An exploration of students' use of digital resources for self-study in anatomy: a survey study.

BACKGROUND: The development of technology has provided new ways for active engagement and for visualizing structures in anatomy education including digital resources that may be used outside of the classroom. To support students' learning, there is a need to better understand students' experiences of using digital resources. This study aimed to identify which resources students use, their preferences, the purpose of using them, and barriers to adopting tools for self-study of anatomy. METHODS: A mixed -methods approach combining qualitative and quantitative data was used to collect and analyse data. Two consecutive cohorts of first-semester medical students (n = 278) were invited to complete an anonymized survey. The survey consisted of itemized questions, free-text space for comments, and one open-ended question. Descriptive statistics were used for demographics and itemized answers. Comments and free-text answers were analysed qualitatively using abductive inference. RESULTS: One hundred and twenty-seven students completed the survey (response rate 45%). Most students (46%) reported that they spend more than 30 h/per week on self-study. They used a variety of digital resources for different purposes. Most students used digital resources to prepare for examinations, when they encountered difficulties and after going through a section. Students reported that they would use digital resources to a greater extent if they were offered an introduction, if resources were more accessible, and if they could interact with a tutor. The free-text responses revealed that digital resources helped students understand anatomy, allowed them to make active choices, provided tools for repetition and memorization, accelerated and simplified the learning process, and complemented other learning resources. CONCLUSIONS: Digital resources may support the understanding of anatomy by offering alternative modes of learning and providing a valuable complement to other learning resources. Educators should consider how digital resources are introduced and offer support and feedback.

Reduction of body weight by increased loading is associated with activation of norepinephrine neurones in the medial nucleus of the solitary tract.

We previously provided evidence supporting the existence of a novel leptin-independent body weight homeostat ("the gravitostat") that senses body weight and then initiates a homeostatic feed-back regulation of body weight. We, herein, hypothesize that this feed-back regulation involves a CNS mechanism. To identify populations of neurones of importance for the putative feed-back signal induced by increased loading, high-fat diet-fed rats or mice were implanted intraperitoneally or subcutaneously with capsules weighing ∼15% (Load) or ∼2.5% (Control) of body weight. At 3-5 days after implantation, neuronal activation was assessed in different parts of the brain/brainstem by immunohistochemical detection of FosB. Implantation of weighted capsules, both subcutaneous and intraperitoneal, induced FosB in specific neurones in the medial nucleus of the solitary tract (mNTS), known to integrate information about the metabolic status of the body. These neurones also expressed tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbH), a pattern typical of norepinephrine neurones. In functional studies, we specifically ablated norepinephrine neurones in mNTS, which attenuated the feed-back regulation of increased load on body weight and food intake. In conclusion, increased load appears to reduce body weight and food intake via activation of norepinephrine neurones in the mNTS.

How students discern anatomical structures using digital three-dimensional visualizations in anatomy education.

Learning anatomy holds specific challenges, like the appreciation of three-dimensional relationships between anatomical structures. So far, there is limited knowledge about how students construct their understanding of topographic anatomy. By understanding the processes by which students learn anatomical structures in 3D, educators will be better equipped to offer support and create successful learning situations. Using video analysis, this study investigates how students discern anatomical structures. Sixteen students at different levels of education and from different study programs were recorded audiovisually while exploring 3D digital images using a computerized visualization table. Eleven hours of recorded material were analyzed using interaction analysis and phenomenography. Seven categories were identified during data analysis, describing the qualitatively different patterns of actions that students use to make sense of anatomy: decoding the image; positioning the body in space; purposeful seeking, using knowledge and experience; making use of and creating variation; aimless exploration, and arriving at moments of understanding. The results suggest that anatomy instruction should be organized to let the students decide how and at what pace they examine visualized images. Particularly, the discovery process of decoding and positioning the body in space supports a deep learning approach for learning anatomy using visualizations. The students' activities should be facilitated and not directed.

Transfer of anatomy during surgical clerkships: an exploratory study of a student-staff partnership.

Objectives: This qualitative study aims to explore how fourth-year medical students on the surgery course perceived a clinical anatomy workshop organised by near-peer student teachers in partnership with faculty. Methods: Forty-seven medical students participated in a workshop on clinical anatomy in the dissection laboratory. A voluntary response sampling method was used. The students' perceptions of the workshop were explored through a thematic content analysis of transcribed, semi-structured group interviews and written comments. Results: A majority of the students had not revisited the dissection laboratory since their second year, and all students described the workshop as a unique opportunity to vertically integrate anatomical knowledge. Four main themes were identified as most valuable for the students' learning experience, namely that the workshop 1) was taught by knowledgeable and friendly near-peer tutors (NPTs), 2) consisted of highly relevant anatomical content, 3) offered a hands-on experience of cadavers in the dissection laboratory, and 4) was taught in a focused session in the middle of the surgery course. Conclusions:  This study shows how hands-on workshops in clinical anatomy, developed in student-staff partnerships and taught by NPTs, can enable senior medical students to recall and vertically integrate anatomical knowledge during surgical clerkships. The results have implications for curriculum design, giving voice to senior students' wishes for spaced repetition and vertical integration of pre-clinical anatomy knowledge during their clinical training. Moreover, this study may inspire other students and faculty to develop similar near-peer teaching activities through student-staff partnerships.

Three-dimensional visualisation of authentic cases in anatomy learning - An educational design study.

BACKGROUND: Many studies have investigated the value of three-dimensional (3D) images in learning anatomy. However, there is a lack of knowledge about students learning processes using technology and 3D images. To understand how to facilitate and support the learning of anatomy, there is a need to know more about the student perspectives on how they can use and benefit from 3D images. METHODS: This study used designed educational sessions informed by Educational Design Research to investigate the role of technology-enhanced 3D images in students' anatomy learning. Twenty-four students representing different health professions and multiple study levels, and one tutor, participated in the study. A visualisation table was used to display the images of real patient cases related to disorders associated with the abdomen and the brain. Students were asked to explore the images on their own and audio/video capture was used to record their words and actions. Directly following the session, students were interviewed about their perceptions and different ways of learning and studying anatomy. The tutor was interviewed about his reflections on the session and his role as a facilitator on two occasions. Content analysis was used in its manifest and latent form in the data analysis. RESULT: Two main categories describing the students' and tutor's accounts of learning using the visualisation table were identified: 1. Interpreting 3D images and 2. Educational sessions using visualisation tables. Each category had signifying themes representing interpretations of the latent meaning of the students' and tutor's accounts. These were: Realism and complexity; Processes of discernment; References to previous knowledge; Exploring on one's own is valuable; Context enhances learning experiences; Combinations of learning resources are needed and Working together affects the dynamics. CONCLUSIONS: This study identifies several important factors to be considered when designing effective and rewarding educational sessions using a visualization table and 3D images in anatomy education. Visualisation of authentic images has the potential to create interest and meaningfulness in studying anatomy. Students need time to actively explore images but also get tutor guidance to understand. Also, a combination of different resources comprises a more helpful whole than a single learning resource.

Delta-like 1 homologue (DLK1) protein in neurons of the arcuate nucleus that control weight homeostasis and effect of fasting on hypothalamic DLK1 mRNA.

Delta-like 1 homologue (DLK1; also called preadipocyte factor 1) is an epidermal growth factor repeat-containing transmembrane protein that is cleaved by tumor necrosis factor-α-converting enzyme to generate a biologically active soluble form. DLK1 is involved in the differentiation of several cell types, including adipocytes. Lack of the dlk1 gene results in adiposity, and polymorphism within the gene encoding DLK1 is associated with human obesity. The dlk1 gene is expressed in restricted areas of the adult brain, with an enrichment of cell bodies expressing DLK1 mRNA in the hypothalamus. Antibodies to DLK1 were used to study the cellular localization and chemical identity of DLK1-immunoreactive neuronal cell bodies in rat hypothalamus. DLK1 immunoreactivity was demonstrated in the cell soma and dendrites of cell bodies in the suprachiasmatic, supraoptic, paraventricular, dorsomedial, arcuate nuclei and in the perifornical/lateral hypothalamic area. In the arcuate nucleus (Arc), DLK1 immunoreactivity was mainly seen in many neurons of the ventromedial and to a lesser extent in its ventrolateral division. Double labeling showed that 93.7% of orexigenic agouti-related peptide (AgRP) and 94.1% of neuropeptide Y (NPY) neurons located in the ventromedial part of the Arc were DLK1 positive, whereas 36.1% of anorexigenic pro-opiomelanocortin and 34.6% of cocaine- and amphetamine-regulated transcript neurons of the Arc contained DLK1 immunoreactivity. DLK1 mRNA was downregulated in the hypothalamus of fasted animals. Presence of DLK1 in the majority of orexigenic Arc NPY/AgRP neurons and regulation of DLK1 mRNA by nutritional challenge suggest that DLK1 has a role in hypothalamic regulation of body weight control. © 2014 S. Karger AG, Basel.

MicroRNAs in the hypothalamus.

MicroRNAs (miRNAs) are short (∼22 nucleotides) non-coding ribonucleic acid (RNA) molecules that negatively regulate the expression of protein-coding genes. Posttranscriptional silencing of target genes by miRNA is initiated by binding to the 3'-untranslated regions of target mRNAs, resulting in specific cleavage and subsequent degradation of the mRNA or by translational repression resulting in specific inhibition of protein synthesis. An increasing amount of evidence shows that miRNAs control a large number of biological processes and there exists a direct link between miRNAs and disease. miRNA molecules are abundantly expressed in tissue-specific and regional patterns and have been suggested as potential biomarkers, disease modulators and drug targets. The central nervous system is a prominent site of miRNA expression. Within the brain, several miRNAs are expressed and/or enriched in the region of the hypothalamus and miRNAs have recently been shown to be important regulators of hypothalamic control functions. The aim of this review is to summarize some of the current knowledge regarding the expression and role of miRNAs in the hypothalamus.

G-protein-gated inwardly rectifying K+ channel 4 (GIRK4) immunoreactivity in chemically defined neurons of the hypothalamic arcuate nucleus that control body weight.

G-protein-gated inwardly rectifying K(+) channels (GIRKs; also called Kir3) are a family of K(+) channels, which are activated (opened) via a signal transduction cascade starting with ligand-stimulated G-protein-coupled receptors (GPCRs). Four GIRK genes have been identified (GIRK1-4). GIRK4 (Kir3.4) has a role in regulating energy homeostasis, since mice with a targeted mutation in the GIRK4 gene exhibit a predisposition to late-onset obesity. GIRK4 mRNA is expressed in hypothalamic regions that harbor neurons involved in the regulation of food intake and body weight. Using goat and rabbit antisera to the GIRK4 protein, the cellular localization and transmitter content of GIRK4-immunoreactive neurons was determined in the hypothalamic arcuate nucleus, a region that contains neurons which are accessible to circulating hormones and is intimately associated with the control of body weight. GIRK4-immunoreactive large cell bodies were demonstrated in the ventrolateral part of the arcuate nucleus, with smaller neuronal cell bodies in the ventromedial part of the nucleus. Double-labeling showed presence of GIRK4 immunoreactivity in large neurons of the ventrolateral arcuate nucleus containing the peptides α-melanocyte-stimulating hormone (α-MSH), a marker for pro-opiomelanocortin (POMC) neurons, and cocaine- and amphetamine-regulated transcript (CART). GIRK4 immunoreactivity was also seen in neurons of the ventromedial part of the arcuate nucleus containing agouti-regulated peptide (AgRP) and neuropeptide Y (NPY). The results suggest that the GIRK4 channel protein plays a role in regulating membrane excitability in chemically defined neurons of the arcuate nucleus that control body weight.

Chemical phenotypes of P2X2 purinoreceptor immunoreactive cell bodies in the area postrema.

Purines such as adenosine 5'-triphosphate (ATP) act as extracellular messengers through specific purinergic receptors. Three different classes of purinergic receptors have been identified and termed P1, P2X, and P2Y. The purinergic receptor subunit P2X2 is a ligand-gated ion channel that is widely expressed by neurons in the CNS. In the brainstem medulla oblongata, the ionotropic P2X2 receptor (P2X2R) is enriched in the area postrema (AP). Two different antisera to P2X2R were used to determine the chemical nature of P2X2R immunoreactive cell bodies in the rat AP, an area lacking a blood-brain barrier. Subcellularly, P2X2R immunoreactivity was located to the periphery of individual cell bodies. The majority of P2X2R-immunoreactive cells were shown to contain tyrosine hydroxylase (TH) (63.5 ± 7.7%) and dopamine ß-hydroxylase (61.5 ± 5.1%). Phenylethanolamine N-methyltransferase (PNMT)-containing cells were not detected in the AP, supporting a noradrenergic nature of P2X2R cells in the AP. There were no P2X2R-immunoreactive cells in the AP that contained the GABA-synthesizing enzyme glutamic acid decarboxylase 65. Only single vesicular glutamate transporter 2-immunoreactive cell bodies that were not P2X2R-positive were demonstrated in the AP. Some P2X2R-positive cells in the AP were immunoreactive for the neuropeptides substance P and pituitary adenylate cyclase-activating polypeptide, whereas dynorphin-, enkephalin-, or cholecystokinin-positive cells were not P2X2R-immunoreactive. Presence of P2X2R in a majority of noradrenergic cells of the AP implies that ATP may have a regulatory action on neuronal noradrenaline release from the AP, a circumventricular organ with a strategic position enabling interactions between circulating substances and the central nervous system.

Protein components of the blood-brain barrier (BBB) in the brainstem area postrema-nucleus tractus solitarius region.

The blood-brain barrier (BBB) prevents entry of circulating substances into the brain. The circumventricular organs (CVOs) lack a BBB and have a direct communication with the circulation blood. One of the CVOs, the area postrema (AP), which has a close relationship with the nucleus of the tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMX), plays a role in controlling the entry of blood-borne substances to neurons of the brainstem. To clarify the cellular localization of protein components of the BBB in the brainstem AP-NTS region, we used antisera to--(1) Tight junctions: claudin-5 and zona occludens-1 (ZO-1). (2) Endothelial cells: (a) all endothelial cells--rat endothelial cell antigen-1 (RECA-1) and (b) endothelial cells at BBB--endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR). (3) Basal lamina--laminin. (4) Vascular smooth muscle cells--smooth muscle actin (SMA). (5) Pericytes--chondroitin sulfate proteoglycan (NG2). (6) Glial cells: (a) astrocytes--glial fibrillary acidic protein (GFAP), (b) tanycytes--dopamine- and cAMP-regulated phosphoprotein of 32 kDA (DARPP-32), and (c) microglia--CD11b. Neuronal cell bodies in the NTS were visualized by antisera to neuropeptide Y (NPY) and alpha-melanocyte-stimulating hormone (alpha-MSH), two peptides regulating energy balance. This study provides a detailed analysis of the cellular localization of BBB proteins in the AP and NTS and shows the existence of vessels in the dorsomedial aspect of the NTS that lack immunoreactivity for the BBB markers EBA and TfR. Such vessels may represent a route of entry for circulating substances to neurons in the NTS that inter alia regulate energy balance.

Protein components of the blood-brain barrier (BBB) in the mediobasal hypothalamus.

The blood-brain barrier (BBB) plays an important role in controlling the access of substances to the brain. Of the circumventricular organs (CVO), i.e. areas that lack a BBB, the median eminence and its close relationship with the hypothalamic arcuate nucleus plays an important role in controlling the entry of blood-borne substances to neurons of the mediobasal hypothalamus. In order to clarify the nature of the BBB in the median eminence-arcuate nucleus complex, we have used immunohistochemistry and antisera to protein components of the BBB-(1) tight junctions, claudin-5 and zona occludens-1 (ZO-1); (2) endothelial cells: (a) all endothelial cells: rat endothelial cell antigen-1 (RECA-1), (b) endothelial cells at BBB: endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR), and (c) endothelial cells at CVOs: dysferlin; (3) basal lamina: laminin; (4) vascular smooth muscle cells: smooth muscle actin (SMA); (5) pericytes: chondroitin sulfate proteoglycan (NG2); (6) glial cells: (a) astrocytes: glial fibrillary acidic protein (GFAP), (b) tanycytes: dopamine- and cAMP-regulated phosphoprotein of 32kDA (DARPP-32), (c) microglia: CD11b. Neuronal cell bodies located in the ventromedial aspect of the arcuate nucleus were visualized by antiserum to agouti-related protein (AgRP). The study provides a detailed analysis on the cellular localization of BBB components in the mediobasal hypothalamus. Some vessels in the ventromedial aspect of the arcuate nucleus lacked the BBB markers EBA and TfR, suggesting an absence of an intact BBB. These vessels may represent a route of entry for circulating substances to a subpopulation of arcuate nucleus neurons.

The role of 5-HT(1A) receptors in learning and memory.

The ascending serotonin (5-HT) neurons innervate the cerebral cortex, hippocampus, septum and amygdala, all representing brain regions associated with various domains of cognition. The 5-HT innervation is diffuse and extensively arborized with few synaptic contacts, which indicates that 5-HT can affect a large number of neurons in a paracrine mode. Serotonin signaling is mediated by 14 receptor subtypes with different functional and transductional properties. The 5-HT(1A) subtype is of particular interest, since it is one of the main mediators of the action of 5-HT. Moreover, the 5-HT(1A) receptor regulates the activity of 5-HT neurons via autoreceptors, and it regulates the function of several neurotransmitter systems via postsynaptic receptors (heteroreceptors). This review assesses the pharmacological and genetic evidence that implicates the 5-HT(1A) receptor in learning and memory. The 5-HT(1A) receptors are in the position to influence the activity of glutamatergic, cholinergic and possibly GABAergic neurons in the cerebral cortex, hippocampus and in the septohippocampal projection, thereby affecting declarative and non-declarative memory functions. Moreover, the 5-HT(1A) receptor regulates several transduction mechanisms such as kinases and immediate early genes implicated in memory formation. Based on studies in rodents the stimulation of 5-HT(1A) receptors generally produces learning impairments by interfering with memory-encoding mechanisms. In contrast, antagonists of 5-HT(1A) receptors facilitate certain types of memory by enhancing hippocampal/cortical cholinergic and/or glutamatergic neurotransmission. Some data also support a potential role for the 5-HT(1A) receptor in memory consolidation. Available results also implicate the 5-HT(1A) receptor in the retrieval of aversive or emotional memories, supporting an involvement in reconsolidation. The contribution of 5-HT(1A) receptors in cognitive impairments in various psychiatric disorders is still unclear. However, there is evidence that 5-HT(1A) receptors may play differential roles in normal brain function and in psychopathological states. Taken together, the evidence indicates that the 5-HT(1A) receptor is a target for novel therapeutic advances in several neuropsychiatric disorders characterized by various cognitive deficits.

Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) defines a subpopulation of hypothalamic POMC neurons.

Several lines of evidence support a role for pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of energy balance. In the present study, we have used fluorescent in situ hybridization and immunohistochemistry to investigate in detail the cellular localization and chemical content of PACAP mRNA- and peptide-containing neuronal cell bodies in the mediobasal hypothalamus of the rat. PACAP mRNA-containing cell bodies were demonstrated in high numbers in the ventromedial hypothalamic nucleus (VMH) and in lower numbers in the arcuate nucleus (Arc). In colchicine-treated rats, PACAP immunoreactivity was demonstrated in many cell bodies of the VMH and several cell bodies of the ARC. Double-labeling revealed that PACAP immunoreactivity was present in approximately 20% of pro-opiomelanocortin (POMC) neurons in the ventrolateral Arc as shown by presence of alpha-melanocyte-stimulating hormone (alpha-MSH), but not in agouti-related peptide (AgRP)-containing neurons in the ventromedial aspect of the Arc. PACAP immunoreactivity was also colocalized with the vesicular acetylcholine transporter (VAChT; a marker for cholinergic neurons) in Arc POMC neurons. Brainstem POMC neurons in the commissural part of the solitary tract nucleus were devoid of PACAP immunoreactivity. However, several VAChT-positive neurons in the dorsal motor nucleus of the vagus nerve were also PACAP immunoreactive, whereas VAChT-positive neurons of the motor nucleus of the hypoglossal nerve were PACAP-negative. The results show presence of PACAP with alpha-MSH in a subpopulation of hypothalamic POMC neurons and point further to the neurochemical heterogeneity of hypothalamic, but not brainstem, POMC neurons.

Neurotransmitters in key neurons of the hypothalamus that regulate feeding behavior and body weight.

During the last two decades attention has been focussed on the role of different neuropeptides in hypothalamic control of feeding behavior. Several hypothalamic peptides that participate in the control of ingestive behavior are produced in neuronal cell bodies of the arcuate nucleus and/or the lateral hypothalamic area. Apart from producing orexigenic or anorexigenic compounds of peptidergic nature, these neurons also produce excitatory and inhibitory amino acid neurotransmitters. The role of GABA and glutamate in regulating energy balance has received less attention in comparison to neuropeptides. The arcuate nucleus-median eminence area, a region with a weak blood-brain barrier, contains at least two neuronal cell populations that exert opposing actions on energy balance. The majority of the neurons located in the ventromedial aspect of the arcuate nucleus, which produce the orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AGRP), contain in addition the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) and the vesicular GABA transporter (VGAT), thereby supporting their GABAergic nature. Some neurons producing pro-opiomelanocortin (POMC)- and cocaine- and amphetamine-regulated transcript (CART), located in the ventrolateral division of the arcuate nucleus have recently been reported to contain the vesicular glutamate transporter 2 (VGLUT2), a marker for glutamatergic neurons, and the acetylcholine (ACh) synthesizing enzyme choline acetyltransferase (ChAT) as well as the vesicular ACh transporter (VAChT), supporting also a cholinergic phenotype. In the lateral hypothalamic area, hypocretin/orexin neurons express VGLUT1 or VGLUT2, but not GAD, whereas some melanin-concentrating hormone (MCH) cells contain GAD. These observations support the view that several classical transmitters, relatively neglected feeding transmitters candidates, are present in key neurons that regulate body weight and consequently may represent important orexigenic/anorexigenic mediators that convey information to other neurons within the hypothalamus as well as from the hypothalamus to other brain regions that participate in regulation of energy balance.

Hypothalamic proopiomelanocortin (POMC) neurons have a cholinergic phenotype.

Neuronal networks originating in the hypothalamic arcuate nucleus play fundamental roles in the control of energy balance. Neuropeptide Y (NPY)-producing neurons in the arcuate nucleus stimulate food intake, whereas arcuate nucleus neurons that release the proopiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone (alpha-MSH) potently reduce food intake. Relatively little attention has been focused on classical neurotransmitters in regulation of food intake. Here, we have investigated the potential presence of acetylcholine (ACh) in NPY- and POMC-containing neuronal populations of the arcuate nucleus. Antisera to proteins required for cholinergic neurotransmission, including choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT), were employed in double-labeling immunohistochemical experiments. In colchicine-treated rats, ChAT- and VAChT-immunopositive cell bodies were located in the ventral aspect of the arcuate nucleus. ChAT and VAChT immunoreactivities were demonstrated in alpha-MSH- and cocaine- and amphetamine-regulated transcript (CART)-containing cell bodies of the arcuate nucleus, whereas cell bodies containing NPY or agouti-related peptide (AGRP) were distinct from VAChT-immunoreactive neuronal perikarya. VAChT immunoreactivity was also present in a large number of alpha-MSH-containing nerve fiber varicosities throughout the central nervous system. In the commissural part of the nucleus tractus solitarius, no alpha-MSH-containing cell bodies were found to have ChAT or VAChT immunoreactivity. The presence of markers for cholinergic neurotransmission in a subpopulation of hypothalamic POMC/CART neurons suggests co-release of ACh with peptides derived from the POMC precursor and CART. The results indicate a role for ACh in control of energy balance, mediating the effects of peripheral hormones such as leptin and insulin.

Tomosyn is expressed in beta-cells and negatively regulates insulin exocytosis.

Tomosyn, a syntaxin-binding protein, is capable of dissociating mammalian homolog of the Caenorhabditis elegans unc-18 gene from syntaxin and is involved in the regulation of exocytosis. We have investigated the expression, cellular localization, and functional role of tomosyn in pancreatic beta-cells. Western blotting revealed a 130-kDa protein corresponding to tomosyn in insulin-secreting beta-cell lines. RT-PCR amplification showed that b-, m-, and s-tomosyn isoform mRNAs are expressed in beta-cell lines and rat pancreatic islets. Immunohistochemistry revealed punctate tomosyn immunoreactivity in the cytoplasm of insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing islet cells. Syntaxin 1 coimmunoprecipitated with tomosyn in extracts of insulin-secreting cells. Overexpression of m-tomosyn in mouse beta-cells significantly decreased exocytosis, whereas inhibition of tomosyn expression by small interfering RNA increased exocytosis. Hence, in the pancreatic beta-cell, tomosyn negatively regulates insulin exocytosis.

5-Hydroxytryptamine 1A receptor blockade facilitates aversive learning in mice: interactions with cholinergic and glutamatergic mechanisms.

The effects of 5-hydroxytryptamine 1A (5-HT(1A)) receptor ligands on aversive learning were examined in the passive avoidance (PA) task in mice. Anxiety and autonomic functions were investigated using the elevated plus-maze and heart rate measurements. The main findings from this study are as follows. 1) Pretraining administration of the 5-HT(1A) receptor agonist 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide] facilitated PA retention at low doses (0.01 and 0.03 mg/kg) but impaired PA retention at higher doses (0.1-1.0 mg/kg), consistent with previous findings in the rat. 2) Similar to the acetylcholinesterase inhibitor physostigmine, pretraining administration of the 5-HT(1A) receptor antagonists [(R)-3-N,N-dicyclobutylamino-8 fluoro-3,4-dihydro-3H-1-benzopyran-5-carboxamide hydrogen(2R,3R)-tartrate monohydrate] NAD-299 (0.1-2 mg/kg) and [N-2-4-(2-methoxyphenyl)-1-piperazinylethyl-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride] WAY-100635 (0.3-3 mg/kg) enhanced PA retention. 3) The impairment (1 mg/kg) but not the facilitation (0.03 mg/kg) induced by 8-OH-DPAT was fully blocked by NAD-299 (0.3 mg/kg). 4) 5-HT(1A) receptor ligands given immediate post-training failed to alter PA retention. 5) NAD-299 (0.3-1 mg/kg) blocked the impairment of PA retention caused by a) the nonselective muscarinic receptor antagonist scopolamine and b) the non-competitive N-methyl-D-aspartate receptor antagonist MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate]. 6) A subthreshold dose of scopolamine completely blocked the facilitatory effect of NAD-299 on PA retention. 7) Anxiety-related behaviors and autonomic function were unchanged by NAD-299. 8) In situ hybridization showed that septal neurons expressing 5-HT(1A) receptor mRNA were codistributed with markers for cholinergic, GABAergic, and glutamatergic neurons. These results indicate that systemic administration of 5-HT(1A) receptor antagonists can facilitate cognitive performance, most likely by enhancing hippocampal/cortical cholinergic and glutamatergic neurotransmissions. Selective 5-HT(1A) receptor antagonists may be useful in the treatment of cognitive deficits such as Alzheimer's disease.

Neuronal calcium sensor-1 potentiates glucose-dependent exocytosis in pancreatic beta cells through activation of phosphatidylinositol 4-kinase beta.

Cytosolic free Ca2+ plays an important role in the molecular mechanisms leading to regulated insulin secretion by the pancreatic beta cell. A number of Ca2+-binding proteins have been implicated in this process. Here, we define the role of the Ca2+-binding protein neuronal Ca2+ sensor-1 (NCS-1) in insulin secretion. In pancreatic beta cells, NCS-1 increases exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in the readily releasable pool. The effect of NCS-1 on exocytosis is mediated through an increase in phosphatidylinositol (PI) 4-kinase beta activity and the generation of phosphoinositides, specifically PI 4-phosphate and PI 4,5-bisphosphate. In turn, PI 4,5-bisphosphate controls exocytosis through the Ca2+-dependent activator protein for secretion present in beta cells. Our results provide evidence for an essential role of phosphoinositide synthesis in the regulation of glucose-induced insulin secretion by the pancreatic beta cell. We also demonstrate that NCS-1 and its downstream target, PI 4-kinase beta, are critical players in this process by virtue of their capacity to regulate the release competence of the secretory granules.

Cyclin-dependent kinase 5 activators p35 and p39 facilitate formation of functional synapses.

Cyclin-dependent kinase 5 (Cdk5) has emerged as a key coordinator of cell signaling in neurite outgrowth. Cdk5 needs to associate with one of the regulatory proteins p35 or p39 to be an active enzyme. To investigate if Cdk5 plays a role in the establishment of functional synapses, we have characterized the expression of Cdk5, p35, and p39 in the neuroblastoma-glioma cell line NG108-15, and recorded postsynaptic activity in myotubes in response to presynaptic overexpression of Cdk5, p35, and p39. Endogenous Cdk5 and p35 protein levels increased with cellular differentiation and preferentially distributed to soluble pools, whereas the level of p39 protein remained low and primarily was present in membrane and cytoskeletal fractions. Transient transfection of a dominant-negative mutant of Cdk5 in NG108-15 cells and subsequent culturing on differentiating muscle cells resulted in a significant reduction in synaptic activity, as measured by postsynaptic miniature endplate potentials (mEPPs). Overexpression of either Cdk5/p35 or Cdk5/p39 resulted in a substantial increase in synaptic structures that displayed postsynaptic activities, as well as mEPP frequency. These findings demonstrate that Cdk5, p35, and p39 are endogenously expressed in NG108-15 cells, exhibit distinct subcellular localizations, and that both Cdk5/p35 and Cdk5/p39 are central in formation of functional synapses.

DARPP-32 and inhibitor-1 are expressed in pancreatic beta-cells.

Insulin secretion from pancreatic beta-cells has to be tightly regulated to ensure accurate glucose homeostasis. The capacity of beta-cells to respond to extracellular stimulation is determined by several signaling pathways. One important feature of these pathways is phosphorylation and subsequent dephosphorylation of a wide range of cellular substrates. Protein phosphatase 1 (PP1) is a major eukaryotic serine/threonine protein phosphatase that controls a multitude of physiological processes. We have investigated the expression and cellular distribution of two endogenous inhibitors of PP1 activity in beta-cells. RT-PCR, Western blotting, and immunohistochemistry showed that DARPP-32 and inhibitor-1 are present in insulin-secreting endocrine beta-cells. Subcellular fractionation of mouse islets revealed that both PP1 inhibitors predominantly localized to cytosol-enriched fractions. Inhibitor-1 was also present in fractions containing plasma membrane-associated proteins. These data indicate a potential role for DARPP-32 and inhibitor-1 in the regulation of PP1 activity in pancreatic beta-cell stimulus-secretion coupling.