Expression of erythropoietin receptor protein in the mouse hippocampus in response to normobaric hypoxia.

Background: Over the past decades, accumulating research on erythropoietin (EPO) and its receptor (EPOR) has revealed various neuroprotective actions and upregulation in hypoxic conditions. To our knowledge, EPOR protein levels in the hippocampus and isocortex have never been measured. Therefore, the aim of this study was to measure EPOR protein in the hippocampus (HPC) and prefrontal cortex (PFC). Further objectives were to examine the effects of exposure to normobaric hypoxia of various degrees and durations on EPOR protein and to explore how long-lasting these effects were. Method: Adult C57BL/6 mice were randomized into a control group (N = 12) or various hypoxia groups (N = 5-11). Mice were exposed to three different O2 concentrations (10 %, 12 %, or 18 %) for 8 h a day for 5 days and sacrificed immediately after the last exposure. The effect of exposure to 12 % O2 for 1 day and 4 weeks (8 h per day) at this survival time was also examined. Additionally, groups of mice were exposed to 12 % O2 for 1 or 5 days (8 h per day) and euthanized at various times (up to 3 weeks) thereafter to examine the duration of EPOR protein regulation in the HPC and the PFC. EPOR protein was detected with a sandwich-ELISA method. Results: EPOR protein was present in the HPC and PFC, at 206.64 ± 43.98 pg/mg and 184.25 ± 48.21 pg/mg, respectively. The highest increase in EPOR protein was observed in the HPC after 5 days of 8 h exposure to 12 % O2 and was most pronounced 24 h after last exposure. The effect of hypoxia normalized within one week after the last exposure. Conclusion: This study successfully measured hippocampal EPOR protein and showed a significant association between normobaric hypoxia and acute EPOR elevation. It is our hope that this study can provide guidance to future research on the neuroprotective effects of EPO.

Glucagon-like peptide 1 receptor activation regulates cocaine actions and dopamine homeostasis in the lateral septum by decreasing arachidonic acid levels.

Agonism of the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. However, the molecular mechanisms and brain circuits underlying the therapeutic effects of GLP-1R signaling on cocaine actions remain elusive. Recent evidence has revealed that endogenous signaling at the GLP-1R within the forebrain lateral septum (LS) acts to reduce cocaine-induced locomotion and cocaine conditioned place preference, both considered dopamine (DA)-associated behaviors. DA terminals project from the ventral tegmental area to the LS and express the DA transporter (DAT). Cocaine acts by altering DA bioavailability by targeting the DAT. Therefore, GLP-1R signaling might exert effects on DAT to account for its regulation of cocaine-induced behaviors. We show that the GLP-1R is highly expressed within the LS. GLP-1, in LS slices, significantly enhances DAT surface expression and DAT function. Exenatide (Ex-4), a long-lasting synthetic analog of GLP-1 abolished cocaine-induced elevation of DA. Interestingly, acute administration of Ex-4 reduces septal expression of the retrograde messenger 2-arachidonylglycerol (2-AG), as well as a product of its presynaptic degradation, arachidonic acid (AA). Notably, AA reduces septal DAT function pointing to AA as a novel regulator of central DA homeostasis. We further show that AA oxidation product γ-ketoaldehyde (γ-KA) forms adducts with the DAT and reduces DAT plasma membrane expression and function. These results support a mechanism in which postsynaptic septal GLP-1R activation regulates 2-AG levels to alter presynaptic DA homeostasis and cocaine actions through AA.

Fluoxetine reverts chronic restraint stress-induced depression-like behaviour and increases neuropeptide Y and galanin expression in mice.

Stressful life events and chronic stress are implicated in the development of depressive disorder in humans. Neuropeptide Y (NPY) and galanin have been shown to modulate the stress response, and exert antidepressant-like effects in rodents. To further investigate these neuropeptides in depression-like behaviour, NPY and galanin gene expression was studied in brains of mice subjected to chronic restraint stress (CRS) and concomitant treatment with the antidepressant fluoxetine (FLX). CRS caused a significant increase in depression-like behaviour that was associated with increased NPY mRNA levels in the medial amygdala. Concomitant FLX treatment reverted depression-like effects of CRS and led to significant increases in levels of NPY and galanin mRNA in the dentate gyrus, amygdala, and piriform cortex. These findings suggest that effects on NPY and galanin gene expression could play a role in the antidepressant effects of FLX.

Electroconvulsive stimulations prevent stress-induced morphological changes in the hippocampus.

Stress can precipitate major depression and other disorders linked to hippocampal shrinkage. It is hypothesized but not established that treatment of these disorders reverses and prevents the hippocampal changes. Dendritic retraction of individual neurons might in concert with other pathophysiological events contribute to the shrinkage phenomenon. Animal studies have shown that various stress paradigms can induce dendritic retraction in the CA3 pyramidal neurons of the hippocampus. Since electroconvulsive treatment is the most effective treatment in humans with major depression, we investigated whether repeated electroconvulsive stimulations (ECSs) could influence such changes in stressed rats. Furthermore, we investigated whether ECSs per se could influence neuronal branching and total length of the CA3 hippocampal neuronal dendritic tree in normal rats. Rats were stressed using the 21-day 6 h daily restraint stress paradigm. The study shows that stress caused remodelling of the pyramidal neurons by significantly reducing the number of dendritic branch points and total length of the apical dendritic tree. Concomitant administration of ECSs prevented these effects. ECSs had no effect on pyramidal neuron dendrites in normal rats.

Abeta(1-42) injection causes memory impairment, lowered cortical and serum BDNF levels, and decreased hippocampal 5-HT(2A) levels.

Aggregation of the beta-amyloid protein (Abeta) is a hallmark of Alzheimer's disease (AD) and is believed to be causally involved in a neurodegenerative cascade. In patients with AD, reduced levels of serum Brain Derived Neurotrophic Factor (BDNF) and cortical 5-HT(2A) receptor binding has recently been reported but it is unknown how these changes are related to beta-amyloid accumulation. In this study we examined in rats the effect of intrahippocampal injections of aggregated Abeta(1-42) (1 microg/microl) on serum and brain BDNF or 5-HT(2A) receptor levels. A social recognition test paradigm was used to monitor Abeta(1-42) induced memory impairment. Memory impairment was seen 22 days after injection of Abeta(1-42) in the experimental group and until termination of the experiments. In the Abeta(1-42) injected animals we saw an abolished increase in serum BDNF levels that was accompanied by significant lower BDNF levels in frontal cortex and by an 8.5% reduction in hippocampal 5-HT(2A) receptor levels. A tendency towards lowered cortical 5-HT(2A) was also observed. These results indicate that the Abeta(1-42) associated memory deficit is associated with an impaired BDNF regulation, which is reflected in lower cortical BDNF levels, and changes in hippocampal 5-HT(2A) receptor levels. This suggests that the BDNF and 5-HT2A changes observed in AD are related to the presence of Abeta(1-42) deposits.

Exacerbated loss of cell survival, neuropeptide Y-immunoreactive (IR) cells, and serotonin-IR fiber lengths in the dorsal hippocampus of the aged flinders sensitive line "depressed" rat: Implications for the pathophysiology of depression?

Impairment of hippocampal neurogenesis has been proposed to provide a cellular basis for the development of major depression. Studies have shown that serotonin (5-HT) and neuropeptide Y (NPY) may be involved in stimulating cell proliferation in the dentate gyrus. The Flinders-sensitive line (FSL) rat represents a genetic model of depression with characterized 5-HT and NPY abnormalities in the hippocampus. Consequently, it could be hypothesized that hippocampal neurogenesis in the FSL rat would be impaired. The present study examined the relationship among 1) number of BrdU-immunoreactive (IR) cells, 2) NPY-IR cells in the dentate gyrus, and 3) length of 5-HT-IR fibers in the dorsal hippocampus, as well as volume and number of 5-HT-IR cells in the dorsal raphé nucleus, in adult and aged FSL rats and control Flinders-resistant line (FRL) rats. Surprisingly, adult FSL rats had significantly more BrdU-IR and NPY-IR cells compared with adult FRL rats. However, aging caused an exacerbated loss of these cell types in the FSL strain compared with FRL. The aged FSL rats also had shortened 5-HT-IR fibers in the dorsal hippocampus, indicative of an impaired 5-HT innervation of this area, compared with FRL. These results suggest that, for "depressed" FSL rats, compared with FRL rats, aging is associated with an excacerbated loss of newly formed cells in addition to NPY-IR cells and 5-HT-IR dendrites in the hippocampus. These observations may be of relevance to the depression-like behavior of the FSL rat and, by inference, to the pathophysiology of depression.

Arrested neuronal proliferation and impaired hippocampal function following fractionated brain irradiation in the adult rat.

The generation of new neurons in the adult mammalian brain has been documented in numerous recent reports. Studies undertaken so far indicate that adult hippocampal neurogenesis is related in a number of ways to hippocampal function.Here, we report that subjecting adult rats to fractionated brain irradiation blocked the formation of new neurons in the dentate gyrus of the hippocampus. At different time points after the termination of the irradiation procedure, the animals were tested in two tests of short-term memory that differ with respect to their dependence on hippocampal function. Eight and 21 days after irradiation, the animals with blocked neurogenesis performed poorer than controls in a hippocampus-dependent place-recognition task, indicating that the presence of newly generated neurons may be necessary for the normal function of this brain area. The animals were never impaired in a hippocampus-independent object-recognition task. These results are in line with other reports documenting the functional significance of newly generated neurons in this region. As our irradiation procedure models prophylactic cranial irradiation used in the treatment of different cancers, we suggest that blocked neurogenesis contributes to the reported deleterious side effects of this treatment, consisting of memory impairment, dysphoria and lethargy.

Model for aging in the basal forebrain cholinergic system.

A key component of the cognitive deficits associated with aging is the loss of function of cholinergic neurons in the basal forebrain due to neuronal losses and decreased cholinergic function of spared neurons. A model to mimic one aspect of this phenomenon is to kill cholinergic neurons selectively in the basal forebrain via administration of the immunotoxin IgG-192-saporin. Here we discuss apoptotic regulators, such as nerve growth factor, in age-associated changes present in the cholinergic system and the role of the NF-kappaB signaling system in cellular commitment to apoptosis. We also examine the age-associated decline in intrinsic response mechanisms, which may account for the age-associated reduction in recovery from both acute and chronic insults to the central nervous system.

Responses of young and aged rat CNS to partial cholinergic immunolesions and NGF treatment.

The cholinergic neurons of the basal forebrain (CNBF) are the major source of cholinergic innervation of the cortex and hippocampus. In Alzheimer's disease and aged brain, there are severe losses of cholinergic neurons in the nucleus basalis of Meynert, leading to a reduction of cortical cholinergic activity which correlates with the severity of cognitive deficits. While there is evidence that aged central nervous system (CNS) displays impaired stress response signaling, pharmacologic treatments with neurotrophic factors appear to ameliorate these age-associated cholinergic deficits. To mimic these cholinergic deficits in experimental animals and study the acute effects of nerve growth factor (NGF), we induced a partial lesion of CBFNs by the intracerebroventricular (i.c.v.) injection of the cholinergic immunotoxin 192IgG-saporin, in groups of 3- and 30-month-old rats. The lesion was followed 14 days later by i.c.v. administration of NGF, known to restore partial immunolesion-induced cholinergic deficits in rat CNS, and all rats were killed 2 days after the NGF treatment. Here we report the effects of partial immunolesions on the levels of choline acetyltransferase (ChAT) activity and NGF receptor mRNA levels in the basal forebrain of 3- and 30-month-old rats. Because of their presence in the promoters of the NGF, NGF receptors, and ChAT genes, we also measured DNA-binding activity of the transcription factors NFB and AP-1 in the cortex and hippocampus. We discuss these findings in the context of endogenous NGF-mediated signal transduction mechanisms and conclude that we have evidence for age-associated decreases in endogenous NGF responses to partial deafferentation of the basal forebrain cholinergic projections.

Cholinergic control of nerve growth factor in adult rats: evidence from cortical cholinergic deafferentation and chronic drug treatment.

It is well documented that nerve growth factor (NGF) plays an important role in maintaining functions of cholinergic basal forebrain neurons. In the present study, we tested the hypothesis that cholinergic activity controls NGF levels in cholinoceptive neurons of the cerebral cortex and hippocampus. To address that question, we used both cholinergic deafferentation of cerebral cortex and hippocampus by cholinergic immunolesion with 192IgG-saporin and chronic pharmacological treatment of sham-treated and immunolesioned rats with the cholinergic agonist pilocarpine and the cholinergic antagonist scopolamine. We observed an increase in NGF protein levels in the cortex and hippocampus after cholinergic immunolesions and also after muscarinic receptor blockade by chronic intracerebroventricular scopolamine infusion in sham-treated rats after 2 weeks. There was no further increase in the accumulation of NGF after scopolamine treatment of immunolesioned rats. Chronic infusion of pilocarpine had no effect on cortical and hippocampal NGF protein levels in sham-treated rats. In rats with cholinergic immunolesions, however, pilocarpine did prevent the lesion-induced accumulation of NGF. There was no effect of cholinergic lesion and drug treatment on cortical or hippocampal NGF mRNA levels, consistent with the importance of NGF retrograde transport as opposed to its de novo synthesis. This study provides strong evidence for the hypothesis that there is cholinergic control of cortical and hippocampal NGF protein but not mRNA levels in adult rats.

Behavioral symptoms in adult rats after postnatal L-nitro-arginine.

We addressed experimentally the suggestion by Gally et al. [Gally J. A., Read Montague P., Reeke G. N. Jr and Edelman G. M. (1990) Proc. Natl Acad. Sci. U.S.A. 87, 3547-3551] that nitric oxide may play a role in the use-dependent modification of synaptic efficacy in the developing nervous system. In a preliminary control experiment, we treated rat pups from postnatal day 8 to postnatal day 22 with a nitric oxide synthase blocker (L-nitro-arginine) and compared their growth curves and brain weights to those of saline injected control pubs. No significant differences were found after the 14 days of nitric oxide synthase inhibition. In the subsequent experiment, we inhibited nitric oxide synthesis in rat pups from postnatal day 8 to day 29 and assessed their place learning ability and open field behavior as adults. We found an increased speed of habituation of locomotion in an open field in 5-month-old rats that had been treated postnatally with a nitric oxide synthase blocker. There was no difference between treated and non-treated rats with respect to place learning in a water maze. We conclude that perturbation of nitric oxide production during early postnatal development does not preclude normal learning and memory function in the adult.

L-nitroarginine reduces hippocampal mediation of place learning in the rat.

Based on previous results it was hypothesized that the neural substrate of the acquisition of place learning during inhibition of the nitric oxide synthesizing enzyme (NOS) by L-nitroarginine (L-N-ARG) differs from the neural substrate of normal task acquisition by a reduced or abolished participation of the hippocampus. This hypothesis was tested in two independent experiments. In Experiment 1 the behavioral consequences of bilateral transection of the fimbria-fornix--a lesion that abolishes normal hippocampal function--were investigated in animals that had acquired the task after either a vehicle control pretreatment or a 5-day pretreatment period during which near-total inhibition of NOS had been accomplished by L-N-ARG injections. While fimbria-fornix transections significantly impaired task performance in normal animals the rats which had acquired the task during NOS inhibition did not reveal a lesion-associated impairment. In Experiment 2 four groups of rats were studied: two groups initially received bilateral transection of the fimbria-fornix, while the two others were subjected to sham surgery. Subsequently, one of the fimbria-fornix-transected and one of the sham-operated groups received a 10-day period of L-N-ARG injections, while the two remaining groups received saline control injections. During the final 5 days of injections the four groups were subjected to training on the place-learning task. While NOS inhibition clearly impaired task acquisition in the sham-operated animals, L-N-ARG administration in fimbria-fornix-transected animals failed to impair place-learning acquisition.(ABSTRACT TRUNCATED AT 250 WORDS)

Place learning by fimbria-fornix transected rats in a modified water maze.

The acquisition of a place learning task in a water maze modified from the "standard" setup by restriction of distal cues was tested in two groups of rats: (1) animals subjected to bilateral transection of the fimbria-fornix--a manipulation that renders the hippocampus dysfunctional--and (2) a sham operated control group. The fimbria-fornix transected animals acquired the task as quickly and to the same level of proficiency as the control group. Upon reaching criterion level performance all animals were tested on "rotation" sessions on which the distal cues were displaced. The outcome of such "rotations" demonstrated that both groups relied upon the distal cues for navigational purposes. Finally, the performance of all animals was "challenged" on two sessions by administration of d-amphetamine and scopolamine, respectively. The outcome of the pharmacological challenges demonstrated that the normal proficiency of place learning by the fimbria-fornix transected rats had been accomplished due to compensatory processes mediated at least partly by the catecholaminergic systems.

Functional and neurochemical profile of place learning after L-nitro-arginine in the rat.

Rats in which near-total inhibition of the nitric oxide synthesizing enzyme (NOS) had been obtained by a 5-day pretreatment during which two daily injections of L-nitro-arginine (50 mg/kg per injection) had been administered were subjected to five sessions of training on a place learning task (one session per day). NOS inhibition was associated with significantly impaired task acquisition but on the fifth training session normal task proficiency was achieved. Subsequent pharmacological and behavioral challenges established that dissimilar neural substrates mediated the task in NOS-inhibited and normal animals. In NOS-inhibited rats the neural substrate of task mediation depended less than normally on cholinergic mechanisms while potentially relying more on catecholaminergic mediation. In a separate experiment rats that had acquired the place learning task to asymptotic quality of performance were subjected to a L-nitro-arginine treatment similar to the pretreatment of the first experiment. Such a NOS inhibition did not impair subsequent task performance. It is concluded that nitric oxide participates in processes subserving acquisition rather than performance of at least some forms of place learning.

Retention and relearning of spatial delayed alternation in rats after ablation of the prefrontal or total non-prefrontal isocortex.

In the present study we addressed the question whether, within the isocortex, the prefrontal area of the rat is uniquely involved in mediation of delayed alternation. In one group of rats the dorsolateral isocortex, from the dorsomedial shoulder to the dorsal lip of the rhinal sulcus was removed bilaterally in a single surgical session. In these rats delayed alternation in a T-maze was significantly less impaired than in rats with one stage bilateral removal of the medial prefrontal cortex. The prefrontal cortex seems not to depend crucially on isocortical input for its medication of delayed alternation.

Cortical area in the rat that mediates visual pattern discrimination.

In Experiment I, bilateral ablations of the caudolateral cortex involving Krieg's area 36 impaired discrimination of visual patterns but not delayed alternation. In Experiment II, the same type of lesions retarded postoperative learning to discriminate embedded visual patterns. In rats from the Experiment II tracers of axonal transport gave no signs of damage of the connections of the primary visual cortex. In agreement with this, Nissl stain of the dorsal lateral geniculate nuclei showed no neuronal loss or gliosis. These results suggest that caudo-lateral cortex in rats corresponds to the inferotemporal cortex of primates.

Vitamin E prevents the place learning deficit and the cholinergic hypofunction induced by AF64A.

The present study examined whether pretreatment with vitamin E would attenuate the place learning impairment and the neurochemical deficits induced by intracerebroventricular (icv) injection of the cholinotoxin ethylcholine mustard aziridinium ion (AF64A). Male Sprague-Dawley rats were pretreated by intramuscular injection of either vitamin E or saline 24 h and 15 min prior to surgery. They were then infused bilaterally into the cerebroventricles with AF64A (3 nmol/side) or artificial cerebrospinal fluid (CSF). Following 14 days of postoperative recovery, the rats were trained to acquire a place learning task in a water maze for two blocks of four trials a day for 5 days. Following the 40th trial a probe trial was used to assess memory for the platform location. After completion of behavioral testing, hippocampal high-affinity choline uptake (HAChU) was assessed. The groups which had received either saline or vitamin E pretreatment followed by icv injection of CSF did not differ significantly on any parameter measured and were therefore pooled as control group. Animals which had been pretreated with vitamin E and had received icv injection of AF64A exhibited neither significant impairment in water maze performance nor significant decrease in HAChU. In contrast, animals which had been pretreated by saline followed by icv injection of AF64A were significantly impaired in acquisition of the place learning task as well as during the probe trial and had reduced HAChU in the hippocampus. These findings indicate that vitamin E may have a neuroprotective effect in the septohippocampal cholinergic system.

Retention and relearning of spatial delayed alternation in rats after combined or sequential lesions of the prefrontal and parietal cortex.

Lesions of the medial prefrontal cortex strongly impaired rats' delayed alternation behaviour in a T-maze, both when the lesion was inflicted after the initial acquisition of the task and when the lesion was added after criterion performance had been reattained following an ablation of the parietal cortex. Lesions of the parietal cortex did not impair this behaviour, either when the parietal lesion was inflicted after the initial acquisition of the task or when it was added to a prefrontal lesion after criterion performance had been reattained. Combined, one stage, parietal and prefrontal lesions did not have stronger effect on delayed alternation than did prefrontal lesions alone. These results indicate that in spite of the strong anatomical connectivity between the prefrontal and parietal "association" cortex the latter is not necessary for the recovery of delayed alternation after prefrontal lesions. In comparison with the parietal cortex, the prefrontal cortex seems to be uniquely involved in mediation of delayed alternation.