Temporal organization of the molecular clockwork and behavioral output were investigated in nocturnal rats housed in constant darkness and synchronized to nonphotic cues (daily normocaloric or hypocaloric feeding and melatonin infusion) or light (light-dark cycle and daily 1-h light exposure). Clock gene (Per1, Per2 and Bmal1) and clock-controlled gene (Vasopressin) expression in the suprachiasmatic nuclei was assessed over 24 h. Light and exogenous melatonin synchronized the molecular clock, signaling, respectively, 'daytime' and 'nighttime', without affecting temporal organization of behavioral output (rest/activity rhythm). By contrast, synchronization to hypocaloric feeding led to a striking temporal change between gene expression in the suprachiasmatic clock and waveform of locomotor activity rhythm, rats then becoming active during the subjective day (diurnal-like temporal organization). When the time of feeding coincided with activity offset, normocaloric feeding also synchronized the locomotor activity rhythm with no apparent switch in temporal organization. Peak of Per2 expression in the piriform cortex occurred between the beginning and the middle of the activity/feeding period, depending on the synchronizer. These data demonstrate that even though the suprachiasmatic clockwork can be synchronized to nonphotic cues, hypocaloric feeding likely acts downstream from clock gene oscillations in the suprachiasmatic nuclei to yield a stable yet opposite organization of the rest/activity cycle.

Under healthy conditions, the blood-brain barrier (BBB) limits the passage of solutes and cells from the blood to the CNS. During neurological diseases, BBB permeability increases dramatically and it has been hypothesized that drug carrier systems such as polymeric nanoparticles could cross the BBB and penetrate into the CNS. PEGylated polyalkylcyanoacrylate nanoparticles (long-circulating carrier) are one such system and have been investigated during experimental allergic encephalomyelitis (EAE). Brain and spinal cord concentrations of [(14)C]-radiolabelled PEGylated polyalkylcyanoacrylate nanoparticles were compared with another blood long-circulating carrier (poloxamine 908-coated polyalkylcyanoacrylate nanoparticles) and with conventional non-long-circulating polyalkylcyanoacrylate nanoparticles. The microscopic localization of fluorescent nanoparticles in the CNS was also investigated in order to further understand the mechanism by which the particles penetrate the BBB. The results demonstrate that the concentration of PEGylated nanoparticles in the CNS, especially in white matter, is greatly increased in comparison to conventional non-PEGylated nanoparticles. In addition, this increase was significantly higher in pathological situations where BBB permeability is augmented and/or macrophages have infiltrated. Passive diffusion and macrophage uptake in inflammatory lesions seems to be the mechanism underlying such particles' brain penetration. Based on their long-circulating properties in blood and on their surface characteristics that allow cell interactions, PEGylated nanoparticles penetrated into CNS to a larger extent than all the other formulations tested. Thus, PEGylated polycyanoacrylate nanoparticles are proposed here as a new brain delivery system for neuroinflammatory diseases.

Relapsing experimental allergic encephalomyelitis (EAE) was induced in DA rats and the ocular pathologic events were examined at the various phases of the illness. About 80% of EAE rats presented anterior uveitis (AU), even after complete EAE recovery. We studied the phenotype and localization of immunocompetent cells, the major histocompatibility complex (MHC) class I and II antigen expression, as well as the chemokine monocyte chemoattractant protein-1 (MCP-1) appearance. In control animals, there were many glial fibrillary acidic protein (GFAP)(+) cells and OX42(+) cells in the ciliary body, retina, optic nerve and chiasma. Except in retina, we observed constitutive MHC class I and II expression. During the EAE acute phase, there was up-regulation of MHC class II and GFAP antigens in iris, ciliary body, limbus, and optic pathways. MHC class I and ED2 antigens were expressed in meninges and in the prechiasmatic cisterna, by cells which could have a role in immune surveillance. MCP-1 mRNA was highly expressed in optic pathways during the acute phase and the protein was expressed by astrocytes, macrophages, and lymphocytes. During the relapsing phase, MCP-1 was weakly expressed to disappear almost completely during the final recovery phase. The expression of MHC class II on astrocytes was increased during the relapsing and final recovery phase in which the inflammatory lesions persisted. These findings suggest that ocular areas and optic pathways, mainly optic chiasma, are important targets in the relapsing EAE.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterised by a disruption of the blood-brain barrier (BBB), demyelination and a relevant inflammatory reaction with an intense infiltration of macrophages. These neurological disorders are similar to those observed in the multiple sclerosis (MS) disease. The use of different liposomes and adeno-associated virus has been proposed for improving the treatment of this pathogenesis. The aim of this work was to evaluate the potential and capacity of albumin nanoparticles to reach the central nervous system (CNS) in EAE-induced rats. For this purpose, the distribution of biotinylated nanoparticles within the CNS was studied. Albumin carriers were mainly found in the lumbar portion of the spinal cord, overlying the meningeal and perivascular areas. The optic chiasma, iris and the area of the Purkinje cells of the cerebellum revealed also an intense presence of these carriers. Finally, immunohistochemical studies also revealed that circulating macrophages (ED1), which migrate to damaged sites, and resident activated microglial cells (OX42) were involved in the distribution of albumin nanoparticles. In summary, the use of nanoparticles may be useful for the design of new pharmaceutical dosage forms able to target the lesions associated with alterations of the BBB.

The effects of enriched environment on short-term memory for event durations and on astrocytes (cell density, cell area and % of GFAP immunoreactivity) in hippocampus (Hi), frontal cortex (FC) and corpus callosum (CC) were analysed in old rats housed from weaning to the end of behavioural testing (23 months) either in standard (SC) or in enriched (EC) conditions and in young adults (5 months) all housed in SC. Old SC and EC and young SC rats trained (for 2 months) or not, in a Symbolic Delayed Matching to Sample Task, had to discriminate and remember two (2- and 10-s) signals after short retention intervals. Results confirm the aging-related acquisition and memory deficit. EC reduced the slowness of acquisition, reversed the short-term memory deficit and promoted the retention of the short signal (choose short effect). Old SC naive rats had many hypertrophied astrocytes with long processes in Hi and CC while old EC rats had decreased astrocytes number and size. The behavioural testing resulted in young adult SC rats in Hi and CC, in increased astrocytes number, size and GFAP% and in their decrease in old SC rats. EC and testing have additive effects (very low astrocytes number, size and GFAP%) to compensate for the aging-induced gliosis, mostly in Hi.

Apoptosis or programmed cell death contributes significantly to the regulation of animal cell populations under physiologic conditions, in contrast to necrosis which is pathological cell death. A distinctive pattern of DNA fragmentation demonstrable using the Tunel technique is seen in apoptosis. In a study performed in rats with EAE, we found apoptotic cells (expression of Fas and Fast) among not only CD4(+) lymphocytes and macrophages but also among neurones and glial cells during the recovery phase of the illness. Also, the proinflammatory cytokine TNFalpha was expressed at a high level during the acute phases of EAE and subsequently persisted after the recovery phase. These results suggest that TNFalpha may contribute to the induction of apoptosis via a mechanism involving ACTH and glucocorticoids. Apoptotic death of striatal neurones has been reported in Huntington's disease.

L'apoptose ou mort programmee joue un role important dans la regulation des populations de cellules animales dans des conditions physiologiques normales, contrairement a la necrose qui est une mort pathologique. L'apoptose est caracterisee par une fragmentation typique du DNA, mise en evidence par la technique du TUNEL. Chez des rats avec une encephalomyelite allergique experimentale (EAE) induite, nous avons mis en evidence des cellules apoptotiques (expression du Fas et du FasL), non seulement parmi les lymphocytes CD4 + et les macrophages, mais aussi dans les neurones et les cellules gliales, et ceci au cours de la phase de recuperation de la maladie. Nous avons aussi montre que le TNFα, une cytokine proinflammatoire, est exprimee de facon intense au cours des phases aigues de l'EAE mais persiste encore apres la phase de recuperation. Ces resultats suggerent que le TNFα participe a l'induction de l'apoptose par l'intermediaire de l'ACTH et des glucocorticoides. Dans la maladie de Huntington, une mort apoptotique des neurones du striatum a ete signalee.

Reactive gliosis is a reaction of glial cells to trauma which is characterized by a phenotypic modification of astrocytes, as well as by a proliferation and a migration of some of these cells to form a glial scar. This scar is currently considered as a physical impediment to neuronal regrowth but it may also be involved in wound healing since the astrocytes beside microglia play a phagocytic role in the clearance of post-traumatic debris. Growth factors are released in the area of the injury and at least some of them could be involved in gliosis. In order to test directly this possibility, we have injected one of them, the basic fibroblast growth factor (bFGF), into several brain areas (cortex, striatum, hippocampus or corpus calIosum) of adult 2-month-old rats in the absence of lesion. A glial reaction was observed after 3 days and was maximum after 7 days. It was characterized by an increase in astrocyte proliferation and in glial fibrillary acidic protein (GFAP) expression, resulting in a higher number of GFAP-positive cells per surface unit, and by an increase in the size and branching of the astroglial processes. The GFAP mRNA levels were also strongly increased following the bFGF injection. These effects resemble the reactive gliosis observed after lesion and suggest that bFGF is actually involved in the triggering of glial reactions which follow brain injury. In further experiments, bFGF was injected in the site of electrolytic lesions made in the same various parts of the brain. These injections did not increase significantly the normal reactive gliosis induced by the lesion alone, but it accelerated some of the effects. It also resulted in a higher labeling index and GFAP mRNA levels were strongly enhanced after a 3-day-post-operative delay. This last observation strengthens the idea that one of the main factors driving the astrogliosis is the bFGF normally released in and around the site of the lesion.

We have examined the regulation of neuron-specific gamma-enolase gene (NSE) expression in oligodendrocytes at various steps of their differentiation/maturation. We have demonstrated for the first time that NSE is expressed in oligodendroglial cells in vitro and in vivo, and only at a certain stage of differentiation. A heterogeneity of the gamma subunit was observed in cultured oligodendrocytes and the same one was found in adult rat brain. The level of gamma mRNA increased when precursor cells differentiated into oligodendrocytes. By contrast, no significant change in alpha-enolase gene expression was observed. High NSE (gamma gamma and alpha gamma) enolase activity was detected in cultured oligodendrocytes. Treatment with basic fibroblast growth factor, which stimulates the proliferation of oligodendrocyte precursor cells and reversibly blocks their differentiation, resulted in lower alpha gamma- and gamma gamma-enolase activities in these cells, but it enhanced alpha alpha-enolase activity slightly. These data indicate that gamma-enolase gene expression is associated with the differentiation of the oligodendrocytes and that it is repressed in adult fully mature cells.

Electrolytic lesions performed in brain cortex of six-day-old or adult rats resulted in the appearance of many reactive astrocytes around the injury site after a postoperative delay of eight days. They were revealed by immunohistochemistry using antibodies against glial fibrillary acidic protein. Injection of tritiated thymidine 24 h prior to autopsy indicated that, in neonates, 50% of the reactive astrocytes were proliferating. Infusion of 2 microliters of liposome suspension made of phosphatidylcholine and a monosialoganglioside, in the injury site, immediately after the electrolytic lesion did not modify the extent of the reactive gliosis. Liposomes containing 3 nmol of either 7 beta-hydroxycholesterol, 7 beta-hydroxycholesteryl-3-stearate or 7 beta-hydroxycholesteryl-3-oleate reduced by about 50% the intensity of the reactive gliosis in the frontal cortex of six-day-old rats and by 40% the number of dividing astrocytes. In the adult rat cortex the intensity of the glial reaction was also decreased by 30% by 15 nmol 7 beta-hydroxycholesteryl-3-oleate. Further investigations demonstrated that it is the 7 beta-hydroxy function which is needed for the biological activity of these oxysterols. These findings, which demonstrate anti-proliferative and anti-inflammatory properties of 7 beta-hydroxycholesterol on astrocytes, facilitate the future investigation of the influence of reactive gliosis on functional recovery following brain injury. This anti-proliferative property could also be used in other kinds of pathologies involving glial cell proliferation, such as glioblastomas.

a single injection of NGF was sufficient to alleviate some behavioral deficits in a long-lasting fashion;

The effects of basic fibroblast growth factor (bFGF) on the morphology and the expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) in cultured astrocytes prepared from various areas of newborn rat brain was studied. The brain was dissected in two ways, either the telencephalon (area A) and the diencephalon (area B) were dissected out of the brain (without olfactory bulbs, mesencephalon and cerebellum) or the brain was cut transversely into 3 parts (areas 1, 2 and 3). Area 1 (the anterior part) included the frontal cortex, the olfactory nuclei, the neostriatum, the accumbens nucleus and the septum; area 2 (the medial part) included the cortex, hippocampus, amygdale, thalamus and hypothalamus, and area 3 (the posterior part) included the occipital cortex, the posterior part of hippocampus and thalamus and the mamillary bodies. Essentially two different morphological aspects were observed. Most cells from areas A, 1 and 3, were flat, large, presented an irregular shape and were loosely arranged; cells from areas B and 2 were essentially polygonal in shape and closely apposed to each other. The various control cultures showed nearly the same immunostaining pattern for GFAP, but different patterns for GS. Most astroglial cells responded to bFGF and became fibrous. The GFAP immunoreaction was intense and localized in the cell bodies and processes of most cells from area A, but essentially in the processes for cells from areas 1 and 2. The immunoreactivity was weaker in cells from areas B and 3. GS-positive cells, heavily and weakly stained, were found in all treated cultures, and very strongly stained cells were located in certain zones of cultures from area A. But GS-negative cells were also seen in these treated cultures as well as in control cultures. Measurements of GS activities revealed no differences. These results indicate that astrocytes from different regions of the brain in primary culture show differences in their responsiveness to bFGF. The astroglial cells from the cerebral cortex and from the thalamus seem to present the highest and the lowest response to bFGF, respectively.

Reactive gliosis was revealed by immunocytochemistry using antibodies against the glial fibrillary acidic protein (GFAP) after a stab or an electrolytic lesion administered to the cerebral cortex, corpus callosum, striatum, or hippocampus of a 6‐day‐old rat. The intensity of the gliosis was about the same in the various structures injured and did not change with the delay of 3, 7, or 20 days between the injury and the sacrifice of the animals. When basic fibroblast growth factor (bFGF) was injected in the lesion locus just after the lesion was performed, it resulted (as soon as 3 days after injury) in a strong astrogliosis that was enhanced after a delay of 7 days, the astrocytes in the lesion area exhibiting enlarged cell processes and intense GFAP‐positive immunoreactivity. After a delay of 20 days, the astrocytes were not dispersed any more but packed in three or four layers along the borders of the lesion, thus reducing its extension. This suggests a possible role for bFGF in promoting scar formation following brain injury.

Adult rats received a partial and unilateral transection of the fimbria. They received then intracerebroventricular (i.c.v.) injections of 5 microliters of Tris, half of them containing 2.5 ng of basic fibroblast growth factor (bFGF). They were injected twice a week for 4 weeks. At the end of this period, choline acetyltransferase (ChAT) activity was measured in the hippocampus. ChAT activity, which was decreased by the fimbria transection, was higher (by about 20%) in medial hippocampus of the bFGF group compared with the Tris group. In addition, bFGF enhanced the lesion-induced astroglial reaction by changing the morphology of the astrocytes and increasing the apparent number of these reactive astrocytes.

Male Wistar rats sustaining prefrontal cortex aspiration or sham operation at 6 days or 30 days of age were submitted to the following behavioural tests: open-field, acquisition and retention of two-way active as well as passive avoidance tasks. In the open-field the locomotor activity proved enhanced in all the aspirated animals and this enhancement lasted for 30 days. In the two-day active avoidance task, an acquisition deficit was observed in both aspirated groups; but when retrained one month later, they were able to acquire the avoidance task like sham-operated rats and no difference appeared between the groups aspirated at 6 or at 30 days of age. Concerning the passive avoidance task, no difference could be detected between aspirated and sham-operated animals of both groups except that the rats aspirated at an early age (6 days) seemed to display a better avoidance ability in the retention test. These behavioural alterations (hyperactivity and impairment of the acquisition of the 2-way active avoidance) resulted from the prefrontal cortex aspiration, at whatever age this aspiration was performed (6 days or 30 days). They disappeared after a postoperative recovery period of about one month, as evidenced by this longitudinal study.

To assess the behavioral effects of preoperative differential housing male rats were placed in either enriched or isolated environments at weaning prior to receiving either sham operations or septal lesions when 57 days of age. Rats with septal lesions showed reduced habituation of ambulation and initially made fewer rears in an empty open field but made more object-contacts coupled with a lack of habituation in the object-filled field. Septal rats also showed severe impairments when tested in a 12-arm radial maze with 7 arms baited and 5 arms unbaited. Preoperative enrichment did not significantly affect these lesion-induced changes. Nevertheless, enrichment significantly lowered ambulation (but did not affect habituation) in the open field and increased the number of manipulatory relative to nonmanipulatory contacts. However, preoperatively enriched septal rats showed a deficit in spontaneous alternation (45%) in contrast to the high levels (83%) shown by intact enriched rats, whereas both intact and septal isolated rats showed similar levels of spontaneous alternation (68%). These results conflict with earlier reports that preoperative enrichment "protects" rats against the deficits produced by septal lesions.

Kant, G. J., R. A. Bauman, J. J. Widholm, S. Ghosh and N. Sharma. Sustained stress and fixed interval performance. Physiol Behav 61(2) 279–284, 1997.—The effects of sustained stress on response rate and temporal patterning (quarter-life) of rats performing either a previously learned fixed-interval schedule (FI 60) or learning an FI 60 simultaneously with stress onset were determined. Rats lived 24 h/day in operant cages, where they earned all of their food via lever-pressing. During the stress portion of each experiment, one group of rats was able to avoid or escape signalled intermittent footshock (Avoidance/Escape Group), a second group (Yoked) did not have control over shock termination, a third group never received shock (Control). Shock trials were presented around the clock at approximately 5-min intervals and the stress portion of each study lasted 1–2 weeks. We have previously reported that rats tolerate this paradigm well and avoid/escape 99% of the shock trials. In rats previously trained on the FI task, both rate of responding and quarter-life values were significantly decreased on the first day of stress for both the Avoidance/Escape and Yoked Groups. Food intakes and quarter-life values were not significantly different from the controls by stress Days 3 and 2, respectively. In the acquisition study, controls learned the FI task by Day 4 as judged by quarter-life of responding. FI task acquisition was significantly impaired in stressed rats compared to controls, not reaching asymptotic performance until Day 9 of stress. There were no major differences between the 2 stress groups in either study. These data demonstrate that stress may impair both the rate and patterning of behavior, and suggest that this rodent paradigm may usefully model some aspects of the effects of stress in humans.

The effects of acute administration of the dihydropyridine calcium channel antagonist, nimodipine, were studied on the actions of ethanol in the radial arm maze and the object recognition test. In the former test, the effects of the drugs were examined on the performance in finding the four baited arms, after previous training in this task. Ethanol, at 1 g/kg, increased both the number of re-entries into baited arms (counted as errors of working memory) and the total number of arm choices required to complete the task. Administration of nimodipine, 10 mg/kg, with the ethanol, completely prevented the deleterious effects on memory in this task, but had no effects on the performance when given in the absence of ethanol. In the object recognition task, ethanol, 1 g/kg, significantly decreased the differences in the time spent exploring novel and familiar objects. Nimodipine, 10 mg/kg, given with the ethanol, completely prevented this effect, but nimodipine alone had no effects. The lack of changes in total exploration times indicated that the effects of ethanol in these tests were not due to loss of motor co-ordination or of alertness. The results are discussed in the light of the known actions of the drugs on brain function.