Delayed developmental changes in neonatal vocalizations correlates with variations in ventral medial hypothalamus and central amygdala development in the rodent infant: effects of prenatal cocaine.

While variations in neonatal distress vocalizations have long been shown to reflect the integrity of nervous system development following a wide range of prenatal and perinatal insults, a paucity of research has explored the neurobiological basis of these variations. To address this, virgin Sprague-Dawley rats were bred and divided into three groups: [1] untreated, [2] chronic-cocaine treated (30 mg/kg/day, gestation days (GDs) 1-20); or [3] chronic saline treated (2 mg/kg/day, GDs 1-20). Pregnant dams were injected with Bromodeoxyuridine (10 mg/kg) on GDs 13-15 to label proliferating cells in limbic regions of interest. Ultrasonic vocalizations (USVs) were recorded on postnatal days (PNDs) 1, 14, and 21, from one male and female pup per litter. Variations in acoustic properties of USVs following cocaine-exposure were age and sex-dependent including measures of total number, total duration and amplitude of USVs, and percent of USVs with at least one harmonic. Following USV testing brains were stained with standard fluorescent immunohistochemistry protocols and examined for variations in neuronal development and if variations were associated with acoustic characteristics. Limbic region developmental differences following cocaine-exposure were sex- and age-dependent with variations in the ventral medial hypothalamus and central amygdala correlating with variations in vocalizations on PND 14 and 21. Results suggest maturation of the ventral medial hypothalamus and central amygdala may provide the basis for variations in the sound and production of USVs. As vocalizations may serve as a neurobehavioral marker for nervous system integrity, understanding the neurobiological basis of neonatal vocalizations may provide the basis for early intervention strategies in high-risk infant populations.

Developmental regulation of neural cell adhesion molecule in human prefrontal cortex.

Neural cell adhesion molecule (NCAM) is a membrane-bound cell recognition molecule that exerts important functions in normal neurodevelopment including cell migration, neurite outgrowth, axon fasciculation, and synaptic plasticity. Alternative splicing of NCAM mRNA generates three main protein isoforms: NCAM-180, -140, and -120. Ectodomain shedding of NCAM isoforms can produce an extracellular 105-115 kilodalton soluble neural cell adhesion molecule fragment (NCAM-EC) and a smaller intracellular cytoplasmic fragment (NCAM-IC). NCAM also undergoes a unique post-translational modification in brain by the addition of polysialic acid (PSA)-NCAM. Interestingly, both PSA-NCAM and NCAM-EC have been implicated in the pathophysiology of schizophrenia. The developmental expression patterns of the main NCAM isoforms and PSA-NCAM have been described in rodent brain, but no studies have examined NCAM expression across human cortical development. Western blotting was used to quantify NCAM in human postmortem prefrontal cortex in 42 individuals ranging in age from mid-gestation to early adulthood. Each NCAM isoform (NCAM-180, -140, and -120), post-translational modification (PSA-NCAM) and cleavage fragment (NCAM-EC and NCAM-IC) demonstrated developmental regulation in frontal cortex. NCAM-180, -140, and -120, as well as PSA-NCAM, and NCAM-IC all showed strong developmental regulation during fetal and early postnatal ages, consistent with their identified roles in axon growth and plasticity. NCAM-EC demonstrated a more gradual increase from the early postnatal period to reach a plateau by early adolescence, potentially implicating involvement in later developmental processes. In summary, this study implicates the major NCAM isoforms, PSA-NCAM and proteolytically cleaved NCAM in pre- and postnatal development of the human prefrontal cortex. These data provide new insights on human cortical development and also provide a basis for how altered NCAM signaling during specific developmental intervals could affect synaptic connectivity and circuit formation, and thereby contribute to neurodevelopmental disorders.

Gestational ethanol and nicotine exposure: effects on maternal behavior, oxytocin, and offspring ethanol intake in the rat.

Alcohol consumption and smoking during pregnancy is common, despite the known adverse effects of these drugs on fetal development. Though studies on the effects of each drug separately are published, little is known about the effect of concurrent use of alcohol and nicotine in humans or in preclinical models. In this report, we examined the impact of continuous gestational exposure to both ethanol via liquid diet and nicotine via an osmotic minipump on maternal behavior, offspring ethanol intake, and oxytocin levels in a rat model. Dams were tested for the onset of maternal behavior with litters of unexposed surrogate pups and then killed to examine oxytocin levels within specific brain regions. Drug-exposed offspring reared by surrogate dams were tested for ethanol intake at either adolescence or adulthood, and oxytocin levels were measured in relevant brain regions after behavioral tests. Dams exhibited minor deficits in maternal care, which were associated with lower oxytocin levels in both the ventral tegmental and medial preoptic areas compared to control dams. Prenatal exposure altered sex-specific ethanol intake, with differential effects at adolescence and adulthood. Oxytocin system changes were also apparent in the ventral tegmental and medial preoptic regions of drug-exposed adolescent and adult offspring. These results suggest that dam treatment with ethanol and nicotine can somewhat negatively affect the early rearing environment, and that prenatal exposure to both of these drugs results in drinking behavior differing from what would be expected from either drug alone. Oxytocin's possible involvement in the mediation of these effects is highlighted.

Impact of gestational cocaine treatment or prenatal cocaine exposure on early postpartum oxytocin mRNA levels and receptor binding in the rat.

Prior research reported decreased oxytocin levels in specific brain regions correlated with disruptions in maternal care following gestational cocaine treatment in rats. Similarly, prenatal exposure to cocaine impaired subsequent maternal behavior in adulthood, but behavioral alterations were not associated with decreases in oxytocin levels in the same brain regions as were found in their cocaine-treated rat dams. To determine if other aspects of the oxytocin system are disrupted by cocaine treatment or prenatal exposure to cocaine during critical time points associated with maternal care, oxytocin mRNA transcription and receptor binding were examined on postpartum day two in relevant brain regions following gestational treatment with, or prenatal exposure to, either cocaine or saline. We hypothesized that oxytocin mRNA levels and receptor binding would be differentially affected by cocaine in the early postpartum period of dams and their offspring. Our findings indicate that gestational cocaine treatment resulted in significant increases in oxytocin mRNA levels in only the paraventricular nucleus of cocaine-treated dams, with almost significant increases in both generations in the supraoptic nucleus, but no significant effects of cocaine on receptor binding in either generation of dams. These findings indicate that in addition to oxytocin levels, cocaine treatment or prenatal exposure primarily affects oxytocin mRNA synthesis, with little effect on receptor binding in specific brain regions associated with maternal behavior in the early postpartum period of the rat.

Structure and expression of a membrane component of the inhibin receptor system.

The purification and cloning of a membrane-anchored proteoglycan with affinity for inhibin A are described. Bovine pituitary membranes were isolated, and membrane-anchored proteins were solubilized and used as an enriched source of inhibin binding protein. The extract was passed over an inhibin A affinity column, and a protein, designated p120, was identified as an inhibin-binding moiety. A partial amino acid sequence was determined for the protein, which matched two human complementary DNAs (cDNAs) in the database. The full-length cDNA predicts a 1336-amino acid glycoprotein. Full-length p120-encoding cDNAs were isolated from human testis RNA and cloned into expression vectors. Two p120 messenger RNA transcripts of 4.6 kb and 2 kb are detected in rat pituitary by RNA blot analysis. Similar analysis of rat testis RNA revealed transcripts of identical molecular mass, albeit at lower abundance. To determine the cellular localization of p120 in pituitary and testis, an antibody directed against the predicted extracellular domain of the protein was generated and used in an immunohistochemical analysis of thin tissue sections. p120 immunostaining is coincident with FSHbeta immunopositive gonadotrope cells in rat pituitary. p120 staining is intense in the testicular Leydig cells, which bind iodinated inhibin but not iodinated activin. In summary, an inhibin-binding protein has been isolated that is produced in tissues that are targets of inhibin action.

Assessing genetic heterogeneity in production cell lines: detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody.

A recombinant antibody directed against the human epidermal growth factor receptor-2 extracellular domain was subjected to detailed structural characterization. Heterogeneity in the heavy chain was demonstrated by recovery of two forms of a tryptic peptide, with either glutamine or the expected tyrosine at residue 376. Subsequent experiments indicated that the Y376Q variant developed during transfection of the antibody heavy and light chain genes into Chinese hamster ovary cells. Levels of the Y376Q variant (range: 27% to 1%) in the purified antibody were inversely proportional to cell age. The established cell line was subcloned and found to be heterogeneous by polymerase chain reaction analysis of cell extracts and protein analysis of the purified antibody. Ten percent of subclones produced high levels of the Y376Q variant while 90% of the subclones produced antibody with only the expected heavy chain sequence. This report demonstrates the utility of peptide mapping as a sensitive tool for assessing genetic heterogeneity of recombinant cell lines.

Identification and characterization of binding proteins for inhibin and activin in human serum and follicular fluids.

Inhibins and activins are produced by a variety of tissues and may have important endocrine and paracrine roles in development, reproduction, and hematopoiesis. However, little is known regarding the physical properties or concentrations of inhibin and activin in biological fluids. Binding proteins for inhibin or activin in serum or at production or target sites may have important implications for restricting the bioactivity of these hormones and may alter the immunoreactivity of these molecules in biological fluids. The objective of this study was to identify inhibin- and activin-binding proteins in human serum (HS) and follicular fluid (hFF) and determine the ability of these proteins to alter biological or immunological activity. In HS, [125I]activin and inhibin bound to a protein identified as alpha 2-macroglobulin (alpha 2M) using three criteria: 1) [125I]inhibin and activin bind purified alpha 2M, but not several other serum proteins tested; 2) complexes formed by [125I]inhibin and activin in HS and in the presence of purified alpha 2M elute with similar retention times on HPLC; and 3) preadsorption of HS with alpha 2M antiserum inhibits inhibin and activin binding to this protein while antiserum directed against follistatin or other serum proteins had no effect. A small amount of a lower mol wt [125I]activin-follistatin complex was also found in HS. This complex eluted with a retention time similar to that of activin bound to purified porcine follistatin. Binding of inhibin to follistatin could not be detected in HS. In contrast, follistatin was the major binding protein of both activin and inhibin in hFF. Concentrations up to 100 micrograms/ml purified alpha 2M had no effect on the bioactivity or immunoreactivity of either inhibin or activin. In contrast, follistatin inhibited both activin-stimulated pituitary FSH release and K562 hemoglobin production as well as antiserum binding in a specific activin-A immunoassay. Follistatin did not interfere with inhibin immunodetection. These data indicate that two inhibin- and activin-binding proteins are present in different relative amounts in HS and hFF, alpha 2M, the primary binding protein in HS, did not alter inhibin or activin bio- or immunoactivity under the conditions of these experiments, while follistatin, the major binding protein in hFF, may mask activin's bio- and immunoactivities.

Stage-specific binding of inhibin and activin to subpopulations of rat germ cells.

Flow cytometry was used to separate and identify Sertoli and germ cell populations in primary rat testicular cultures derived from animals of different ages on the basis of cell size and DNA and lipid content. Multiparameter fluorescent evaluation of each cell preparation resulted in the assignment of specific staining patterns to Sertoli cells (diploid, high lipid content), spermatogonia (diploid, low lipid content), spermatocytes (large, tetraploid, high lipid content), and round spermatids (haploid, low lipid content). Each field was separately analyzed for inhibin and activin binding. Fluorescein isothiocyanate-conjugated activin bound with greatest intensity to spermatogonia, with little binding to leptotene or zygotene spermatocytes. Fluorescein isothiocyanate-conjugated inhibin bound to all stages of germ cells tested. Cross-competition data indicate that at least two and probably three distinct receptors exist for these peptides.

Assay requirements for cell culture process development.

Because the structural parameters which influence product "quality" will vary from protein to protein, the screening methods used during development of each production process will also differ. Thus, not all of the methods described above will be needed for each product. One of the challenges of cell culture process development is choosing the correct structural features to monitor. The next level of process development may involve on-line control of many of the parameters discussed above. This would allow product quality-driven manipulations of culture conditions. The major factor limiting this advance may not be the techniques for real-time analysis, but rather a more thorough understanding of the structural features which describe a high-quality pharmaceutical protein.