ABSTRACT
Molecules that govern the formation, integrity, and function of the hippocampus remain an important area of investigation. Here we show that absence of the proneuropeptide processing enzyme, carboxypeptidase E (CPE) in CPE knock-out (KO) mice had a profound effect on memory, synaptic physiology, and the cytoarchitecture of the hippocampus in these animals. Adult CPE-KO mice displayed deficits in memory consolidation as revealed by the water-maze, object preference, and social transmission of food preference tests. These mice also showed no evoked long-term potentiation. Additionally, CPE-KO mice at 4 weeks of age and older, but not at 3 weeks of age, exhibited marked degeneration specifically of the pyramidal neurons in the hippocampal CA3 region which normally expresses high levels of CPE. Immunohistochemistry revealed that the neuronal marker, NeuN, was reduced, while the glial marker, GFAP, was increased, characteristic of gliosis in the CA3 area of CPE-KO mice. Calbindin staining indicated early termination of the mossy fibers before reaching the CA1 region in these mice. Thus, absence of CPE leads to degeneration of the CA3 neurons and perturbation of the cytoarchitecture of the hippocampus. Ex vivo studies showed that overexpression of CPE in cultured hippocampal neurons protected them against H(2)O(2) oxidative-stress induced cell death. These findings taken together indicate that CPE is essential for the survival of adult hippocampal CA3 neurons to maintain normal cognitive function.
Subject(s)
Carboxypeptidase H/metabolism , Memory Disorders/enzymology , Nerve Degeneration/enzymology , Pyramidal Cells/enzymology , Animals , Carboxypeptidase H/genetics , Gliosis/enzymology , Hippocampus/enzymology , Hippocampus/pathology , Immunohistochemistry , Long-Term Potentiation/physiology , Maze Learning/physiology , Mice , Mice, Knockout , Microscopy, Confocal , Nerve Degeneration/pathology , Oxidative Stress/physiology , Pyramidal Cells/pathology , Rats , Synapses/ultrastructure , Synaptic Transmission/physiologyABSTRACT
Pharmacological studies indicate that vasoactive intestinal peptide (VIP) may be necessary for normal embryonic development in the mouse. For example, VIP antagonist treatment before embryonic day 11 resulted in developmental delays, growth restriction, modified adult brain chemistry and reduced social behavior. Here, developmental milestones, growth, and social behaviors of neonates of VIP-deficient mothers (VIP +/-) mated to VIP +/- males were compared with the offspring of wild type mothers (VIP +/+) mated to VIP +/+ and +/- males, to assess the contributions of both maternal and offspring VIP genotype. Regardless of their own genotype, all offsprings of VIP-deficient mothers exhibited developmental delays. No delays were seen in the offspring of wild type mothers, regardless of their own genotype. Body weights were significantly reduced in offspring of VIP-deficient mothers, with VIP null (-/-) the most affected. Regardless of genotype, all offspring of VIP-deficient mothers expressed reduced maternal affiliation compared with wild type offspring of wild type mothers; +/- offspring of wild type mothers did not differ in maternal affiliation from their wild type littermates. Play behavior was significantly reduced in all offsprings of VIP-deficient mothers. Maternal behavior did not differ between wild type and VIP-deficient mothers, and cross-fostering of litters did not change offspring development, indicating that offspring deficits were induced prenatally. This study illustrated that the VIP status of a pregnant mouse had a greater influence on the growth, development and behavior of her offspring than the VIP genotype of the offspring themselves. Deficiencies were apparent in +/+, +/- and -/- offspring born to VIP-deficient mothers; no deficiencies were apparent in +/- offspring born to normal mothers. These results underscore the significant contribution of the uterine environment to normal development and indicate a potential usefulness of the VIP knockout mouse in furthering the understanding of neurodevelopmental disorders with social behavior deficits such as autism.
Subject(s)
Behavior, Animal/physiology , Social Behavior , Vasoactive Intestinal Peptide/physiology , Analysis of Variance , Animals , Body Weight/genetics , Body Weight/physiology , Female , Genotype , Male , Mice , Mice, Knockout , Vasoactive Intestinal Peptide/deficiency , Vasoactive Intestinal Peptide/geneticsABSTRACT
Vasoactive intestinal peptide (VIP) is an important mediator of development during the neural tube closure period of embryogenesis and may regulate, in part, the expression of activity-dependent neuroprotective protein (ADNP), which is essential for neural tube closure and embryogenesis. To evaluate the impact of VIP expression in vivo on ADNP and the related protein ADNP2 the current study examined gene expression in adult wild-type (VIP +/+) and VIP null (VIP -/-) offspring of VIP deficient mothers (VIP+/-) comparing them to wild-type offspring of wild-type mothers. Quantitative real time polymerase chain reaction (PCR), using an ABI Prisma cycler revealed regionally specific reductions of ADNP mRNA in the brains of VIP null mice compared with the brains of wild-type offspring of a wild-type mother. ADNP was significantly reduced in the cortex and hypothalamus of VIP null mice, but not in the hippocampus or thalamus. ADNP2 exhibited a similar pattern but reached a statistically significant reduction only in the hypothalamus. The mRNA for ADNP and ADNP2 also tended to be reduced in the cortex and hippocampus of the wild-type littermates of the VIP null mice, indicating that the VIP genotype of the mother may have had an impact on the ADNP expression of her offspring, regardless of their own VIP genotype. These results showed that VIP regulated brain ADNP expression in a regionally specific manner and indicated that both maternal and offspring VIP genotype may influence ADNP expression in the brain.
Subject(s)
Homeodomain Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neuroprotective Agents/metabolism , Neurulation/physiology , Protein Isoforms/metabolism , Vasoactive Intestinal Peptide/metabolism , Animals , Brain/anatomy & histology , Brain/metabolism , Female , Homeodomain Proteins/genetics , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Pregnancy , Protein Isoforms/genetics , Vasoactive Intestinal Peptide/geneticsABSTRACT
Vasoactive intestinal peptide (VIP) regulates growth and development during the early postimplantation period of mouse embryogenesis. Blockage of VIP with a VIP antagonist during this period results in growth restriction, microcephaly, and developmental delays. Similar treatment of neonatal rodents also causes developmental delays and impaired diurnal rhythms, and the adult brains of these animals exhibit neuronal dystrophy and increased VIP binding. These data suggest that blockage of VIP during the development of the nervous system can result in permanent changes to the brain. In the current study, pregnant mice were treated with a VIP antagonist during embryonic days 8 through 10. The adult male offspring were examined in tests of novelty, paired activity, and social recognition. Brain tissue was examined for several measures of chemistry and gene expression of VIP and related compounds. Glial cells from the cortex of treated newborn mice were plated with neurons and examined for VIP binding and their ability to enhance neuronal survival. Treated adult male mice exhibited increased anxiety-like behavior and deficits in social behavior. Brain tissue exhibited regionally specific changes in VIP chemistry and a trend toward increased gene expression of VIP and related compounds that reached statistical significance in the VIP receptor, VPAC-1, in the female cortex. When compared to control astrocytes, astrocytes from treated cerebral cortex produced further increases in neuronal survival with excess synaptic connections and reduced VIP binding. In conclusion, impaired VIP activity during mouse embryogenesis resulted in permanent changes to both adult brain chemistry/cell biology and behavior with aspects of autism-like social deficits.
Subject(s)
Behavior, Animal/physiology , Brain Chemistry , Embryonic Development/physiology , Vasoactive Intestinal Peptide , Animals , Astrocytes/cytology , Astrocytes/metabolism , Brain/anatomy & histology , Brain/metabolism , Cells, Cultured , Choline O-Acetyltransferase/metabolism , Female , Homeodomain Proteins/metabolism , Male , Mice , Nerve Tissue Proteins/metabolism , Neurons/cytology , Neurons/metabolism , Pregnancy , Social Behavior , Synaptophysin/metabolism , Vasoactive Intestinal Peptide/antagonists & inhibitors , Vasoactive Intestinal Peptide/chemistry , Vasoactive Intestinal Peptide/metabolismABSTRACT
Oxytocin mediates social affiliation behaviors and social memory in rodents. It has been suggested that disruptions in oxytocin contribute to the deficits in reciprocal social interactions that characterize autism. The present experiments employed a new social approach task for mice which is designed to detect low levels of sociability, representing the first diagnostic criterion for autism. Two lines of oxytocin knockout mice were tested, the National Institute of Mental Health line in Bethesda, and the Baylor/Emory line at the University of North Carolina in Chapel Hill. Similar methods were used for each line to evaluate tendencies to spend time with a stranger mouse versus with an inanimate novel object with no social valence. Adult C57BL/6J males were tested identically, as controls to confirm the robustness of the methods used in the social task. Comprehensive phenotyping of general health, neurological reflexes, olfactory and other sensory abilities, and motor functions was employed to assess both lines. No genotype differences were detected in any of the control measures for either line. Normal sociability, measured as time spent with a novel stranger mouse as compared to time spent with a novel object, was seen in both the NIMH and the Baylor/Emory lines of oxytocin null mutants, heterozygotes, and wild-type littermate controls. Normal preference for social novelty, measured as time spent with a second novel stranger as compared to time spent with a more familiar mouse, was seen in both the NIMH and the Baylor/Emory lines of oxytocin null mutants, heterozygotes, and wild-type littermate controls, with minor exceptions. Similar behavioral results from two independent targeted gene mutations, generated with different targeting vectors, bred on different genetic backgrounds, and tested in different laboratory environments, corroborates the negative findings on sociability in oxytocin mutant mice. Intact tendencies to spend time with another mouse versus with a novel object, in both lines of oxytocin knockouts, supports an interpretation that oxytocin plays a highly specific role in social memory, but is not essential for general spontaneous social approach in mice.
Subject(s)
Oxytocin/genetics , Oxytocin/physiology , Social Behavior , Animals , Behavior, Animal/physiology , Exploratory Behavior/physiology , Feeding Behavior/physiology , Handling, Psychological , Health Status , Heterozygote , Locomotion/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation/physiology , Phenotype , Reflex/genetics , Reflex/physiology , Reverse Transcriptase Polymerase Chain Reaction , Smell/genetics , Smell/physiology , Vocalization, Animal/physiologyABSTRACT
The Ts65Dn segmental mouse model of Down syndrome (DS) possesses a triplication of the section of chromosome 16 that is most homologous to the human chromosome 21 that is trisomic in DS. This model exhibits many of the characteristics of DS including small size, developmental delays, and a decline of cholinergic systems and cognitive function with age. Recent studies have shown that vasoactive intestinal peptide (VIP) systems are upregulated in aged Ts65Dn mice and that VIP dysregulation during embryogenesis is followed by the hypotonia and developmental delays as seen in both DS and in Ts65Dn mice. Additionally, astrocytes from aged Ts65Dn brains do not respond to VIP stimulation to release survival-promoting substances. To determine if VIP dysregulation is age-related in Ts65Dn mice, the current study examined VIP and VIP receptors (VPAC-1 and VPAC-2) in postnatal day 8 Ts65Dn mice. VIP and VPAC-1 expression was significantly increased in the brains of trisomic mice compared with wild-type mice. VIP-binding sites were also significantly increased in several brain areas of young Ts65Dn mice, especially in the cortex, caudate/putamen, and hippocampus. Further, in vitro treatment of normal neurons with conditioned medium from VIP-stimulated Ts65Dn astrocytes from neonatal mice did not enhance neuronal survival. This study indicates that VIP anomalies are present in neonatal Ts65Dn mice, a defect occurs in the signal transduction mechanism of the VPAC-1 VIP receptor, cortical astrocytes from neonatal brains are dysfunctional, and further, that VIP dysregulation may play a significant role in DS.
Subject(s)
Astrocytes/physiology , Down Syndrome/physiopathology , Vasoactive Intestinal Peptide/metabolism , Vasoactive Intestinal Peptide/pharmacology , Animals , Animals, Newborn , Astrocytes/drug effects , Cerebral Cortex/drug effects , Cerebral Cortex/physiopathology , Chromosomes, Human, Pair 21 , DNA Primers , Diploidy , Disease Models, Animal , Down Syndrome/genetics , Humans , In Situ Hybridization, Fluorescence , Mice , Polymerase Chain Reaction , Receptors, Vasoactive Intestinal Peptide/genetics , Vasoactive Intestinal Peptide/geneticsABSTRACT
NAP is a short octapeptide sequence (single letter code, NAPVSIPQ) that protects neurons against a wide variety of insults. The NAP sequence was identified by peptide structure/function scanning of activity-dependent neuroprotective protein (ADNP), a gene product essential for brain formation. To further evaluate the in vivo efficacy of NAP neuroprotection we used a mouse model of head trauma; a condition that presents a risk factor for the development of Alzheimer's disease in injured patients. In the mouse model, NAP treatment (prophylactic or curative) indicated improvement in longitudinal clinical, biochemical and anatomical outcomes. Furthermore, closed head injury was associated with a delayed increase in the expression of the immune cell surface glycoprotein Mac-1 (CD11B antigen) at the injury site that was decreased in NAP-treated mice. Additional experiments with Mac-1-deficient mice suggested partial protection against death related to severe head injury. NAP protection in Mac-1-deficient mice against adverse clinical outcome was concomitant with the time period when increases in Mac-1 transcripts were observed in the Mac-1 expressing mice ( approximately four weeks after the injury). The expression of ADNP (the NAP parent protein) was also increased at the injured brain site four weeks after the traumatic event, only in Mac-1 expressing mice. Here, using immunocytochemistry, we localized the increase in ADNP to microglia and astrocyte-like cells. The increase in ADNP in injured brains is now suggested to be a part of an endogenous compensatory mechanism and NAP treatment provides an additional protection. Toxicology studies suggest NAP as safe for further clinical development.
Subject(s)
Brain Injuries/drug therapy , Brain Injuries/metabolism , Homeodomain Proteins/biosynthesis , Hypoxia, Brain/drug therapy , Hypoxia, Brain/metabolism , Nerve Tissue Proteins/biosynthesis , Oligopeptides/therapeutic use , Animals , Brain Injuries/genetics , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Homeodomain Proteins/genetics , Hypoxia, Brain/genetics , Male , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Oligopeptides/pharmacologyABSTRACT
Activity-dependent neuroprotective protein (ADNP) is a highly conserved vasoactive intestinal peptide (VIP) responsive gene that is expressed abundantly in the brain and in the body and is essential for brain formation and embryonic development. Since, VIP exhibits sexual dimorphism in the hypothalamus, the potential differential expression of ADNP in male and female mice was investigated. Real-time polymerase chain reaction revealed sexual dimorphism in ADNP mRNA expression as well as fluctuations within the estrus cycle. Immunohistochemistry with an antibody to ADNP showed specific staining in the arcuate nucleus of the hypothalamus. ADNP-like immunoreactivity in the arcuate nucleus also exhibited fluctuations during the estrus cycle. Here, brain sections at proestrus were the most immunoreactive and brain sections at estrus--the least. Furthermore, male arcuate nucleus ADNP-like immunoreactivity was significantly lower than that of the female estrus. Many neuropeptides, neurotransmitters and proteins are localized to the arcuate nucleus where they contribute to the regulation of reproductive cyclicity and energy homeostasis. The results presented here suggest that ADNP has a part in the estrus cycle as an affecter or an effector.
Subject(s)
Arcuate Nucleus of Hypothalamus/metabolism , Homeodomain Proteins/metabolism , Nerve Tissue Proteins/metabolism , Sex Characteristics , Animals , Estrous Cycle/physiology , Female , Hypothalamus/metabolism , Immunohistochemistry , Male , Mice , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A carboxypeptidase E (CPE) knockout (KO) mouse was generated by deletion of exons 4 and 5 from the CPE gene, and its phenotype was characterized. KO mice became obese by 10-12 wk of age and reached 60-80 g by 40 wk. At this age, body fat content was more than double that in the wild-type (WT) controls. The null animals consumed more food overall, were less physically active during the light phase of the light-dark cycle, and burned fewer calories as fat than WT littermates. Fasting levels of glucose and insulin-like immunoreactivity in plasma were elevated in both male and female KO mice at approximately 20 wk; males recovered fully and females partially from this state by 32 wk. At this time, insulin-like immunoreactivity in the plasma, identified as proinsulin, was 50-100 times higher than that of the WT animals. The KO mice showed impaired glucose clearance and were insulin resistant. High levels of leptin and no circulating fully processed cocaine- and amphetamine-related transcript, a peptide that is responsive to leptin-induced feedback inhibition of feeding, were found in serum. The KO mice were subfertile and showed deficits in GnRH processing in the hypothalamus. Behavioral analyses revealed that KO animals showed diminished reactivity to stimuli and had reduced muscle strength and coordination, as well as visual placing and toe-pinch reflexes. These data demonstrate that CPE KO mice display a wide range of neural and endocrine abnormalities and suggest that CPE may have additional physiological roles beyond those ascribed to peptide processing and sorting of prohormones in cells.
Subject(s)
Carboxypeptidase H/genetics , Endocrine System Diseases/genetics , Endocrine System Diseases/physiopathology , Obesity/genetics , Obesity/physiopathology , Adipose Tissue/anatomy & histology , Animals , Behavior, Animal , Body Size/genetics , Body Weight/genetics , Calorimetry, Indirect , Chromatography, High Pressure Liquid , Drinking , Eating , Endocrine System Diseases/mortality , Female , Fertility , Glucose Intolerance/genetics , Glucose Intolerance/metabolism , Glucose Intolerance/mortality , Insulin Resistance/genetics , Leptin/blood , Male , Mice , Mice, Knockout , Nerve Tissue Proteins/blood , Obesity/mortality , Phenotype , Proinsulin/bloodABSTRACT
The effects of vasoactive intestinal peptide (VIP) on the proliferation of central nervous system (CNS) and cancer cells were investigated. VIP has important actions during CNS development. During neurogenesis, VIP stimulates the proliferation and differentiation of brain neurons. Addition of VIP to embryonic mouse spinal cord cultures increases neuronal survival and activity dependent neurotrophic factor (ADNF) secretion from astroglial cells. VIP is an integrative regulator of brain growth and development during neurogenesis and embryogenesis. Also, VIP causes increased proliferation of human breast and lung cancer cells in vitro. VIP binds with high affinity to cancer cells, elevates the cAMP and increases gene expression of c-fos, c-jun, c-myc and vascular endothelial cell growth factor. The effects of VIP on cancer cells are reversed by VIPhybrid, a synthetic VPAC(1) receptor antagonist. VIPhyb inhibits the basal growth of lung cancer cells in vitro and tumors in vivo and potentiates the ability of chemotherapeutic drugs to kill cancer cells. Due to the high density of VPAC(1) receptors in cancer cells, VIP has been radiolabeled with 123I, 18F and 99mTc to image tumors. It remains to be determined if radiolabeled VIP analogs will be useful agents for early detection of cancer in patients.
Subject(s)
Central Nervous System/physiology , Neoplasms/physiopathology , Vasoactive Intestinal Peptide/metabolism , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Second Messenger Systems , Sequence Homology, Amino Acid , Vasoactive Intestinal Peptide/chemistryABSTRACT
Vasoactive intestinal peptide (VIP), a regulator of embryonic growth, increased the concentration of nerve growth factor (NGF)-like immunoreactivity in the conditioned medium of cultured explanted embryonic day (E) 9.5 neural tube preparations compared to control preparations. VIP treatment also induced an increase of NGF-like immunoreactivity (NGF-IR) within the neural tube preparation tissue. A 60 kDa isoform was the primary form of NGF detected. VIP is shown to be a regulator of NGF in the E9.5 embryonic mouse and stimulates the release of a high molecular weight isoform of NGF.
Subject(s)
Nerve Growth Factor/physiology , Vasoactive Intestinal Peptide/pharmacology , Animals , Culture Media, Conditioned , Embryo, Mammalian , Gene Expression Regulation, Developmental , L-Lactate Dehydrogenase/analysis , Mice , Nerve Growth Factor/chemistry , Nerve Growth Factor/genetics , Protein Isoforms/chemistry , Protein Isoforms/drug effects , Protein Isoforms/genetics , Protein Isoforms/physiologyABSTRACT
Vasoactive intestinal peptide (VIP) regulates growth in the early post-implantation embryo. Previous work has demonstrated that peptide agonists (SALLRSIPA and NAPVSIPQ) from downstream mediators that are regulated by VIP were able to prevent the alcohol-induced fetal death, growth restriction and microcephaly associated with fetal alcohol syndrome. Here we evaluated the role of VIP in this mouse model of fetal alcohol syndrome, to determine if fetal or maternal levels of VIP are altered. In addition, we evaluated whether peptide treatment would alter the effects of alcohol on VIP levels. Treatment groups included control, alcohol, and alcohol+peptides. VIP levels were measured with enzyme immunoassay [EIA] (Peninsula Laboratories, Belmont, CA). Quantitation of VIP expression was measured with rt-PCR using mimic cDNA primers. Embryo/decidual VIP levels were similar in control and alcohol-treated groups 6 h after treatment. However, in the embryo/deciduas at 12 and 24 h, VIP levels were below the EIA's detection limit in the alcohol-treated groups, and significantly lower than the control or peptide-pretreated groups (p<0.05). Maternal cortex VIP levels were undetectable and significantly lower in the alcohol-treated group than control or peptide+alcohol group at 6 and 12 h (p<0.001). VIP mRNA expression was quantitated in the embryo and deciduas, with a significant decline noted at 6 h to 58% of control levels (p=0.02). Pretreatment with the peptides attenuated the alcohol-induced decrease in VIP mRNA. These studies demonstrate that treatment with alcohol can decrease the expression and immunoreactivity of VIP in both maternal and fetal tissues. This alcohol-induced loss of a recognized regulator of embryonic growth and differentiation may contribute to the sequelae of toxicity observed in fetal alcohol syndrome.
Subject(s)
Fetal Alcohol Spectrum Disorders/genetics , Gene Expression Regulation, Developmental , Transcription, Genetic , Vasoactive Intestinal Peptide/metabolism , Animals , Disease Models, Animal , Female , Fetal Death , Gene Expression Regulation, Developmental/physiology , Gestational Age , Maternal-Fetal Exchange , Mice , Pregnancy , RNA, Messenger/geneticsABSTRACT
A complex mixture of five cytokines has been shown to be released by vasoactive intestinal peptide (VIP). Cytokines were measured in paired samples of culture medium and astroglial cytosol by capillary electrophoresis. This is the first description of VIP-mediated release for TNF-alpha, IL-3, G-CSF and M-CSF from astrocyte cultures. Kinetic studies after VIP treatment demonstrated a gradual but incomplete depletion of cytosolic cytokine levels, with differences observed among the cytokines. Significant increases in release were apparent within 15-30 min for all cytokines. As the recognized VIP receptors (VPAC1 and VPAC2) are linked to adenylate cyclase and also interact with pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), both this homologous peptide and 8-bromo cAMP were investigated and compared to VIP-mediated release. Treatment with 1 mM 8-bromo cAMP produced cytokine release similar in amount to 0.1 nM PACAP-38, but significantly less (<50%) in comparison to 0.1 nM VIP. PACAP-38 and VIP exhibited similar EC(50)'s for the release of G-CSF and TNF-alpha; however, the maximal release was 4-6 times greater for VIP than for PACAP-38. This similarity in potency suggested a VPAC-like receptor; however, the greater efficacy for VIP in comparison to PACAP-38, combined with a lack of cAMP production at subnanomolar concentrations of VIP, suggested a mechanism not currently associated with VPAC receptors. For M-CSF, IL-3 and IL-6, the EC(50)'s of VIP were 3-30 times more potent than those of PACAP-38 in producing release. These studies suggested that multiple mechanisms mediate cytokine release in astrocytes: (1) a low efficacy release produced by PACAP-38 that is cAMP-mediated and (2) a high efficacy, VIP-preferring mechanism that was not linked to cAMP. In summary, subnanomolar concentrations of VIP released a complex array of cytokines from astrocytes that may contribute to the mitogenic and neurotrophic properties of this neuropeptide in the central nervous system.
Subject(s)
Cytokines/metabolism , Vasoactive Intestinal Peptide/pharmacology , Adenylyl Cyclases/metabolism , Animals , Animals, Newborn , Astrocytes/drug effects , Astrocytes/metabolism , Cells, Cultured , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Interleukin-3/metabolism , Kinetics , Macrophage Colony-Stimulating Factor/metabolism , Neuroglia/metabolism , Neuropeptides/pharmacology , Pituitary Adenylate Cyclase-Activating Polypeptide , RatsABSTRACT
Blockage of vasoactive intestinal peptide (VIP) receptors during early embryogenesis in the mouse has been shown to result in developmental delays in neonates, and social behavior deficits selectively in adult male offspring. Offspring of VIP deficient mothers (VIP +/-) also exhibited developmental delays, and reductions in maternal affiliation and play behavior. In the current study, comparisons among the offspring of VIP deficient mothers (VIP +/-) mated to VIP +/- males with the offspring of wild type (WT) mothers mated to VIP +/- males allowed assessment of the contributions of both maternal and offspring VIP genotype to general health measures, social behavior, fear conditioning, and spatial learning and memory in the water maze. These comparisons revealed few differences in general health among offspring of WT and VIP deficient mothers, and all offspring exhibited normal responses in fear conditioning and in the acquisition phase of spatial discrimination in the water maze. WT mothers produced offspring that were normal in all tests; the reduced VIP in their VIP +/- offspring apparently did not contribute to any defects in the measures under study. However, regardless of their own VIP genotype, all male offspring of VIP deficient mothers exhibited severe deficits in social approach behavior and reversal learning. The deficits in these behaviors in the female offspring of VIP deficient mothers were less severe than in their male littermates, and the extent of their impairment was related to their own VIP genotype. This study has shown that intrauterine conditions had a greater influence on behavioral outcome than did genetic inheritance. In addition, the greater prevalence of deficits in social behavior and the resistance to change seen in reversal learning in the male offspring of VIP deficient mothers indicate a potential usefulness of the VIP knockout mouse in furthering the understanding of neurodevelopmental disorders such as autism.
Subject(s)
Learning Disabilities/genetics , Reversal Learning/physiology , Sex Characteristics , Social Behavior , Vasoactive Intestinal Peptide/deficiency , Analysis of Variance , Animals , Behavior, Animal , Conditioning, Psychological/physiology , Exploratory Behavior/physiology , Fear/physiology , Female , Habituation, Psychophysiologic/genetics , Male , Maze Learning , Mice , Mice, Knockout , Olfactory Pathways/physiology , Reflex/genetics , Spatial Behavior/physiology , Time FactorsABSTRACT
Vasoactive intestinal peptide (VIP) mediates important events during the development of the nervous system. VIP can stimulate neuronogenesis as well as differentiation and neurite outgrowth; it can promote the survival of neurons and assist in neuronal repair; it is also anti-inflammatory and can modulate immune responses. In addition, VIP is necessary for the normal growth and development of the early postimplantation mouse embryo during the period when the major embryonic events are neural tube formation, neuronogenesis and expansion of the vascular system. Receptors for VIP appear during early postimplantation embryogenesis in the rodent and exhibit changing localization patterns throughout the development of the brain. During embryogenesis, unregulated VIP may have major and permanent consequences on the formation of the brain and may be a participating factor in disorders of neurodevelopment. VIP has been linked to autism, Down syndrome and fetal alcohol syndrome. This paper will review the role of VIP in neurodevelopment, its known involvement in neurodevelopmental disorders and propose ways in which VIP might be of therapeutic value.
Subject(s)
Nervous System Diseases/metabolism , Vasoactive Intestinal Peptide/physiology , Vasoactive Intestinal Peptide/therapeutic use , Animals , Central Nervous System/embryology , Central Nervous System/metabolism , Central Nervous System Vascular Malformations/embryology , Central Nervous System Vascular Malformations/prevention & control , Humans , Mice , Nervous System Diseases/drug therapy , Nervous System Diseases/embryologyABSTRACT
Vasoactive intestinal peptide (VIP) is a regulator of rodent embryogenesis during the period of neural tube closure. VIP enhanced growth in whole cultured mouse embryos; treatment with a VIP antagonist during embryogenesis inhibited growth and development. VIP antagonist treatment during embryogenesis also had permanent effects on adult brain chemistry and impaired social recognition behavior in adult male mice. The neurological deficits of autism appear to be initiated during neural tube closure and social behavior deficits are among the key characteristics of this disorder that is more common in males and is frequently accompanied by mental retardation. The current study examined the blockage of VIP during embryogenesis as a model for the behavioral deficits of autism. Treatment of pregnant mice with a VIP antagonist during embryonic days 8 through 10 had no apparent effect on the general health or sensory or motor capabilities of adult offspring. However, male offspring exhibited reduced sociability in the social approach task and deficits in cognitive function, as assessed through cued and contextual fear conditioning. Female offspring did not show these deficiencies. These results suggest that this paradigm has usefulness as a mouse model for aspects of autism as it selectively impairs male offspring who exhibit the reduced social behavior and cognitive dysfunction seen in autism. Furthermore, the study indicates that the foundations of some aspects of social behavior are laid down early in mouse embryogenesis, are regulated in a sex specific manner and that interference with embryonic regulators such as VIP can have permanent effects on adult social behavior.
Subject(s)
Autistic Disorder/metabolism , Brain/embryology , Cognition Disorders/metabolism , Mental Disorders/metabolism , Prenatal Exposure Delayed Effects/metabolism , Vasoactive Intestinal Peptide/metabolism , Aging/physiology , Animals , Animals, Newborn , Autistic Disorder/etiology , Autistic Disorder/physiopathology , Avoidance Learning/drug effects , Avoidance Learning/physiology , Brain/drug effects , Brain/physiopathology , Cognition/drug effects , Cognition/physiology , Cognition Disorders/chemically induced , Cognition Disorders/physiopathology , Disease Models, Animal , Female , Habituation, Psychophysiologic/drug effects , Habituation, Psychophysiologic/physiology , Male , Mental Disorders/chemically induced , Mental Disorders/physiopathology , Mice , Peptides/pharmacology , Pregnancy , Prenatal Exposure Delayed Effects/chemically induced , Prenatal Exposure Delayed Effects/physiopathology , Sex Characteristics , Smell/drug effects , Smell/physiology , Social Behavior , Vasoactive Intestinal Peptide/antagonists & inhibitorsABSTRACT
Cocaine has become a popular illicit drug in our society, and pregnant women are not immune from this epidemic. Recently, there have been several references in the literature describing an association between prenatal cocaine exposure and the subsequent development of necrotizing enterocolitis in the neonate, but the mechanism underlying this relationship remains speculative. Because alpha-2 adrenergic receptors are thought to play a role in the autoregulatory mechanism in the newborn intestine that responds to hypoxia and ischemia, we examined the expression of this receptor in the intestine of embryonic rats exposed to low- and high-dose cocaine in utero. Pregnant Sprague Dawley rats were injected daily with either saline, low-dose cocaine, or high-dose cocaine beginning on embryonic d 5 (E 5) and continuing to E 20. Mothers were killed on E 16, E 17, E 18, E 19, and E 20. Embryos were frozen and stored at -80 degrees C. In situ hybridization was performed on 20- micro m sections with 35S-labeled oligonucleotide probes specific for the alpha-2A adrenergic receptor subtype. Densitometric analysis revealed a significant decrease in the alpha-2A receptor expression in the intestine of both the low-dose and high-dose cocaine-exposed animals compared with controls. This down-regulation was demonstrated by E 17, and continued through the remainder of gestation. These changes may limit the normal adaptation to vasoconstriction, thus exacerbating the already insufficient compensatory mechanisms for responding to ischemic injury, and thus may be one of the important factors predisposing cocaine-exposed infants to necrotizing enterocolitis.
Subject(s)
Cocaine/pharmacology , Intestines/drug effects , Receptors, Adrenergic, alpha-2/genetics , Animals , Base Sequence , DNA Primers , Female , In Situ Hybridization , Intestinal Mucosa/metabolism , Intestines/embryology , Pregnancy , Rats , Rats, Sprague-DawleyABSTRACT
OBJECTIVE: Fetal alcohol syndrome (FAS) results in stillbirth, fetal growth restriction, and mental retardation with injury attributed to oxidative stress. Our objective was to identify signal transduction pathways expressed in a model of FAS and to quantify expression of c-fos, a gene in the stress signal pathway. STUDY DESIGN: Timed, pregnant C57Bl6/J mice were injected on E8 with saline solution or alcohol. RNA was extracted from decidua and embryo 6 and 24 hours later. Microarray analysis was used to screen gene pathways. Differential gene expression was confirmed using real-time polymerase chain reaction with results presented as the ratio of c-fos concentration to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: Differential gene expression between alcohol and control was noted for stress signal pathway genes including c-fos. Real-time polymerase chain reaction demonstrated that c-fos messenger RNA expression was greater in the alcohol than control decidua at 6 hours after injection (P<.01). This effect persisted at 24 hours (P<.01). There was no difference in c-fos expression in embryos whose mothers received alcohol versus control after 6 hours (P=.12) or 24 hours (P=.89). CONCLUSION: Alcohol administration during pregnancy results in differential gene expression in the stress signal pathway, particularly in c-fos. C-fos expression in the decidua increases from 6 to 24 hours after alcohol injection, but does not change in the embryo, which may contribute to alcohol-induced damage in FAS.
Subject(s)
Disease Models, Animal , Fetal Alcohol Spectrum Disorders/genetics , Gene Expression , Genes, fos , Animals , Decidua/chemistry , Embryo, Mammalian/chemistry , Ethanol/administration & dosage , Female , Gestational Age , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Humans , Mice , Mice, Inbred C57BL , Polymerase Chain Reaction , Pregnancy , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Signal TransductionABSTRACT
OBJECTIVE: Fetal alcohol syndrome (FAS) is the most common nongenetic cause of mental retardation. Peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL), related to activity-dependent neuroprotective protein (ADNP), prevent alcohol-induced damage in a mouse model of FAS. Our objective was to characterize ADNP in this model to relate this protein to the mechanisms of damage and peptide neuroprotection. STUDY DESIGN: Timed, pregnant C57Bl6/J mice were treated on day 8. Groups were control, alcohol, peptide pretreatment, or peptide alone. Embryo and decidua were harvested at 6 and 24 hours and 10 days. To evaluate ADNP expression, real-time polymerase chain reaction was performed with results presented as the ratio of ADNP-to-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) concentration. Analysis of variance was performed for overall comparisons with P<.05 considered significant. RESULTS: At 6 hours, there was no difference in ADNP between alcohol-exposed embryos compared with control embryos. At 24 hours, there was an increase in ADNP in alcohol-exposed embryos compared with controls (P<.001); these findings persisted at 10 days (P<.001). In the decidua at 6 hours, there was no difference between alcohol and control. At 24 hours, there was greater ADNP in alcohol-exposed decidua compared with controls (P<.001), which did not persist at 10 days (P=.97). Peptide pretreatment did not prevent the alcohol-induced increase in ADNP in embryo or decidua. CONCLUSION: Alcohol increased embryonic and decidual ADNP expression at 24 hours and it persisted in the embryo for 10 days. Because ADNP is a known neuroprotectant, these findings suggest that it may be released as a protective mechanism in FAS. Changes in the embryo were persistent suggesting that the embryo is more vulnerable to alcohol-induced damage than the mother.
Subject(s)
Disease Models, Animal , Fetal Alcohol Spectrum Disorders/physiopathology , Homeodomain Proteins/physiology , Nerve Tissue Proteins/physiology , Animals , Decidua/chemistry , Embryo, Mammalian/chemistry , Ethanol/administration & dosage , Female , Gene Expression , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Homeodomain Proteins/administration & dosage , Homeodomain Proteins/genetics , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/administration & dosage , Nerve Tissue Proteins/genetics , Pregnancy , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The most common genetic cause of mental retardation is Down syndrome, trisomy of chromosome 21, which is accompanied by small stature, developmental delays, and mental retardation. In the Ts65Dn segmental trisomy mouse model of Down syndrome, the section of mouse chromosome 16 most homologous to human chromosome 21 is trisomic. This model exhibits aspects of Down syndrome including growth restriction, delay in achieving developmental milestones, and cognitive dysfunction. Recent data link vasoactive intestinal peptide malfunction with developmental delays and cognitive deficits. Blockage of vasoactive intestinal peptide during rodent development results in growth and developmental delays, neuronal dystrophy, and, in adults, cognitive dysfunction. Also, vasoactive intestinal peptide is elevated in the blood of newborn children with autism and Down syndrome. In the current experiments, vasoactive intestinal peptide binding sites were significantly increased in several brain areas of the segmental trisomy mouse, including the olfactory bulb, hippocampus, cortex, caudate/putamen, and cerebellum, compared with wild-type littermates. In situ hybridization for VIP mRNA revealed significantly more dense vasoactive intestinal peptide mRNA in the hippocampus, cortex, raphe nuclei, and vestibular nuclei in the segmental trisomy mouse compared with wild-type littermates. In the segmental trisomy mouse cortex and hippocampus, over three times as many vasoactive intestinal peptide-immunopositive cells were visible than in wild-type mouse cortex. These abnormalities in vasoactive intestinal peptide parameters in the segmental trisomy model of Down syndrome suggest that vasoactive intestinal peptide may have a role in the neuropathology of Down-like cognitive dysfunction.