 441) is gratefully acknowledged.
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Present address: Pavlov Institute of Physiology, Russian Academy of Sciences, nab. Makarova 6, 197034 St. Petersburg, Russia.