Following the human point: Research with nonhuman animals since Povinelli, Nelson, and Boysen (1990).

For this special issue in honor of Dr. Sarah (Sally) Boysen's career, we review studies on point following in nonhuman animals. Of the 126 papers that we documented on this topic published since the publication of Povinelli, Nelson, and Boysen (1990, Journal of Comparative Psychology, 104, 203-210), 94 (75%) were published in the past 15 years, including 22 in the past 5 years, indicating that this topic is still an active area of interest in the field of animal behavior and cognition. We present results of a survey of publication trends, discussing the species tested and the sample sizes, and we note methodological considerations and current multilaboratory approaches. We then categorize and synthesize the research questions addressed in these studies, which have been at both the ultimate level (e.g., questions related to evolutionary adaptiveness and phylogenetic differences) and proximate level (e.g., questions related to experiential and temperamental processes). Throughout, we consider future directions for this area of research.

Insulin-like growth factor mRNA in Barramundi (Lates calcarifer): alternative splicing and nonresponsiveness to growth hormone.

Barramundi (Lates calcarifer) is a teleost of the superorder Acanthopterygii. Barramundi IGF-I cDNA was cloned and the distribution of alternative transcripts in various barramundi tissues was investigated using rt-PCR and RPA. It was demonstrated that in barramundi tissues, IGF-I mRNAs were represented by two transcripts corresponding to the reported salmonid Ea-2 and Ea-4. The acute effect of GH on hepatic IGF-I mRNA levels was investigated. Seven hours after intraperitonal administration of either 6 micrograms of recombinant bream GH/g body weight or saline, no significant increase in the levels of either of the two transcripts could be observed. The presence of GH receptors in the barramundi liver was demonstrated in binding assays using recombinant bream GH and liver membrane preparations. An analysis of a barramundi IGF-I genomic sequence encompassing the three exons that encode the E domain suggested that the pattern of splice site utilization is determined by the degree of homology of the splicing signals to the consensus splice site sequences.

Growth hormone treatment in young children with Down's syndrome: effects on growth and psychomotor development.

BACKGROUND: Learning disability and short stature are cardinal signs of Down's syndrome. Insulin-like growth factor I (IGF-I), regulated by growth hormone (GH) from about 6 months of age, may be involved in brain development. AIMS: To study long term effects of GH on linear growth and psychomotor development in young children with Down's syndrome. Study design-Fifteen children with Down's syndrome were treated with GH for three years from the age of 6 to 9 months (mean, 7.4). Linear growth, psychomotor development, skeletal maturation, serum concentrations of IGF-I and its binding proteins (BPs), and cerebrospinal fluid (CSF) concentrations of IGF-II were studied. RESULTS: The mean height of the study group increased from -1.8 to -0.8 SDS (Swedish standard) during treatment, whereas that of a Down's syndrome control group fell from -1.7 to -2.2 SDS. Growth velocity declined after treatment stopped. Head growth did not accelerate during treatment. No significant difference in mental or gross motor development was found. The low concentrations of serum IGF-I and IGFBP-3 became normal during GH treatment. CONCLUSIONS: GH treatment results in normal growth velocity in Down's syndrome but does not affect head circumference or mental or gross motor development. Growth velocity declines after treatment stops.

Nutritional regulation of insulin-like growth factor-I mRNA expression in barramundi, Lates calcarifer.

The effect of nutritional status on IGF-I mRNA expression in the liver and brain of juvenile barramundi (Lates calcarifer) was investigated. Fish were either fed a satiety ration (SAT) or starved (STV) for 6 weeks. Starved fish demonstrated significantly lower condition factor and hepatic IGF-I mRNA expression at 3 and 6 weeks, when compared with the SAT group. IGF-I mRNA expression in the brain was 10 fold lower than the liver and was not affected by ration size. These results suggest the liver is the major site of IGF-I mRNA synthesis and hepatic but not brain IGF-I mRNA expression is regulated by food availability in juvenile barramundi.

Characterization of an insulin-like growth factor (IGF) receptor and the insulin-like effects of IGF-1 in the bony fish, Lates calcarifer.

The present work is part of a broad phylogenetic study of the insulin superfamily of peptides in lower vertebrates. In the bony fish barramundi (Lates calcarifer), the presence of IGF receptors were investigated in the liver by means of competitive binding studies. The results suggested the presence of a type 1-like but no type 2-like IGF receptor. We also demonstrated insulin-like effects of intraperitoneally injected recombinant human (rh)-IGF-1 in barramundi with rh-IGF-1 and rh-insulin showing similar effects with respect to induction of hypoglycemia and stimulation of incorporation of [14C]-glucose into muscle glycogen.

The effects of insulin/IGF-I on glucose and leucine metabolism in the redclaw crayfish (Cherax quadricarinatus).

In recent years, invertebrate peptides have been identified which share substantial homologies with vertebrate insulin and insulin-like growth factors (IGFs), indicating a high degree of conservation of insulin/IGF systems through animal evolution. In a previous study, we provided evidence for the presence of IGF-I-like peptides in the redclaw (Cherax quadricarinatus), a species of freshwater crayfish endemic to northern Australia river systems which has attained support as a culture species. The general aim of the current study was to elucidate the functional significance of IGF-I-like peptides in this species by examining the effects of mammalian IGF-I on glucose and leucine metabolism. Juvenile redclaw were injected with a single dose of purified human insulin, recombinant human (rh) IGF-I, or Des-1-3-IGF-I. Glucose levels in redclaw tissues were then determined over an 8-hr period using enzymatic approaches. It was shown that injection of rhIGF-I induced an acute increase in free glucose content in hepatopancreas while Des-1-3-IGF-I and insulin raised free glucose levels in abdominal muscle. Radiolabel tracer approaches also demonstrated that injection of rhIGF-I increased glycogen synthesis in abdominal muscle and elevated the incorporation of leucine into protein in both abdominal muscle and hepatopancreas. Taken together, the findings of this study suggest that IGF-I-like peptides are biologically active in this species and may be of significance to metabolic and growth-related processes.

Localisation of insulin-like growth factor-I (IGF-I) immunoreactivity in the olivocerebellar system of developing and adult rats.

The molecular mechanisms which underlie the development of the olivocerebellar topography are not fully understood. Insulin-like growth factor-I (IGF-I) is a growth factor known to play important roles in neural development and it has been identified within the cerebellum and the inferior olive. To assess the contribution of IGF-I to the development of climbing fibre topography, the distribution of IGF-I-like immunoreactivity (IGF-I IR) was identified in the cerebellar cortex and inferior olive of rats, 0, 3, 5, 7, 10, 15, 21, 28 and 90 days old. In the cerebellar cortex, IGF-I IR was localised solely to Purkinje cells and its distribution was spatially and temporally regulated in a manner which coincides with climbing fibre development. At birth, weak IGF-I IR was detected in a few Purkinje cells in the ventral vermis. More Purkinje cells became positive until at postnatal day 7(P7) all Purkinje cells displayed IGF-I IR. Subsequently, a subpopulation of Purkinje cells lost their reactivity for IGF-I to leave IGF-I-positive cells organised into sagittal bands by P15. IGF-I IR was also seen in all subdivisions of the inferior olive between birth and P10 in a distribution which paralleled the maturation of the inferior olive. The Purkinje cell and inferior olivary IGF-I IR parallels climbing fibre development and thus the results of this study support the hypothesis that IGF-I is involved in the development of climbing fibre topography.

Increase of insulin-like growth factor (IGF)-1, IGF binding protein-2 and -4 mRNAs following cerebral contusion.

The insulin-like growth factor (IGF) system has a role in repair following hypoxic-ischemic injury in many tissues including the brain. To study the involvement of the IGF system following head trauma, we used a rat contusion model, which produces a focal lesion of the cerebral cortex. Molecules in the IGF system were analyzed using in situ hybridization at different times following impact. We observed a dramatic up-regulation of insulin-like growth factor binding protein-2 (IGFBP-2) mRNA in cortical areas adjacent to the injury 24 h after impact, with a peak 10-fold increase engaging most of the ipsilateral cortex 2 and 3 days post-contusion. Seven days after the contusion, IGFBP-2 expression was only moderately up-regulated and again concentrated around the injury. IGFBP-4 mRNA levels increased 4-fold ipsilateral to the site of injury, with retained pattern of cortical expression. IGFBP-3, IGFBP-5 and IGFBP-6 mRNA all displayed distinct expression patterns in the brain but no significant changes were observed following injury. In contrast, IGF-1 mRNA levels were very low prior to contusion, but increased markedly at the site of injury with a peak at day 3. We were unable to detect any changes in the type 1 IGF-receptor or IGF-2 mRNA following contusion. The neuropeptide cholecystokinin (CCK) mRNA was clearly up-regulated following contusion, with an even distribution over the ipsilateral cortex. The expression pattern of molecules in the IGF system post-contusion differs in part to changes observed following hypoxic-ischemia or ischemia alone, perhaps reflecting different regulatory mechanisms depending on the type of injury.

Increased amounts of a high molecular weight insulin-like growth factor II (IGF-II) peptide and IGF-II messenger ribonucleic acid in pancreatic islets of diabetic Goto-Kakizaki rats.

Insulin-like growth factor II (IGF-II), a member of the insulin family, regulates cell growth and differentiation. The IGF-II gene is localized close to the insulin gene in man and rat. IGF-II peptide binds weakly to the insulin receptor and exerts insulin-like effects on the blood glucose level. We studied IGF-II in endocrine pancreas in an animal model of noninsulin-dependent diabetes mellitus, the Goto-Kakizaki (GK) rat. At the age of 2 months, these rats have structural islet changes, with fibrosis and irregular configuration, so-called starfish-shaped islets. Immunohistochemical investigation revealed IGF-II immunoreactivity in the beta-cells in both GK and control rats. Pancreatic extraction, followed by size separation using gel chromatography, disclosed a high mol wt form of IGF-II in all animals, and RIA measurements revealed a considerably larger amount of the IGF-II peptide in the 2-and 6-month-old GK rats than in the 1-month GK and control rats. In situ hybridization of 3-month-old GK rats showed increased IGF-II messenger RNA expression in the starfish-shaped islets of GK rats than in the islets with normal structure in both diabetic and control animals. The reason for the increased amount of IGF-II is unclear. As the animals are diabetic before the islet changes occur, it might be a compensatory effect in response to hyperglycemia, but could also be a cause of the islet fibrosis.

Localization of insulin-like growth factor-I immunoreactivity in larval and juvenile barramundi (Lates calcarifer).

Antisera to mammalian IGFs cross-react with several fish species, suggesting a long phylogenetic history of IGF-like peptides and their functional importance to all vertebrates. In this study, the tissue distribution of IGF-I-immunoreactivity (IGF-I-IR) was studied at various larval and juvenile stages of the life cycle of barramundi (Lates calcarifer). It was shown that the distribution of IGF-I-IR in this species was tissue-specific and age-dependent. In newly hatched larvae, presumptive musculature in the trunk and the pectoral fin rudiments reacted positively for IGF-I. As specimen age increased, however, IGF-I-IR in these tissues became less evident. In the retinas of barramundi 42 hr and older, a distinct band of IGF-I-IR was consistently detected between the presumptive outer nuclear and bacillary layers. Examination of sections from specimens 9 days and older revealed strong reactivity for IGF-I in a large proportion of renal tubule epithelial cells. In sections from larvae 13 to 28 days old, diffuse cytoplasmic IGF-I-IR was identified in cells lining the gill structures. At these same developmental stages, IGF-I reactive cells were also observed in the islets of Langerhans. In young juveniles (22 to 28 days posthatching), sparsely scattered clusters of neurons in the lower brain were observed which exhibited granular IGF-I-IR in perikarya. In all instances reported, IGF-I-IR in barramundi tissue was abolished by replacing antisera with normal rabbit serum or by preabsorption of antisera with purified IGF-I peptide, indicating the specificity of the reactions obtained. The distribution patterns of IGF-I-IR in barramundi tissues were broadly consistent with the reported distributions of IGF-I-like peptides and transcripts in other teleost species. The findings of this study are in general agreement with the hypothesis that IGF-I-like peptides may be involved in the regulation of tissue growth, differentiation, and function during early barramundi development.

IGF-2-like peptides are present in insulin cells of the elasmobranchian endocrine pancreas: an immunohistochemical and chromatographic study.

Evidence for the presence of peptides, related to insulin-like growth factor 2 (IGF-2), has been obtained in the endocrine pancreas of the elasmobranchian species Raja clavata, the sting ray. By radioimmunoassay, IGF-2-like immunoreactivity was detected in Raja pancreas extract. Further characterization of this activity by acid gel chromatography revealed two distinct peaks of IGF-2-like immunoreactivity with apparent molecular weights of approximately 8.2 kDa and 4.5 kDa. Using the same IGF-2 antibody as well as antisera specific for mammalian IGF-1, insulin, glucagon, somatostatin and pancreatic polypeptide in double immunofluorescence studies, IGF-2-like immunoreactivity was located exclusively in insulin-immunoreactive cells. In contrast, IGF-1-like immunoreactivity was mainly observed in somatostatin- and glucagon-immunoreactive cells. A varying proportion (0-70%) of insulin-immunoreactive cells, however, displayed both IGF-1- and IGF-2-like immunoreactivity. Absorption studies indicated that the IGF-2-like peptides in Raja are different from mammalian and submammalian insulin and mammalian IGF-1, but similar to mammalian IGF-2. Thus, IGF-2-like peptides seem to occur during evolution as early as the phylogenetic development of the elasmobranchians. Furthermore, the results indicate a particularly conservative evolution of the islet IGF-2 system.

The branching of insulin-like growth factor 1 and insulin: an immunohistochemical analysis during phylogeny.

The co-existence of insulin-like growth factor 1 (IGF-1) with the classical islet hormones insulin (INS), glucagon (GLUC), somatostatin (SOM) and pancreatic polypeptide (PP) in the endocrine pancreas of representative species of cyclostomes (Myxine glutinosa), cartilaginous fish (Raja clavata, Squalus acanthias) and bony fish (Cottus scorpius, Carassius auratus, Cyprinus carpio, Anguilla anguilla) was studied by the use of monoclonal and polyclonal antisera and the double immunofluorescence technique. In all species investigated, IGF-1-like-immunoreactive cells were found in the endocrine pancreas, however, in varying localization. In Myxine glutinosa, all INS-immunoreactive cells and some of the SOM-immunoreactive cells contained IGF-1-like-immunoreactivity. In Raja and Squalus, only a minority of the INS-immunoreactive cells also displayed IGF-1-like-immunoreactivity. The majority of the IGF-1-like-immunoreactivity was observed in SOM- and in GLUC-immunoreactive cells. Different results were obtained in bony fish. In Cottus, in the Brockmann bodies and the small islets IGF-1-like- and INS-immunoreactivities co-existed to 100%. In contrast, in the other bony fish studied IGF-1-like-immunoreactivity was not observed in INS-immunoreactive cells: in Cyprinus, IGF-1-like-immunoreactivity was found in GLUC-, PP- and SOM-immunoreactive cells and in Carassius and Anguilla, in SOM-immunoreactive cells only. Thus, in all bony fish species with the exception of Cottus, IGF-1 and insulin display a distinct cellular distribution, similar to that of mammals. The present results, thus, may indicate that the branching of IGF-1 and insulin has occurred at the phylogenetic level of bony fish.

A high-molecular IGF-2 immunoreactive peptide (pro-IGF-2?) in the insulin cells of the islets of Langerhans in pancreas of man and rat.

Histopathologically normal pancreatic parenchyma from 12 adult men and women, as well as that from 14 adult rats (Sprague-Dawley and Wistar strains), were investigated immunohistochemically with a mouse monoclonal antibody, raised against recombinant human pro-IGF-2. The antiserum showed no crossreactivity with insulin; IGF-1 had 0.1% of the reactivity of IGF-2. The immunohistochemical observations were checked by means of a radioimmunoassay (RIA), based on the same antibody, of an extract of a sample of one of the human pancreatic glands. Analogous investigations for insulin were made in parallel, using polyclonal insulin antisera. A high-molecular (12 kDa) IGF-2-like peptide was found in the islets of Langerhans, being localized to the insulin cells. These cells were identified as beta-cells by immunohistochemistry with insulin antisera on adjacent paraffin sections. From observations made by means of acid-gel-chromatography, the peptide was tentatively supposed to represent either pro-IGF-2, or a partially processed form of it.

Effects of IGF-1, truncated IGF-1 and the tripeptide Gly-Pro-Glu on acetylcholine release from parietal cortex of rat brain.

The effects of intact IGF-1, truncated IGF-1 and Gly-Pro-Glu (GPE), the aminoterminal tripeptide of IGF-1, on the potassium (35 mM K+) stimulated release of acetylcholine (ACh) from rat cortical slices were investigated. GPE significantly increased the release of ACh in the dose range of 10(-10)-10(-6) M, while IGF-1 significantly enhanced the release of ACh only at 4 x 10(-8) M. The truncated form of IGF-1, lacking the tripeptide GPE, did not effect the release of ACh in rat cortex. Binding experiments also showed that truncated IGF-1 was less available to the brain slices. The possible underlying mechanisms of action of GPE in the cholinergic synapse were investigated. GPE (10(-5) M) significantly (40%) displaced [3H]nicotine from its binding sites in rat cortex. In the concentration range of 10(-10)-10(-5) M, GPE did not interact with the choline uptake sites ([3H]hemicholinium binding) or the muscarinic ([3H]QNB) receptor binding sites in rat cortex. The mechanism of action behind GPEs enhancement of cholinergic transmission is therefore still unknown.

Normalized growth velocity in children with Down's syndrome during growth hormone therapy.

Between 6 months and 3 years of age, growth velocity in children with Down's syndrome (DS) is markedly reduced in comparison to that of healthy children. However, after 3 years of age, it is almost normal. Thus, growth retardation becomes pronounced during the period when growth hormone (GH) starts to regulate growth. The present authors report the long-term effects of GH-therapy in 16 children with DS, who are being treated for 3 years from the age of 6-9 months. The treatment, Genotropin, 0.1 U kg-1 BW day-1, was started at a mean age of 7.4 (6-9) months. The results after 12 (n = 16), 24 (n = 12) and 30 (n = 8) months are presented. The mean height standard deviation score, SDS (range; Swedish standard), before therapy was -1.8 (-0.5 to -3.1) and the mean head circumference was -1.2 (-0.4 to -3.5). After 12, 24 and 30 months, the mean height SDS were -1.1 (-0.8 to -1.9), -0.9 (0 to -1.5) and -0.9 (0.1 to -1.5) and the mean head circumference SDS were -1.1 (0 to -2.5), -1.1 (0 to -2.2) and -1.2 (-0.5 to -2.0), respectively. During hGH-treatment, the children with DS thus gained height during the first year, and then followed the growth rate of healthy Swedish children. When compared to growth charts for children with DS the mean height of these children started at the fiftieth centile and reached the ninety-fifth centile after 24 months of treatment. Head circumference only slightly increased during the therapy, and not to the same extent as height. This indicates that small head circumference in DS is not only an effect of growth retardation, but also due to microcephaly.

Occurrence of members of the insulin superfamily in central nervous system and digestive tract of protochordates.

Antisera specific for mammalian insulin-like growth factor 1 (IGF-1) and mammalian insulin and the double immunofluorescence technique were used for this study. IGF-1-like-immunoreactivity was localized in entero-endocrine cells in the gastro-intestinal tract of the protochordates Ciona intestinalis and Branchiostoma lanceolatum. Some of the specimens also showed IGF-1-like-immunoreactive (-IR) perikarya and fibers in the central nervous system. Whilst in rat endocrine pancreas, IGF-1-IR and insulin-IR occurred in different cell populations, in Ciona and Branchiostoma the vast majority of entero-endocrine cells and central neurons were IGF-1-like- +insulin-IR. A minor portion exhibited IGF-1-like-IR alone. For further characterization of the IGF-1-like-IR material, in Ciona intestinalis, peptides related to IGF-1 were identified by radioimmunoassay and gel chromatography. In accordance with the immunohistochemical results, IGF-I-like-IR was detected both in cerebral ganglion and in gastro-intestinal tract. Using acid gel chromatography, in Ciona gastro-intestinal tract the IGF-1-like-IR was found to occur in two peaks, with apparent molecular weights of approximately 16 kDa and 3 kDa. Absorption studies with insulin- and IGF-related peptides, with crude extracts and the peak material obtained after gel chromatography, indicated that the IGF-1-like peptides in Ciona are different from mammalian insulin and IGF-1. The findings are in accordance with the presence of a common insulin/IGF precursor molecule in protochordates.

Insulin-like growth factors and insulin-like growth factor binding proteins in cerebrospinal fluid and serum of patients with dementia of the Alzheimer type.

After acid gel-chromatography cerebrospinal fluid and serum levels of immunoreactive insulin-like growth factor 1 and 2 (IGF-1 and IGF-2) were determined in patients with dementia of the Alzheimer type (AD) and in healthy subjects. The AD CSF levels of immunoreactive IGF-1 did not differ from the subjects but the levels of immunoreactive IGF-2 was significantly elevated in both serum and CSF in the AD patient group. Additionally immunoreactive IGF-1 in AD serum was found to be significantly elevated. To characterize the CSF IGF binding protein activity (IGFBP), ligand blotting was performed on whole CSF from AD patients and subjects. The results demonstrate two major forms of IGFBP in CSF with approximate molecular weights of 33 KDa and 30 KDa. The two IGFBP forms are suggested to represent IGFBP-2 and IGFBP-6. A highly significant increase in both the IGFBPs was observed in the CSF of the AD patients compared to the healthy subjects.

Identification of IGF-1 receptors in primitive vertebrates.

This is the first report of the existence of insulin-like growth factor (IGF-1) receptors in three representatives of lower vertebrates: the osteichtyes, chondrichtyes and cyclostomi. Competitive binding studies and affinity labelling of brain membranes from Cottus scorpius (sea scorpion), Raja clavata (ray) and Myxine glutinosa (atlantic hagfish) identified a mammalian type 1 or IGF-1 receptor by its binding specificity and the molecular size of its alpha-subunit. IGF-1 and IGF-2 are almost equally potent in displacing receptor-bound 125I-IGF-1 or 125I-IGF-2, and the proteins labeled with both tracers have a molecular size of 100,000-120,000 under reducing conditions. There was no evidence for the presence of a mammalian type 2 or IGF-2/mannose 6-phosphate receptor in brains of Cottus, Raja or Myxine. In all three species the binding of 125I-IGF-1 and 125I-IGF-2 was significantly higher in brain compared with liver and gastrointestinal tract, and the IGF-1 receptor could only be identified with certainty in Raja liver. It is concluded that the brain of three lower vertebrates express mammalian IGF-1 receptors, whereas IGF-2-mannose 6-phosphate receptors could not be detected.