Control of water intake by a pathway from the nucleus of the solitary tract to the paraventricular hypothalamic nucleus.

Several brain areas have been shown to participate in thirst and control of fluid intake. An understanding of how these circuits interact, and their roles in the activation, maintenance, and termination of fluid intake remains incomplete. Central glucagon-like peptide-1 (GLP-1) receptor activation appears to be an important part of the termination of drinking, but the site(s) of action for this suppression has not yet been determined. In an attempt to use GLP-1 responsiveness as a means to screen targets of hindbrain cells that participate in the termination of thirst and the resultant water intake, we injected the GLP-1 receptor agonist exendin-4 (Ex-4) into three brain areas known to express GLP-1 receptors, and measured subsequent water intake. Ex-4 reduced water consumption when injected into the paraventricular hypothalamic nucleus (PVH) and nucleus of the solitary tract (NTS), but not when injected into the nucleus accumbens (NAc). Using the effective response after injection into the PVH as a guide, we examined the connection between the NTS - the site of endogenous central GLP-1 production - and the PVH. Retrograde tracing combined with Fos immunohistochemistry suggested intake-induced activity in PVH-projecting NTS cells. To test the hypothesis that this pathway is important in the termination of drinking, we chemogenetically activated PVH-projecting hindbrain cells. Interestingly, activation of this population of cells increased water intake, calling into question the heterogeneity of the pathway with respect to the control of fluid intake. Taken together, we conclude that the PVH is a site of action for GLP-1 receptor activation in the inhibition of water intake, but suspect that endogenous GLP-1 in NTS-to-PVH projections may be counterbalanced by a parallel pathway that either activates or maintains already activated water intake.

Symposium review: Integration of postweaning nutrient requirements and supply with composition of growth and mammary development in modern dairy heifers.

To optimize first lactation and lifetime milk yield, growth benchmarks were established to help meet the appropriate growth objectives of breeding weight and age at an economically viable time and to achieve the optimum body size and composition at first calving. These guidelines provide a framework that helps to minimize overfeeding and, thus, potential overconditioning of heifers, which can lead to postpartum metabolic issues and reduced milk yield. Concerns still exist that mammary development is impaired when body weight gain exceeds a certain threshold, which would negatively affects milk yield. The objective of this review was to integrate concepts of nutrient requirements, body growth and composition, mammary development, and milk yield to provide a systems-based perspective on first-lactation milk differences that have been associated with mammary development. Work in the early 1980s described the effect of high energy intake on mammary development and the relationship with circulating growth hormone linked the relationship between prepubertal growth, mammary development, and future milk yield. The primary outcome of that research was to provide an intuitive mechanism to explain why rapid growth during the prepubertal phase resulted in reduced milk yield. The observation of reduced mammary development could be repeated in almost every experiment, leading to the conclusion that high energy intake and increased average daily gain reduced mammary development through altered hormone status or some signaling processes. However, further work that looked at mammary development over the entire prepubertal growth phase recognized that mammary development was not reduced by high energy intake, and instead accumulated at a constant rate; thus, overall mammary parenchymal growth was a function of the time to reach puberty and the associated signals to change from allometric mammary growth. The mammary gland, similar to most reproductive organs, grows in proportion to the size of the body and not in proportion to nutrient intake during the postweaning, prepubertal phase. First-lactation milk yield, mammary development, and body composition will be further discussed in the context of mechanisms and opportunities.

Effects of body weight and nutrition on histological mammary development in Holstein heifers.

Our objective was to determine the effects of rate of gain and body weight (BW) on development of the mammary parenchyma. Mammary tissue samples were collected from heifers (n = 72) reared on 1 of 2 dietary treatments (restricted, 650 g/d of daily gain; or elevated, 950 g/d of daily gain) and slaughtered at 100, 150, 200, 250, 300, or 350 kg of BW. Mammary samples were excised, preserved, prepared for histology, and stained with hematoxylin and eosin. Digital images of tissue sections were captured for analysis. Tissue areas occupied by the interlobular and intralobular stroma, epithelium, and lumen were measured (mum(2)). The numbers of epithelial and luminal structures per image were tabulated to measure the complexity of ductal development. Mean percentages of mammary parenchyma occupied by the interlobular stroma, epithelium, lumen, and intralobular stroma were 29, 20, 7, and 43%, respectively. Percentage of area occupied by the intralobular stroma was affected by BW and was lower for 100-kg heifers compared with heifers 200 kg and heavier (33 +/- 4 vs. 46 +/- 4), but the percentage of area occupied by other tissue elements did not differ by BW or treatment, nor was there an interaction. However, the numbers of both epithelial (8.3 +/- 4 vs. 47 +/- 4) and luminal-containing (6 +/- 4 vs. 38 +/- 4) structures per image increased markedly between 100 and 350 kg of BW, irrespective of diet. For heifers slaughtered between 100 and 350 kg of BW, alterations in the rate of gain between 650 and 950 g/d, accomplished by feeding varying amounts of the same diet, had no significant effect on tissue characteristics or the pattern of mammary parenchymal development. These data emphasize the importance of BW and age in determining developmental characteristics of the heifer mammary parenchyma and suggest that the rate of gain per se has a minimal impact on histological development, and thus do not support the hypothesis that rate of gain has a direct negative impact on ductal development.

Ontogenic and nutritional regulation of steroid receptor and IGF-I transcript abundance in the prepubertal heifer mammary gland.

In prepubertal cattle, mammary development is characterized by the growth of an epithelial-rich parenchyma (PAR) into the mammary fat pad (MFP). This proliferation and accumulation of mammary epithelial cells require estrogen. Paradoxically, both epithelial cell proliferation and PAR accumulation rate decline with rising plasma estrogen as puberty approaches. The possibility that variation in abundance of estrogen receptors (ERs) in PAR or MFP accounts for a portion of these effects has not been examined in cattle. Additionally, we recently demonstrated that MFP is highly responsive to exogenous estrogen, suggesting that this tissue may play a role in coordinating estrogen's effects on PAR; however, the developing bovine MFP has yet to be studied in detail. To address these hypotheses, Holstein heifers were assigned to planes of nutrition supporting body growth rates of 950 (E) or 650 (R) g/day and harvested every 50 kg from 100 to 350 kg body weight (BW). Post-harvest, their mammary glands were dissected into PAR and MFP compartments. Transcript abundance of genes encoding members of the ER family (ERalpha, ERbeta, and estrogen-related receptor alpha-1 (ERRalpha)) and estrogen-responsive genes (IGF-I and progesterone receptor (PR)) were measured in both mammary compartments by quantitative real-time RT-PCR. Significant expression was detected for all genes in both compartments, with the exception of the ERbeta gene. Transcript abundance of both ERalpha and IGF-I decreased linearly with increasing BW within both compartments. ERRalpha and PR expressions decreased with increasing BW in PAR but not in MFP. Nutrition stimulated ERalpha and ERRalpha expression in the PAR but had no effect on IGF-I or PR in either PAR or MFP. Overall, ERalpha and IGF-I transcript abundance are consistent with the drop in mammary epithelial cell proliferation and PAR accretion observed over development, but do not support a negative effect of nutrition on PAR growth.

Effect of estrogen on leptin and expression of leptin receptor transcripts in prepubertal dairy heifers.

Plasma leptin concentrations increase as growing dairy heifers approach puberty and have greater plasma estrogen. In intact and ovariectomized rodents, estrogen has been shown to modulate expression of leptin and its receptor (Ob-R). To determine if estrogen regulates the bovine leptin system, prepubertal dairy heifers were ovariectomized at 140 d of age or left intact. A month later, both groups received a subcutaneous injection of excipient or 17beta-estradiol for 3 consecutive days. Neither ovarian status nor 17beta-estradiol injection altered plasma leptin or leptin mRNA abundance in adipose tissue depots. To assess whether these factors affected Ob-R expression, we tested 20 bovine tissues for leptin receptor (Ob-R) by using quantitative real-time PCR assays for the short receptor isoform (Ob-Ra), the long receptor isoform (Ob-Rb), and all receptor isoforms (Ob-R(TOTAL)). Ob-R(TOTAL) was detected in all tissues, with copy numbers covering 3 orders of magnitude between the lowest and highest expressing tissues (kidney cortex vs. liver). The Ob-Rb isoform accounted for 40% of Ob-R(TOTAL) in the hypothalamus, but averaged less than 3% of Ob-R(TOTAL) in peripheral tissues. Reciprocally, Ob-Ra accounted for only 19% of Ob-R(TOTAL) in the hypothalamus and for nearly all of Ob-R(TOTAL) in most peripheral tissues. Finally, we evaluated the effects of ovarian status and 17beta-estradiol on Ob-R expression in selected tissues. Treatment with 17beta-estradiol reduced Ob-R(TOTAL), Ob-Rb, and Ob-Ra expression by 70% in the uterine endometrium and tended to do the same in mammary adipose tissue. There was no effect of 17beta-estradiol on Ob-R in the hypothalamus, liver, soleus muscle, or subcutaneous adipose tissue. We conclude that greater estrogen secretion does not cause increased plasma leptin in prepubertal dairy heifers but estradiol can modulate Ob-R expression in some estrogen-responsive tissues.

Regulation of gene expression in the bovine mammary gland by ovarian steroids.

It is well established that estrogen is required for mammary epithelial cell proliferation and ductal development in the growing animal, and that lobuloalveolar development during gestation is dependent on progesterone. The effects of these steroid hormones on gene expression in the mammary gland are mediated primarily by their respective nuclear hormone receptors, which function as hormone-bound transcription factors. To gain insight into how estrogen and progesterone regulate mammary gland growth and function in cattle, we and others have characterized the expression patterns of their cognate nuclear hormone receptors in the bovine mammary gland throughout development, pregnancy, and lactation. This work has identified a lack of expression of estrogen receptor beta and a greater abundance of progesterone receptor during lactation in the bovine mammary gland, compared with the rodent gland. We speculate that interactions among the estrogen receptor isoforms that regulate progesterone receptor expression may contribute to these species differences. Further, demonstrated expression of substantial quantities of estrogen receptor within the prepubertal bovine mammary fat pad, along with coordinated insulin-like growth factor-I expression, suggests that this tissue may stimulate parenchymal growth via an estrogen-responsive paracrine mechanism. In addition, the recent availability of bovine genomic sequence information and microarray technologies has permitted the study of global gene expression in the mammary gland in response to the steroid environment. We have identified more than 100 estrogen-responsive genes, of which the majority are novel estrogen gene targets. Estrogen-induced changes in gene expression were consistent with increased mammary epithelial cell proliferation, increased extracellular matrix turnover in parenchyma, and increased extracellular matrix deposition in the fat pad. A comparison of estrogen-responsive genes in the mammary glands of humans, mice, and cattle suggests considerable variation among species, as well as potential differences in regulatory elements in common estrogen receptor gene targets. Continuing studies using advanced molecular techniques should assist in elucidating the complex regulation of mammary function at the transcript level.

Estrogen-dependent responses of the mammary fat pad in prepubertal dairy heifers.

Ovaries are absolutely required for development of the mammary parenchyma (PAR) in cattle, reflecting estrogen-dependent epithelial cell proliferation. However, the estrogen receptor (ER) that mediates the mammary estrogen effects, ERalpha, is absent in proliferating epithelial cells. In the mouse, this discrepancy is explained in part by the ability of the mammary fat pad (MFP) to synthesize epithelial cell mitogens such as IGF-I in response to estrogen. Consistent with a similar role for the bovine MFP, 30% of its fibroblasts and adipocytes were immunoreactive for ERalpha in prepubertal dairy heifers. To assess estrogen-dependent gene expression in the MFP, 16 prepubertal dairy heifers were randomly assigned to a 2x2 factorial. The first factor was ovarian status, with heifers undergoing bilateral ovariectomy or left intact at 4.6 months of age. The second factor was applied 30 days after surgery and consisted of injection of estrogen or excipient. After 3 days of injection, heifers were administered an intrajugular bolus of bromodeoxyuridine (BrdU) and slaughtered 2 h later. The estrogen injection, but not ovarian status, caused significant increases in the fraction of epithelial cells labeled with BrdU and produced tissue-specific effects on gene expression. In the PAR, estrogen injection increased IGF-I gene expression by twofold despite reductions of 50% or more in ERalpha mRNA abundance and the fraction of epithelial cells immunoreactive for ERalpha. The estrogen-dependent increase in IGF-I mRNA was greater in the MFP, presumably because estrogen failed to downregulate ERalpha expression in this mammary compartment. Finally, estrogen-responsiveness of the MFP appears unique among the bovine fat depots as estrogen injection did not induce IGF-I expression in its s.c. counterpart. Our data demonstrate that the bovine MFP is highly responsive to exogenous estrogen, consistent with a role for this tissue compartment in communicating its effects on epithelial cell proliferation.

Developmental and nutritional regulation of the prepubertal heifer mammary gland: I. Parenchyma and fat pad mass and composition.

Prior to puberty, elevated nutrient intake has been shown to negatively affect prepubertal mammary development in the heifer. The objective of this study was to evaluate the effects of increased nutrient intake on mammary development in Holstein heifers at multiple body weights from birth through puberty. Specifically, this study evaluated the effects of nutrient intake and body weight at harvest on 1) total weight and DNA content of the parenchyma (PAR) and mammary fat pad (MFP) and 2) PAR and MFP composition. Starting at 45 kg of body weight, heifers (n = 78) were assigned to either a restricted (R) or elevated (E) level of nutrient intake supporting 650 (R) or 950 (E) g/d of body weight gain. Heifers were harvested at 50-kg increments from 100 to 350 kg of body weight. Mammary fat pad weight and DNA content were greater in E- than in R-heifers. Additionally, E-heifers had a greater fraction of lipids and a smaller fraction of protein in their MFP than did R-heifers. Parenchyma weight and DNA were lower in E- than in R-heifers; however, when analyzed with age as a covariate term, treatment was no longer a significant term in the model. Level of nutrient intake had no effect on the lipid, protein, or hydroxyproline composition of the PAR. Collectively, these data demonstrate that PAR is refractory to the level of nutrient intake whereas MFP is not. Furthermore, the covariate analysis demonstrated that age at harvest, not the level of nutrient intake, was the single greatest determinant of total PAR DNA content.

Developmental and nutritional regulation of the prepubertal bovine mammary gland: II. Epithelial cell proliferation, parenchymal accretion rate, and allometric growth.

It is well documented that elevated nutrient intake prior to puberty reduces prepubertal mammary development in the bovine. The companion paper demonstrated that age at harvest is a primary determinant of parenchymal (PAR) mass and that any effects of elevated energy intake on mechanisms regulating mammary development are dwarfed by this effect of time. Therefore, it is hypothesized that while causing a decrease in prepubertal PAR mass, elevated nutrient intake will have no effect on growth characteristics of the mammary gland. The objectives of this experiment were to evaluate the effects of increased nutrient intake from early in life on 1) mammary epithelial cell proliferation, 2) mammary PAR DNA accretion rates, and 3) the dynamics of prepubertal allometric PAR growth. Holstein heifers (n = 78) were fed from 45 kg of body weight either elevated (E) or restricted (R) levels of nutrients to support 950 (E) or 650 (R) g/d of body weight gain. Six heifers per treatment were harvested at 50-kg increments from 100 to 350 kg of body weight. Heifers on the E plane of nutrition had higher plasma leptin and less PAR DNA than their body weight-matched R-intake cohorts. Despite this reduction in PAR DNA, treatment did not negatively influence mammary epithelial cell proliferation or the PAR DNA accretion rate. Dynamics of allometric and isometric mammary growth were also unaffected by the level of nutrient intake, as was exit from allometric growth. This work represents the first demonstrating that the level of nutrient intake and the concomitant increase in plasma leptin have no measurable influence on 1) the rate of PAR DNA accretion, 2) mammary epithelial cell proliferation, or 3) total PAR mass and, by default, the local or systemic controls that coordinate these processes.

Effects of body weight and nutrition on mammary protein expression profiles in Holstein heifers.

A proteomics approach was used to characterize biochemical and cellular mechanisms governing effects of peripubertal feeding on heifer mammary development. Mammary parenchymal tissue from 24 Holstein heifers randomly assigned to treatments arranged in a 2 x 2 factorial design was used to generate 2-dimensional protein maps of mammary tissue extracts. Heifers were reared on 1 of 2 dietary treatments, restricted (650 g/ d of daily gain) or elevated (950 g/d of daily gain) and killed at 1 of 2 body weights (BW, 200 or 350 kg). Cytosolic mammary gland extracts were prepared from frozen mammary parenchyma. Proteome maps of extracts were constructed using PDQuest software. Densities of 820 protein spots were analyzed using the MIXED procedure of SAS. Protein spots were characterized by changes in profiles of expression in response to increased BW, dietary treatment, or both. Dietary treatment influenced the expression of 131 protein spots, whereas heifer BW influenced the expression of 108 spots. The 22 most highly influenced (statistically) spots were excised and submitted for mass spectrometric analyses. Returned protein names and accession numbers were used in National Center for Biotechnology Information database searches to obtain information on the identified proteins. For example, one of the proteins that differed by dietary treatment, transferrin, a binding protein of insulin-like growth factor binding protein-3, was identified via these methods. Possible roles of this and other proteins in mammary development are described. We concluded that a proteomic approach is an effective tool for identifying the proteins involved in bovine mammary development.

Protein adduct biomarkers: state of the art.

Covalent protein adducts formed after exposure to xenobiotics may provide readily measurable indicators of these exposures. After adequate characterization of the dose-dependent formation of a specific adduct, the adduct can often be used as a quantitative marker for exposure, DNA adduct formation, or, possibly, risk of disease. By elucidating the structure of an adduct and studying the conditions under which it forms, information about the reactions that lead to its formation can be obtained. Continuing work in this area includes methods to expand the number, types, and levels of chemical exposures that can be studied by covalent adduct formation. In addition to the use of this technology in the field of occupational health, basic research in this area provides insights into metabolic pathways and biochemistry, as well.

Training future leaders of academic medicine: internal programs at three academic health centers.

The authors review the need for internal programs for leadership training at academic health centers and then describe in detail three programs of this type that have operated during the 1990s: (1) the Allegheny Leadership Institute, founded by the Allegheny Health, Education and Research Foundation, Pittsburgh, Pennsylvania; (2) the Physician Executive Management Development Program (PEMDP) of Saint Louis University School of Medicine; and (3) the University of Nebraska Medical Center Leadership Institute. Educational elements common to these programs include having a small class size and participants from many areas of academic medicine and health care, focusing on educational strategies that draw on participants' experiences and training, conducting the training away from the participants' institutions, having short sessions, using faculty from both within and outside the participants' institutions, and creating strategies to reinforce learning. Lessons learned reflect the unique context of each institution; the authors list the major lessons learned by each of the three programs they surveyed (e.g., leaders of the Saint Louis University PEMDP program believe that it is important to help participants implement desired changes in their work areas once they return to work, and are investigating how to do this). The authors conclude with an extensive list of recommendations to optimize the effects of leadership development training carried out in AHCs' internal programs (e.g., "Focus on specific skills that can be learned, and link the learning experiences to real work situations in health care and higher education") and explain why they think internal leadership institutes have at least three distinct advantages over external programs.

Oral lesions in patients with psoriasis: clinical presentation and management.

Psoriasis is a chronic inflammatory skin disease that rarely involves the oral cavity. In this report we describe 2 cases, initially diagnosed with cutaneous psoriasis, that present with oral lesions on the attached gingiva. The clinical appearance and differential diagnosis are presented and discussed. Case 1 describes the non-surgical management of intraoral psoriasiform lesions and the use of a free gingival graft to restore an area of gingival recession resulting from an oral lesion. The second case outlines the use of topical corticosteroid therapy as an adjunct to non-surgical periodontal therapy. Although patients with cutaneous psoriasis rarely present with oral involvement, the clinician should be aware that oral lesions may occur. Accurate diagnosis is dependent on a thorough clinical examination, a biopsy of the oral lesions, and a history of cutaneous psoriasis.

Self-regulation of stimulation for ADD-H children during reading and vigilance task performance.

The present study examines the hypothesis that motor responses added into rote tasks would modulate the sensation-seeking activity and impulsive errors of hyperactive (ADD-H) children. To this purpose 22 ADD-H and 25 comparison children were administered two repetitive tasks (word decoding and an auditory vigilance task) under both an active response and a passive response condition. Findings were that the impulsive errors, talking/noisemaking, and activity of ADD-H children was normalized (i.e., did not differ from comparison children) only in the high stimulation active response conditions. Behavioral improvements for ADD-H children were documented in both tasks in the active condition, but performance gains were found only in the vigilance task. The findings supported predictions derived from the optimal stimulation theory that the excessive activity and attraction to novel stimuli of ADD-H children can be channeled into appropriate instrumental motor and attention responses.

Histological evaluation of sinus augmentation using platelet rich plasma (PRP): a case series.

The use of Platelet Rich Plasma (PRP) in conjunction with autogenous bone graft materials has recently been advocated for use in sinus augmentation surgery as a means of enhancing both quantity and quality of newly forming bone. The use of PRP is based on the premise that autogenous plasma, rich in platelets, contributes large quantities of mitogenic polypeptides such as Platelet Derived Growth Factor (PDGF), Transforming Growth Factor-b (TGF-b) and Insulin-like Growth Factor-I (IGF-I), thereby enhancing osteogenesis. The aim of this study was to evaluate the potential of PRP to enhance bone formation following sinus augmentation with different bone derivative/substitute materials (DFDBA, FDBA, Xenograft, Bioactive Glass). This study presents histology of trephine-obtained core samples from five clinical cases in which sinus augmentation was performed with PRP combined with bone derivatives/substitutes. Histological evaluation of this case series consistently revealed the presence of residual graft particles surrounded by loose connective tissue, with a limited amount of newly formed bone. The findings suggest that the addition of PRP to bone derivative/substitute materials may not significantly enhance bone formation in the maxillary sinus area.

Localized ridge augmentation using GBR and platelet-rich plasma: case reports.

Combining platelet-rich plasma (PRP) with autogenous bone graft materials has recently been advocated as a means of enhancing rate and quality of new bone formation in regenerative procedures. The aim of this case series was to evaluate the potential of PRP in combination with bone allograft to enhance bone regeneration in alveolar ridge defects exhibiting both vertical and horizontal loss prior to the placement of dental implants. Augmentation resulted in clinical and radiographic gains in both vertical and horizontal components of the osseous defects, thereby facilitating subsequent placement of dental implants. Histologic evaluation of the cases revealed the presence of residual allograft particles surrounded by connective tissue as well as newly formed bone within the grafted areas. However, the addition of PRP did not appear to enhance the quality or quantity of new bone formation over that reported in comparable guided bone regeneration (GBR) studies without PRP.

Guided tissue regeneration in the management of severe periodontal-endodontic lesions.

Diagnosis of combined periodontal-endodontic lesions can prove difficult and frustrating. They are often characterised by extensive loss of periodontal attachment and alveolar bone, and their successful management depends on careful clinical evaluation, accurate diagnosis, and a structured approach to treatment planning for both the periodontic and endodontic components. Recent advances in regenerative periodontics have led to improved management of periodontal-endodontic lesions. This paper reviews the management of such lesions in light of these recent advances and illustrates this through reports of two patients who had severe periodontal involvement.

Familial gingival fibromatosis: a report of two patients.

This report describes two patients with familial gingival fibromatosis. Treatment for both patients involved gingivectomies to provide acceptable gingival function and appearance. The first patient has been monitored for only a short period, the second patient for 11 years. Neither patient has shown any recurrence of the condition.

Implant site development using orthodontic extrusion: a case report.

One of the most important factors in the successful placement of endosseous implants is the presence of adequate alveolar bone at the recipient site. Alveolar bone loss associated with destructive periodontal disease frequently results in osseous defects that may complicate subsequent implant placement. Typically, such defects are treated prior to or at the time of implant surgery using the principles of guided bone regeneration. Under certain circumstances, however, such defects may be managed non-surgically by orthodontic extrusion. Orthodontic extrusion can be used to increase the vertical bone height and volume and to establish a more favourable soft-tissue profile prior to implant placement. The addition, the increase in the vertical osseous dimension at interproximal sites may assist in the preservation of the interdental papillae and can further enhance gingival aesthetics. This report illustrates the treatment sequence for site development with orthodontic extrusion prior to immediate implant placement.

Perceptual differences in fetal alcohol effect boys performing a modeling task.

Perceptual differences were examined for four boys identified with fetal alcohol effect (FAE) and labeled learning disabled for educational purposes. Without a model, all FAE boys built asymmetrical building block designs with no stacking of blocks. When the boys were shown a short videotape of a peer building a simple symmetrical structure with one-layer stacking of blocks, none of the FAE boys successfully imitated. They continued to place blocks randomly on the floor surface with no stacking or layering. This limited study suggests modeling perceptual tasks may not be an effective teaching strategy for children identified with fetal alcohol effect and such children may not be able to learn new skills using demonstration or modeling.