Male-specific deficits in natural reward learning in a mouse model of neurodevelopmental disorders.

Neurodevelopmental disorders, including autism spectrum disorders, are highly male biased, but the underpinnings of this are unknown. Striatal dysfunction has been strongly implicated in the pathophysiology of neurodevelopmental disorders, raising the question of whether there are sex differences in how the striatum is impacted by genetic risk factors linked to neurodevelopmental disorders. Here we report male-specific deficits in striatal function important to reward learning in a mouse model of 16p11.2 hemideletion, a genetic mutation that is strongly associated with the risk of neurodevelopmental disorders, particularly autism and attention-deficit hyperactivity disorder. We find that male, but not female, 16p11.2 deletion animals show impairments in reward-directed learning and maintaining motivation to work for rewards. Male, but not female, deletion animals overexpress mRNA for dopamine receptor 2 and adenosine receptor 2a in the striatum, markers of medium spiny neurons signaling via the indirect pathway, associated with behavioral inhibition. Both sexes show a 50% reduction of mRNA levels of the genes located within the 16p11.2 region in the striatum, including the kinase extracellular-signal related kinase 1 (ERK1). However, hemideletion males show increased activation in the striatum for ERK1, both at baseline and in response to sucrose, a signaling change associated with decreased striatal plasticity. This increase in ERK1 phosphorylation is coupled with a decrease in the abundance of the ERK phosphatase striatum-enriched protein-tyrosine phosphatase in hemideletion males. In contrast, females do not show activation of ERK1 in response to sucrose, but notably hemideletion females show elevated protein levels for ERK1 as well as the related kinase ERK2 over what would be predicted by mRNA levels. These data indicate profound sex differences in the impact of a genetic lesion linked with neurodevelopmental disorders, including mechanisms of male-specific vulnerability and female-specific resilience impacting intracellular signaling in the brain.

Offspring neuroimmune consequences of maternal malnutrition: Potential mechanism for behavioral impairments that underlie metabolic and neurodevelopmental disorders.

Maternal malnutrition significantly increases offspring risk for both metabolic and neurodevelopmental disorders. Animal models of maternal malnutrition have identified behavioral changes in the adult offspring related to executive function and reward processing. Together, these changes in executive and reward-based behaviors likely contribute to the etiology of both metabolic and neurodevelopmental disorders associated with maternal malnutrition. Concomitant with the behavioral effects, maternal malnutrition alters offspring expression of reward-related molecules and inflammatory signals in brain pathways that control executive function and reward. Neuroimmune pathways and microglial interactions in these specific brain circuits, either in early development or later in adulthood, could directly contribute to the maternal malnutrition-induced behavioral phenotypes. Understanding these mechanisms will help advance treatment strategies for metabolic and neurodevelopmental disorders, especially noninvasive dietary supplementation interventions.

The hypothalamic transcriptional response to stress is severely impaired in offspring exposed to adverse nutrition during gestation.

Gestation is a time of profound vulnerability, as insults during pregnancy increase the lifelong risk of morbidity for the offspring. Increasingly, maternal diet is recognized as a key factor influencing the developing fetus. Poor-quality maternal diets, whether they provide an excess or an insufficiency of nutrients, lead to overt gestational growth disturbances in the offspring, and elevated risk for a common cluster of metabolic and mental disorders. Metabolic disturbances, particularly a substantially increased risk of obesity, have been linked in both maternal overnutrition and maternal undernutrition with abnormal development of the offspring hypothalamus, which serves a vital role in the central regulation of feeding. Additionally, the hypothalamus also coordinates physiological responses to stressors, and may thus play a role in vulnerability to psychiatric disease in these offspring. We examined hypothalamic molecular and endocrine responses to a psychological stressor (restraint) and a physiological stressor (lipopolysaccharide; LPS) in adult offspring from dams fed a high-fat diet or a low-protein diet during gestation and lactation. Targeted gene expression in the hypothalamus for 26 genes of interest sorted via hierarchical clustering revealed that the vast majority of these transcripts were substantially upregulated by both stressors. In contrast, offspring of maternal high-fat and low-protein diets mounted essentially no gene expression response to either stressor. However, male and female offspring of all conditions showed elevated hypothalamic-pituitary-adrenal glucocorticoid responses to both stressors, though the recovery of corticosterone responses after stress termination was significantly impaired in offspring of poor-quality maternal diets. Overall, it appears that the ability of the hypothalamus to respond in the immediate aftermath of stressful experiences is severely impaired in offspring of poor-quality maternal diets, regardless of whether the diet provided insufficient nutrients or excessive nutrients.

Metabolic adaptations to early life protein restriction differ by offspring sex and post-weaning diet in the mouse.

BACKGROUND AND AIMS: Low birth weight affects 1 in every 7 babies born globally and can predict a lifetime of increased risk for adverse health outcomes, including cardiovascular disease, hypertension, obesity, diabetes, and metabolic syndrome. Maternal low protein diet during pregnancy and lactation is a well-characterized rat model for low birth weight and the subsequent increase in chronic disease risk. However, mice have been relatively understudied in this paradigm and represent a critical resource for investigating the underlying molecular mechanisms that link adverse early life experience and the development of chronic disease. METHODS AND RESULTS: The present manuscript describes a mouse model of low birth weight (maternal consumption of low protein diet (8% protein) through pregnancy and lactation) and characterizes metabolic adaptations (food intake, locomotor activity, oxygen consumption, and glucose tolerance) in male and female offspring. At weaning, mice were maintained either on the control diet or a high fat diet. Notable sex differences were observed, with male mice from the low protein pregnancies showing increased food intake, hyperactivity and increased metabolic rate only when weaned to the high fat diet, while female mice consistently showed increased food intake and were hypometabolic, regardless of post-weaning diet. CONCLUSION: These data identify offspring sex and post-weaning diet as critical variables in the metabolic adaptations to early life protein deficiency, and suggest that females may be more vulnerable to the adverse long-term health consequences of low birth weight.

High-fat diet alters the dopamine and opioid systems: effects across development.

Consumption of a high-fat diet has been linked to obesity, dyslipidemia and cardiovascular disease. Less well appreciated are adverse effects on the brain and behavior. Recent research has shown that consumption of a high-fat diet can alter gene expression within the brain, and the dopamine and opioid neurotransmitter systems appear to be vulnerable to dysregulation. This review will focus on recent reports in both humans and animal models that describe adverse effects of high-fat diet consumption on the central reward circuitry. In addition, the importance of different development windows will be discussed, with effects observed in both the prenatal/perinatal time period and with chronic high-fat diet consumption in adulthood.

Early life protein restriction alters dopamine circuitry.

Adverse prenatal environment, such as intrauterine growth retardation (IUGR), increases the risk for negative neurobehavioral outcomes. IUGR, affecting approximately 10% of all US infants, is a known risk factor for attention deficit hyperactivity disorder (ADHD), schizophrenia spectrum disorders and addiction. Mouse dams were fed a protein deficient (8.5% protein) or isocaloric control (18% protein) diet through pregnancy and lactation (a well validated rodent model of IUGR). Dopamine-related gene expression, dopamine content and behavior were examined in adult offspring. IUGR offspring have six to eightfold over-expression of dopamine (DA)-related genes (tyrosine hydroxylase (TH) and dopamine transporter) in brain regions related to reward processing (ventral tegmental area (VTA), nucleus accumbens, prefrontal cortex (PFC)) and homeostatic control (hypothalamus), as well as increased number of TH-ir neurons in the VTA and increased dopamine in the PFC. Cyclin-dependent kinase inhibitor 1C (Cdkn1c) is critical for dopaminergic neuron development. Methylation of the promoter region of Cdkn1c was decreased by half and there was a resultant two to sevenfold increase in Cdkn1c mRNA expression across brain regions. IUGR animals demonstrated alterations in dopamine-dependent behaviors, including altered reward-processing, hyperactivity and exaggerated locomotor response to cocaine. These data describe significant dopamine-related molecular and behavioral abnormalities in a mouse model of IUGR. This animal model, with both face validity (behavior) and construct validity (link to IUGR and dopamine dysfunction) may prove useful in identifying underlying mechanisms linking IUGR and adverse neurobehavioral outcomes such as ADHD.

Diminished metabolic responses to centrally-administered apelin-13 in diet-induced obese rats fed a high-fat diet.

The central administration of apelin, a recently identified adipokine, has been shown to affect food and water intake. The present study investigated whether body weight could affect an animal's response to apelin. The effects of centrally-administered apelin-13 on food and water intake, activity and metabolic rate were investigated in adult male diet-induced obese (DIO) rats fed either a high fat (32%) or control diet. Rats were administered i.c.v. apelin-13, 15-30 min prior to lights out, and food and water intake, activity and metabolic rate were assessed. Intracerebroventricular administration of apelin-13 decreased food and water intake and respiratory exchange ratio in DIO rats on the control diet, but had no effect in DIO rats on the high-fat diet. In an effort to identify potential central mechanisms explaining the observed physiological responses, the mRNA level of the apelin receptor, APJ, was examined in the hypothalamus. A high-fat diet induced an up-regulation of the expression of the receptor. Apelin induced a down-regulation of the receptor, but only in the DIO animals on the high-fat diet. In conclusion, we have demonstrated a diminished central nervous system response to apelin that is coincident with obesity.

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor.

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors.

Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A)(+) RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1-10 microg of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.

Brain endothelial cell production of a neuroprotective cytokine, interleukin-6, in response to noxious stimuli.

Brain endothelial cells (BECs), specialized cells of the blood-brain barrier (BBB), are ideally positioned to monitor and respond to events in the periphery. The present study examined their potential role in transducing immune signals to the brain and in responding to noxious stimuli. BECs were isolated from rhesus monkeys at 3 age points (fetal/neonatal, adult, and very old animals). Cells were then challenged in vitro with either an immune stimulus (interleukin-1 beta (IL-1 beta), or lipopolysaccharide (LPS)) or an oxidative challenge (hypoxia). BECs released interleukin-6 (IL-6), which is known to have neurotrophic and neuroprotective functions. Furthermore, higher amounts of IL-6 were released in both baseline and stimulated conditions by BECs derived from aged animals. This research indicates a pathway whereby immune signals may be communicated to the CNS and has revealed one way that the BBB may protect neuronal survival under challenge conditions.

Resistance of central nervous system interleukin-6 to glucocorticoid inhibition in monkeys.

The ability of both exogenous and endogenous glucocorticoids (GCs) to inhibit proinflammatory cytokine production was investigated in vivo. Specifically, we investigated the effects of elevated GC levels on interleukin (IL)-1-induced release of IL-6 into both blood and cerebrospinal fluid (CSF). Three experiments were conducted in rhesus macaques to elevate corticoid levels for at least 4 h before administration of IL-1beta. The first study used dexamethasone pretreatment, the second utilized ACTH to stimulate endogenous cortisol release, while the third relied on a psychological challenge to stimulate the hypothalamic-pituitary-adrenal axis. Contrary to our a priori predictions, none of these treatments attenuated the IL-1-induced release of IL-6 into CSF. Additionally, the pattern in the blood response was similar, such that the IL-6 response was not blocked, although there was a trend toward a reduction of this response. These data indicated that the IL-1-induced IL-6 response is for the most part resistant to corticosteroid influence, such that even when a partial inhibition was sometimes evident in blood, cytokine release in the central nervous system was not affected.

The proinflammatory cytokine network: interactions in the CNS and blood of rhesus monkeys.

Proinflammatory cytokines [interleukin (IL)-1 and -6 and tumor necrosis factor-alpha] function within a complex network, stimulating the release of one another, as well as other cytokine agonists and antagonists. These interactions have not been as widely studied in vivo. Therefore, the following studies measured cytokines in blood and cerebrospinal fluid (CSF) from juvenile rhesus monkeys after intravenous administration of cytokines. IL-1 alpha and IL-1 beta were equally effective in elevating blood levels of IL-6. In contrast, IL-1 beta was the only cytokine that significantly elevated IL-6 levels in the CSF. Interestingly, both IL-1 and IL-6 increased levels of IL-1 receptor antagonist in the blood and comparably stimulated the release of cortisol. A second study confirmed that the IL-1-induced IL-6 in CSF was brain derived and not a result of diffusion from blood. This research extends studies of the cytokine cascade to the central nervous system (CNS), highlighting the brain response to peripheral activation.

Prenatal manipulations reduce the proinflammatory response to a cytokine challenge in juvenile monkeys.

Two studies were conducted to assess the potential long-term effects of prenatal stress on the cytokine-related inflammatory response in juvenile rhesus monkeys. Subjects were derived from two different pregnancy conditions. Study 1 involved endocrine activation of the pregnant female by daily adrenocorticotropic hormone (ACTH) injection across a 2-week period (days 120-133 post-conception). Pregnant females in Study 2 experienced a psychological stressor, 10 minutes per day, for a 6-week period (days 106-147 post-conception). When the offspring from these pregnancies were 1.5-2 years of age, they were administered recombinant human interleukin-1beta (rhIL-1beta) to stimulate the release of endogenous cytokines, elicit fever, and activate the hypothalamic-pituitary-adrenal (HPA) axis. Cerebrospinal fluid (CSF) and blood levels of interleukin-6 (IL-6) were measured, as well as cortisol levels and body temperature. The prenatal ACTH treatment altered the postnatal response to IL-1beta in juvenile offspring. These monkeys showed a significantly blunted response to the IL-1beta, with smaller increments in blood and CSF levels of IL-6 and diminished temperature responses to the IL-1beta. In contrast, the prenatal psychological stressor was not as potent and did not have lasting effects on this physiological response in juvenile monkeys. IL-1beta also induced significant increases in cortisol secretion, but this adrenal response was comparable in all monkeys. These data suggest that differences in the prenatal environment could have a selective effect on cytokine physiology accounting for individual differences in the inflammatory response.

Quinolinic acid and lymphocyte subsets in the intrathecal compartment as biomarkers of SIV infection and simian AIDS.

Cerebrospinal fluid (CSF) samples were collected from monkeys infected with SIVmac251 (SIV) or HIV-1/SIVmac chimeric viruses (SHIV(HXBc2) and SHIV(89.6P)) to investigate quinolinic acid (QUIN) levels in the intrathecal compartment. CSF levels of QUIN were elevated in the SIV-infected monkeys, especially in animals with end-stage disease, and in those infected with pathogenic SHIV(89.6P), but not after infection with the nonpathogenic construct SHIV(HXBc2). QUIN elevations occurred in association with reduced CD4+ and increased CD8+ lymphocytes, cellular alterations that were more pronounced in CSF than in the blood. These findings support the view that the intrathecal compartment provides a unique window on viral infection, and are in keeping with the a priori prediction that QUIN increases primarily in response to more pathogenic viral strains.

Dissociation of LPL and LDL: effects of lipoproteins and anti-apoB antibodies.

We have shown previously that the activity of lipoprotein lipase (LPL), the major enzyme responsible for hydrolysis of triglyceride contained in circulating lipoproteins, is associated with lipoproteins in postheparin plasma. In other studies, microtiter plate assays showed that LPL interaction with low density lipoprotein (LDL) and very low density lipoprotein (VLDL) was decreased by antibodies to apolipoprotein (apo)B. To test whether antibodies to apoB affected LPL-LDL association in solution, two types of assays were performed, gel filtration and coprecipitation. First we showed that LPL activity and immunoreactive mass co-eluted during gel filtration of normal postheparin plasma, approximately with the peak of low density lipoproteins. Then LPL was used for gel filtration studies in the presence and absence of LDL and anti-apoB monoclonal antibodies. LPL association with LDL was diminished by antibodies to the amino-terminal region of apoB; antibodies to the carboxyl-terminal LDL receptor binding region of apoB were less effective. LDL binding to LPL containing heparin-agarose was also disrupted by the amino-terminal antibodies to apoB. To determine the LPL-lipoprotein association in situations in which the distribution of plasma lipoproteins was altered, we studied plasma from two types of subjects with dyslipidemias. The addition of 125I-labeled LPL to type 1 postheparin plasma produced two peaks of radioactivity, one peak eluted in the void volume of the column (with the chylomicrons) and a second peak eluted just prior to the normal elution of low density lipoproteins. In postheparin plasma from an abetalipoproteinemic subject, LPL eluted with HDL. We conclude that LPL associates primarily with apoB-containing lipoproteins. The reason for this appears to be that LPL interacts with the apoB.

Context-dependent behavioral effects of interleukin-1 in the rhesus monkey (Macaca mulatta).

The behavioral effects of recombinant human interleukin-1 alpha (IL-1) in rhesus monkeys (Macaca mulatta) were assessed in 3 experimental paradigms: (1) a testing situation in which an initial quiescent period was followed by a challenge designed to evoke agitation; (2) a novel environment with a social partner; and (3) a working memory-dependent nonspatial cognitive task. In the first two experiments we replicated our previous observations that a high IL-1 dose (25 micrograms) induces somnolence in a quiet setting within 1 h. A lower IL-1 dose (1 ng) did not have these sedative properties, but both IL-1 doses significantly reduced the number of vocalizations made by the monkeys. In contrast, when the monkeys were challenged through direct eye contact with a human experimenter, the 25 micrograms IL-1 dose significantly increased agonistic behavior. Finally, performance on a working memory-dependent task (delayed non-matching-to-sample) was unaffected by doses of IL-1 ranging from 1 to 25 micrograms, possibly because the monkeys were tested after learning the task rather than during the acquisition phase. These results demonstrate that high levels of IL-1 in peripheral circulation can have potent behavioral effects in the nonhuman primate, but that the nature of the influence will depend on the context in which the animal is evaluated. Manifestation of cytokine-induced 'sickness behavior' appears to require a permissive environment.

Interleukin-1 beta differentially affects interleukin-6 and soluble interleukin-6 receptor in the blood and central nervous system of the monkey.

Two studies were conducted to investigate whether behavioral and physiological responses induced by administration of interleukin-1 beta (IL-1 beta) were also associated with changes in interleukin-6 (IL-6) and soluble IL-6 receptor levels (sIL-6R). Following intravenous injection of rhIL-1 beta, blood and cerebrospinal fluid (CSF) samples were collected from juvenile rhesus monkeys. Marked increases in IL-6 levels were evident at 1 h in both blood and intrathecal compartments. IL-1 beta also induced significant elevations in the release of ACTH and cortisol into the blood stream, and following high doses, the monkeys evinced signs of sickness behavior. The second study characterized the IL-beta dose-response relationship showing that these physiological changes were most evident at doses between 0.5 microgram and 1.0 microgram IL-1/kg body weight. Soluble IL-6 receptor concentration was also increased, but only in plasma. There was no detectable sIL-6R in CSF. The large release of IL-6 into CSF suggests that some behavioral symptoms may be due to intrinsic changes in central nervous system activity concomitant with the alterations in peripheral physiology.

Rehabilitation in the Philippines.

The international community has perspective and experience that will freshen our approaches to rehabilitation. Martin Grabois, MD(*), editor of this special section, has gathered articles written by experts from other countries. The intention is to stimulate thought, discussion, and action-and to broaden horizons.

The effects of malnutrition on the motor, perceptual, and cognitive functions of Filipino children.

The motor, perceptual, and cognitive abilities of 99 Filipino children, aged 4-6 years with a documented history of malnutrition from a nutritionally depressed area of Manila were determined using the Revised Manila Motor-Perceptual Screening Test. They were classified into four groups of: (1) normal; (2) acutely malnourished; (3) stunted but not malnourished; and (4) chronically malnourished using the Waterlow classification. Thirty-one normal children of comparable ages and background from a nationwide pool were similarly tested and served as the control group. Motor (p = 0.001) and perceptual skill (p less than 0.03 to less than 0.001) scores were significantly lower than in their normal counterparts, especially in the chronically malnourished children. Cognitive abilities were not evidently affected by malnutrition.

Energy cost of ambulation in healthy and disabled Filipino children.

The energy expenditures (Ee) for locomotion by nondisabled and disabled Filipino children aged 7 to 13 were determined and compared using indirect calorimetry. Forty-one controls (20 boys and 21 girls) ambulated at a comfortable pace; 16 children (eight boys and eight girls) with lower extremity poliomyelitis of varying severity ambulated by (1) wheelchair propulsion, (2) bilateral axillary crutches, (3) unilateral lower extremity ankle-foot orthoses or knee-ankle-foot orthoses, and (4) unassisted. Disabled children, regardless of their mode of ambulation, had to expend significantly more energy to ambulate than normal children (p less than 0.05). Wheelchair propulsion cost 16% more energy than the normal gait; crutch ambulation cost 41% more than the control. Children using unilateral braces sacrificed speed to attain near-normal Ee. When they ambulated without orthoses, their Ee increased by 109% over the control. In ascending order, the least energy was expanded by normal ambulation followed by disabled ambulation with unilateral brace, disabled propelling a wheelchair, disabled ambulation with bilateral axillary crutches, and disabled ambulation without brace. Efficiency of locomotion was reflected in the values obtained for Ee in terms of kcal x 10(-3)/kg/m, as demonstrated by the lower Ee but slower ambulation of children with braces, as compared to the nondisabled children.