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Background Increased catabolism has recently been recognized as a clinical manifestation of amyotrophic lateral sclerosis (ALS). The hypothalamic systems have been shown to be involved in the metabolic dysfunction in ALS, but the exact extent of hypothalamic circuit alterations in ALS is yet to be determined. Here we explored the integrity of large-scale cortico-hypothalamic circuits involved in energy homeostasis in murine models and in ALS patients. Methods The rAAV2-based large-scale projection mapping and image analysis pipeline based on Wholebrain and Ilastik software suites were used to identify and quantify projections from the forebrain to the lateral hypothalamus in the SOD1(G93A) ALS mouse model (hypermetabolic) and the FusΔNLS ALS mouse model (normo-metabolic). 3 T diffusion tensor imaging (DTI)-magnetic resonance imaging (MRI) was performed on 83 ALS and 65 control cases to investigate cortical projections to the lateral hypothalamus (LHA) in ALS. Results Symptomatic SOD1(G93A) mice displayed an expansion of projections from agranular insula, ventrolateral orbitofrontal and secondary motor cortex to the LHA. These findings were reproduced in an independent cohort by using a different analytic approach. In contrast, in the FusΔNLS ALS mouse model hypothalamic inputs from insula and orbitofrontal cortex were maintained while the projections from motor cortex were lost. The DTI-MRI data confirmed the disruption of the orbitofrontal-hypothalamic tract in ALS patients. Conclusion This study provides converging murine and human data demonstrating the selective structural disruption of hypothalamic inputs in ALS as a promising factor contributing to the origin of the hypermetabolic phenotype.

Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS., Freischmidt et al. show direct interaction of fragile X proteins with a subset of microRNAs that share a common sequence motif and that are downregulated in the majority of patients with ALS. Neuropathology suggests contribution of the fragile X proteins to a convergent disease mechanism implicated in many cases of ALS.

Scientific competences as defined in the German competency framework describes the ability to think independently and act scientifically, and forms a central component of medical education. This report describes its integration into anatomical teaching. On the basis of the findings in dissection courses from two consecutive years, students worked on either a case report (n = 70) or an original work (n=6) in the format of a scientific poster while learning to use primary literature. Posters were evaluated by juror teams using standardized evaluation criteria. Student perception of the project was estimated by quantitative and qualitative data obtained from the faculty´s course evaluation and an online-survey. Overall, students worked collaboratively and invested extra-time (median [MD] 3.0 hours) in poster creation. Primary literature was integrated in 90.8% of the posters. Overall poster quality was satisfactory (46.3 ±8.5 [mean ±standard deviation] out of 72 points), but several insufficiencies were identified. Students integrated information gained from the donor´s death certificate, post-mortem full-body computer tomography (CT) scan (22.4%) and histopathological workup (31.6%) in their case reports. Students were positive about the experience of learning new scientific skills (MD 4 on a six-point Likert scale), but free text answers revealed that some students experienced the project as an extra burden in a demanding course. In summary, it was feasible to introduce students to science during the dissection course and to increase interest in science in approximately a third of the survey respondents. Further adjustments to ensure the posters´ scientific quality might be necessary in the future.

While most German anatomy institutes provide only limited information about body donors and their lives, students have expressed a desire to learn more about these individuals, especially about their motivations to donate their bodies for the sake of medical education. In order to gratify this wish, as well as to further humanize body donors, an educational film was compiled, and a study designed to capture the film's effects on medical students. This is the first study using standardized, validated psychological tools to evaluate the impact of an educational film about body donors on students' empathy and psychological stress levels. The study followed a longitudinal, controlled, and cluster randomized design, including 77 (48 females/29 males) participants who watched the video either before, midway, or after the dissection course. Questionnaires were completed at four points in time applying the Jefferson Scale for Empathy (JSPE-S) and the Interpersonal Reactivity Index (IRI) to measure empathy. Psychological stress levels were recorded by the Brief Symptom Inventory (BSI). Overall, students recommended the film to be shown to all students (median 6.0; maximum on the six-point Likert scale). Viewing the film revealed no significant changes between study groups or over time in JSPE-S sum scores. All groups demonstrated a significant reduction of BSI values before the dissection course actually started and increased values during the course, but both developments appeared not to be associated with the intervention. Overall, the educational film did not correlate with any negative effects on students' empathy and psychological stress levels, and it was strongly approved of by students, as it provided more humanizing personal information about body donors without violating their anonymity.

Hand-held devices have revolutionized communication and education in the last decade. Consequently, mobile learning (m-learning) has become popular among medical students. Nevertheless, there are relatively few studies assessing students' learning outcomes using m-learning devices. This observational study presents an anatomy m-learning tool (eMed-App), an application developed to accompany an anatomy seminar and support medical students' self-directed learning of the skeletal system. Questionnaire data describe where, how frequently, and why students used the app. Multiple choice examination results were analyzed to evaluate whether usage of the app had an effect on test scores. The eMed-App application was used by 77.5% of the students, mainly accessed by Android smartphones, and at students' homes (62.2%) in order to prepare themselves for seminar sessions (60.8%), or to review learning content (67%). Most commonly, students logged on for less than 15 minutes each time (67.8%). Frequent app users showed better test results on items covering eMed-App learning content. In addition, users also achieved better results on items that were not related to the content of the app and, thus, gained better overall test results and lower failure rates. The top quartile of test performers used the eMed-App more frequently compared to students in lower quartiles. This study demonstrated that many students, especially the high-performing ones, made use of the eMed-App. However, the app itself did not result in better outcomes, suggesting that top students might have been more motivated to use the app than students who were generally weak in anatomy.

Synopsis image The lack of an effective treatment for ALS is calling for the development of novel therapeutic strategies. By using hiPSC���derived motoneurons and focusing on synapse���related processes, we provide new molecular targets rescuing the degenerative processes and neuronal loss in ALS. Human C9orf72���mutant motoneurons are characterized by reduced expression of synaptic gene, progressive loss of CREB activity and synapse loss. Similar alterations are observed also in motoneurons harboring TBK1 pathogenic mutations, and in primary neurons upon overexpression of poly(GA) aggregates. The K+ channel blockers Apamin and XE991 revert the CREB���dependent loss of synaptic contacts and rescue the degenerative phenotypes of ALS motoneurons., publishedVersion

Peer-teaching is widely established in anatomy teaching and offers well-described advantages. Nevertheless, at Ulm University, Germany, a reduction in the number of peer teacher applicants for the dissection course was observed. This study examined factors related to the attractiveness of a position as a peer teacher for Generation Z students. Participants of the gross anatomy course were asked to evaluate factors influencing the attractiveness of a peer teacher position using a six-point Likert scale. Additionally, open-ended questions were analyzed qualitatively. Sex-specific subgroup analysis was performed comparing students of low and high motivation to apply for a tutorship. Of the 374 students who participated in this study (response rate 53%), 38% stated that they were intending to apply as peer teachers. Data indicated that students displayed intrinsic motivation to apply for a tutorship because of the opportunity to improve their anatomy knowledge and/or their pleasure in teaching. In contrast, extrinsic factors like remuneration of the tutorship and its relevance for their curriculum vitae were least important. Anatomy educators underestimated the demotivating factor of the workload associated with the tutorship and encouraged students less frequently to apply than peer teachers. Only minor sex-specific differences could be identified. Nevertheless, female students were encouraged less frequently to apply than their male peers. In summary, Generation Z students apply as peer teachers because they are enthusiastic about the task. To motivate students to commit to extracurricular activities like a tutorship, anatomy educators should actively encourage students-particularly females-more frequently to apply.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is still missing effective therapeutic strategies. Although manipulation of neuronal excitability has been tested in murine and human ALS models, it is still under debate whether neuronal activity might represent a valid target for efficient therapies. In this study, we exploited a combination of transcriptomics, proteomics, optogenetics and pharmacological approaches to investigate the activity‐related pathological features of iPSC‐derived C9orf72‐mutant motoneurons (MN). We found that human ALSC9orf72 MN are characterized by accumulation of aberrant aggresomes, reduced expression of synaptic genes, loss of synaptic contacts and a dynamic “malactivation” of the transcription factor CREB. A similar phenotype was also found in TBK1‐mutant MN and upon overexpression of poly(GA) aggregates in primary neurons, indicating a strong convergence of pathological phenotypes on synaptic dysregulation. Notably, these alterations, along with neuronal survival, could be rescued by treating ALS‐related neurons with the K+ channel blockers Apamin and XE991, which, respectively, target the SK and the Kv7 channels. Thus, our study shows that restoring the activity‐dependent transcriptional programme and synaptic composition exerts a neuroprotective effect on ALS disease progression., The lack of an effective treatment for ALS is calling for the development of novel therapeutic strategies. By using hiPSC‐derived motoneurons and focusing on synapse‐related processes, we provide new molecular targets rescuing the degenerative processes and neuronal loss in ALS.

Axonal trafficking deficits and neurodegeneration in C9ORF72 motoneurons are mediated by GOF and LOF mechanisms with RNA foci and DPRs as upstream events, whereas DNA damage appears downstream., Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis, a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOFs) through DPRs versus loss of C9ORF72 functions (LOFs). Through multiparametric high-content (HC) live profiling in spinal MNs from induced pluripotent stem cells and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA double-strand breaks (DSBs), RNA foci, DPRs, and apoptosis. We show that both GOFs and LOFs were necessary to yield the overall C9ORF72 pathology. Increased RNA foci and DPRs concurred with onset of axon trafficking defects, DSBs, and cell death, although DSB induction itself did not phenocopy C9ORF72 mutants. Interestingly, the majority of LOF-specific DEGs were shared with HRE-mediated GOF DEGs. Finally, C9ORF72 LOF was sufficient—albeit to a smaller extent—to induce premature distal axonal trafficking deficits and increased DSBs.

Ethanol intoxication is a risk factor for traumatic brain injury (TBI) but clinical evidence suggests that it may actually improve the prognosis of intoxicated TBI patients. We have employed a closed, weight-drop TBI model of different severity (2cm or 3cm falling height), preceded (-30min) or followed (+20min) by ethanol administration (5g/Kg). This protocol allows us to study the interaction of binge ethanol intoxication in TBI, monitoring behavioral changes, histological responses and the transcriptional regulation of a series of activity-regulated genes (immediate early genes, IEGs). We demonstrate that ethanol pretreatment before moderate TBI (2cm) significantly reduces neurological impairment and accelerates recovery. In addition, better preservation of neuronal numbers and cFos+cells was observed 7days after TBI. At transcriptional level, ethanol reduced the upregulation of a subset of IEGs encoding for transcription factors such as Atf3, c-Fos, FosB, Egr1, Egr3 and Npas4 but did not affect the upregulation of others (e.g. Gadd45b and Gadd45c). While a subset of IEGs encoding for effector proteins (such as Bdnf, InhbA and Dusp5) were downregulated by ethanol, others (such as Il-6) were unaffected. Notably, the majority of genes were sensitive to ethanol only when administered before TBI and not afterwards (the exceptions being c-Fos, Egr1 and Dusp5). Furthermore, while severe TBI (3cm) induced a qualitatively similar (but quantitatively larger) transcriptional response to moderate TBI, it was no longer sensitive to ethanol pretreatment. Thus, we have shown that a subset of the TBI-induced transcriptional responses were sensitive to ethanol intoxication at the instance of trauma (ultimately resulting in beneficial outcomes) and that the effect of ethanol was restricted to a certain time window (pre TBI treatment) and to TBI severity (moderate). This information could be critical for the translational value of ethanol in TBI and for the design of clinical studies aimed at disentangling the role of ethanol intoxication in TBI.

Many Anglo-American universities have undertaken a paradigm shift in how the dissection of human material is approached, such that students are encouraged to learn about the lives of body donors, and to respectfully "personalize" them as human beings, rather than treating the specimens as anonymous cadavers. For the purposes of this study, this provision of limited personal information regarding the life of a body donor will be referred to as "personalization" of body donors. At this time, it is unknown whether this paradigm shift in the personalization of body donors can be translated into the German-speaking world. A shift from donor anonymity to donor personalization could strengthen students' perception of the donor as a "first patient," and thereby reinforce their ability to empathize with their future patients. Therefore, this study aimed to collect data about the current status of donation practices at German-speaking anatomy departments (n = 44) and to describe the opinions of anatomy departments, students (n = 366), and donors (n = 227) about possible donor personalization in medical education. Anatomy departments in Germany, Austria, and Switzerland were invited to participate in an online questionnaire. One-tenth of registered donors at Ulm University were randomly selected and received a questionnaire (20 items, yes-no questions) by mail. Students at the University of Ulm were also surveyed at the end of the dissection course (31 items, six-point Likert-scale). The majority of students were interested in receiving additional information about their donors (78.1%). A majority of donors also supported the anonymous disclosure of information about their medical history (92.5%). However, this information is only available in about 28% of the departments surveyed and is communicated to the students only irregularly. Overall, 78% of anatomy departments were not in favor of undertaking donor personalization. The results appear to reflect traditional attitudes among anatomy departments. However, since students clearly preferred receiving additional donor information, and most donors expressed a willingness to provide this information, one could argue that a change in attitudes is necessary. To do so, official recommendations for a limited, anonymous personalization of donated cadaveric specimens might be necessary. Anat Sci Educ 11: 282-293. © 2017 American Association of Anatomists.

The preclinical compulsory elective course "Ready for the Operating Room (OR)!?" [in German]: "Fit fur den OP (FOP)"] was implemented for students in their second year, who were simultaneously enrolled in the gross anatomy course. The objective of the study was to determine whether the direct practical application of anatomical knowledge within the surgical context of the course led to any improvement in learning motivation, learning orientation, and ultimately examination results in the gross anatomy course, as compared with a control group. Within the scope of five teaching sessions, the students learned surgical hand disinfection, suturing techniques, and the identification of commonly used surgical instruments. In addition, the students attended five surgical demonstrations performed by surgical colleagues on cadavers. Successful learning of these basic skills was then assessed based on an Objectively Structured Practical Examination. Learning motivation and learning orientation in both subgroups was determined using the SELLMO-ST motivation test and the Approaches and Study Skills Inventory test. While a significant increase in work avoidance was identified in the control group, this was not the case for FOP participants. Similarly, an increase in the "deep approach" to learning, as well as a decrease in the "surface approach," was able to be documented among the FOP participants following completion of the course. The results suggest that students enrolled in the gross anatomy course, who were simultaneously provided with the opportunity to learn in clinical context, were more likely to be successful at maintaining learning motivation and learning orientation required for the learning process, than students who attended the gross anatomy course alone.

The dissection course (here abbreviated: PK) is still an obligatory part of medical schools in Germany. In this study we investigated the experiences and burdens of medical students in gross anatomy, especially "distancing from the human body".This study was carried out three times with the self-composed questionnaire BF-PK: before, while and after PK. In total 371 students participated in the PK. 297 students participated at measurement 1. In advance 25-30% of the medical students reported anxiety and emotional inhibition, during the course only 7-10%. The coping strategy "distancing from the human body" was prominent. Anxiety, emotional inhibition and disgust remained for 5-10% of the participants.The gross anatomy course causes emotional stress for a considerable amount of medical students. For those students that were not able to overcome the mental stress themselves service offers should be implemented.

Background To improve the ease and safety of cricothyroidotomy especially in the hand of the inexperienced, new instruments have been developed. In this study, we compared a new indicator-guided puncture technique (PCK) with standard surgical technique (ST) regarding success rate, performance time and complications. Methods Cricothyroidotomy in 30 human cadavers performed by 30 first year anaesthesia residents. The set chosen for use was randomised: PCK-technique (n=15) and ST (n=15). Success rates, insertion times and complications were compared. Traumatic lesions were anatomically confirmed after dissection. Results The ST-group had a higher success rate (100% vs 67%; p=0.04). There was no difference in time taken to complete the procedure (PCK 82 s. vs ST 95 s.; p=0.89). There was a higher complication rate in the PCK-group (67% vs 13%; p=0.04). Most frequent complication in the PCK-group was injury to the posterior tracheal wall (n=8), penetration to the oesophageal lumen (n=4) and injury to the thyroid and/or cricoid cartilage (n=5). In the ST-group in only 2 cases minor complications were observed (small vessel injury). Conclusions In this human cadaver study the PCK technique produced more major complications and more failures than the ST. In the hand of the inexperienced operator the standard surgical approach seems to be a safe procedure, which can successfully be performed within an adequate time. The PCK technique cannot be recommended for inexperienced operators.

Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.

The main aim of this study was to provide anatomical data on the heights of the human intervertebral discs for all levels of the thoracic spine by direct and radiographic measurements. Additionally, the heights of the neighboring vertebral bodies were measured, and the prediction of the disc heights based only on the size of the vertebral bodies was investigated. The anterior (ADH), middle (MDH) and posterior heights (PDH) of the discs were measured directly and on radiographs of 72 spine segments from 30 donors (age 57.43 ± 11.27 years). The radiographic measurement error and the reliability of the measurements were calculated. Linear and non-linear regression analyses were employed for investigation of statistical correlations between the heights of the thoracic disc and vertebrae. Radiographic measurements displayed lower repeatability and were shorter than the anatomical ones (approximately 9% for ADH and 37% for PDH). The thickness of the discs varied from 4.5 to 7.2 mm, with the MDH approximately 22.7% greater. The disc heights showed good correlations with the vertebral body heights (R(2), 0.659-0.835, P-values < 0.005; anova), allowing the generation of 10 prediction equations. New data on thoracic disc morphometry were provided in this study. The generated set of regression equations could be used to predict thoracic disc heights from radiographic measurement of the vertebral body height posterior. For the creation of parameterized models of the human thoracic discs, the use of the prediction equations could eliminate the need for direct measurement on intervertebral discs. Moreover, the error produced by radiographic measurements could be reduced at least for the PDH.

Transgenic mice expressing human mutated superoxide dismutase 1 (SOD1) linked to familial forms of amyotrophic lateral sclerosis are frequently used as a disease model. We used the SOD1G93A mouse in a cross-breeding strategy to study the function of physiological prion protein (Prp). SOD1G93APrp-/- mice exhibited a significantly reduced life span, and an earlier onset and accelerated progression of disease, as compared with SOD1G93APrp+/+ mice. Additionally, during disease progression, SOD1G93APrp-/- mice showed impaired rotarod performance, lower body weight, and reduced muscle strength. Histologically, SOD1G93APrp-/- mice showed reduced numbers of spinal cord motor neurons and extended areas occupied by large vacuoles early in the course of the disease. Analysis of spinal cord homogenates revealed no differences in SOD1 activity. Using an unbiased proteomic approach, a marked reduction of glial fibrillary acidic protein and enhanced levels of collapsing response mediator protein 2 and creatine kinase were detected in SOD1G93APrp-/- versus SOD1G93A mice. In the course of disease, Bcl-2 decreases, nuclear factor-kappaB increases, and Akt is activated, but these changes were largely unaffected by Prp expression. Exclusively in double-transgenic mice, we detected a significant increase in extracellular signal-regulated kinase 2 activation at clinical onset. We propose that Prp has a beneficial role in the SOD1G93A amyotrophic lateral sclerosis mouse model by influencing neuronal and/or glial factors involved in antioxidative defense, rather than anti-apoptotic signaling.

The modulation of cell proliferation in neural progenitor cells (NPCs) is believed to play a role in neuronal regeneration. Recent studies showed that K(+) channel activity influenced cell proliferation. Therefore, we examined NPCs for K(+) channels and tested whether NPC self renewing can be modulated by synthetic K(+) channel modulators. The whole-cell K(+) current was partly K(+) dependent and showed a cumulative inactivating component. Two tetra-ethyl-ammonium ion (TEA)-sensitive K(+) currents with different voltage dependencies ( = 65 microm, E(50) = -0.3 +/- 1.3 mV and = 8 mm, E(50) = -15.2 +/- 2.8 mV) and an almost TEA-insensitive current were identified. Kaliotoxin blocked approximately 50% of the entire K(+) currents (IC(50) = 0.25 nm). These properties resembled functional characteristics of K(v)1.4, K(v)1.3 and K(v)3.1 channels. Transcripts for these channels, as well as proteins for K(v)1.3 and K(v)3.1, were identified. Immunocytochemical staining revealed K(v)1.3 and K(v)3.1 K(+) channel expression in almost all NPCs. The blockage of K(v)3.1 by low concentrations of TEA, as well as the blockage of K(v)1.3 by Psora-4, increased NPC proliferation. These findings underline the regulatory role of K(+) channels on the cell cycle and imply that K(+) channel modulators might be used therapeutically to activate endogenous NPCs.

PSD95-DLG-ZO1 domain-containing proteins of the ProSAP/Shank family are major scaffolding proteins of the neuronal postsynaptic density which play a pivotal role in the linkage of membrane receptors to downstream signal effectors and the actin-based cytoskeleton. Recently, ProSAP1/Shank2 has also been localized in various non-neuronal cells where it may fulfill similar functions as in neurons. We now complement these data by the study of ProSAP/Shank expression at the mRNA and protein level in a primary lymphoid organ, i.e., the thymus. Transcripts for ProSAP1/Shank2, the spliceoform Shank2E, and ProSAP2/Shank3 could be clearly detected in the thymus. Western blot and immunocytochemical analyses verified the presence of ProSAP1/Shank2 and ProSAP2/Shank3 proteins in thymic tissue. Immunoreactivity was concentrated in the whole peripheral cytoplasm of thymocytes underneath the plasma membrane. Discrete subplasmalemmal areas of pronounced ProSAP/Shank immunoreactivity could be demonstrated inside several thymocytes by confocal laser scanning microscopy. Our results establish ProSAP/Shank as a constituent of the cell cortex of thymocytes and thus lead to the hypothesis that ProSAP/Shank proteins serve as a platform for the coordination of membrane receptor-dependent signal transduction in immune cells.

The four members of the family of synapse-associated protein 90/postsynaptic density-95-associated proteins (SAPAP1–4) are adapter proteins of postsynaptic density (PSD). They interact with different synaptic scaffolding proteins, cytoskeletal components, and signalling components, and are therefore considered to assemble functional multiprotein units at synapses. Here, we analyzed the spatiotemporal expression of SAPAP1–SAPAP4 genes in postnatal rat brain by in situ hybridization. All four genes are expressed in many brain areas, leading to overlapping yet distinct mRNA distribution patterns. Moreover, two mRNAs encoding distinct SAPAP3 isoforms exhibit basically identical postnatal expression patterns. In the hippocampus, SAPAP1, SAPAP2, and SAPAP4 transcripts are restricted to cell body zones, whereas SAPAP3 mRNAs are also detected in molecular layers. Thus, SAPAP3 is one of the few PSD components whose local synthesis in dendrites may contribute to an input-specific adaptation of dendritic spine function.

Shank proteins are scaffolding proteins in the postsynaptic density of excitatory synapses in the mammalian brain. In situ hybridization revealed that Shank1/SSTRIP and Shank2/ProSAP1 mRNAs are widely expressed early in postnatal brain development whereas Shank3/ProSAP2 expression increases during postnatal development especially in the cerebellum and thalamus. Shank1 and Shank3 (but not Shank2) mRNAs are present in the molecular layers of the hippocampus, consistent with a dendritic transcript localization. Shank1 and Shank2 transcripts are detectable in the dendritic fields of Purkinje cells, whereas Shank3 mRNA is restricted to cerebellar granule cells. The appearance of dendritic Shank mRNAs in cerebellar Purkinje cells coincides with the onset of dendrite formation. Expression of reporter transcripts in hippocampal neurons identifies a 200-nucleotide dendritic targeting element (DTE) in the Shank1 mRNA. The widespread presence of Shank mRNAs in dendrites suggests a role for local synthesis of Shanks in response to stimuli that induce alterations in synaptic morphology.

The establishment of retino-collicular topography is a well-investigated model of axon pathfinding and it was believed that this topography is irreversibly fixed throughout life. We now report that, after partial crush of the adult rat optic nerve, the anterograde transport of intravitreally-injected tracers via axons of surviving retinal ganglion cells (RGC) in all retinal quadrants is confined to the rostro-medial part of the superior colliculus (SC). This indicates that the retino-collicular topography is rearranged after partial crush of the adult rat optic nerve. The reorganization starts in the injured optic nerve where surviving axonal fibres are demyelinized and bundled in the periphery of the optic nerve distal to the crush site. This is followed by a displacement of surviving axons to the medial part of the optic tract (OT) within 2 weeks. The infiltration of macrophages with the subsequent production of tumour necrosis factor-alpha at the lesion site is a prerequisite for the altered retino-collicular projection as blockade of tumour necrosis factor-alpha signalling with the neutralizing antibody Infliximab abolishes reorganization in the SC and lateralization of RGC axons in the optic nerve and OT. This suggests that optic nerve inflammation is necessary for a progressive bundling of surviving RGC axons, probably via clearance of cellular debris which, in turn, may lead to a redistribution of RGC axons to the medial OT and rostro-medial SC.

The axons of neurons in the CNS with their delicate ramification patterns and terminal boutons can be visualized with conventional neuroanatomical techniques with a high degree of accuracy. Whether identified terminal boutons form synaptic contacts with target neurons identified by a second and different marker needs resolution beyond that offered by conventional light microscopy. The morphological elements associated with synaptic connectivity consist of specialized pre- and post-synaptic junctional complexes known as the pre- and post-synaptic densities. Electron microscopy of these junctional complexes consumes much time and resources. In an attempt to increase the speed with which we can analyze networks of neurons we developed a high-resolution triple-fluorescence approach including neuroanatomical tracing, immunofluorescence, confocal laserscanning and 3D-computer reconstruction to pinpoint at the light microscopic level the three elements involved in synaptic connectivity: afferent fibers and their terminal boutons, close apposition with neurons identified by the presence of a fluorescent marker, and sandwiched in between a post-synaptic density marker. We used morphological criteria for the detection of axon terminals (swellings on fibers). Antibodies against ProSAP2/Shank3, a post-synaptic density-associated scaffolding protein, were used to pinpoint the location of the synaptic junctions. The results show the existence of sandwich-like configurations: pre-synaptic fiber, ProSAP2/Shank3, post-synaptic neuron. Thus we feel that we can minimize (and perhaps completely eliminate) the need for electron microscopy and hence dramatically increase the overall efficiency of neuroanatomical tracing and network analysis.

Caldendrin defines a novel family of neuronal calcium-sensor proteins, the C-terminal moiety of which displays high similarity to calmodulin. We now report that the protein is recruited to the postsynaptic density (PSD) of cortical and hippocampal neurons in response to kainate-induced epileptic seizures, an animal model of human temporal lobe epilepsy. The translocation of caldendrin to the PSD did not occur in kainate-treated rats that did not develop seizures. The enhanced PSD levels of caldendrin are not due to increased protein synthesis and most likely reflect a recruitment from the soluble caldendrin protein pool. These findings suggest that the transduction of dendritic Ca2+-signals via caldendrin is altered by epileptic seizures and that caldendrin might be involved in the pathophysiology of temporal lobe epilepsy.

Caldendrin is a neuronal calcium-binding protein, which is highly enriched in the postsynaptic density fraction and exhibits a prominent somato-dendritic distribution in brain. Two additional splice variants derive from the caldendrin gene, which have unrelated N-termini and were previously only de- tected in the retina. We now show that these isoforms are present in neurohypophyseal axons and on secretory granules of endocrine cells. In light of the described interaction of the Caldendrin C-terminus with Q-type Cav2.1 calcium channels these data suggest that this interaction takes place in neuro- hypophyseal axons and pituitary cells indicating functions of the short splice variants in triggering Ca 2+ transients to a vesic- ular target interaction. ! 2003 Federation of European Biochemical Societies. Pub- lished by Elsevier Science B.V. All rights reserved.

Neuroplastin (np) 55 and 65 are immunoglobulin superfamily members that arise by alternative splicing of the same gene and have been implicated in long-term activity-dependent synaptic plasticity. Both biochemical and immunocytochemical data suggest that np55 is the predominant isoform (>95% of total neuroplastin) in cerebellum. Neuroplastin immunoreactivity is concentrated in the molecular layer and synaptic glomeruli in the granule cell layer. Expression in the molecular layer appears to be postsynaptic. First, neuroplastin is associated with Purkinje cell dendrites in two mouse granuloprival cerebellar mutants, disabled and cerebellar deficient folia. Second, in an acid sphingomyelinase knockout mouse with widespread protein trafficking defects, neuroplastin accumulates in the Purkinje cell somata. Finally, primary cerebellar cultures show neuroplastin expression in Purkinje cell dendrites and somata lacking normal histotypic organization and synaptic connections, and high-magnification views indicate a preferential association with dendritic spines. In the molecular layer, differences in neuroplastin expression levels present as a parasagittal array of stripes that alternates with that revealed by the expression of another compartmentation antigen, zebrin II/aldolase c. Neuroplastin immunoreactivity is first detected weakly at postnatal day 3 (P3) in the anterior lobe vermis. By P5, parasagittal stripes are already apparent in the immature molecular layer. At this stage, punctate deposits are also localised at the perimeter of the Purkinje cell perikarya; these are no longer detected by P15. The data suggest a role for neuroplastins in the development and maintenance of normal synaptic connections in the cerebellum.

The ProSAP/Shank family of multidomain proteins of the postsynaptic density (PSD) can either directly or indirectly interact with NMDA-type and metabotropic glutamate receptors and the actin-based cytoskeleton. In a yeast two hybrid screen utilizing a proline-rich domain that is highly conserved among the ProSAP/Shank family members, we isolated several cDNA clones coding for the insulin receptor substrate IRSp53. The specificity of this interaction was confirmed in transfected COS cells. Co-immunoprecipitation of IRSp53 and ProSAP2 solubilized from rat brain membranes indicates that the interaction occurs in vivo. The C-terminal SH3 domain of IRSp53 is responsible for the interaction with a novel proline-rich consensus sequence of ProSAP/Shank that was characterized by mutational analysis. IRSp53 is a substrate for the insulin receptor in the brain and acts downstream of small GTPases of the Rho family. Binding of Cdc42Hs to IRSp53 induces actin filament assembly, reorganization and filopodia outgrowth in neuronal cell lines. Our data suggest that IRSp53 can be recruited to the PSD via its ProSAP/Shank interaction and may contribute to the morphological reorganization of spines and synapses after insulin receptor and/or Cdc42Hs activation.

Limited optic nerve crush is a model of diffuse mechanical axon injury, the most prevalent cause of secondary neurodegeneration after closed head neurotrauma. In this report, a protocol is presented which allows for the rapid screening of differential gene expression in the inner retina, as well as the optic nerve, in response to partial nerve crush. To prove the reliability of the method, prototypically, the differential expression profiles of three candidate genes (kinesin light chain, ferritin, RYB-A) were verified. The method seems to be suitable to address the question of how differential gene expression contributes to degeneration, survival and axonal repair after partial nerve crush.

The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the N-terminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein α-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of α-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.

The pars tuberalis (Pt) of most mammalian species contains specific cells which are structurally and functionally different from the pars distalis (Pd) cells. Pt-specific cells possess melatonin receptors and reveal morphological changes dependent on the duration of the photoperiod. Furthermore, in hamsters the transmission of photoperiodic stimuli to the endocrine system is influenced by melatonin, an effect which is likely to be mediated by Pt-specific cells. In monkeys, however, only little is known about this cell type. Therefore, we studied the ultrastructural differentiation of Pt-specific cells and describe the expression of different hormones and their mRNA by immunohistochemistry and in situ hybridization. Apparently the Pt consists of (1) cells similar to gonadotropic cells of the Pd, (2) folliculostellate cells and (3) a cell population which is morphologically and functionally clearly distinct from all other cell types found in the Pd. Morphologically they resemble the Pt-specific cells found in other species. Regarding the expression of secretory products there is evidence that they transcribe and translate the β-TSH subunit. Although there is a strong signal for the mRNA of the common α-chain, protein staining is much weaker. POMC mRNA is expressed in the Pt while there is no evidence for PRL mRNA. The present results lead to the conclusion that the Pt of the monkey contains Pt-specific cells which express different hormonal subunits as was already shown for other species. In context with previous findings of melatonin receptors in the monkey Pt further investigations are necessary to establish the possible role of Pt-specific cells in the photoperiod-dependent generation of endocrine rhythms.

Neuroplastin-65 and -55 (previously known as gp65 and gp55) are glycoproteins of the Ig superfamily that are enriched in rat forebrain synaptic membrane preparations. Whereas the two-Ig domain isoform neuroplastin-55 is expressed in many tissues, the three-Ig domain isoform neuroplastin-65 is brain-specific and enriched in postsynaptic density (PSD) protein preparations. Here, we have assessed the function of neuroplastin in long-term synaptic plasticity. Immunocytochemical studies with neuroplastin-65-specific antibodies differentially stain distinct synaptic neuropil regions of the rat hippocampus with most prominent immunoreactivity in the CA1 region and the proximal molecular layer of the dentate gyrus. Kainate-induced seizures cause a significant enhancement of neuroplastin-65 association with PSDs. Similarly, long-term potentiation (LTP) of CA1 synapses in hippocampal slices enhanced the association of neuroplastin-65 with a detergent-insoluble PSD-enriched protein fraction. Several antibodies against the neuroplastins, including one specific for neuroplastin-65, inhibited the maintenance of LTP. A similar effect was observed when recombinant fusion protein containing the three extracellular Ig domains of neuroplastin-65 was applied to hippocampal slices before LTP induction. Microsphere binding experiments using neuroplastin-F c chimeric proteins show that constructs containing Ig1–3 or Ig1 domains, but not Ig2–3 domains mediate homophilic adhesion. These data suggest that neuroplastin plays an essential role in implementing long-term changes in synaptic activity, possibly by means of a homophilic adhesion mechanism.

The expression of c-fos, c-jun, jun-b, jun-d, srf and pc4 mRNA was examined after partial optic nerve crush in the adult rat retina by in situ hybridization. Optic nerve injury led exclusively to the upregulation of c-jun, with cellular label indicative for c-jun mRNA in the retinal ganglion cell layer after two days, three days and one week post-injury. This expression pattern was in accordance with the appearance of c-Jun immunoreactivity in retinal flat mounts. Injection of an antisense but not a missense oligonucleotide against c-jun after partial crush resulted in a reduced number of connected retinal ganglion cells (RGCs) as shown by retrograde labeling. Prelabeling of RGCs with fluorogold before optic nerve section and subsequent antisense targeting against c-jun, however, led to a slightly higher number of surviving but axotomized RGCs. C-Jun antibody staining of retinal whole mounts pre- or postlabeled after crush by intracollicular administration of fluorogold showed strong c-Jun immunoreactivity in connected RGCs and also in a population of disconnected RGCs. Double labeling with an antibody directed against the transcription factor ATF-2 revealed strong co-expression of c-Jun and ATF-2 in connected RGCs but not in axotomized cells. Taken together these data indicate that both RGCs in continuity and those in discontinuity with the superior colliculus respond both equally to the noxious stimulus with c-Jun expression. Moreover, the co-expression of c-Jun with high levels of ATF-2 appears to be essential for either the continuity or survival of RGCs which remain connected with their target. In disconnected RGCs, however, low levels of ATF-2 and the co-expression of c-Jun may be related to cell death.

We have investigated time course and characteristics of retinal ganglion cell (RGC) death after partial optic nerve injury. In situ end labeling of DNA fragments with the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine (dUTP)-biotin nick end labeling (TUNEL) method revealed the presence of apoptotic cells on as early as 5 days postcrush with a very high number of TUNEL-positive cells 1 week postinjury. At the ultrastructural level, features of apoptosis were clearly present in the ganglion cell layer at this time point. Moreover, TUNEL-positive cells could be identified as retinal ganglion cells by retrograde labeling with fluorogold. In addition, DNA laddering characteristic for apoptosis was found 1 week postinjury. A considerable number of TUNEL-labeled cells was still found after 2 weeks postinjury. Retinal whole mounts prepared at postlesion days 2-5, however, revealed that many cell bodies with ruptured membranes as evidenced by nucleosomal Sytox staining were present. These cells were also identified as retinal ganglion cells by retrograde labeling with fluorogold. Moreover, at this early stages of RGC degeneration necrotic cellular profiles could be detected by electron microscopic analysis. Thus, evidence is provided that necrosis and apoptosis follow a distinctly different time course after partial optic nerve injury.

Brevican is a member of the aggrecan/versican family of proteoglycans. In contrast to the other family members, brevican occurs both as soluble isoforms secreted into the extracellular space and membrane-bound isoforms which are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety. Expression of both variants, which are encoded by two differentially processed transcripts from the same gene, is confined to the nervous system. In the current study, we have used in situ hybridization to examine the cellular sites of synthesis for both mRNAs during postnatal development of the rat brain. Whereas the 3.6-kb transcript encoding secreted brevican displays a widespread distribution in grey matter structures, including cerebellar and cerebral cortex, hippocampus and thalamic nuclei with silver grains accumulating over neuronal cell bodies, the smaller transcript (3.3 kb) encoding GPI-anchored isoforms appears to be largely confined to white matter tracts and diffusely distributed glial cells. This expression pattern is further confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) experiments with RNA from different glial cell cultures, and by biochemical data demonstrating that the crude membrane fraction from isolated optic nerve contains high amounts of phosphatidylinositol-specific phospholipase C (PI-PLC)-sensitive brevican immunoreactivity. During ontogenetic development, both brevican transcripts are generally up-regulated. However, the expression of glypiated brevican is delayed by about 1 week, compared with the expression of the secreted isoform. This late appearance of GPI-linked brevican, its predominant expression in glial cells and its tight association with brain myelin fractions suggest a functional role in neuroglia.

Recently emerging evidence suggests important roles for inositol polyphosphates and inositol phospholipids in neuronal Ca2+ signalling, membrane vesicle trafficking and cytoskeletal rearrangement. A prerequisite for a detailed physiological characterization of the signalling of both potential second messengers inositol-(1,3,4,5)-tetrakisphosphate (InsP4) and phosphatidylinositol-3,4,5-trisphosphate (PtdInsP3) in the nervous system is the precise cellular localization of their receptors. Based on the cDNA sequence of a recently cloned brain-specific receptor with high affinity for both InsP4 and PtdInsP3 (InsP4-PtdInsP3R), p42IP4/centaurin-alpha, we localized the mRNA and the protein in rat brain. In situ hybridization revealed a widespread expression of the InsP4-PtdInsP3R with prominent labelling in cerebellum, hippocampus, cortex and thalamus, which moreover is developmentally regulated. Using peptide-specific antibodies, the immunoreactivity was localized in the adult brain in the vast majority of neuronal cell types and probably also in some glial cells. Prominent immunoreactivity was found in axonal processes and in cell types characterized by extensive neurites. In the hypothalamus a subpopulation of parvocellular neurons in the peri- and paraventricular nuclei was most heavily labelled. This was confined by strong immunoreactivity in the lamina externa of the median eminence in close proximity to portal plexus blood vessels. Electron microscopy revealed that the InsP4-PtdInsP3R was frequently associated with presynaptic vesicular structures. Further studies should identify the role of the InsP4-PtdInsP3R in cellular neural processes.

When exposed to short-day conditions, hamsters and other long-day breeders undergo gonadal regression. With chronic exposure to short days, however, the animals become photorefractory and gonadal recrudescence occurs. The underlying mechanism for this insensitivity is still unknown. There is growing evidence, however, that specific cells of the pituitary pars tuberalis (PT) mediate these photoperiod/nonphotoperiod-dependent changes as a direct or indirect "Zeitgeber" for the endocrine system. We investigated messenger RNA (mRNA)/protein formation for several hypophyseal hormones (beta-TSH, beta-LH, PRL, common alpha-chain) in the pars distalis (PD) and PT of female Djungarian hamsters in long photoperiod (LP) and after 18, 28, and 38 weeks of short photoperiod (SP). As indicated by gonadal and body weight, the hamsters displayed gonadal regression after 18 and 28 weeks of SP; after 38 weeks of SP, all animals showed recrudescence. At 18 and 28 weeks of SP, only PRL mRNA and protein levels were significantly reduced in the PD and returned to LP values after 38 weeks of SP. The expression of hypothalamic tyrosine hydroxylase in the arcuate nucleus that was determined by immunocytochemistry and by in situ hybridization was also down-regulated in SP18 and SP28 with increasing levels at SP38. Urinary 6-sulfatoxymelatonin levels were elevated in SP with highest levels in the SP18 group. In the PT, beta-TSH mRNA and protein were not detectable in all SP groups compared with the moderate signal intensity in LP. The common alpha-chain mRNA and protein, however, which were also reduced in the animals of the SP18 group, were already elevated after 28 weeks of SP and nearly reached LP-levels after 38 weeks of SP. These results show that, in contrast to LH and TSH, PRL expression in the PD is a sensitive indicator for photoperiod dependent changes of the endocrine system and seems to be tyrosine hydroxylase independent. The increase of common alpha-chain expression in PT-specific cells depending upon duration of SP that precedes the hormonal changes in the PD leads us to speculate that PT-specific cells initiate spontaneous recrudescence via a PT-PD pathway.

Several hormones not only regulate the activity of endocrine cells and non-endocrine tissues but also serve as neuronal transmitters or modulators of neuronal activity. Accordingly, the expression and physiological significance of hormonal receptors in the central nervous system (CNS) could be demonstrated for a whole set of hormones (e. g. hCG/LH, GH, T3, CRF, TRH). The G-protein coupled TSH receptor is densely expressed in the thyroid gland and mediates the production and secretion of thyroid hormones. Not all TSH effects, especially in neurological and psychiatric disease states, can readily be explained by the action of the hormone on the thyroid gland and/or TRH levels. Therefore, it has been suggested that TSH might exert its effects directly in the CNS, although no direct proof for a TSH receptor in the human brain has been provided yet. Here we describe the cloning of a TSH receptor from an ovine hypothalamic cDNA library that is similar to thyroid derived cDNA clones. The comparison of amino acid sequences indicates that several protein domains important for the function and activity of the receptor are highly conserved. RT-PCR and RNA protection assay demonstrated that the TSH receptor mRNA is widely expressed throughout the ovine brain. The expression of a TSH receptor in the CNS indicates that TSH is not only a hormonal messenger for the thyroid gland but can also act directly in the brain. Further studies should focus on the physiological role of TSH in the CNS and the regulation of TSH receptor expression in the mammalian brain.

Purpose This preliminary study was performed to evaluate a proposed maxillary expansion treatment method for adults with fused intermaxillary sutures. Materials and methods This study was performed in three Thiel-fixed skulls from older female cadavers with a microimplant-supported expansion screw. This modified expansion screw was mounted on the palatine process with microimplants and activated every 15 to 20 seconds with an activation key until the intermaxillary suture ruptured. A strain gauge was bonded to the expansion screw and calibrated so it could be used as a force sensor device. Rupture of the intermaxillary suture was indicated by a sudden drop in the registered force, through visible opening of the suture, and via computed tomographic data. Finite element simulations were performed, which led to the experimental testing. Results Rupture of the intermaxillary suture was achieved in all three experiments with the microimplant-supported screw. The strain measurement on one of the expansion screws resulted in an expansion force of 86 N. Finite element simulations showed a high tensile stress concentration exerted by the microimplant-supported expansion screw on the intermaxillary suture. Conclusion The applied expansion force led to high tensile stress concentrations, mainly on the intermaxillary sutures, resulting in the opening of fused intermaxillary sutures. This method may help adults to be treated by an orthodontist, thereby avoiding surgical intervention.

Hamster hypophyseal pars tuberalis (PT)-specific cells are characterized by the expression of common alpha-chain and TSH beta. Immunoreactivity for these subunits and the morphology of these cells are known to exhibit remarkable seasonal changes. The high density of melatonin (Mel) receptors on PT-specific cells leads to the supposition that fluctuations in circulating Mel levels induced by photoperiodic signals are a crucial factor for the morphological alterations. To more closely investigate transcriptional and translational activities in PT-specific cells, we cloned and sequenced hamster alpha and TSH beta complementary DNA fragments and assessed messenger RNA/protein formation by in situ hybridization and immunocytochemistry under short and long photoperiod and in pinealectomized animals kept in short photoperiod. Hamster common alpha-chain and TSH beta exhibited high sequence homology with the corresponding rat hormones [94% (alpha-chain) and 90% (TSH beta) on the nucleotide level and 100% (alpha-chain) and 96% (TSH beta) on the amino acid level]. Immunocytochemical staining with antibodies directed against the common alpha-chain and TSH beta revealed a reduced immunoreactivity of PT-specific cells under short photoperiod, but this was not altered in pinealectomized animals exposed to short photoperiod. In situ hybridization against both hormonal subunits paralleled these changes, with a dramatic decrease in hormonal messenger RNA in short photoperiod. This regulatory influence was also blocked in pinealectomy. Taken together, these results demonstrate that transcription and translation of hormonal subunits are regulated by photoperiod in hamster PT-specific cells, whereas expression remained unchanged in short photoperiod if pinealectomy was performed. We, therefore, conclude that in hamsters, the Mel Signal not the light regimen per se, is a direct or indirect Zeitgeber for the transduction of photoperiodic information to the secretory activity in this pituitary cell type.

Specific cells of the hypophyseal pars tuberalis (PT) have been associated with the transmission of photoperiodic stimuli to the endocrine system. However, their principal secretory products have not been identified yet. Therefore we studied the expression of several adenohypophyseal hormones and their subunits (TSH, FSH, LH, common alpha-chain, GH, ACTH, PRL, alpha- and gamma-MSH, beta-lipotropin) by immunocytochemistry, Northern blot analysis and in situ hybridization in the sheep pituitary. Only the common alpha-chain of glycoprotein hormones could be detected in ovine PT-specific cells by immunocytochemistry while antibodies directed against the beta-chains of LH, FSH, TSH and beta-lipotropin labeled single cells in the PT but failed to detect these antigens in PT-specific cells. In situ hybridization and Northern blot analysis with antisense oligonucleotides against the common alpha-chain, beta-LH, beta-FSH, beta-TSH, PRL and POMC revealed the expression of these subunits in the ovine PT. The mRNA of the common alpha-chain, beta-TSH and, to a far lower extent, PRL and POMC were found throughout the entire pars tuberalis while beta-FSH and beta-LH could only be detected in cells of the caudal PT. Hence, GH-mRNA and GH immunoreactivity were exclusively found in the pituitary pars distalis. Compared to these results--obtained under the short photoperiod (winter)--we found clear ultrastructural signs of altered secretory activity in PT-specific cells of animals exposed to the long photoperiod (summer); the common alpha-chain immunoreactivity was nearly absent in PT-specific cells of summer animals. However, no seasonal influence on gene transcription or translation for other adenohypophyseal hormone was observed. These findings suggest that ovine PT-specific cells, which are only immunopositive for the common alpha-chain, are capable to express different mRNAs of adenohypophyseal hormones. Although it remains elusive how gene transcription and translation are related in this cell type, the presence of an mRNA pool for hormone subunits leads to the speculation that--at least in the sheep--hormone synthesis is mainly regulated at the translational level and that secretion of hormones may be primarily constitutive.

BACKGROUND Gender medicine has gained importance over the past 20 years. Nevertheless, the scientific findings concerning gender- and sex-specific patient care have not been sufficiently integrated into the education of physicians. It was therefore our aim, against initial resistance in our school, to integrate clinically relevant aspects of gender medicine into the existing medical curriculum. This paper describes the implementation process of a lecture-based interdisciplinary, longitudinal, basic gender curriculum and evaluates students' attitudes in relation to sex and semester level. METHODS The curriculum encompasses 15 lecture sessions scheduled in years 1 through 5 of the medical curriculum at Ulm University, Germany. Prospectively gathered evaluation data of two cross-sectional analyses of this basic curriculum in the first and fifth semesters are analyzed by sex. RESULTS More than 80% of the students have registered for this new curriculum. Evaluation data show a predominantly positive (75.5%) student response; however, only about half of those surveyed indicated that they had learned new material or judged the content on gender to be relevant to their practice of medicine. Students at a more advanced semester level (88.2% vs. 55.2%) and male participants more than female participants (36.7% vs. 62.4%) showed lower acceptance. DISCUSSION It was possible to integrate gender issues into the existing medical student curriculum. Despite the overall positive rating, our evaluation data identified the aspects of rejection and resistance in some students, particularly male and more advanced students. Further studies on the development of student attitudes toward gender issues are needed.

Hypophyseal pars tuberalis (PT)-specific cells are known to exhibit remarkable seasonal changes in morphology especially in photoperiodic animals like the Djungarian hamster Phodopus sungorus. Their high density of melatonin-receptors leads to the supposition that fluctuations in circulating melatonin levels are a crucial factor for the morphological alterations induced by photoperiodic signals. To prove this hypothesis the nocturnal elevation of melatonin in long photoperiods was prolonged by late afternoon administration of melatonin. We investigated whether this treatment induces cytological changes usually observable under short photoperiod. Electron microscopy revealed that in contrast to hamsters maintained in long photoperiods PT-specific cells of hamsters injected with melatonin or those kept in short photoperiods appear inactive, containing a relatively high number of secretory granules, sparse endoplasmatic reticulum, irregularly outlined and invaginated cell nuclei and a high amount of glycogen. Furthermore immunoreactivity for the common alpha-chain of glycoprotein hormones and beta-TSH was significantly weaker in hamsters kept in short photoperiods or daily injected with melatonin than untreated or vehicle injected controls in long photoperiod. These results demonstrate that an exogenous prolongation of the elevated nocturnal melatonin levels causes a similar morphological appearance of PT-specific cells as observed in short photoperiods. It is tempting to speculate that the melatonin signal is a direct 'Zeitgeber' for the transduction of photoperiodic information to the secretory activity in this cell type.

The expression of the glutaminyl cyclase (QC), an enzyme responsible for the post-translational modification of N-terminal pyroglutamyl residues of neuropeptide precursors, was examined in bovine/porcine hypothalamic and pituitary tissue by means of immunocytochemistry and in situ hybridization. In the anterior pituitary a distinct pattern of QC immunoreactivity and mRNA expression was found associated exclusively with somatotrophs. In corticotrophs of the pars intermedia QC expression was undetectable, whereas a small portion of putative pars tuberalis cells in the rostral part of the pars distalis were heavily labelled. The neurointermediate lobe was devoid of signals for QC mRNA, but showed significant QC-immunoreactivity on secretory granules of axonal nerve endings. Also nuclei of the hypothalamus were found to be positive for QC-immunoreactivity. Intense labelling was observed in the nucleus supraopticus and nucleus paraventricularis. Staining of the nucleus periventricularis was found to be moderate, whereas no labelling of perikarya in the nucleus arcuatus, the preoptic area and the nucleus suprachiasmaticus was detectable. Moreover, varicose fibers stained positive for QC-immunoreactivity and could be identified in the main transport route from the hypothalamus to the pars neuronalis (tractus hypothalamo hypophysialis). These results suggest that the enzyme is transported via the same routes as its substrate/product to the median eminence or the neural lobe. Furthermore, the mapping of the cellular QC distribution reveals a strikingly distinctive expression pattern, that should be useful for the identification of yet undiscovered places of peptide synthesis and processing.