"Anatomy and imaging": 10 years of experience with an interdisciplinary teaching project in preclinical medical education - from an elective to a curricular course.

PURPOSE: Presentation of an interdisciplinary, interactive, tutor-based preclinical teaching project called "Anatomy and Imaging". MATERIALS AND METHODS: Experience report, analysis of evaluation results and selective literature review. RESULTS: From 2001 to 2012, 618 students took the basic course (4 periods per week throughout the semester) and 316 took the advanced course (2 periods per week). We reviewed 557 (return rate 90.1 %) and 292 (92.4 %) completed evaluation forms of the basic and the advanced course. Results showed overall high satisfaction with the courses (1.33 and 1.56, respectively, on a 5-point Likert scale). The recognizability of the relevance of the course content for medical training, the promotion of the interest in medicine and the quality of the student tutors were evaluated especially positively. CONCLUSION: The "Anatomy and Imaging" teaching project is a successful concept for integrating medical imaging into the preclinical stage of medical education. The course was offered as part of the curriculum in 2013 for the first time. "Anatomia in mortuis" and "Anatomia in vivo" are not regarded as rivaling entities in the delivery of knowledge, but as complementary methods.

Increased inorganic phosphate induces human endothelial cell apoptosis in vitro.

Chronic kidney disease with hyperphosphatemia is associated with accelerated atherosclerosis and endothelial dysfunction. However, the contribution of high serum phosphate levels to endothelial injury is incompletely understood. The aim of this work was to evaluate the responses of endothelial cells to elevated levels of extracellular phosphate in vitro. High phosphate in concentrations similar to those observed in uremia-associated hyperphosphatemia (>2.5 mM) induced apoptosis in two endothelial cell lines (EAhy926 cells and GM-7373 cells). This effect was enhanced when cells were incubated for 24 h in the presence of 2.8 mM calcium instead of 1.8 mM. By treating cells with 0.5 or 1.0 mM phosphonoformic acid, an inhibitor of the phosphate transporter, death was completely prevented. The process of phosphate-induced apoptosis was further characterized by increased oxidative stress, as detected by increased ROS generation and disruption of the mitochondrial membrane potential at approximately 2 h after treatment, followed by caspase activation. These findings show that hyperphosphatemia causes endothelial cell apoptosis, a process that impairs endothelial integrity. Endothelial cell injury induced by high phosphate concentrations may be an initial event leading to vascular complications in patients with chronic kidney disease.

Flow-cytometric identification, enumeration, purification, and expansion of CD133+ and VEGF-R2+ endothelial progenitor cells from peripheral blood.

A flow cytometric method for identifying, purifying, and expanding endothelial progenitor cells (EPC) derived from peripheral blood is reported. During our experiments, we found that isolation of viable EPC is not possible by using the standard flow cytometric protocols, since erythrocyte lysing influences cell survival. Furthermore, erythrocyte lysing has a high impact on quantative analysis of EPC with 20% lower numbers compared to no-lyse data. The viability of EPCs was tested with a colony forming test after both lysis based FACS of EPCs and without lysing. CD133 and VEGF-R2 revealed as positive markers for EPC selection and 7-amino actinomycin D (7-AAD) to eliminate dead cells. The few purified CD133+ and VEGF-R2+ cells showed strong colony-forming capacity in a human stem cell methylcellulose based medium (colony assay) when isolated by the no-lyse protocol. The colonies showed the typical shape of early EPC-colonies with round immature cells in the centre and dendritic or spindelcell-shaped peripheral cells, which were also immunologically identified as EPC-derived. Compared to this, erythrocyte lysing reagents destroyed even all sorted EPCs. Summarizing the presented data suggest that the use of erythrocyte lysing reagents is neither suitable for cloning nor for counting of endothelial progenitor cells, and no-lyse protocols should be used.

Copper and zinc dismetabolism in the mouse brain upon chronic cuprizone treatment.

Recent reports describe successful treatment using copper chelation therapy in neurodegenerative animal models. However, the success claimed for chelation therapy in neurodegenerative diseases is still rather controversial. To acquire new information on copper metabolism/homeostasis, we utilized cuprizone, a very sensitive and selective copper-chelating agent with well-known neurotoxic properties, as a relevant chemical model in mice. Upon cuprizone treatment, mice developed a pronounced astrocytosis, with brain oedema and spongiosis characterised by vacuolisations of the neuropil predominantly in the white matter. In addition, cuprizone treatment severely altered copper and zinc homeostasis in the central nervous system (CNS) as well as in all other tissues examined, with increasing metal ion concentrations particularly in the CNS. Concomitant with this increase in the Cu and Zn concentration in the brain, metallothionein-I and -II were also highly immunoreactive in astrocyte, consistent with the astrocytosis and demyelination observed in our and other laboratories.

Influence of hypergravity on hypothalamic vasopressin and oxytocin neurons in rats.

This study was aimed to evaluate the reaction of the vasopressin (VP) and oxytocin (OT) neurons of the supraoptic nucleus (SON) in rats to single or repeated hypergravity (HG). Special attention was paid to the tyrosine hydroxylase (TH) expression in VP neurons as a marker of the neuron activation. Rats were revolved in a centrifuge with overloading 2G for 5 days or 34 days as well as for 34 days plus 5 days with an interval of 39 days between two rotations. Control rats were kept in a centrifuge room. Radioimmununoassay, quantitative and semi-quantitative immunocytochemistry and in situ hybridization were used to evaluate: a) VP concentration in the pituitary posterior lobe (PL) and in plasma; b) the number of VP-, OT- and TH-immunoreactive neurons in the SON; c) the optic density of VP-, OT- and TH-immunoreactive materials in cell bodies (SON) and distal axons (PL), d) the optic density of VP and OT mRNAs signals (S35) in the whole SON on microfilms. According to our data, VP neurons were strongly activated during HG (5 days or 34 days) that was manifested in the functional hypertrophy of the neurons, greatly increased concentrations of VP mRNA in the SON and VP in plasma, the onset of the TH expression. The neurons showed initially (5 days) the functional insufficiency (VP release > VP synthesis) followed by their adaptation (subsequent 29 days) to the increased need in VP (VP release < VP synthesis). No reaction of VP neurons was observed to repeated HG. In contrast to VP neurons, OT neurons did not react to short-term HG or showed functional depression after the long-term treatment.

The Pars tuberalis of the monkey (Macaca fascicularis) hypophysis: cell types and hormone expression.

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 beta-TSH subunit. Although there is a strong signal for the mRNA of the common alpha-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.

Expression and functional characterization of the mt1 melatonin receptor from rat brain in Xenopus oocytes: evidence for coupling to the phosphoinositol pathway.

Melatonin-sensitive receptors were expressed in Xenopus laevis oocytes following an injection of mRNA from rat brain. The administration of 0.1-100 micromol/L melatonin to voltage-clamped oocytes activates calcium-dependent chloride currents via a pertussis toxin-sensitive G protein and the phosphoinositol pathway. To determine which melatonin receptor type (mt1, MT2, MT3) is functionally expressed in the Xenopus oocytes, we used (i) agonists and antagonists of different receptor types to characterize the pharmacological profile of the expressed receptors and (ii) a strategy of inhibiting melatonin receptor function by antisense oligonucleotides. During pharmacological screening administration of the agonists 2-iodomelatonin and 2-iodo-N-butanoyl-5-methoxytryptamine (IbMT) to the oocytes resulted in oscillatory membrane currents, whereas the administration of the MT3 agonist 5-methoxycarbonylamino-N-acetyltryptamine (GR135,531) exerted no detectable membrane currents. The melatonin response was abolished by a preceding administration of the antagonists 2-phenylmelatonin and luzindole but was unaffected by the MT3 antagonist prazosin and the MT2 antagonist 4-phenyl-2-propionamidotetralin (4-P-PDOT). In the antisense experiments, in the control group the melatonin response occurred in 45 of 54 mRNA-injected oocytes (83%). Co-injection of the antisense oligonucleotide, corresponding to the mt1 receptor mRNA, caused a marked and significant reduction in the expression level (13%; P < 0.001). In conclusion, the results demonstrate that injection of mRNA from rat brain in Xenopus oocytes induced the expression of the mt1 receptor which is coupled to the phosphoinositol pathway.

Localization of metallothionein I-II immunoreactivity in bovine pituitary gland.

Metallothioneins belong to a family of shock proteins characterized by an unusual high content of cystein, absence of aromatic amino acids and high metal content (Zinc and Copper). Metallothioneins are ubiquitously present in a large variety of prokaryotic and eukaryotic species as well as in all mammalian organs and tissues examined thus far. To the best of our knowledge this is the first report describing the presence of metallothioneins in the pituitary gland. Metallothioneins were identified immunohistochemically and chromatographically both in the neuro and adenohypophysis of the bovine pituitary gland. Metallothioneins are highly expressed in the neurohypophyseal glial cells, and in a subpopulation of folliculo-stellate cells located in the pars intermedia of the adenohypophysis. While the specific role of these proteins in the pituitary gland remains to be established, we hypothesize that, besides their protective action against free radicals, hypophyseal metallothioneins might be involved in the regulation of metal ion homeostasis with putative implication in release of hypothalamic peptide hormones in the neurohypophysis and synthesis/release of alpha-MSH by POMC-cells located in the pars intermedia of the adenohypophysis.

Cutting of living hippocampal slices by a highly pressurised water jet (macromingotome).

Living brain slices are usually cut with razor blades, which compress a ca. 50-microm-thick layer of tissue. This results in cell debris and lesioned cells which, e.g. form diffusion barriers between the bath and living neurons underneath, thereby prolonging response times of neurons to drugs in the bath saline and impeding the experimental access to intact neurons. To avoid such drawbacks, a macromingotome was developed which cuts nervous tissue with water jets. Physiological saline under pressures of 100-1800 bar was ejected through nozzles of 35-100 microm to cut 300-500-microm-thick hippocampal slices. Systematic variations of pressure and nozzle diameter revealed best results at 400-600 bar and with nozzle diameters of 60-80 microm. Under these conditions, intact CA1- and CA3-neurons as well as granule cells were detected with infrared microscopy at less than 10 microm underneath the surface of the slice. Superficial neurons with intact fine structures were also seen when the slices were studied by light-microscopy. Intra- and extracellular recordings from superficial neurons showed normal membrane- and full action potentials and the development of stable epileptiform discharges in 0 Mg(2+)-saline. These results indicate that the macromingotome offers an alternative way of cutting slices which may facilitate electrophysiological/neuropharmacological or fluorometric studies on superficial neurons.

Melatonin in epilepsy: first results of replacement therapy and first clinical results.

At a single evening dose of 5-10 mg, melatonin (MLT), the pineal gland hormone, can exert a positive effect on the frequency of epileptic attacks in children with sleep disturbances of various etiologies. We have shown that the sleep behavior can be normalized and an existing epilepsy can be favorably influenced. Pretherapeutic MLT secretion profiles can provide new information concerning the origin and treatment of these disturbances. In vitro experiments suggest that this effect might be the result of the interaction between MLT and MLT-specific receptors in the neocortex. Due to its favorable safety profile, MLT can be liberally administered in the specified doses and be considered as a useful antiepileptic drug.

Cell and molecular biology of the pars tuberalis of the pituitary.

The pars tuberalis of the adenohypophysis is mainly composed of a special type of endocrine cells, pars tuberalis-specific cells, lining the primary capillary plexus of the hypophysial portal system. Dense expression of melatonin receptors and marked changes in morphological appearance, production pattern, and secretory activity during annual cycle show that these cells are highly sensitive to changes in photoperiod. This leads to the hypothesis that the pars tuberalis is involved in the transmission of photoperiodic stimuli to endocrine targets. Several investigations support the theory that pars tuberalis-specific cells are multipotential cells exerting a modulatory influence on the secretory activity of the pars distalis. Specifically, there is accumulating evidence that seasonal modulation of prolactin secretion, independent of hypothalamic input, is due to melatonin-regulated activity of pars tuberalis-specific cells. The exact nature of secretory products and their effects within neuroendocrine regulation, however, remain rather enigmatic. Accordingly, molecular mechanisms regulating gene expression under the influence of photoperiod, respectively, circulating melatonin levels are still incomplete. Recent cloning of melatonin receptor genes and new data on intracellular signal transduction will probably lead to new insights on melatonin action and pars tuberalis-specific cell physiology.

Tanycytes and pituicytes: morphological and functional aspects of neuroglial interaction.

The hypothalamo-hypophyseal system is supplied with two types of specialized glial cells that interact in neuroendocrine functional dynamics: the tanycytes and the pituicytes. Tanycytes are the dominating glial cells within the median eminence. Similar to radial glia, they extend from the floor of the third ventricle to the neurohemal surface of the median eminence. Pituicytes, as specialized astrocytes, are the main glial cells of the neural lobe. They are in intimate contact with the perivascular space of the sinusoidal vessels. Morphological similarities between the two cell types focus on their interaction with terminal branches of hypothalamic neurons in both regions of the neurohypophysis, the median eminence and the neural lobe. Release of hypothalamic hormones is apparently influenced by pituicytes and tanycytes. For instance, both types of cells are capable of closing or opening the access to the vessels. Thereby, in contrast to the "blood-brain-barrier" function of astrocytes, pituicytes and tanycytes display "brain-blood-barrier" functions. Pituicytes are characterized by the expression of specific membrane-bound receptors for opioids, vasopressin, and beta-adrenoceptors, indicating that they receive input by numerous neuroactive substances. Integration of these incoming signals may result in a regulation of neurosecretion, especially by morphological changes and by modulation of extracellular ion concentrations. Comparable modulatory mechanisms of tanycytes have not yet been elucidated in a convincing manner. Besides possible regulatory functions, tanycytes are considered to possess guiding functions for hypothalamic axons and to be involved in transport mechanisms between ventricle and blood vessels of the portal system.

Microcutting of living brain slices by a pulsed ultrafine water jet which allows simultaneous electrophysiological recordings (micromingotome).

Up to now microsurgical dissections in living nervous tissue (e.g. in slices or cell cultures) are performed either by micro-scalpels or by laser beams. As an alternative technique, a device for cutting with an ultrafine pulsed water jet was developed to allow precise, visually controled dissections in neuronal circuits even during electrophysiological recordings. Water is ejected by pressure (20-30 bar) from patch pipettes with tip diameters of 10-12 microm. By means of a piezo-element the pipette and the water jet are forced to oscillate vertically with a frequency of 200-400 Hz with an adjustable amplitude. These oscillations facilitate the transsection of neuronal connections even in thick slice preparations. Best results were obtained when the tip of the pipette was about 500 microm above the surface of the submerged slice tissue. This micromingotome offers the following advantages: (i) histological studies show that the water jet cleans the cutting surface, thus avoiding debris and its uncontrolable effects on cells underneath; (ii) the arrangement enables ongoing electrophysiological recordings from hippocampal slices during the cutting procedure and thus facilitates studies of the functions of neuronal connections; (iii) the device allows even disconnection in cultured nervous tissue overgrowing polyamid grids with 50 microm wide meshes.

Initial expression of the common alpha-chain in hypophyseal pars tuberalis-specific cells in spontaneous recrudescent hamsters.

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.

Cloning and expression of a brain-derived TSH receptor.

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.

Thyrotropin expression in hypophyseal pars tuberalis-specific cells is 3,5,3'-triiodothyronine, thyrotropin-releasing hormone, and pit-1 independent.

The expression of TSH subunit genes (TSH alpha and -beta) in pituitary thyrotropes is primarily regulated via circulating thyroid hormone levels (T3) and the hypothalamic TRH. Hypophyseal pars tuberalis (PT)-specific cells also express both hormonal subunits of TSH, but do not resemble thyrotropes of the pars distalis (PD) with respect to their distinct morphology, secretion, and direct modulation of TSH expression by photoperiodic inputs and melatonin. To investigate whether this distinct regulation of TSH is related to a different molecular structure or different signaling cascades, we analyzed PT-specific TSH and its transcriptional regulation in ovine PT-specific cells. After construction of PT- and PD-specific complementary DNA (cDNA) libraries, the cloning and sequencing of several TSH alpha and -beta subunit clones revealed identical sizes and sequences for the translated and untranslated regions in both hypophyseal compartments. Transcription start site analysis also displayed three identical start sites for the transcription of TSH beta in PT and PD. After cloning of the ovine TRH receptor cDNA and a partial T3 receptor cDNA, in situ hybridization. Northern blot analysis, and PCR experiments showed that TRH and T3 receptors are not expressed in specific cells of the PT. The transcription factor Pit-1 that is involved in TSH expression of thyrotropes could only be detected in the PD. In additional experiments rats were treated with T4 or TRH, and subsequent in situ hybridization studies showed that TSH beta messenger RNA (mRNA) formation was not altered in the PT. In the PD, however, TSH beta mRNA was significantly reduced in the T4-treated group, but was enhanced in the TRH-treated group. We conclude that PT-specific cells of the pituitary are characterized by the transcription of TSH subunits that are identical to TSH expressed in thyrotropes of the PD. The absence of TRH, T3 receptor mRNA, and Pit-1, respectively, as well as the different reactions compared to PD thyrotropes in in vivo experiments lead to the conclusion that the expression of TSH in PT-specific cells of the pituitary is not regulated via the classical thyrotrope receptors and their intracellular pathways, but through a novel, photoperiod-dependent mechanism.

TSH expression in murine hypophyseal pars tuberalis-specific cells.

Several experiments in photoperiod-dependent species suggest that the hypophyseal pars tuberalis (PT) plays a key role in transducing light/dark information to the endocrine system. In rat and hamster it has been shown that both TSH subunits (TSH-alpha and -beta) are expressed in PT-specific cells, a morphologically distinct cell type which does not resemble thyrotropes of the pars distalis (PD). In order to investigate whether TSH expression is a characteristic and common feature of PT-specific cells we studied cellular morphology and TSH subunit expression in mouse pars tuberalis by electron microscopy, immunocytochemistry and in situ hybridization. In contrast to all other species investigated so far the number and size of secretory granules in mouse PT-specific cells is enlarged. As in rat and hamster, however, TSH subunit expression (mRNA and protein) was found in thyrotropes of the PD and throughout the whole extent of the PT cell layer. We conclude that although mouse PT-specific cells display an ultrastructural morphology that is different from other species, they are nevertheless characterized by TSH subunit expression. Further studies are needed to determine the physiological role of 'PT-specific cell TSH' and to elucidate whether TSH is the only PT-specific cell secretory product.

Alterations of neuronal fibers after epileptic activity induced by pentylenetetrazole: fine structure investigated by calcium cytochemistry and neurobiotin labeling (buccal ganglia, Helix pomatia).

The influence of epileptic activity on both the fine structure of neuronal processes and the subcellular distribution of calcium-binding sites was investigated in an epileptic model system, the buccal ganglion of Helix pomatia. Pentylenetetrazole was used to induce epileptic activity. Calcium-binding sites were visualized as electron-dense precipitates. Epileptic and control activity was intracellularly recorded from neuron B3 labeled with neurobiotin. After epileptic treatment, many processes contained vacuolated or electron-lucent areas next to morphologically intact areas. Most of these areas were enveloped by layers of endoplasmic reticulum. Lamellar formations of membranes occurred frequently. Calcium cytochemistry revealed a high content of dense precipitates within these formations of the endoplasmic reticulum. Local accumulations of diffuse precipitates were more frequent after epileptic activity than in controls. In contrast, structures such as lamellar bodies, cytosomes, and synapse-like formations, all of which contained many electron-dense precipitates, were apparently unchanged after epileptic activity. This study demonstrates that epileptic activity can lead to local degeneration of neuronal fibers and an associated increase in calcium-binding sites. It is suggested that calcium sequestration is locally increased within neuronal processes during epileptic activity.

Short photoperiod-dependent down-regulation of thyrotropin-alpha and -beta in hamster pars tuberalis-specific cells is prevented by pinealectomy.

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.