Normalization of NF-κB activity in dorsal root ganglia neurons cultured from diabetic rats reverses neuropathy-linked markers of cellular pathology.

AIMS/HYPOTHESIS: Dorsal root ganglia (DRG) sensory neurons cultured from 3 to 5 month streptozotocin (STZ)-induced diabetic rats exhibit structural and biochemical changes seen in peripheral nerve fibers in vivo, including axonal swellings, oxidative damage, reduced axonal sprouting, and decreased NF-κB activity. NF-κB is a transcription factor required by DRG neurons for survival and plasticity, and regulates transcription of antioxidant proteins (e.g. MnSOD). We hypothesized that the diabetes-induced decrease in NF-κB activity in DRG contributes to pathological phenomena observed in cultured DRG neurons from diabetic rats. METHODS: NF-κB localization was assessed in intact DRG and neuron cultures using immunostaining. NF-κB activity was manipulated in sensory neuron cultures derived from age-matched normal or 3-5 month STZ-diabetic rats using pharmacological means and lentiviral expression of shRNA. The impact of diabetes and altered NF-κB activity on neuronal phenotype involved analysis of neurite outgrowth, neurite morphology, oxidative stress (lipid peroxidation) and expression of MnSOD. RESULTS: STZ-induced diabetes caused a significant decrease in nuclear localization of NF-κB subunits p50 and c-rel, but no change in p65 in intact DRG. Inhibition of NF-κB in normal neuron cultures significantly increased axonal swellings and oxidative stress, and reduced both neurite outgrowth and expression of MnSOD. These phenomena mimicked markers of pathology in cultured DRG neurons from diabetic rats. Enhancement of NF-κB activity in cultured diabetic DRG neurons ameliorated the sub-optimal neurite outgrowth and MnSOD levels triggered by diabetes. Exogenous insulin enhanced nuclear localization of p50 and c-rel but not p65 in diabetic neuronal cultures. CONCLUSION/INTERPRETATION: The diabetes-induced decrease of nuclear localization of NF-κB subunits p50 and c-rel in DRG contributes to development of in vitro markers of peripheral neuropathy, possibly through impaired mitochondrial ROS scavenging by deficient MnSOD.

Evidence for the involvement of calbindin D28k in the presenilin 1 model of Alzheimer's disease.

Pathological hallmarks of Alzheimer's disease include memory deficits, accumulation of amyloid beta (Abeta) plaques, the appearance of neurofibrillary tangles, and dysregulation of calcium homeostasis, which has been linked to mutations in the presenilin gene that code for presenilin (PS) proteins. PSs are a family of multi-pass transmembrane proteins where normal presenilins (PS1 and PS2) are highly localized in the endoplasmic reticulum (ER). Several past studies have explored alterations in long-term potentiation (LTP), a proposed molecular correlate of memory, and in behavioral tests of spatial memory in a variety of PS1 models. These reports suggest that calcium plays a role in these alterations, but mechanistic explanations for changes in LTP and in behavioral tests of memory are still lacking. To test the hypothesis that calcium-related mechanisms, such as changes in calcium buffering, are associated with alterations in LTP and memory, we utilized in vitro experimental paradigms of LTP in hippocampal slices obtained from the PS1-M146V transgenic mouse model of Alzheimer's disease (AD). We also used the in vivo Morris water maze (MWM), a test for hippocampal dependent spatial memory. In addition, we used cellular assays to explore molecular mechanisms. We confirm that PS1 mutations (M146V) enhance LTP. We also find increases in some parameters of the MWM, and alterations in other parameters, such as path length indicating impairment in cognitive functioning in PS1-M146V mice. In addition, these findings are observed in association with increased calbindin D28K expression in the CA1 hippocampus of PS1-M146V mice.

Xestospongin C empties the ER calcium store but does not inhibit InsP3-induced Ca2+ release in cultured dorsal root ganglia neurones.

The action of Xestospongin C (XeC) on calcium concentration in the cytosol ([Ca2+]i) and within the lumen of endoplasmic reticulum (ER) ([Ca2+]L) was studied using cultured dorsal root ganglia (DRG) neurones. Application of 2.5 microM of XeC triggered a slow [Ca2+]i transient as measured by Fura-2 video-imaging. The kinetics and amplitude of XeC-induced [Ca2+]i response was similar to that triggered by 1 microM thapsigargin (TG). The [Ca2+]L was monitored in cells loaded with low-affinity Ca2+ indicator Mag-Fura-2. The cytosolic portion of Mag-Fura-2 was removed by permeabilisation of the plasmalemma with saponin. Application of XeC to these permeabilised neurones resulted in a slow depletion of the ER Ca2+ store. XeC, however, failed to inhibit inositol 1,4,5-trisphosphate (InsP3)-induced [Ca2+]L responses. We conclude that XeC is a potent inhibitor of sarco(endo)plasmic reticulum calcium ATPase, and it cannot be regarded as a specific inhibitor of InsP3 receptors in cultured DRG neurones.

Endoplasmic reticulum D-myo-inositol 1,4,5-trisphosphate-sensitive stores regulate nuclear factor-kappaB binding activity in a calcium-independent manner.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays critical roles in neuronal survival and plasticity and in activation of immune responses. The activation of NF-kappaB has been closely associated with changes in intracellular calcium levels, but the relationship between the two remains unclear. Here we report that inhibition of endoplasmic reticulum (ER) d-myo-inositol 1,4,5-trisphosphate (IP(3))-gated calcium release caused decreased basal NF-kappaB DNA-binding activity in cultured rat cortical neurons. Activation of NF-kappaB in response to tumor necrosis factor-alpha and glutamate was completely abolished when IP(3) receptors were blocked, and NF-kappaB activation in response to depletion of ER calcium by thapsigargin treatment was also decreased by IP(3) receptor blockade. We further investigated the relationship between IP(3) receptor activation and NF-kappaB activity using a cell-free system. Microsomes enriched in the ER were isolated from adult rat cerebral cortex, resuspended, and treated with agents that induce or inhibit ER calcium release. They were then recentrifuged, and the supernatant was added to cytoplasmic extract isolated from the same source tissue. We found that microsomes released an NF-kappaB-stimulating signal in response to activation of IP(3) receptors or inhibition of the ER Ca(2+)-ATPase, but not in response to ryanodine. Studies of intact cells and cell-free preparations indicated that the signal released from the ER was not calcium and was heat- and trypsin-sensitive. Our data suggest that activation of IP(3) receptors is required for a major component of both constitutive and inducible NF-kappaB binding activity in neurons and that decreasing ER intraluminal calcium levels triggers release of a diffusible NF-kappaB-activating signal from the ER.

Nuclear factor-kappaB mediates the cell survival-promoting action of activity-dependent neurotrophic factor peptide-9.

Activity-dependent neurotrophic factor (ADNF) is produced by astrocytes in response to neuronal depolarization and, in turn, promotes neuronal survival. A nineamino acid ADNF peptide (ADNF9) exhibits full neurotrophic activity and potently protects cultured embryonic rat hippocampal neurons from oxidative injury and apoptosis. Picomolar concentrations of ADNF9 induced an increase in nuclear factor-kappaB (NF-kappaB) DNA-binding activity within 1 h of exposure, with a maximum increase of approximately 10-fold by 6 h. Activation of NF-kappaB was correlated with increased resistance of neurons to apoptosis induced by exposure to Fe(2+). The antiapoptotic action of ADNF9 was abolished when NF-kappaB activation was specifically blocked with kappaB decoy DNA. Oxidative stress was attenuated in neurons pretreated with ADNF9, and this effect of ADNF9 was blocked by kappaB decoy DNA, suggesting that ADNF9 suppresses apoptosis by reducing oxidative stress. ADNF9 also prevented neuronal apoptosis following trophic factor withdrawal via an NF-kappaB-mediated mechanism. Thus, NF-kappaB mediates the neuron survival-promoting effects of ADNF9 in experimental models relevant to developmental neuronal death and neurodegenerative disorders.

Caspase-mediated degradation of AMPA receptor subunits: a mechanism for preventing excitotoxic necrosis and ensuring apoptosis.

Activation of ionotropic glutamate receptors of the AMPA and NMDA subtypes likely contributes to neuronal injury and death in various neurodegenerative disorders. Excitotoxicity can manifest as either apoptosis or necrosis, but the mechanisms that determine the mode of cell death are not known. We now report that levels of AMPA receptor subunits GluR-1 and GluR-4 are rapidly decreased in cultured rat hippocampal neurons undergoing apoptosis in response to withdrawal of trophic support (WTS), whereas levels of NMDA receptor subunits NR1, NR2A, and NR2B are unchanged. Exposure of isolated synaptosomal membranes to "apoptotic" cytosolic extracts resulted in rapid degradation of AMPA receptor subunits. Treatment of cells and synaptosomal membranes with the caspase inhibitors prevented degradation of AMPA receptor subunits, demonstrating a requirement for caspases in the process. Calcium responses to AMPA receptor activation were reduced after withdrawal of trophic support and enhanced after treatment with caspase inhibitors. Vulnerability of neurons to excitotoxic necrosis was decreased after withdrawal of trophic support and potentiated by treatment with caspase inhibitors. Our data indicate that caspase-mediated degradation of AMPA receptor subunits occurs during early periods of cell stress and may serve to ensure apoptosis by preventing excitotoxic necrosis.

Differential effects of BDNF, ADNF9, and TNFalpha on levels of NMDA receptor subunits, calcium homeostasis, and neuronal vulnerability to excitotoxicity.

Calcium influx through N-methyl-d-aspartate (NMDA) receptors can result in neuronal apoptosis or necrosis and may play a pivotal role in neuronal death in many different neurodegenerative diseases. In the present study we employed primary neuronal cultures and three different excitoprotective factors, brain-derived neurotrophic factor (BDNF), activity-dependent neurotrophic factor (ADNF9), and tumor necrosis factor alpha (TNFalpha), to elucidate the mechanisms whereby trophic factors modify the excitotoxic process. Neurons pretreated with BDNF exhibited increased levels of the NMDA receptor subunits NR1 and NR2A, which was associated with increased calcium responses to NMDA and vulnerability to excitotoxic necrosis and reduced vulnerability to apoptosis. ADNF9 and TNFalpha suppressed calcium responses to glutamate and protected neurons against both excitotoxic necrosis and apoptosis, but had no effect on levels of NMDA receptor subunits. Inhibition of phosphorylation and DNA binding of NF-kappaB, by H7 and kappaB decoy DNA, respectively, suggest that the excitotoxic-modulating actions of BDNF are mediated by kinases, while those of ADNF9 and TNFalpha are mediated by both kinases and the transcription factor NF-kappaB. Our data show that, whereas BDNF increases neuronal responses to glutamate while ADNF9 and TNFalpha decrease the same, all three protect against excitotoxic apoptosis.

Activity-dependent neurotrophic factor peptide (ADNF9) protects neurons against oxidative stress-induced death.

Activity-dependent neurotrophic factor (ADNF) and a 14-amino acid fragment of this peptide (sequence VLGGGSALLRSIPA) protect neurons from death associated with an array of toxic conditions, including amyloid beta-peptide, N-methyl-D-aspartate, tetrodotoxin, and the neurotoxic HIV envelope coat protein gp120. We report that an even smaller, nine-amino acid fragment (ADNF9) with the sequence SALLRSIPA potently protects cultured embryonic day 18 rat hippocampal neurons from oxidative injury and neuronal apoptosis induced by FeSO4 and trophic factor withdrawal. Among the characteristics of this protection are maintenance of mitochondrial function and a reduction in accumulation of intracellular reactive oxygen species.

Activity-dependent neurotrophic factor: a potent regulator of embryonic growth and development.

Activity-dependent neurotrophic factor is a potent, neuroprotective molecule released from astroglia following stimulation by vasoactive intestinal peptide and, at least in part, accounts for the neuroprotective actions of vasoactive intestinal peptide. As well as enhancing neuronal survival, vasoactive intestinal peptide is known to regulate embryonic growth during the early postimplantation period of development. The current study was designed to assess activity-dependent neurotrophic factor's role in the growth-regulatory properties of vasoactive intestinal peptide. Treatment of whole cultured day-9 mouse embryos with activity-dependent neurotrophic factor (10(-13) M) resulted in a growth of 3.1 somites, compared with 1.6 somites in control embryos after a 4 h incubation period. Significant increases were also seen in cross-sectional area, protein and DNA content and bromodeoxyuridine incorporation. Activity-dependent neurotrophic factor-treated embryos were morphologically indistinguishable from control embryos of the same size. Anti-activity-dependent neurotrophic factor ascites significantly inhibited growth. In addition, co-treatment of embryos with anti-activity-dependent neurotrophic factor ascites inhibited vasoactive intestinal peptide-stimulated growth. Although anti-vasoactive intestinal peptide treatment inhibited growth, it did not inhibit activity-dependent neurotrophic factor-induced growth. These data indicate that an activity-dependent neurotrophic factor-like substance is an endogenous and potent growth-promoting factor in the early postimplantation embryo and that vasoactive intestinal peptide-regulated growth of embryos occurs, at least in part, through the action of activity-dependent neurotrophic factor.

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation.

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer's disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer's disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer's disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid beta-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide.

The vulnerability of neurons and the irreversibility of loss make discoveries of neuroprotective compounds fundamentally important. Here, the complete coding sequence of a novel protein (828 amino acids, pI 5.99), derived from mouse neuroglial cells, is revealed. The sequence contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor; (2) a glutaredoxin active site; and (3) a zinc binding domain. Gene expression was enriched in the mouse hippocampus and cerebellum and augmented in the presence of the neuropeptide vasoactive intestinal peptide, in cerebral cortical astrocytes. In mixed neuron-astrocyte cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the beta-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E-deficient mice accelerated the acquisition of developmental reflexes and prevented short-term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E-deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function.

Vasoactive intestinal peptide regulates embryonic growth through the action of activity-dependent neurotrophic factor.

Activity-dependent neurotrophic factor is a potent, neuroprotective protein released from astroglia by VIP and accounts in part for the neuroprotective properties of this neuropeptide. The growth-regulatory actions of VIP during embryogenesis may also occur indirectly through the release of activity-dependent neurotrophic factor. Whole cultured day-9 mouse embryos treated with activity-dependent neurotrophic factor (10(-13) M) for 4 hr grew 3.1 somites, compared with 1.6 somites in control embryos. Treated embryos appeared morphologically normal and exhibited significant increases in cross-sectional area, protein, and DNA content and bromodeoxyuridine incorporation. Anti-activity-dependent neurotrophic factor significantly inhibited growth. Co-treatment of embryos with anti-activity-dependent neurotrophic factor inhibited VIP-stimulated growth; however, anti-VIP did not inhibit activity-dependent neurotrophic factor-induced growth. These data indicate that an activity-dependent neurotrophic factor-like substance is an endogenous embryonic growth factor and that VIP-regulated growth occurs, at least in part, through activity-dependent neurotrophic factor.

A novel signaling molecule for neuropeptide action: activity-dependent neuroprotective protein.

The complete coding sequence of a novel protein (828 amino acids, pI 5.99), a potential new mediator of vasoactive intestinal peptide (VIP) activity was recently revealed. The expression of this molecule, activity-dependent neuroprotective protein (ADNP), was augmented in the presence of VIP, in cerebral cortical astrocytes. The mRNA transcripts encoding ADNP were enriched in the mouse hippocampus and cerebellum. The protein deduced sequence contained the following: (1) a unique peptide, NAPVSIPQ, sharing structural and immunological homologies with the previously reported, activity-dependent neurotrophic factor (ADNF) and exhibiting neuroprotection in vitro and in vivo; (2) a glutaredoxin active site; and (3) a classical zinc binding domain. Comparative studies suggested that the peptide, NAPVSIPQ (NAP), was more efficacious than peptides derived from ADNF. ADNP, a potential mediator of VIP-associated neuronal survival, and the new peptide, a potential lead compound for drug design, are discussed below.

Localization of glial cell line-derived neurotrophic factor receptor alpha and c-ret mRNA in rat central nervous system.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor that influences the survival and function of several neuronal populations in the central (CNS) and peripheral nervous systems. The actions of GDNF are mediated by a multicomponent receptor complex composed of the tyrosine kinase product of c-ret and the ligand-binding protein GDNF receptor alpha (GDNFR-alpha). In the present study, we used in situ hybridization to localize cells expressing the mRNA for these GDNF receptor subunits in rat CNS. As reported previously, GDNFR-alpha and c-ret mRNA are present in the substantia nigra and ventral tegmental area, regions containing GDNF-responsive dopamine neurons. However, both mRNA were found in motor neurons of spinal cord and brainstem nuclei that innervate skeletal muscle. These areas include alpha motor neurons in the ventral horn of spinal cord and neurons in hypoglossal, facial, trigeminal, and abducens nuclei. In areas rostral to the substantia nigra, c-ret mRNA is not detected, whereas GDNFR-alpha is found in numerous brain structures, including the hippocampus, cortex, medial geniculate, and the medial habenula, the latter area expressing the highest levels of GDNFR-alpha mRNA in brain. These results provide evidence that c-ret and GDNFR-alpha mRNA are expressed in neuronal populations involved in motor function and provides further support for GDNF as a target-derived neurotrophic for these motor neurons. The observation that GDNFR-alpha mRNA is localized in several brain structures that do not contain detectable levels of c-ret mRNA indicates that either GDNFR-alpha utilizes signal transduction molecules other than c-ret in these areas or that other GDNF-like ligands that utilize GDNFR-alpha as a receptor may be present.

VIP neurotrophism in the central nervous system: multiple effectors and identification of a femtomolar-acting neuroprotective peptide.

Vasoactive intestinal peptide has neurotrophic and growth-regulating properties. As in the case of many neurotrophic molecules, VIP also has neuroprotective properties, including the prevention of cell death associated with excitotoxicity (NMDA), beta-amyloid peptide, and gp120, the neurotoxic envelope protein from the human immunodeficiency virus. The neurotrophic and neuroprotective properties are mediated in part through the action of glial-derived substances released by VIP. These substance include cytokines, protease nexin I, and ADNF, a novel neuroprotective protein with structural similarities to heat-shock protein 60. Antiserum against ADNF produced neuronal cell death and an increase in apoptotic neurons in cell culture. A 14 amino acid peptide (ADNF-14) derived from ADNF has been discovered that mimics the survival-promoting action of the parent protein. These studies support the conclusion that VIP, PACAP, and associated molecules are both important regulators of neurodevelopment and strong candidates for therapeutic development for the treatment of neurodegenerative disease.

Inhibition of murine embryonic growth by human immunodeficiency virus envelope protein and its prevention by vasoactive intestinal peptide and activity-dependent neurotrophic factor.

Intrauterine growth retardation and neurodevelopmental handicaps are common among infants born to HIV-positive mothers and may be due to the actions of virions and/or maternally derived viral products. The viral envelope protein, gp120, is toxic to neurons, induces neuronal dystrophy, and retards behavioral development in neonatal rats. Vasoactive intestinal peptide, a neuropeptide regulator of early postimplantation embryonic growth, and the neuroprotective protein, activity-dependent neurotrophic factor, prevent gp120-induced neurotoxicity. Whole embryo culture of gestational day 9.5 mouse embryos was used to assess the effect of gp120 on growth. Embryos treated with gp120 exhibited a dose-dependent inhibition of growth. gp120-treated embryos (10(-8) M) grew 1.2 somites in the 6-h incubation period, compared with 3.9 somites by control embryos. Embryos treated with gp120 were significantly smaller in cross-sectional area and had significantly less DNA and protein than controls. Growth inhibition induced by gp120 was prevented by cotreatment with vasoactive intestinal peptide or activity-dependent neurotrophic factor. gp120 may play a role in the growth retardation and developmental delays experienced by infants born to HIV-positive mothers. Vasoactive intestinal peptide and related factors may provide a therapeutic strategy in preventing developmental deficits.

Cholinergic stimulation increases thrombin activity and gene expression in cultured mouse muscle.

Activity-dependent synapse reduction is a major determinant of neuromuscular innervation. Previous research has shown that nanomolar concentrations of hirudin, a specific thrombin antagonist, significantly attenuates this reduction, and protease nexin 1 (PN1), an endogenous thrombin inhibitor closely localized to the neuromuscular synapse, can inhibit synapse reduction at similar concentrations. Protease inhibitors which do not inhibit thrombin, including cystatin and aprotinin, had no effect on synapse reduction. We present a series of experiments examining whether prothrombin and/or PN1 gene expression, as well as thrombin activity, are regulated in muscle cultures by acetylcholine (ACh) receptor activation. We also studied the effect of exogenous thrombin on synapse elimination in co-cultures of muscle and cholinergic neurons. Cultured muscle cells were electrically blocked with tetrodotoxin (TTX), or co-treated with ACh in order to isolate ACh receptor activation. Electrical blockade resulted in a decrease in thrombin release to about two-thirds of control values. The application of ACh to electrically blocked muscle cultures resulted in a 2.5-fold increase in thrombin activity released into the medium and a 2-fold increase in prothrombin gene expression. In contrast, ACh treatment in the presence of TTX had no effect on PN1 gene expression compared to treatment with TTX alone. In addition, exogenous thrombin significantly increased synapse elimination in unstimulated muscle/cholinergic neuron co-cultures. These results suggest that thrombin or a thrombin-like molecule released from muscle is required for activity-dependent synapse elimination and is regulated by neuromuscular activity.

VIP regulation of embryonic growth.

Vasoactive intestinal peptide (VIP) plays a regulatory role in the growth of early postimplantation rodent embryos through its action on receptors localized to the central nervous system (CNS). However, the origin of the VIP influencing embryonic growth is unknown. VIP binding sites have been found prenatally; however, VIP mRNA was not detected in the rat CNS before birth and has been detected in peripheral organs only during the final third of gestation. Recent studies have revealed that VIP receptors were limited to the CNS in the embryonic day 11 (E11) rat embryo/trophoblast, which, in addition, had almost four times the VIP concentration of the E17 fetus. However, neither in situ hybridization or reverse transcriptase-polymerase chain reaction methods detected VIP mRNA in the E11 rat embryo or embryonic membranes. Rat maternal serum revealed a peak in VIP concentration at days E10-E12 of pregnancy, with VIP levels 6- to 10-fold higher than later during pregnancy. Radiolabeled VIP, administered intravenously to pregnant female mice, was found in the E10 embryo. These results suggest that VIP produced by extraembryonic tissues may regulate embryonic growth during the early postimplantation stage of development in the rodent.

Maternal vasoactive intestinal peptide and the regulation of embryonic growth in the rodent.

Vasoactive intestinal peptide (VIP) has been shown to regulate early postimplantation growth in rodents through central nervous system receptors. However, the source of VIP mediating these effects is unknown. Although VIP binding sites are present prenatally, VIP mRNA was not detected in the rat central nervous system before birth and was detected in the periphery only during the last third of pregnancy. In the present study, the embryonic day (E11) rat embryo/trophoblast was shown to have four times the VIP concentration of the E17 fetus and to have VIP receptors in the central nervous system. However, no VIP mRNA was detected in the E11 rat embryo or embryonic membranes by in situ hybridization or reverse transcriptase-PCR. RIA of rat maternal serum revealed a peak in VIP concentration at days E10-E12 of pregnancy, with VIP rising to levels 6-10-fold higher than during the final third of pregnancy. After intravenous administration of radiolabeled VIP to pregnant female mice, undegraded VIP was found in the E10 embryo. These results suggest that maternal tissues may provide neuroendocrine support for embryonic growth through a surge of VIP during early postimplantation development in the rodent.