Acute oral Bryostatin-1 administration improves learning deficits in the APP/PS1 transgenic mouse model of Alzheimer's disease.

BACKGROUND: Previous studies showed that Bryostatin-1, a potent PKC modulator and alphasecretase activator, can improve cognition in models of Alzheimer's disease (AD) with chronic (>10 weeks), intraperitoneal (i.p.) administration of the drug. We compared learning and spatial memory in the APPswe, PSEN1dE985Dbo (APP/PS1) mouse model of AD and studied the ability of acute intraperitoneal and oral Bryostatin-1 to reverse cognitive deficits in this model. Compared to wild-type (WT) mice, APP/PS1 mice showed significant delays in learning the location of a submerged platform in the Morris water maze. Bryostatin-1 was administered over a 2-week course prior to and during water maze testing. RESULTS: Acute i.p. Bryostatin-1 administration did not improve latency to escape but oral Bryostatin-1 significantly improved memory (measured by a reduction in latency to escape). This benefit of oral Bryostatin-1 administration was most apparent during the first 3 days of testing. These findings show that: 1) Bryostatin-1 is orally active in models of learning and memory, 2) this effect can be produced in less than 2 weeks and 3) this effect is not seen with i.p. administration. We conclude that oral Bryostatin-1 represents a novel, potent and long-acting memory enhancer with future clinical applications in the treatment of human AD.

Embryonic "binge" cocaine exposure alters neural-immune and neural-endocrine interactions in young chickens: involvement of serotonin(2) receptors.

As part of our characterization of the developmental consequences of prenatal cocaine exposure, cocaine was injected into eggs containing viable chicken embryos on embryonic day (E) 18 and the fever response to the endotoxin lipopolysaccharide (LPS) and a delayed-type hypersensitivity response to phytohemagglutinin (PHA) were assessed postnatally. E18 cocaine exposure did not affect basal body temperature. LPS induced a fever in the chicks at 4 h post-injection on post-hatch day (D) 4 and 2 h post-injection on D24. E18 cocaine exposure suppressed the peak LPS-induced fever by 50% at both ages. E18 cocaine exposure also suppressed the hypersensitivity reaction to an intradermal injection of PHA on D17, while having no effect on the response to a saline injection. To determine the importance of serotonin(2) (5-HT(2)) receptors in the developmental toxicity of cocaine, varying doses of the 5-HT(2) antagonist ritanserin were injected on E17 followed by cocaine on E18. Ritanserin, like cocaine, did not alter basal temperature, but it dose-relatedly attenuated or blocked cocaine's effect on LPS-induced fever on both D4 and D24. Ritanserin pretreatment was also able to block the blunted isolation stress response seen in D16 chicks following E18 cocaine exposure. Thus, late prenatal cocaine exposure significantly alters adaptive fever and hypersensitivity responses, and embryonic 5-HT(2) receptors played a mediating role in the fever effect.

Embryonic cocaine exposure and corticosterone: serotonin(2) receptor mediation.

Cocaine activates the mature hypothalamic-pituitary-adrenal (HPA) axis, increasing corticosterone concentrations in animals and humans and serotonin(2) receptors (5-HT(2)) are involved in this effect. Although prenatal cocaine exposure is associated with altered responsiveness of the HPA axis to "stress" and serotonergic compounds postnatally, it is unknown whether cocaine directly activates the embryonic HPA axis or if 5-HT(2) receptors are involved. Domestic chicken eggs with viable embryos were exposed to either the 5-HT(2) receptor agonist dimethoxyiodophenylaminopropane (DOI: 0.4, 0.8, or 1.2 mg/kg egg) or saline on embryonic day 18 (E18). In a second study, the 5-HT(2) antagonist ritanserin (0.3 mg/kg egg, a dose found effective against other effects of DOI or cocaine) or vehicle was administered on E17, prior to treatment on E18 with either saline or cocaine (5 injections of 12 mg/kg egg, equivalent to a total dose of 3.5 mg/egg). Radioimmunoassay was used to measure serum corticosterone from blood samples taken approximately 1-2 h after drug injections. DOI significantly raised corticosterone in a dose-related fashion. Cocaine-induced corticosterone elevations were blocked by pretreatment with ritanserin, whereas ritanserin by itself did not affect corticosterone concentrations. These data indicate that 5-HT(2) receptors are involved in cocaine's effect on the HPA axis during late chicken embryogenesis.

Late embryonic ritanserin exposure fails to alter normal responses to immune system stimulation in young chicks.

Prior studies in our laboratory have demonstrated that prenatal treatment with the serotonin2 (5-HT2) antagonist ritanserin is effective in blocking some of the lethal, dysmorphic, cardiovascular, and behavioral consequences of excessive direct or indirect stimulation of 5-HT2 receptors in the developing chicken. The efficacious dose range for ritanserin in these studies had very little or no effect on the above measures of toxicity when administered alone. In the present study, we extend our characterization of ritanserin's potential toxicity, or lack thereof, to include the normal behavioral and endocrine responses to immune system stimulation by the endotoxin lipopolysaccharide (LPS). LPS administration induces a syndrome collectively known as sickness behavior, manifest as altered thermoregulatory processes leading to fever, and increased serum concentrations of neuroendocrine hormones, including corticosterone. These survival-promoting responses to LPS were assessed in young chickens that had been treated with doses of ritanserin ranging from 0 to 2.7 mg/kg on embryonic day 17 (E17). When sickness behavior was assessed in 5-7-day-old chicks 1 h post-LPS injection, E17 ritanserin-treated subjects did not differ from controls. At 4-6 h post-LPS, 4-day-old chicks displayed a robust fever, and E17 ritanserin did not affect the magnitude of this response. Similarly, E17 ritanserin treatment failed to affect corticosterone concentrations 2 h post-LPS in 14-day-old chicks. Thus, ritanserin treatment during late embryogenesis, a time when it is effective against direct and indirect acting 5-HT2 agonists, failed to modify the survival promoting and beneficial interactions between the nervous, endocrine, and immune systems that are elicited following immunostimulation.

Open-field and LPS-induced sickness behavior in young chickens: effects of embryonic cocaine and/or ritanserin.

Exposure to drugs of abuse during embryogenesis may adversely affect nervous, immune, and endocrine systems development. We compared exposure on embryonic day 18 (E18) by single or multiple cocaine (COC) injections (56.25 mg/kg total dose for both) or saline on hatching and activity measures. In saline-exposed controls, repeated testing, age, and gender affected activity levels. A single or multiple COC injections increased the median latency to explore and multiple COC injections decreased the median number of lines crossed by female chicks in the open field. We also determined if pretreatment with the serotonin2 (5-HT2) receptor antagonist ritanserin could attenuate COC's effects on open-field behavior as well as behaviors sensitive to immune system stimulation (lipopolysaccharide (LPS)-induced sickness behavior). Eggs containing embryos were pretreated on E17 with 0.4 mg ritanserin/kg or its vehicle followed by multiple COC injections or saline on E18. E18 COC treatment decreased the median number of lines crossed and distress vocalizations in females. Ritanserin pretreatment mitigated the COC induced effects. E18 COC exposure also suppressed LPS-induced sickness behaviors in both males and females, increasing food consumption and the time spent awake and active, as well as decreasing the time spent sleeping. Ritanserin alone had no effect on the food consumed or time spent active, nor did this dose affect COC-induced alterations in sickness behavior. Ritanserin alone decreased time spent sleeping and also failed to affect the COC-induced suppression. Thus, embryonic COC exposure can suppress open field and LPS-induced sickness behavior in the young chick, and ritanserin pretreatment can block the former, but not the latter effects at the dose chosen for these experiments.

Attenuation of behavioral abnormalities in autoimmune mice by chronic soluble interferon-gamma receptor treatment.

NZB x NZW F1 hybrid (B/W) mice develop altered behavior in the elevated plus maze and novel object tasks between 6 and 12 weeks of age in parallel with lupus-like autoimmune disease. To confirm the relationship between disease progression and development of behavioral abnormalities, B/W and nonautoimmune NZW mice received chronic treatment with a soluble IFN gamma receptor (sIFN gamma R), a treatment known to retard autoimmune disease progression, or vehicle, beginning at 6 weeks of age. After 6 weeks of treatment, elevated plus maze and novel object testing revealed that although sIFN gamma R treated B/W mice still differed from NZW mice, chronic sIFN gamma R treatment significantly retarded the development of behavioral abnormalities in the B/W mice, while the NZW mice were not affected by this treatment. sIFN gamma R treated B/W mice were more active in both the plus maze and novel object tasks, and displayed less plus maze anxiety behavior and more exploratory activity in the novel object task compared to vehicle treated B/W mice. To clarify the role of acute action of the sIFN gamma R on the elevated IFN gamma levels of B/W mice, a second experiment examined the effects of a single injection of sIFN gamma R on B/W and NZW mice. Unlike chronic treatment, acute treatment with the same dose of sIFN gamma R did not affect plus maze or novel object behavior in 12-week-old mice. These results add to the growing evidence that lupus-associated behavioral abnormalities are a direct effect of the autoimmune disease.

Evidence for a serotonin-mediated effect of cocaine causing vasoconstriction and herniated umbilici in chicken embryos.

Some of cocaine (COC)'s pathophysiological effects on exposed embryos likely result from its vasoconstrictive action, and serotonin2 (5-HT2) agonists such as dimethoxyiodophenylaminopropane (DOI) can mimic these effects. Infusions of COC (5 mg/kg/min) or DOI (0.5 mg/kg/min) for 15 min into chicken eggs with embryos on E15 caused a significant reduction in blood vessel diameters (14 and 30%, respectively). Pretreatment with the 5-HT2 antagonist ritanserin (RIT, 0.9 mg/kg) 18-22 h earlier blocked the effect of COC and blocked or attenuated the effect of DOI. In separate groups of chicken embryos exposed to multiple injections of low doses of COC on E18, herniated umbilici were prominent in hatchlings. A single bolus injection of the same absolute amount of COC did not cause herniated umbilici. An additional experiment replicated the induction of herniated umbilici by multiple injections of COC and demonstrated the probable involvement of 5-HT2 receptors because RIT blocked COC's ability to induce this anomaly. These data suggest that COC's vasoconstrictive effect, via 5-HT2 receptors, may play a mechanistic role in some adverse outcomes in embryos exposed to COC.

Ontogeny of glucocorticoid receptors in the hyperstriatum-hippocampus-parahippocampal area and optic tectum of the embryonic chicken (Gallus domesticus) brain.

The importance of glucocorticoids and their perturbation during development is an active research area. Developmental insults, including direct and indirect consequences of exposure to drugs of abuse or withdrawal from them, may act upon or via the neuroendocrine axis of the pregnant experimental subject (e.g. rat) and/or directly upon the neuroendocrine axis of the embryo or fetus. The use of the domestic chicken embryo may constitute a good experimental subject for studying these effects in the absence of maternal influences. Thus, the pattern of brain glucocorticoid cytosolic receptors were characterized in an early developing brain region, the optic tectum (OT) and a later developing region with a different function, the hyperstriatum-hippocampus-parahippocampal (HHP) area, on embryonic days (E) 11, 15, 18 and on the day of hatching (HD). The influence of the glucocorticoid synthesis inhibitor metyrapone, injected into eggs on E14 and on E17, upon glucocorticoid receptors (on E15 and E18) was also studied to determine effects of a 'chemical adrenalectomy'. Receptors for this steroid are high on E11 and E15, decreasing as they approach the time of hatching, with the HHP generally showing greater numbers of specific binding sites for [3H]-corticosterone (CORT). Although metyrapone treatment did not alter the apparent number of receptors on E15, on E18 it unmasked receptors otherwise occupied by endogenous ligand(s) and/or induced their synthesis, resulting in significantly more receptors identified with [3H]-CORT. Nevertheless, the HHP continued to display more of these receptors than the OT on E15 and E18 after injection of metyrapone. These observations are consistent with the hypotheses that the HHP of embryos of this species contains a higher density of glucocorticoid receptors than does the OT; that glucocorticoid receptor quantification is related to steroid synthesis inhibition in late embryonic development; and that neuroendocrine feedback control of serum glucocorticoids may become functional between E15 and E18. The results also suggest the use of this experimental approach for assessing the effects of developmental insults with drugs, other than metyrapone, as a marker for altered neuroendocrine development and/or function.

Effect of training and environment on brain morphology and behavior.

Using defined rearing or training paradigms, environmental stimulation has been found to increase brain weight (especially forebrain), cortical thickness, the number of glial cells, the glia to neuron ratio, neuronal cell body and nucleus size, and to alter synaptic profiles by increasing dendritic branching, dendritic spine density and the number of discontinuous synapses. Examples will be given from both animal and human studies that document these profound changes. Controversy exists as to whether enriched environments and/or training can compensate for neural deficits produced earlier in life. Examples will be given from animal studies with induced cortical lesions and prenatal genetic neural anomalies that support a role for environmental manipulations ameliorating earlier central nervous system damage.

Anxiety behavior, exploratory behavior, and activity in NZB x NZW F1 hybrid mice: role of genotype and autoimmune disease progression.

Behavioral changes often accompany the autoimmune disease systemic lupus erythematosus (SLE) in humans and animals. In a mouse model of SLE, the NZB x NZW F1 (B/W) hybrid, 12-week-old mice display more anxiety behavior, less activity, and less exploratory behavior than non-autoimmune female NZW mice. To determine whether these behaviors result from the autoimmune disease or genetic differences between B/W and NZW mice, they were assessed prior to and during disease emergence (6 and 12 weeks of age, respectively). B/W mice were less active at both ages, suggesting a genetic component to this behavioral difference. Anxiety behavior and exploratory behavior did not differ between B/W and NZW mice at 6 weeks; however, at 12 weeks B/W mice displayed more anxiety behavior and less exploratory behavior, indicating that these behaviors were related with the development of autoimmune disease. Prior experience with these tasks increased anxiety behavior in B/W but not NZW mice, suggesting that B/W mice may be more sensitive to anxiogenic experiences.

The role of performance factors in the active avoidance-conditioning deficit in autoimmune mice.

Prior studies with autoimmune mice demonstrated deficits in 2-way active avoidance conditioning that correlated with the degree of autoimmunity. In this study, autoimmune female NZB x NZW F1 hybrid (B/W) mice were tested in shock-motivated discrimination learning, 1-way avoidance conditioning, and a modified 2-way avoidance task and compared to nonautoimmune female NZW mice. The discrimination and 1-way conditioning results indicated that B/W mice can learn shock-motivated tasks that involve minimal fatigue and no conflict. B/W mice were also able to learn the 2-way avoidance task when it was made easier by increasing conditioned-stimulus cue salience, clarifying contingencies, and increasing trial spacing to decrease possible cognitive, emotional, and physical fatigue. Thus, poor performance in 2-way avoidance appears to be a consequence of altered attention, motivation, or emotionality and can be overcome by altering task parameters.

Increased anxiety behaviors in autoimmune mice.

The human autoimmune disorder systemic lupus erythematosus (SLE) is often accompanied by psychiatric manifestations including anxiety. In this study, the performance of SLE-prone NZB x NZW F1 (B/W) hybrid mice was compared with nonautoimmune NZW control mice on 3 anxiety tasks: the elevated plus maze, the open-field drink test, and the novel-object task. B/W mice displayed decreased activity as well as an anxiety profile in all 3 tasks, which was characterized by avoidance of open and exposed places even when the motivation to explore these areas was high. Cytokines are overexpressed in autoimmune disease, and NZW controls injected with the cytokine interferon-alpha displayed an anxiety profile in the plus maze. Thus, cytokines may play a role in the genesis of the behavioral manifestations of autoimmune disease.

Effects of embryo transfer and cortical ectopias upon the behavior of BXSB-Yaa and BXSB-Yaa + mice.

The BXSB-Yaa and BXSB-Yaa + inbred strains of mice differ primarily with respect to the Y chromosome, although there is evidence that they differ on several autosomal genes as well. Each strain has ectopic collections of neurons in neocortical layer I (ectopias), with a higher occurrence in males (58%) than females (42%). Conventionally reared mice from these strains were compared to mice that were transferred, as 8-cell embryos, into the uteri of non-autoimmune recipients, who gave birth to and reared the offspring. The transfer procedure did not change the incidence of ectopias in either sex. There were, however, major differences in behavior. Compared to conventionally reared controls, embryo transfer mice had greater behavioral asymmetry, poorer performance in a black-white discrimination, poorer Morris maze learning, better Lashley maze learning, and better performance in a two-way shuttlebox. Within the transfer groups, females differed as much as males, confirming our prior findings and supporting our thesis that the two strains differ on several autosomal genes in addition to the Y chromosome. These findings show that the intra-uterine environment can powerfully and selectively affect later behavior. When ectopic and non-ectopic mice were compared, BXSB-Yaa mice with neocortical ectopias were better able to learn the Morris spatial maze than non-ectopic controls; this was true whether the mice were conventionally reared or embryo transferred. In contrast, BXSB-Yaa + ectopic mice did not differ from their controls if conventionally reared, but were much worse than controls if embryo transferred.

Sensitivity to foot shock in autoimmune NZB x NZW F1 hybrid mice.

Prior studies have demonstrated deficits in active avoidance learning in young (12-week-old) mice that develop lupus-like autoimmunity. Because foot shock is the motivating stimulus in this task, sensitivity to foot shock was assessed in autoimmune NZB x NZW F1 hybrid (B/W) and nonautoimmune NZW female mice. Responses to shock at levels ranging from 0.05 to 1.6 mA were recorded twice during the development of autoimmunity. At 12 weeks of age, B/W mice did not differ from NZW mice in sensitivity to shock. However, at 24 weeks of age, when antibody levels were elevated, sensitivity to foot shock decreased in B/W mice at low shock levels and increased at high shock levels. A neurological battery revealed no deficits that could account for these effects. However, IgM anti-DNA antibody levels were positively correlated with responsiveness to high levels of shock. The change in the pattern of sensitivity at 24 weeks may be due to a combination of disease-related sensory impairment at low shock levels and hyperalgesia at high shock levels. The response to high levels of shock may also be an indication of enhanced emotionality, an interpretation consistent with reports in other lupus-prone strains and affective disorders in humans with lupus.

Autoimmunity and avoidance learning in NXRF recombinant inbred strains.

Four recombinant inbred (RI) strains, generated from NZB and RF progenitors, were tested on a two-way shuttlebox and their sera were assayed for serological factors. The four RI strains differed with respect to number of null responses and escape time. The strain with the poorest performance displayed the greatest degree of autoimmunity. The avoidance measures were correlated against the autoimmune serological parameters for the four RI strains and the two progenitors. The number of avoidances was negatively correlated with anti-DNA antibody titer, while the number of null responses showed a positive association with immune complex level. Escape time had positive correlations with IgG, IgM-RF, and immune complexes. These findings are consistent with, and extend, our prior reports that avoidance learning is inversely related to the degree of autoimmunity.

Behavior, cortical ectopias, and autoimmunity in BXSB-Yaa and BXSB-Yaa+ mice.

The BXSB-Yaa recombinant inbred strain was created by crossing a male SB/Le with a female C57BL/6J. A Y chromosome factor derived from the SB/Le male, known as the autoimmune accelerator (Yaa), leads to an earlier onset and greater severity of autoimmune disease in males. In contrast, male BXSB mice, which lack the Yaa gene (called BXSB-Yaa+) because their Y chromosome is derived from the C57BL/6J, do not develop an autoimmune condition. To examine the influence of the Y chromosome on behavior, cortical ectopia incidence, and immune functioning, males and females of these two strains were compared. Significant strain differences (for both sexes) were found for behavioral measures including discrimination, spatial and avoidance learning, and activity. For immunological parameters, a sex difference was seen in the BXSB-Yaa (males more autoimmune), but not in the BXSB-Yaa+ strain. As expected, male BXSB-Yaas were more autoimmune than male BXSB-Yaa+s. However, there was also a strain difference for IgG in the females (BXSB-Yaa+ greater). No strain difference was found for the presence of ectopias. However, there was a sex difference across both strains, with males having a higher incidence. BXSB-Yaa and BXSB-Yaa+ mice have behavioral and immunological differences greater than would be predicted by their known genetic differences. The significant differences between the two female groups suggest that the two strains differ with respect to autosomal genes, in addition to the Y chromosome. The incidence of ectopias is independent of this genetic difference and is influenced by the subject's sex.

Ovarian estrogen acts to feminize the female rat's corpus callosum.

The rat corpus callosum (CC) is sexually dimorphic, with the male CC being larger. Ovariectomy (Ovx) on day 12 has been shown to eliminate this sex difference, with callosal values of Ovx females approaching those of male controls. This suggested that postnatal ovarian estrogen affects the size of the female CC. In the present experiment, one group of female rats received Ovx on day 12, and a second group received Ovx followed by chronic implantation of a silastic tube containing beta-estradiol on day 25. Unmanipulated males and sham females served as controls. Examination of the CC at 110 days confirmed our prior findings that males have larger callosa than females and that the Ovx group had increased CC's compared to sham controls. Our new finding was that estrogen treatment was capable of reversing the effects of Ovx. Ovx+estrogen-treated females had decreased CC size as compared to Ovx alone. Indeed, they also had smaller CC values than control females. These findings indicate that ovarian estrogen plays a role in determining CC morphology and that estrogen in the female acts to inhibit overall callosal growth as measured by changes in gross callosal size.

Lashley maze learning deficits in NZB mice.

In a prior study we found excellent Lashley III maze learning in BXSB mice and poor learning in NZB mice, despite the fact that both strains are autoimmune and develop cortical ectopias. This prompted us to examine NZB Lashley maze performance in detail, including comparisons to other strains and attempts to improve performance by giving additional trials with or without additional intramaze visual cues. In conventional Lashley testing (10 trials), RF mice (non-autoimmune and nonectopic) and BXSBs performed well in the Lashley maze. They had high learning indices and few errors. NZB mice performed poorly, with low learning indices and many errors. Even with additional trials or additional trials plus intramaze cues, NZB performance remained poor. The number of backward and forward errors stayed high; learning indices were low. Since both BXSB and NZB mice develop autoimmune disorders and cortical ectopias, it is unlikely that differential Lashley performance is the result of the presence of these phenomena. NZB mice are known to have alterations in their hippocampal morphology, and this is a possible mediator of the Lashley deficit.

Environmental enrichment, neocortical ectopias, and behavior in the autoimmune NZB mouse.

New Zealand Black (NZB) mice have severe autoimmune disease and approximately 40% have cortical ectopias in layer I of sensorimotor cortex. Because the ectopias are similar to those found in dyslexics, NZB mice have been used as an animal model for developmental learning disorders. In addition, these mice have been used as a model of learning deficits associated with autoimmune disease. To determine whether early intervention would affect learning processes in NZB mice, they were reared after weaning in standard cages or enriched environments. They were given a battery of behavioral tests to measure learning, laterality, and activity, after which they were sacrificed and their brains examined for cortical ectopias. The tests sorted into two behavioral sets. Ectopia-associated behaviors included black-white discrimination learning and the Morris spatial maze. As a group, the mice performed well on these tasks. Ectopic mice had poorer performance than non-ectopics on these measures, and environmental enrichment countered the effects of the ectopias. Autoimmune-associated behavior involved two-way avoidance learning in a shuttlebox. Mice were uniformely poor on this task, ectopias did not affect behavior, and environmental enrichment was without benefit. Evidence from this and other studies shows that poor shuttlebox performance is related to the presence of autoimmune disease. Thus, autoimmune disease and cortical ectopias each appear to affect a separate set of behavioral processes. Environmental enrichment is most effective for behavioral impairments mediated via cortical ectopias, but is much less effective, if at all, if autoimmunity is the primary mediator of the impairments.

Behavior, ectopias and immunity in BD/DB reciprocal crosses.

In a previous study, in which fertilized DBA ova were transferred into an autoimmune female, and NZB ova were transferred into a non-autoimmune female, we found that (1) the maternal environment affected the degree of autoimmunity, (2) the incidence of cortical ectopias was not affected by the maternal environment (3) DBA and NZB females had greater paw asymmetry if reared in an autoimmune uterus, and (4) avoidance learning scores were inversely related to degree of autoimmunity. In the present experiment, reciprocal crosses of DBA and BXSB mice were studied to confirm and extend the original findings. DB mice (DBA female x BXSB male) had greater immune activity than the BD animals, had poorer avoidance learning, but were better on black-white discrimination learning and the Lashley III maze. The BD mice had greater paw asymmetry. Only one of 38 animals had a cortical ectopia. The results lead to the following conclusions: (1) there is an inverse relationship between amount of immune activity and active avoidance learning; (2) some uterine factor in autoimmune mice causes females to have greater paw asymmetry; (3) cortical ectopias are under genetic control; and (4) the lesser immune activity of the BD mice suggests that they developed a suppressor system following early exposure to autoimmunity in the uterine/maternal environment.