Effects of prenatal stress on maternal behavior in the rat.

Some authors reported a link between maternal stress and disturbances in their infants. Because of difficulties due to human research, the effects of prenatal stress have to be examined in animal models. Our approach was original in that the stressor was an ecological one and was applied at a given gestational day. Indeed, the stressor was a cat and the effects of stress on maternal behavior were investigated in five groups of 10 female rats: two groups were composed of females which were acutely stressed either at the 10th or the 14th gestational day; two other groups were composed of females which were repeatedly stressed either at the 10th or the 14th gestational day; the fifth group comprised non-stressed females. Plasma corticosterone concentrations measured in blood samples collected from dams just after stress were significantly higher than in controls showing that cat represents an efficient stressor for rats. Maternal behavior was recorded during 30 min at the 2nd, 4th, and 6th postnatal days. In all cases, stressed dams' activities directly directed towards the pups (retrieving, sniffing and licking), those non-directly directed towards the pups (carrying its tail and digging the sawdust), and those directed towards themselves (eating, drinking and resting) were altered to different degrees. These alterations in maternal behavior can explain, at least in part, the mortality and the low growth rate observed in pups born from stressed dams.

The effects of a mild stressor on spontaneous alternation in mice.

Adult mice of the C57BL/6J strain were subjected to an acute mild stress at different periods before the choice trial of a spontaneous alternation test. The stressful event was either inescapable (forced exploration in a brightly lit open field) or escapable (temporary entries into an adjacent dark chamber). While unstressed control mice alternated above chance in each condition, mice subjected to inescapable stress did not alternate when forced exposure to the open field occurred during the entire retention interval. The same effect was seen when forced exposure to the open field occurred after a post-forced trial delay period. However, no change in the alternation rate was observed when the inescapable stress occurred before the forced trial, or if the delay intervened between the stressful event and the choice trial. The escapable stressful event had no effect on spontaneous alternation. These results indicate the role of cognitive mediation in the behavioral effects of inescapable stress, causing either a retrieval deficit or neophobia.

The endozepine ODN stimulates [3H]thymidine incorporation in cultured rat astrocytes.

High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10(-14) to 10(-11) M), ODN caused a dose-dependent increase of [3H]thymidine incorporation. At higher doses (10(-10) to 10(-5) M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10(-6) M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10(-6) M) had no effect. The ODN-induced stimulation of [3H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The GABAA receptor antagonist bicuculline (10(-4) M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABAA agonist 3APS (10(-10) to 10(-4) M) also induced a dose-related increase of [3H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABAA receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation.

GABA inhibits endozepine release from cultured rat astrocytes.

In the mammalian brain, the endogenous ligands for benzodiazepine receptors (also called endozepines) are predominantly synthesized by glial cells. It has recently been reported that rat astrocytes in primary culture release substantial amounts of endozepines. The aim of the present study was to investigate the possible involvement of GABA in the control of endozepine release. Exposure of cultured rat astrocytes to GABA (10(-7) to 10(-5) M) induced a dose-related inhibition of endozepine secretion. At higher doses (3 x 10(-5) to 10(-3) M), the effect of GABA gradually diminished. The inhibitory effect of GABA (10(-5) M) was mimicked by the GABA(B) receptor agonist baclofen (10(-5) M). In contrast, the GABA(A) receptor agonists 3APS and isoguvacine (10(-5) M each) did not modify endozepine release. The inhibition of endozepine secretion evoked by GABA and baclofen (10(-5) M each) was totally abrogated by the specific GABA(B) receptor antagonist phaclofen (10(-4) M). GABA and baclofen caused a significant inhibition of forskolin-evoked production of cAMP in astrocytes and this effect was abolished in the presence of phaclofen. In contrast, isoguvacine had no effect on cAMP production. Exposure of astrocytes to dbcAMP induced a time- and dose-dependent stimulation of endozepine release. These data indicate that GABA, acting through GABA(B) receptors negatively coupled to adenylyl cyclase, inhibits endozepine release from cultured rat astrocytes. The secretion of endozepines thus appears to be a valuable marker to monitor astrocyte activity.

Structure-activity relationships of a series of analogues of the octadecaneuropeptide ODN on calcium mobilization in rat astrocytes.

The octadecaneuropeptide ODN (QATVGDVNTDRPGLLDLK), originally characterized as an endogenous ligand for central-type benzodiazepine receptors, increases intracellular calcium concentration ([Ca2+]i) in rat astroglial cells. A series of ODN analogues was synthesized, and each compound was studied for its ability to induce Ca2+ mobilization in cultured rat astrocytes. Replacement of each amino acid by an L-alanine residue (AlaScan) showed that the N-terminal region of the molecule was relatively tolerant to alanine substitution (2-8, 10), except for the Ala9-substituted analogue (9) which was totally devoid of activity. Pyroglutamization (21) and acetylation (22) of the Gln1 residue reduced the Ca2+ response suggesting that a free N-terminal amine function is required for full activity of ODN. Alanine substitution of the residues in the C-terminal region of the molecule (11-14, 16-18) significantly reduced the biological activity of ODN. In particular, modifications of the Leu15 residue (15, 20) abolished the Ca2+-mobilizing activity. The analogues [Ala9]ODN (9), [Ala15]ODN (15), [D-Thr9]ODN (19), and [D-Leu15]ODN (20) partially antagonized the Ca2+ response evoked by ODN. Most importantly, the octapeptide ODN11-18 (OP, 24) produced a dose-response curve that was superimposable to that obtained with ODN, indicating that the C-terminal region of the molecule possesses full biological activity. Finally, the AlaScan of OP revealed that replacement of the Leu5 residue by Ala (29) or D-Leu (33) totally suppressed the calcium response, confirming the crucial contribution of the Leu15 residue of ODN to the biological activity of the neuropeptide.

Immunocytochemical localization of enkephalins in the brain of the African lungfish, Protopterus annectens, provides evidence for differential distribution of Met-enkephalin and Leu-enkephalin.

The distribution of various opioid peptides derived from proenkephalin A and B was studied in the brain of the African lungfish Protopterus annectens by using a series of antibodies directed against mammalian opioid peptides. The results show that both Metenkephalin- and Leu-enkephalin-immunoreactive peptides are present in the lungfish brain. In contrast, enkephalin forms similar to Met-enkephalin-Arg-Phe, or Met-enkephalin-Arg-Gly-Leu, as well as mammalian alpha-neoendrophin, dynorphin A (1-8), dynorphin A (1-13), or dynorphin A (1-17) were not detected. In all major subdivisions of the brain, the overwhelming majority of Met-enkephalin- and Leu-enkephalin-immunoreactive cells were distinct. In particular, cell bodies reacting only with Leu-enkephalin antibodies were detected in the medial subpallium of the telencephalon, the griseum centrale, the reticular formation, the nucleus of the solitary tract, and the visceral sensory area of the rhombencephalon. Cell bodies reacting only with Met-enkephalin antibodies were found in the lateral subpallium of the telencephalon, the caudal hypothalamus, and the tegmentum of the mesencephalon. The preoptic periventricular nucleus of the hypothalamus exhibited a high density of Metenkephalin-immunoreactive neurons and only a few Leu-enkephalin-immunoreactive neurons. The distribution of Met-enkephalin- and Leu-enkephalin-immunoreactive cell bodies and fibers in the lungfish brain showed similarities to the distribution of proenkephalin A-derived peptides described previously in the brain of land vertebrates. The presence of Met-enkephalin- and Leu-enkephalin-like peptides in distinct regions, together with the absence of dynorphin-related peptides, suggests that, in the lungfish, Met-enkephalin and Leu-enkephalin may originate from distinct precursors.

The stimulatory effect of the octadecaneuropeptide (ODN) on cytosolic Ca2+ in rat astrocytes is not mediated through classical benzodiazepine receptors.

Diazepam-binding inhibitor has been initially isolated from the rat brain from its ability to compete with benzodiazepines for their receptors. We have recently shown that the octadecaneuropeptide (diazepam-binding inhibitor-(33-50) or ODN) induces an increase in cytosolic free Ca2+ concentration ([Ca2+]i) in astroglial cells. The purpose of the present study was to determine whether central-type benzodiazepine receptors or peripheral-type benzodiazepine receptors are involved in the response of cultured rat astrocytes to ODN. The mixed central-/peripheral-type benzodiazepine receptor ligand flunitrazepam (10(-10) to 10(-6) M), the specific peripheral-type benzodiazepine receptor agonist Ro5-4864 (10(-10) to 10(-6) M) and the peripheral-type benzodiazepine receptor 'antagonist' PK 11195 (10(-9) to 10(-6) M) all induced a dose-dependent increase in [Ca2+]i. At high doses (10(-7) to 10(-5) M), the central-type benzodiazepine receptor agonist clonazepam also mimicked the stimulatory effect of ODN on [Ca2+]i. However, the [Ca2+]i rise induced by ODN was blocked neither by PK 11195 nor by the central-type benzodiazepine receptor antagonist flumazenil (10(-6) M each). Binding of [3H]flunitrazepam to intact astrocytes was displaced by low concentrations of the peripheral-type benzodiazepine receptor ligands flunitrazepam, Ro5 4864 and PK 11195, and by high concentrations of clonazepam. In contrast, ODN did not compete for [3H]flunitrazepam binding in intact cells. These data indicate that the effect of ODN on Ca2+ mobilization in rat astrocytes is mediated by high affinity receptors which are not related to classical benzodiazepine receptors.

Neuropeptide tyrosine in the brain of the African lungfish, Protopterus annectens: immunohistochemical localization and biochemical characterization.

Lungfishes, which share similarities with both fishes and amphibians, represent an interesting group in which to investigate the evolutionary transition from fishes to tetrapods. In the present study, we have investigated the localization and biochemical characteristics of neuropeptide Y (NPY)-immunoreactive material in the central nervous system of the African lungfish, Protopterus annectens. NPY-immunoreactive cell bodies were found in various regions of the brain, most notably in the telencephalon (septal area, ventral striatum, and nucleus accumbens), in the diencephalon (preoptic nucleus, periventricular region of the hypothalamus, and ventral thalamus), and in the tegmentum of the mesencephalon. A strong immunoreaction was also detected in cell bodies of the nervus terminalis. Immunoreactive nerve fibers were particularly abundant in the ventral striatum, the nucleus accumbens, the diagonal band of Broca, the hypothalamus, and the mesencephalic tegmentum. Positive fibers were also seen in the median eminence and in the neural lobe of the pituitary. The NPY-immunoreactive material localized in the brain and pituitary was characterized by combining high-performance liquid chromatography (HPLC) analysis and radioimmunological quantitation. The displacement curves obtained with synthetic porcine and frog NPY and serial dilutions of brain and pituitary extracts were parallel. Reversed-phase HPLC analysis of telencephalon, diencephalon, and pituitary extracts resolved a major NPY-immunoreactive peak that coeluted with frog NPY. The similarity between the distribution of NPY-containing neurons and the biochemical characteristics of the immunoreactive peptide in the brain of lungfish and frog strongly favors a close phylogenetic relationship between dipnoans and amphibians.

Distribution of FMRFamide-like immunoreactivity in the brain of the lungfish Protopterus annectens.

The distribution of FMRFamide-like immunoreactive peptides was studied in the brain of the African lungfish, Protopterus annectens, using the indirect immunofluorescence technique. The main populations of FMRFamide-positive cell bodies were detected in the forebrain and in the mesencephalic tegmentum. In the telencephalon, only a small number of FMRFamide-immunoreactive neurons was localized at the level of the subpallium, in the nucleus septi medialis. The diencephalon contained two prominent groups of FMRFamide-positive cell bodies located in the preoptic and periventricular preoptic nuclei. The thalamus exhibited only scattered FMRFamide-immunoreactive perikarya in its ventral part. In the mesencephalon, a group of positive cell bodies was identified in the caudal region of the tegmentum. A strong immunoreaction was also detected in the nervus terminalis. In the pituitary, most of the cells of the intermediate lobe were brightly stained. FMRFamide-like immunoreactive fibers and nerve terminals were widely distributed in the brain. In the telecephalon, numerous fibers were observed in several regions of the pallium and subpallium. A dense plexus of fibers was found in the hypothalamus and the thalamus. Immunoreactive fibers were seen coursing along the ventral wall of the infundibular cavity and terminating in the pars nervosa of the pituitary. The tectum and the ventral mesencephalon were also densely innervated. In contrast, the caudal brainstem only showed scarce immunoreactive processes. Coexistence of FMRFamide- and neuropeptide Y-like immunoreactivity was observed in the preoptic nucleus and in the nervus terminalis. The widespread distribution of FMRFamide-immunoreactive neurons in the brain and pituitary of P. annectens suggests that the peptide may exert both neuromodulator and neuroendocrine functions. The similarity between the distribution patterns of FMRFamide and neuropeptide Y in the brain of lungfish and amphibians supports the concept of a close phylogenetic link between these two groups.