Spatial learning and the hippocampal corticosterone receptor system of old rats: effect of the ACTH4–9 analogue ORG 2766

Old (26 months) and young (6 months) male Wistar rats were treated chronically for 2 weeks with ORG 2766 or with vehicle, delivered via subcutaneously implanted minipumps (0.5 μg peptide/0.5 μl/h). Learning of a spatial task was not impaired in the old animals, except for one measure, i.e. the latency to find the goal ☐. In neither age group did ORG 2766 influence behavioral performance. The number of corticosterone receptor sites was decreased in the hippocampus of senescent rats, but restored to the level observed in young rats following ORG 2766 treatment. It is concluded that the number of hippocampal corticosterone receptor sites is a sensitive index of brain aging and effectiveness of ORG 2766.     

Cerebral 2-deoxyglucose accumulation in mice following chronic treatment with an ACTH4–9 analog, ORG 2766

Mice were treated with [MET(O2)4, D-Lys8,Phe9]ACTH4-9 (ORG 2766, 100 micrograms/kg per day SC) for 10 days. On the tenth day mice were injected with [3H]2-deoxyglucose and its cerebral accumulation determined in 17 brain areas. The combined results of four experiments indicated a significant decrease of the 2-deoxyglucose accumulation in the septum. Changes observed in other brain areas were not statistically significant in the combined analysis of all four experiments. This selective change in the septum is consistent with selective uptake of ORG 2766 in this region, and with the lack of behavioral activity of ACTH in septal lesioned animals. It also suggests that the behavioral activity of this peptide, generally considered to be on arousal, vigilance, and/or selective attention, may be mediated through the septum, a limbic system structure.     

Autoradiographic studies with a behaviorally potent H-ACTH4–9 analog in the brain after intraventricular injection in rats

Autoradiographic studies aimed at identifying target cells in the brain for ACTH-like peptides were performed using (³H-7-Phe)-4-Met(O₂),8-D-Lys, 9-Phe-ACTH_4–9, a behaviorally potent analog of ACTH_4–9. The ³H-peptide was injected into the lateral ventricle of hypophysectomized rats that were sacrificed 5, 30, 60, 180, and 240 min later. Dry-mount autoradiograms of brain showed the highest density of silver grains in the ventricular lumen and choroid plexus. In addition, radioactivity penetrated brain tissue as far as 1000 μm from the ventricles, and was distributed predominantly over neuropil. Within 5 min after the injection, an intracellular concentration of radioactivity above back-ground levels was observed in a small proportion of cells near the ventricles in the septum, caudate-putamen, preoptic area, hypothalamus, thalamus, amygdala, and hippocampus. The cellular labeling decreased in intensity at greater distances from the injection site and at longer survival intervals, and was no longer evident 4 hr after the injection. The labeled cells were usually small, dark, and often elongated, suggesting that ACTH peptides may act preferentially upon a morphologically distinct class of cells in the brain.     

Effect of ACTH-like peptides on morphine-induced hypothermia in unrestrained guinea pigs

Peripheral treatment with adrenocorticotropin (1-24) (ACTH1-24), at different doses and sequences, consistently antagonized the decrease in body temperature produced by morphine in the freely moving guinea pig, whereas adrenocorticotropin (4-10) (ACTH4-10), which lacks corticotrophic activity, was partially effective only when it was administered in a high dose 24 h prior to morphine. Centrally administered ACTH1-24 completely prevented the hypothermic effect of intracerebroventricularly (i.c.v.)-injected morphine. Likewise, the i.c.v. administration of ACTH4-10 was equally effective in blocking the i.c.v. morphine-induced hypothermia. Neither ACTH1-24 nor ACTH4-10 did produce changes in body temperature. These results suggest that peripherally administered ACTH1-24 antagonizes indirectly the actions of morphine through the release of adrenal corticosteroids, whereas centrally injected ACTH1-24 or ACTH4-10 act as direct antagonists of morphine effects through opioid receptors.     

Cholinotoxic effects of β-amyloid(1–42) peptide on cortical projections of the rat nucleus basalis magnocellularis

β-Amyloid_(1–42) peptide (βAP) was injected into the right nucleus basalis magnocellularis (nbm) of rats. After a 14-day survival time, the acetylcholinesterase and choline acetyltransferase activities and the number of muscarinic receptors were found biochemically to be significantly reduced in the ipsilateral frontal cortices. Confirmation of these data with silver staining also revealed degeneration of the projective fibers of the nbm to the frontal cortex. These results demonstrate the cholinotoxicity of βAP in an in vivo animal model.     

The time course of excessive grooming after neuropeptide administration

In this paper we review the temporal pattern of excessive grooming in the hour or so following the central injection of ACTH1-24 in the rat. Changes in the grooming pattern after specific neuropharmacological manipulations of dopaminergic and opiate-related systems are presented which indicate a differential sensitivity of the grooming responses at different times after injection. The grooming affected by dopaminergic antagonists and opiate agonists and antagonists occurs in the last 30 min of the observation period while that found earlier is unaffected. It is also the grooming in this last 30 min of the observation period term tolerance to central administration of ACTH1-24. In contrast lesions of the central nervous system that affect excessive grooming, i.e., the substantia nigra and the hippocampus, reduce grooming throughout the observation period. The present analysis has provided evidence for dopamine/opiate insensitive and sensitive systems in excessive grooming, and thus temporal aspects are of extreme importance to the understanding of central neuropeptide influences on behavior.     

Electroencephalographic spreading depression and concomitant behavioral changes induced by intrahippocampal injections of ACTH1–24 andd-Ala-met-enkephalinamide in the rat

ACTH_1–24 (0.5or10 μg 0.17or3.45nmol) andd-Ala²-Met-enkephalinamide (DAME; 10 μg 17.05nmol) were injected unilaterally into the hippocampus of freely moving rats to examine their effects on EEG activity, DC potentials and behavior. In 85% of the rats DAME elicited spreading depression (SD) with epileptiform discharges preceding and following the wave of SD. The following behavioral changes were recorded. DAME- and KCl-induced SD were accompanied by an increase in locomotor activity and wet-dog shaking behavior, which occurred only during the period of SD. After a wave of SD induced by DAME a biphasic pattern of activity, consisting of an initial depression in locomotion followed by hyperactivity, appeared in 59% of the rats.ACTH_1–24 elicited SD in 13% of the rats tested. Neither the dosage of ACTH_1–24 nor the strain of rats influenced the occurrence of SD and the incidence of ACTH-induced grooming behavior. SD induced by KCl also resulted in excessive grooming comparable to that induced by ACTH_1–24. In the case of KCl-induced SD, grooming began directly after the injection of KCl and was frequently interrupted by short periods of locomotion. ACTH-induced grooming had a later onset and episodes of stretching and yawning were observed. It can be concluded that the behavioral effects of the injection of DAME are unspecific responses to SD and seizure activity. However, ACTH-induced grooming is not solely a byproduct of SD, since it occurred also in the absence of SD.     

Effects of ACTH4−10 on vestibular compensation

ACTH4-10, a fragment of the adrenocorticotropic hormone (ACTH) molecule, has marked effects on the compensation process following unilateral labyrinthectomy. In Rana temporaria ACTH4-10-treatment (5-250 micrograms/kg) influences both the acquisition and the maintenance of the compensated state. The compensation process is slowed down by hypophysectomy but can then be restored by the administration of ACTH4-10. It is concluded that ACTH-like neuropeptides might physiologically be involved in the plastic processes underlying functional recovery from CNS lesions.     

Influence of a 1 h immobilization stress on sleep states and corticotropin-like intermediate lobe peptide (CLIP or ACTH18–39, Ph-ACTH18–39) brain contents in the rat

A 1 h immobilization stress (IS) was imposed to rats at the beginning of the dark period, i.e., when the animals start to be active. The IS was accompanied by an intense polygraphic waking and followed, over 12 h of the dark period, by a significant rebound of slow-wave sleep (SWS, +17%) and paradoxical sleep (PS, +57%). In order to estimate the IS-related changes in the endogenous concentrations of corticotropin-like intermediate lobe peptide (CLIP, ACTH_18–39) and related compounds, a specific radioimmunoassay (RIA) was used. Assays performed in cerebral biopsies taken from arcuate (AN) and raphe dorsalis (nRD) nuclei led to the obtention of 2 main immunoreactive peaks, corresponding to CLIP and its phosphorylated form Ph-CLIP. Just after the end of the IS and within the nRD, Ph-CLIP immunoreactivity increased by about 95%. Four hours later, i.e., when PS rebound was maximal, a 37% increase in Ph-CLIP immunoreactivity was measured in the AN. These observations have never been described before. In the blood, at the end of the restraint, CLIP/ACTH_1–39 total immunoreactivity was increased by 330%. It returned to baseline level 4 h later. Blood concentration of corticosterone was also increased by 56% at the end of the IS and was close to baseline level 4 h later. Data reported here indicate that the IS first triggers an increase in Ph–CLIP within the nRD. Since the nRD contains sleep permissive components, this increase might be determinant for the SWS and PS rebound induction. The changes observed in the blood as regards CLIP/ACTH_1–39 total immunoreactivity and corticosterone concentration testify to the efficacy of the IS and are part of the conventional picture accompanying such a situation. Finally, the increase in Ph-CLIP, occurring in the AN 4 h after the end of the restraint, might be part of the restorative processes necessary to compensate the stress overshoot.© 1997 Elsevier Science B.V. All rights reserved.     

Beneficial effect of chronic treatment with Org 2766 and α-MSH on impaired reversal learning of rats with bilateral lesions of the parafascicular area

The effects of chronic treatment with the ACTH-(4–9) analogue Org 2766, α-MSH and γ₂-MSH were studied on T-maze reversal learning and on behavior assessed on the basis of open-field and other gross behavioral activities, grasping responses, inspection of various reflexes and electrical footshock sensitivity of rats with parafascicular lesions or sham-lesions. Repeated administration of Org 2766 and α-MSH to parafascicular area-lesioned rats resulted in functional recovery of impaired T-maze reversal learning. The structurally related neuropeptide γ₂-MSH was without any effect. The α-MSH effect did not depend on time after lesioning as treatments during the first or second post-operative week were equally effective. Chronic peptide treatments did not change disturbed motor functions of parafascicular-lesioned rats, as measured by open-field activity, other gross behavioral activities and grasping responses. Since acute peptide treatments did not affect the impaired reversal learning performance of lesioned rats, the beneficial effect of Org 2766 and α-MSH could not be explained as a short-term effect on attention and motivation. It was more likely to be an accelerated recovery of cognitive function as a result of long-term neurotropic influences.     

Increased NPY activity in the PVN contributes to food-restriction induced reductions in blood pressure in aortic coarctation hypertensive rats

We hypothesized that hypothalamic NPYergic mechanisms mediate the blood pressure lowering effect of caloric restriction in hypertensive rats. Aortic coarctation-induced (AC) hypertensive rats (n=25) were assigned to either an ad libitum fed control group (AL) or food restricted group (FR; 60% of AL consumption) for 3 weeks. Rats were instrumented chronically with vascular catheters and bilateral guide cannulae directed at the paraventricular hypothalamic nuclei (PVN). Blood pressure (BP) and heart rate (HR) responses to bilateral PVN microinjection of saline (200 nl) or the putative NPY receptor antagonists [D-Trp32]NPY(1-36) (3.3 micrograms/200 nl) and [D-Tyr27,36 Thr32]NPY(27-36) (D-NPY(27-36); 3.3 micrograms/200 nl) were determined. The FR rats were then refed and cardiovascular responses to PVN injections of NPY receptor antagonists were again determined. FR rats had significantly reduced resting BP (159+/-4 vs. 129+/-4 mmHg) and HR (360+/-11 vs. 326+/-9 bpm) compared to AL controls. Refeeding restored BP and HR of FR rats to levels similar to AL (BP=153+/-4 mmHg, HR=359+/-11 bpm). PVN administration of [D-Trp32]NPY produced foraging behavior and concurrent increases in BP and HR in FR, AL and Re-fed rats. The behavioral activation suggests that [D-Trp32]NPY(1-36) produced activation of NPY receptors. In contrast, D-NPY (27-36) did not produce any behavioral response or affect BP or HR in AL or Re-fed rats. In FR rats, D-NPY (27-36) produced significant increases in BP (peak=15+/-3 mmHg) which partially reversed the effect of FR on BP. Thus, in FR rats with reduced BP, PVN administration of an NPY receptor antagonist increases BP. NPY blockade in the PVN accounted for about 50% of the BP effect of food restriction, thus other mechanisms are likely to be involved. These findings are consistent with the hypothesis that NPYergic mechanisms may contribute to the reduction of BP produced by food restriction.     

Neuropeptides derived from prosomatostatin that do not contain the somatostatin-14 sequence

Two neuropeptides reacting with antibodies directed against the C-terminal region of somatostatin 28(1-12) were purified to homogeneity from rat brain extracts. Amino acid analysis revealed that the larger peptide (8 kdaltons) consisted of 76 amino acids. Microsequencing established its aminoterminal structure as: Ala-Pro-Ser-Asp-Pro-Arg-Leu-Arg-Gln-Phe-X-Gln-Lys. The 8 kdalton somatostatin 28(1-12)-like peptide corresponds to the whole prosomatostatin molecule without Arg-Lys-somatostatin-14. The smaller peptide (5 kdaltons) consists of 44 amino acids and is generated after cleavage of a Leu-Leu bond at position 56-57 of pre-prosomatostatin. Both 8 kdalton and 5 kdalton somatostatin 28(1-12)-like peptides contain somatostatin 28(1-12) at their C-termini. The 4 most abundant neuropeptides derived from pre-prosomatostatin (pre-proSS) and presently characterized are: somatostatin-14, somatostatin 28(1-12), somatostatin 28 and pre-proSS25-100.     

Selective FSH-releasing activity of [D-Trp]GAP1–13: Comparison with gonadotropin-releasing abilities of analogs of GAP and natural LHRHs

We had previously shown that fragments of human gonadotropin-releasing hormone associated peptide (GAP) stimulated FSH and LH release in vivo. In particular, GAP₁₋₁₃ had a preferential FSH-releasing activity. To decrease enzymatic degradation, analogs of GAP_1–13 with D-amino acid substitutions were synthesized. The activities were tested in ovariectomized, estrogen-progesterone primed (OEP) rats and compared with those of GAP₁₋₁₃, mammalian (m), chicken II (eII), and lamprey (1) LHRH. The peptides were injected (IV) into conscious, OEP rats and blood samples were obtained via the jugular catheter. [D-Trp⁹]GAP_1–13 selectively stimulated FSH release at a dose of 1 μg. Multiple injections of this analog (10 μg every 30 min for 5 injections) induced a marked elevation of plasma FSH values which peaked (p < 0.001) after the third injection. By contrast, [D-Trp⁹]GAP_1–13 had no effect on LH and prolactin (PRL) release after either single or multiple injections. These doses of [DAla⁴] GAP_1–13 had no effect on the release of FSH, LH or PRL. Both human GAP_1–13 and its [D-Trp⁹] analog exerted a selective FSH-releasing effect at a dose of 10 μg, however, the [D-Trp⁹] analog was more potent than GAP_1–13 on FSH release. The potency of [D-Trp⁹]GAP_1–13 in releasing FSH was approximately that of mLHRH. Chicken II LHRH had slightly selective FSH-releasing activity with a potency that of mLHRH. Lamprey LHRH had a preferential LH-releasing activity and a potency 1000 times less than mLHRH. In conclusion, [D-Trp⁹]GAP_1–13 is a selective FSH-releasing peptide of potential clinical value.     

Morphine and ACTH1–24: correlative behavioral excitations following microinjections in rat periaqueductal gray

Rats implanted with bilateral cannulas in the periaqueductal gray exhibited similar behavioral excitations following microinjections of morphine sulphate and ACTH_1–24. Injections were more effective when the sites were located within rather than below the periaqueductal gray. Analgesia was observed following morphine but not ACTH microinjection. These results confirm that morphine exerts a dual action, inhibitory (i.e. analgesic) and excitatory, with ACTH mimicking only the latter action.     

Corticotropin-releasing factor peptide antagonists: Design,characterization and potential clinical relevance

Elusive for more than half a century, corticotropin-releasing factor (CRF) was finally isolated and characterized in 1981 from ovine hypothalami and shortly thereafter, from rat brains. Thirty years later, much has been learned about the function and localization of CRF and related family members (Urocortins 1, 2 and 3) and their 2 receptors, CRF receptor type 1 (CRFR₁) and CRF receptor type 2 (CRFR₂). Here, we report the stepwise development of peptide CRF agonists and antagonists, which led to the CRFR₁ agonist Stressin₁; the long-acting antagonists Astressin₂-B which is specific for CRFR₂; and Astressin B, which binds to both CRFR₁ and CRFR₂.This analog has potential for the treatment of CRF-dependent diseases in the periphery, such as irritable bowel syndrome.     

Effects of β-endorphin2–9 on arginine-8-vasopressin and oxytocin levels in hypothalamic and limbic brain regions

Immunoreactive arginine-8-vasopressin (AVP) and oxytocin (OXT) were measured in rat hypothalamic and limbic brain regions after the intracerebroventricular administration of β-endorphin fragment 2–9 (βE_2–9). The peptide decreased the AVP content of the hippocampus and the OXT levels in the septum and amygdala. The present data favor the view that βE_2–9 interacts with limbic AVP- and OXT-systems.     

Immunohistochemical localization of the sigma1-receptor in oligodendrocytes in the rat central nervous system

By using a new polyclonal antibody raised against a 21-amino acid peptide sequence corresponding to the fragment 138-157 of the cloned rat sigma(1)-receptor, we demonstrated by immunoperoxidase and double immunofluorescence techniques, that rat oligodendrocytes express the sigma(1)-receptor. Experiments in vivo and in vitro showed that sigma(1)-receptor colocalized with specific markers of progenitor (A2B5) and mature oligodendrocytes (GalC, RIP). These results suggest that sigma(1)-receptor in oligodendrocytes might be involved in myelination by direct implication in cholesterol biosynthesis or by interaction with endogenous ligands such as neurosteroids.     

Two-Step Spreading Mode of Human Glioma Cells on Fibrin Monomer: Interaction of with the Substratum Followed by Interaction of with Endogenous Cellular Fibronectin Secreted in the Extracellular Matrix

Glioma cells, a human astrocyte-derived glioma cell line, were found to spread on immobilized fibrin monomer but not on fibrinogen. As a synthetic RGD-containing peptide GRGDSP blocked the spreading of glioma cells on fibrin monomer concentration-dependently, the spreading was thought to be mediated by their cell surface receptors. In fact, both the β₁- and β₃-integrins were located at 3 hours of incubation in the cytoplasmic areas and at 24 hours in the peripheral areas as well, although their distribution profiles were not necessarily identical with each other by immunohistochemical studies. By cytometry analysis utilizing respective monoclonal antibodies against α₅- and α_v-integrins, we were able to show expression of α₅ (α₅β₁) but not α_V on the surface of glioma cells at 24 hours of incubation on immobilized fibrin monomer. A 50-kDa transmembrane protein designated as integrin-associated protein (IAP) known to be closely associated with the β₃-integrin was also located in the cytoplasmic and apical areas of spreading glioma cells, but its specific antibody B6H12 failed to inhibit the spreading. Thus, the IAP-dependent involvement of β₃-integrin may not be predominantly involved in the glioma cell spreading on fibrin monomer. As an anti-α_Vβ₃ antibody LM 609 inhibited the spreading of glioma cells partially at approximately 35%, the spreading seems to proceed in a two-step mode, i.e., via α_Vβ₃ with its ligand exposed in fibrin monomer, and then via α₅β₁ with endogenous cellular fibronectin secreted from the glioma cells themselves. In fact, the cellular fibronectin was clearly visualized by confocal microscopic observation. Thus, upon contact with fibrin in clots formed at traumatized areas in the brain, for example, glioma cells may have a chance to adhere to and spread via α_Vβ₃ with fibrin monomer and then via α₅β₁ with endogenous cellular fibronectin in the extracellular matrices.     

Alpha-adrenergic agonists inhibit the dipsogenic effect of angiotensin II by their stimulation of atrial natriuretic peptide release

Angiotensin II (ANG-II) and atrial natriuretic peptide (ANP) have opposing actions on water and salt intake and excretion. Within the brain ANP inhibits drinking induced by ANG-II and blocks dehydration-induced drinking known to be caused by release of ANG-II. Alpha-adrenergic agonists are known to release ANP and antagonize ANG II-induced drinking. We examined the hypothesis that alpha agonists block ANG-II-induced drinking by stimulating the release of ANP from ANP-secreting neurons (ANPergic neurons) within the brain that inhibit the effector neurons stimulated by ANG-II to induce drinking. Injection of ANG-II (12.5 ng) into the anteroventral region of the third ventricle (AV3V) at the effective dose to increase water intake increased plasma ANP concentrations (P<0.01) within 5 min. As described before, previous injection of phenylephrine (an alpha(1)-adrenergic agonist) or clonidine (an alpha(2)-adrenergic agonist) into the AV3V region significantly reduced ANG-II-induced water intake. Their injection also induced a significant increase in plasma ANP concentration and in ANP content in the olfactory bulb (OB), AV3V, medial basal hypothalamus (MBH) and median eminence (ME). These results suggest that the inhibitory effect of both alpha-adrenergic agonists on ANG-II-induced water intake can be explained, at least in part, by the increase in ANP content and presumed release from these neural structures. The increased release of ANP from the axons of neurons terminating on the effector neurons of the drinking response by stimulation of ANP receptors would inhibit the stimulatory response evoked by the action of ANG-II on its receptors on these same effector neurons.     

Disruption of mdr1a p-glycoprotein gene results in dysfunction of blood–inner ear barrier in mice

P-glycoprotein (p-gp), a drug transporter in multidrug-resistant cancer cells, is a transmembrane protein encoded by mdr1a, mdr1b and mdr2 genes in mice. In our previous report, high level p-gp was immunohistochemically detected in capillary endothelial cells of the guinea pig inner ear, supporting a possible role as an extrusion pump in the blood–inner ear barrier (BIB). We investigated the functional involvement of p-gp in the inner ear using mdr1a gene knock-out mice [mdr1a(−/−) mice]. Pharmacokinetic analyses showed that mdr1a(−/−) mice displayed obviously increased accumulations of the p-gp-transported drugs doxorubicin (adriamycin, ADM) and vinblastine in the inner ear tissues compared with those in mdr1a(+/+) mice. Subsequent functional studies using auditory-evoked brainstem responses showed hearing impairment only in mdr1a(−/−) mice after administering these drugs. Furthermore, inhibition of p-gp function by co-administration of cyclosporin A (CsA) with doxorubicin (ADM) in mdr1a(+/+) mice resulted in increased accumulation of ADM in inner ear tissues and hearing impairment similar to that noted in mdr1a(−/−) mice. We conclude that mdr1a p-gp, which acts as an efflux pump in the inner ear, prevents ototoxicity induced by p-gp substrate drugs and contributes to a new functional mechanism in the BIB.