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We report a hitherto unknown radiation within the clingfishes (Gobiesocidae), discovered in one of the best-studied marine biomes, the Mediterranean Sea. The monotypic genus Gouania is a Mediterranean endemic inhabiting the interstices of gravel beaches. Using geometric morphometric analyses, we identified two distinct morphotypes (characterized by a slender and a stout body shape, respectively) among Gouania willdenowi sampled from the three major Mediterranean basins (Eastern, Western and Adriatic). Slender and stout G. willdenowi occurred sympatrically in the Adriatic and the Eastern Mediterranean basins. Only the stout morphotype was found in the Western Mediterranean. Morphotypes were further distinguished by relative eye size and number of vertebrae. Based on mitochondrial and nuclear gene sequences, five highly divergent clades (COI-based K2P distances of 8 to 15%) were identified. The clades represented, respectively, stout Adriatic, slender Adriatic, stout Eastern, slender Eastern and stout Western Mediterranean Gouania. This suggests that the genus Gouania comprises at least five different species. Phylogenetic relationships among clades furthermore imply repeated evolution of convergent morphotypes. The onset of the Gouania radiation was dated to 3.23 (95% HPD 2.08-5.90) mya, and may therefore have overlapped with or followed the Messinian salinity crisis.

We present a review of galanin in the brain from a historical perspective of the development of "chemoarchitectonics" and "brain cartography" accomplished in the Histopharmacology Section at the National Institutes of Health. It was the mapping of potential brain neuroregulators that served as a springboard of ideas from which behavioral studies emanate. The integration of the known localization of neurotransmitter/neuromodulatory nerves ("chemoarchitectonic maps") and receptor binding sites with biochemical data derived from brain micropunches coupled with behavioral analysis at the level of discrete brain allows one to define the anatomical circuits which support behavioral changes and which ultimately will improve our understanding of mental disorders.

PROBLEM: The scarce data on glucose transporter expression of leukocytes are contradictory and nothing is known about changes accompanying physiological leukocytosis during pregnancy, which imposes acute metabolic demands on the cells. METHOD OF STUDY: Cytospin preparations of intravascular leukocytes were searched immunocytochemically for the high affinity glucose transporters GLUT1, 3 and 4. Pregnancy-associated quantitative changes in transporter expression were assessed by flow cytometry. RESULTS: Granulocytes and monocytes stained for GLUT1, 3 and 4. Major changes in cell surface transporter expression during pregnancy were a 36% (P

Glutamate (1 mM), iodoacetate (0.01 mM) and ionomycin (0.25 micro M) are reported to induce several characteristics of ischemia and neuronal degeneration in vitro, e.g. glutamate and ionomycin lesion result in a disturbance of Ca(2+) homeostasis, iodoacetate impairment leads to an inhibition of energy metabolism, suppression of protein synthesis and generation of oxygen free radicals. In this study these three lesion models were used to investigate the effects of the nootropic drug Cerebrolysin (Cere) on the survival of cortical neurons in culture and on the occurrence of apoptosis. The viability of the cells was evaluated with the colorimetric MTT-reduction assay. Apoptosis was detected with Bisbenzimide (Hoechst:33258), a fluorescent DNA stain. Administration of Cere resulted in dose dependent neuroprotection independent from the kind of lesion. In the glutamate model the drug almost doubled neuronal viability compared to lesioned controls. After acute glutamate exposure Cere reduced the number of apoptotic cells significantly. In spite of the protective efficacy after cytotoxic hypoxia induced by iodoacetate, the drug significantly increased the number of apoptotic neurons, indicating a shift from necrosis to apoptosis. In contrast to previous studies investigating acute ionomycin lesions, the chronic Ca(2+)-overload used here did not increase the abundance of apoptosis compared to the unlesioned control. Summarizing the findings it can be suggested that Cere is able to stabilize Ca(2+) homeostasis, to protect protein synthesis and to counteract neuronal death in different in vitro medels of ischemia.

Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryo's production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.

Cerebrolysin (EBEWE Arzneimittel, Austria, Europe) is a widely used drug relieving the symptoms of a variety of neurological disorders, particularly of neurodegenerative dementia of the Alzheimer's type. It consists of approximately 25% of low molecular weight peptides (

In the present study, the facilitative D-glucose transporter protein GLUT 1 was localised by immunohistochemistry in the placenta of human, marmoset (Callithrix jacchus) and rat at different developmental stages. A polyclonal antiserum against a 13-amino-acid peptide of the GLUT 1 carboxy terminus was used. It identified a protein of around 50 kDa molecular weight in immunoblotting of the placental tissues. GLUT 1 was located in the syncytiotrophoblast, in cytotrophoblast cells and in fetal endothelium. Similar staining patterns, except in human extravillous cytotrophoblast cells, were observed at all differentiation stages, despite differences in the internal placental architecture of the species. In the marmoset placenta, GLUT 1 was undetectable in endothelial cells of maternal vessels. In rat placentae, trophoblastic giant cells, epithelial cells of both visceral and parietal yolk sac, yolk sac vessels and the stratum spongiosum were stained. Reichert's membrane did not immunoreact. Preadsorption of the antiserum with a 13-amino-acid peptide resulted in the loss of immunoreactivity. The results suggest that GLUT 1 is a prominent isoform of glucose transporters in mammalian placentae. It is generally abundant in placental cell populations bordering on the maternal and fetal circulations and may therefore facilitate an effective glucose supply to the fetus and placenta.

The localization of CGRP mRNA in neurons of the rat brain and spinal cord was assessed by in situ hybridization histochemistry (ISH) using a radiolabeled synthetic 57-mer oligodeoxynucleotide probe complementary to the rat prepro CGRP mRNA. Results were compared with previously published findings of CGRP-immunoreactive (CGRP-IR) cell bodies revealed by an indirect immunofluorescence technique. The highest numbers of CGRP mRNA expressing neurons as well as the greatest intensity of staining were found in the lateral hypothalamic area, the parabrachial nuclei, and among the cranial motor nuclei, especially in the nuclei of the 7th and 12th nerve and the ambiguus nucleus, which is generally in good agreement with findings assessed by immunocytochemistry (ICH). However, some mismatches between the localization of the peptide by ICH and the localization of the CGRP mRNA were also observed. Thus, ISH was not able to confirm CGRP-IR in cells of the amygdaloid complex and parts of the medial hypothalamus, the central gray, and the inferior colliculus, but ISH revealed considerably more CGRP mRNA expressing cells in the lateral hypothalamic area, arcuate nucleus, posterior and peripeduncular thalamic nuclei, and all cranial motor nuclei than CGRP-IR containing cells found by ICH. Moreover, ISH also revealed CGRP mRNA synthesis in the nucleus of the lateral olfactory tract and in the perihypoglossal nuclei that were devoid of CGRP-IR. The reasons for the observed mismatches still remain to be elucidated; however, intracerebroventricular colchicine pretreatment used to increase immunocytochemical signals also might have induced or suppressed gene expression in certain brain regions in an unpredictable matter. On the other hand, detection of only the mRNA in a certain region does not necessarily mean that also the active peptide is synthesized there.

Medullary thyroid carcinoma (MTC) is a calcitonin-producing tumor of the parafollicular C-cells, accounting for 5-10% of all thyroid tumors. To date, the only effective treatment is the early and total surgical removal of all neoplastic tissue. As the prognosis of patients with advanced MTC, unresectable or distant metastases is poor, and chemotherapy or irradiation is of no significant value, alternative strategies have been sought.A promising treatment approach for human MTC, that has already been introduced at our facility, is based on vaccination with autologous dendritic cells (DCs). Strong evidence that vaccination with autologous tumor lysate-pulsed DCs induces a specific immune response in vivo has been provided. However, the therapeutic success of this approach is sometimes critically limited by the small amount of tumor material available, especially from patients operated at an early tumor stage. Thus, it would be to the best advantage to have sufficient amounts of autologous tumor cells available for DC pulsing.A method to generate viable autologous tumor cell cultures from a variety of MTC tissue samples, even when the sample size is small, has been successfully established. These cell lines maintain their neuroendocrine phenotype. In addition, it can be shown that these cells also display the biological features of neuroendocrine tumor cells at the molecular level.The unlimited availability of these MTC cell lines makes it possible to specify cancerogenesis of MTC. In addition, the availability of sufficient amounts of tumor lysate from these cell lines offers the advantage of prolonged immunotherapy. Finally, these cell lines could be elegantly used as read-out system to monitor the in vivo immune response during immunotherapy with DC cell-based vaccination in patients suffering from MTC.

The aim of the study was to investigate neurochemical changes in a kainic acid (KA; 10 mg/kg, s.c.)-induced spontaneous recurrent seizure model of epilepsy, 6 months after the initial KA-induced seizures. The neuronal markers of cholinergic and gamma-aminobutyric acid (GABA)ergic systems, i.e. choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) activities, and a marker for neuropeptide, i.e. level of somatostatin, have been investigated. The brain regions investigated were the hippocampus, amygdala/piriform cortex, caudate nucleus, substantia nigra and the frontal, parietal, temporal and occipital cortices. Six months after KA injection, reduced ChAT activity was observed in the amygdala/piriform cortex (47% of control; p0.001), increased ChAT activity in the hippocampus (119% of control; p0.01) and normal ChAT activity in the other brain regions. The activity of GAD was significantly increased in all analysed cortical regions (between 146 and 171% of control), in the caudate nucleus (144% of control; p0.01) and in the substantia nigra (126% of control; p0.01), whereas in the amygdala/piriform cortex, the GAD activity was moderately lowered. The somatostatin level was significantly increased in all cortical regions (between 162 and 221% of control) as well as in the hippocampus (119% of control), but reduced in the amygdala/piriform cortex (45% of control; p0.01). Six months after KA injection, the somatostatin:GAD ratio was lowered in the amygdala/piriform cortex (49% of control) and in the caudate nucleus (41% of control), whereas it was normal in the hippocampus and moderately increased in the cortical brain regions. A positive correlation was found between seizure severity and the reduction of both ChAT activities and somatostatin levels in the amygdala/piriform cortex. The results show a specific pattern of changes for cholinergic, GABAergic and somatostatinergic activities in the chronic KA model for epilepsy. The revealed data suggest a functional role for them in the new network that follows spontaneous repetitive seizures.

The scarce data on glucose transporter expression of leukocytes are contradictory and nothing is known about changes accompanying physiological leukocytosis during pregnancy, which imposes acute metabolic demands on the cells.Cytospin preparations of intravascular leukocytes were searched immunocytochemically for the high affinity glucose transporters GLUT1, 3 and 4. Pregnancy-associated quantitative changes in transporter expression were assessed by flow cytometry.Granulocytes and monocytes stained for GLUT1, 3 and 4. Major changes in cell surface transporter expression during pregnancy were a 36% (P0.05) down-regulation of granulocyte GLUT1 at term, and an increase in monocyte GLUT3 levels to 137% (P0.05), paralleled by a 24% (P0.05) decrease in GLUT4 content in second trimester. Apart from a minor subpopulation, lymphocytes were negative for these carriers.GLUT1, 3 and 4 are abundantly expressed in granulocytes and monocytes. The particular isoforms are differentially regulated during pregnancy, suggesting an individual functional significance.

The scarce data available on leukocyte glucose transporter expression are contradictory and nothing is known about its regulation by glycemic state. Therefore, cytospin preparations of blood leukocytes were searched immunocytochemically for the high-affinity glucose transporters GLUT1, 3, and 4. Hypoglycemia-associated quantitative changes in transporter expression were assessed by flow cytometry. Granulocytes and monocytes stained for GLUT1, 3, and 4. Granulocyte GLUT4 levels were increased by 73% (P < 0.05) under hypoglycemic conditions, which was paralleled by a reduction in GLUT1 and a rise in GLUT3. In monocytes, GLUT3 was elevated by 134% (P < 0.05), whereas GLUT1 and GLUT4 remained unaffected upon hypoglycemia. Apart from a minor subpopulation, lymphocytes were negative for these carriers. In conclusion, GLUT1, 3, and 4 are abundantly expressed in granulocytes and monocytes. The differential response of individual isoforms to hypoglycemia may represent a mechanism to protect the cells from the stress of glucose deprivation.

The effects of two toxins, sodium cyanide (NaCN) and ionomycin (IM), on neuronal viability and on the expression of the microtubule-associated proteins MAP1, MAP2, and tau were studied in isolated chick cortical neurons. Cytotoxic hypoxia due to NaCN treatment was performed to mimic acute neuronal damage, whereas long-term IM treatment was used as a model for chronic neuronal impairment. After 5 days in vitro, a cytotoxic lesion was induced either by addition of NaCN (0.01-10 mM) or IM (0.01-10 microM). The NaCN solution was aspirated after 30 min and cells were allowed to regenerate for 6 h, 24 h, 48 h, or 72 h; whereas the permanent IM lesions were left undisturbed during the same periods of time. Neuronal viability was assessed by MTT assay. The abundance of MAP1, MAP2, and tau was evaluated by immunoblotting and, for MAP2, by immunohistochemistry also. Results showed that NaCN and IM lesions dose-dependently decreased viability. Irreversible cell damage occurred after impairment with 10 mM NaCN and 1 microm or 10 microm IM, while neurons lesioned with lower concentrations regenerated partially or adapted to the toxic environment. However, the same level of viability as of untreated cells was never reached. Furthermore abundance of MAPs was changed after both lesions. But while after extended recovery from NaCN lesion protein expression was normalizing (MAP2) or at least still detectable (MAP1A, tau), the consequences of a permanent IM lesion were more severe, since neurons were not able to maintain or even restore their MAP expression. Immunohistochemical experiments for MAP2 revealed that, compared with controls, NaCN and, to a much higher extent, IM treatment resulted in a loss of immunoreactivity in neurites due to progressing cell death.

Nerve growth factor (NGF) has the ability to increase the content of peptide transmitter in intact primary sensory afferents of the adult rat. We have previously shown that NGF can also induce a refill of peptide transmitters in capsaicin-depleted peptidergic nerve terminals of the rat paw skin upon intraplantar injection. The present study was aimed at investigating the neurochemical, immunohistochemical and functional recovery of peripheral and central terminals of capsaicin-lesioned afferents following administration of recombinant human NGF-beta (rhNGF-beta). The systemic capsaicin treatment in adult rats by 50 mg/kg s.c. (day 0) was followed by intraplantar rhNGF-beta injections (4 micrograms each) into one hind paw on days 1, 2, 3, 5, 6 and by the analysis on day 8. The content of the marker peptide calcitonin gene-related peptide (CGRP) showed a 100% NGF-induced recovery in the peripheral (sciatic nerve) and central axons (lumbar dorsal roots) on the side of the NGF treatment and also in the contralateral sciatic nerve and lumbar dorsal roots. In the terminals of the hind paw skin, the recovery of the CGRP content, as measured by radioimmunoassay, was 100% in the plantar and 80% in the dorsal skin ipsilaterally, and 55% in the dorsal and plantar hind paw skin contralaterally. In the lumbar dorsal spinal cord, CGRP content recovered by 85% bilaterally. The morphological appearance of the sensory nerve terminals was visualized by CGRP-immunohistochemistry. In the paw skin, the CGRP-immunoreactive (CGRP-IR) nerve endings were restricted to a fragmentary subepidermal plexus after the capsaicin treatment, whereas the subsequent NGF treatment caused a bilateral recovery of the subepidermal plexus and an intact reinnervation of the epidermis and blood vessels with free nerve terminals. The capsaicin-induced fragmentation of the CGRP terminal plexus in laminae I and II of the lumbar spinal dorsal horn was also markedly repaired on both sides by the intraplantar NGF injections. The NGF treatment caused the CGRP nerve terminals in the spinal cord to regain their ability of releasing transmitter upon capsaicin stimulation as shown in tissue slice superfusion experiments. These results show that within one week, rhNGF-beta can induce a complete reinnervation of skin and spinal cord with intact CGRP-IR nerve terminals after an acute capsaicin lesion.

Neonatal denervation of primary afferents with capsaicin leads to increased sympathetic innervation of the rat ureter. In the present study the development and the immunohistochemical characterization of this sympathetic hyperinnervation as well as the specific involvement of nerve growth factor (NGF) was investigated. Noradrenaline levels were found elevated in neonatally capsaicin-treated rats by 2 weeks of age and remained at that high level into adulthood. Injections of an anti-NGF antiserum during postnatal days (PN) PN 8–14, PN 13–19 or during PN 17–23 counteracted the capsaicin effect and reduced noradrenaline towards control levels. Immunohistochemical localization of tyrosine hydroxylase (TH), a marker for sympathetic nerve fibres, revealed that the capsaicin-induced hyperinnervation was mainly represented by fibres in deeper muscle layers and to a smaller extent by fibres in the submucosa. In control animals and in rats treated with capsaicin and anti-NGF antiserum fibres were mainly distributed in the adventitia and in the outer part of the smooth muscle layer. These results show that NGF is responsible for the development of an increased noradrenergic innervation in the rat ureter after neonatal capsaicin treatment.

In the present study the ability of nerve growth factor (NGF) to facilitate the recovery of peptidergic primary sensory C-fibers after an acute capsaicin treatment (50 mg/kg s.c.) was investigated in adult rats. NGF (4 μg 1 /day for 3 days) was injected into the plantar of one hind paw starting 24 h after the capsaicin treatment. Without NGF, there was a significant reduction of calcitonin gene-related peptide (CGRP) and substance P content of the paw skin and the sciatic nerve. CGRP and substance P levels were completely replenished in the NGF-treated paw skin and in the innervating sciatic nerve they even increased over control levels as determined 40 h after the last injection of NGF. CGRP levels also recovered in the contralateral paw and sciatic nerve, but no recovery was observed in other tissues such as the front paw, the auricle, or the urinary bladder. Mustard oil-induced neurogenic plasma extravasation, taken as a functional parameter for peptidergic primary sensory C-fibers, was significantly decreased after the capsaicin treatment and showed a complete recovery by NGF in the injected paw as well as in the contralateral paw skin. These results show that NGF not only was able to reverse the decrease of transmitter content caused by capsaicin but also restored the peripheral function of primary afferent neurons.

Maternal glucose is a major substrate for placental and fetal metabolism. The kinetics of its uptake into placental trophoblast cells has not been characterised yet and was therefore investigated in the present study. In addition to trophoblast cells isolated from human term placentae, JEG-3 and JAR choriocarcinoma cells were used. Measurements were carried out in 5 s intervals until 30 s with the non-metabolisable glucose analogue 3-O-[14C]methyl-D-glucose using confluent cells adhering to glass coverslips. L-[1-14C]glucose was used to correct for extracellular trapped tracer and diffusion. The uptake was rapid and saturable. It reached equilibrium after 30 s at 20 degrees C and could be inhibited by 0.4 mmol/l cytochalasin B up to 98%. The choriocarcinoma cells took up twice as much glucose as trophoblast cells. Fitting the experimental data to the Michaelis-Menten equation by non-linear regression failed to adequately describe the data, even when a contribution of diffusion to total uptake was considered. Introducing the Hill coefficient n into the Michaelis-Menten equation significantly improved the quality of the fits as was assessed by three statistical criteria. Using this equation modified for allosteric kinetics (v = k[To] [S]n)/(Km + [S]n)), parameters were calculated as Km = 12 mmol/l, Vmax = 17 fmol/l s-1 per cell, n = 1.1 for trophoblast cells; Km = 13 mmol/l, Vmax = 27 fmol/l s-1 per cell, n = 1.2 for JEG-3 cells and Km = 29 mmol/l, Vmax = fmol/l s-1 per cell, n = 1.4 for JAR cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A general and simple autoradiographic procedure to visualize neuropeptide receptor binding sites in rat brain tissue sections is described in detail. The method utilizes unfixed slide-mounted cryostat tissue sections of the rat brain which are incubated with 125I labeled neuropeptides. Visualization of binding sites is achieved by direct exposure of radiolabeled tissue sections to X-ray film in autoradiography cassettes. For example, binding of 125I-porcine galanin to coronal sections of the rat brain is shown. Details of the autoradiographic procedure are discussed.

The location and physiological functions of acetylcholinesterase and butyrylcholinesterase in the placenta are still debated. In the present study the activities of both enzymes were studied histochemically in the rat and human placenta, using an optimized Karnovsky/ Roots method. Additionally, they were measured biochemically. Acetylcholinesterase was active in the syncytiotrophoblast, cytotrophoplast cells and the visceral and parietal yolk sac epithelial cells of the rat (n = 10) and in the syncytiotrophoblast, cytotrophoblast cells, endothelial cells and the media of fetal blood vessels of the human placenta (n = 9). Butyrylcholinesterase could not be detected histochemically. Biochemically measured levels at certain developmental stages of the placenta revealed maximum acetylcholinesterase activity in the 8th week p.m. human placentae (102.9 nmol·min−1 per mg protein), 35% lower activity in the 12th week p.m., and minimum (44.1 nmol-min−1 per mg protein) in term placentae. In contrast, maximum butyrylcholinesterase activity was measured in week 12 p.m. (106.9 nmol·min−1 per mg protein). In rat placentae, butyrylcholinesterase activity on gestational day 21 reached 150% of the level on gestational day 16. Acetylcholinesterase activity remained constant. In placentae of pre-eclamptic patients, acetylcholinesterase and butyrylcholinesterase activities were found to be increased by 16% and 45%, respectively. The results suggest that placental acetylcholinesterase can no longer be considered as derived from maternal blood, but is primarily located within rat and human placental tissue.

The heterogeneity of endothelial cell surface antigen expression was studied in 5 human full-term placentae by means of indirect immunohistochemistry using 9 monoclonal antibodies and by staining with fluorescent-conjugated Ulex europaeus lectin, both of which are widely used endothelial cell markers. (1) A highly specific, homogeneous staining of fetal and maternal placental vessels of all sizes and anatomical regions was observed by the monoclonal antibodies PAL-E, QBEND10 and 1F10. These antibodies were even more specific than Ulex europaeus lectin, factor VIII antibody and von Willebrand factor antibody, which cross-reacted with some non-endothelial cells and structures. The reactivity of PAL-E, QBEND10 and 1F10 with residual surface cells of the basal plate strongly suggests an endothelial origin of these cells. (2) In contrast to other organs, PAL-E, QBEND10 and HM15/3 strongly stained endothelial cells of the macrovascular system in the human placenta. This might indicate an organ-associated heterogeneity of fetal endothelial cells. (3) Monoclonal antibodies against receptors for transferrin and IgG (Fc gamma RII) labeled the endothelial cells of fetal placental vessels with increasing intensity distal to the insertion of the umbilical cord. The vessels of the umbilical cord itself were unreactive. This might suggest a heterogeneity of macro- and microvascular endothelial cells.

CGRP in the amygdala is concentrated in an area close to the central nucleus. High potassium releases CGRP, and this release, as well as the tissue concentration of CGRP in the amygdala, can be influenced by neuroleptic drugs. Both molecular forms, alpha- and beta-CGRP, are present in the amygdala in a ratio of approximately 3:1. CGRP influences social behavior in chicken. Therefore, CGRP may have a prominent role in psycho-behavioral function and may be a target for action and/or side effects of antipsychotic drugs.

In 1983 a 29 amino acid peptide was isolated and sequenced from the small intestine of pigs and has been named galanin (GAL: Tatemoto et al., 1983). Synthesis of this peptide was reported shortly thereafter (Yajima et al., 1985, 1986). A cDNA library and nucleotide sequence of different clones encoding the preproGAL-precursor has been demonstrated for porcine medullary GAL (Rokaeus and Brownstein, 1986). Recently cDNA libraries of GAL have been published of rat hypothalamic tissue (Kaplan et al., 1988) and oestrogen induced prolactin secreting pituitary tumours (Vrontakis et al., 1987).

The distribution of melanin-concentrating hormone-like immunoreactivity (MCH-LI) in 41 microdissected brain and spinal cord regions was determined using radioimmunoassay with antibodies to salmon MCH. The highest concentration of MCH-LI was detected just ventral to the zona incerta (subzona incerta) (2923.2 fmol/mg protein). Very high concentrations of MCH-LI (greater than 1000 fmol/mg protein) were detected also in the nucleus of the diagonal band, medial forebrain bundle, posterior hypothalamic nucleus and medial mammillary nucleus. High concentrations of the peptide (between 500-1000 fmol/mg protein) were measured in 11 brain regions, including bed nucleus of stria terminalis, paraventricular nucleus, anterior hypothalamic nucleus, median eminence, parabrachial nucleus. Moderate concentrations of MCH-LI (between 250-500 fmol/mg protein) were measured in 16 brain regions, such as frontal cortex, central amygdaloid nucleus, medial septum, periventricular nucleus (preoptic) and nucleus of the solitary tract. Low concentrations of MCH-LI (less than 250 fmol/mg protein) were measured in 9 brain regions such as cortical areas, hippocampus, caudate nucleus and substantia nigra. Cervical spinal cord and neurointermediate lobe of the pituitary gland contain low concentrations of the peptide.

Recently it has been shown that calcitonin gene-related peptide (CGRP) and substance P (SP) are coexisting in central and peripheral nerve endings of sensory neurons. In the present study we compared the vasodepressor and plasma extravasating activity of CGRP with that of SP. Systemic administration of CGRP to pithed, vagotomized rats evoked a dose dependent, long lasting vasodilation accompanied by a parallel rise in heart rate. The tachycardic response to CGRP may indicate a direct positive chronotropic action on the heart since this effect could not be blocked by β-adrenoceptor blockade. For any equimolar dose the hypotensive effect of CGRP was much larger than that of SP. Both, CGRP and SP, showed a more pronounced decrease in mean arterial blood pressure after elevation of basal blood pressure levels by constant infusion of either phenylephrine, arginine-vasopressin, or angiotensin II. After systemic administration in equimolar doses CGRP was much less effective in producing plasma extravasation than SP. In conclusion, at equimolar doses CGRP is 10 times more potent than SP in producing vasodilatation but it possesses less than a third of the potency of equimolar doses of SP in producing plasma extravasation.

Plasma extravasation was induced in rats or guinea-pigs by intravenous injections of (1) substance P (SP), (2) the C-terminal SP-hexapeptide SP(6--11), (3) serotonin (5-HT), (4) histamine, (5) bradykinin, (6) capsaicin and (7) by antigen challenge. Plasma extravasation induced by SP, SP(6--11), by 5-HT and by capsaicin was, with few exceptions, observed in the same tissues. The effect of SP was not blocked by H1 and H2 histamine receptor antagonists. The effect of i.v. capsaicin was absent in capsaicin desensitized animals. Plasma extravasation upon i.v. SP, SP(6--11), 5-HT and capsaicin was seen in the skin and in all organs containing mucous membranes except the intestinal mucosa. Plasma extravasation by histamine, bradykinin, and antigen challenge of sensitized guinea-pig was, in addition, also observed in the stomach and intestine. Plasma extravasation and bronchoconstriction by antigen challenge with 20 micrograms/kg ovalbumin was completely blocked by combined H1 and H2 histamine receptor blockade. Both responses were reduced to about the half capsaicin desensitized guinea-pigs, although the reduction of the permeability response was statistically not significant in all organs. In conclusion, several substances including anaphylaxis induce protein leakage in many tissues with differing selective distribution patterns. Anaphylactic histamine release leads to protein leakage partly via activation of sensory neurons. SP is a likely mediator of neurogenic protein leakage in many organs.

Using antisera generated in rabbits against rat atriopeptin III [α-rANP(5–28)] and human α-atrial natriuretic polypeptide we mapped the distribution of atrial natriuretic factor-like immunoreactivity throughout the rat central nervous system. Cell bodies were observed in the telencephalon (nucleus interstitialis striae terminalis and between the amygdala centralis and medialis), throughout the diencephalon in all nuclei of the “anteroventral third ventricle”, the base of the hypothalamus, the subzona incerta area, the medial forebrain bundle and the medial habenula, in the mesencephalon (mamillary body, substantia nigra lateralis, dorsal and ventral parabrachial nuclei) and very sparse in the medulla oblongata along the fourth ventricle towards the vestibular nuclei, the nucleus tractus solitarii and nervi trigemini. Fibers were present wherever cell bodies were located. The highest relative densities were observed in the anteroventral third ventricle area and the medial habenula. Sparse fibers were also seen in the spinal cord (dorsal and ventral horn and around the central canal) and in the posterior pituitary. The predominance of the atrial natriuretic factor-like perikarya and fibers in the anteroventral third ventricle area suggests an involvement of this peptide in central blood pressure control.

Treatment of newborn rats with capsaicin was shown to cause a disappearance of corticotropin-releasing factor immunoreactive nerve fibers in the dorsal horn of the spinal cord (laminae I and II), the spinal trigeminal nucleus and tract, and the nucleus tractus solitarius, but not in the median eminence and the nucleus amygdaloideus centralis. Since it is well known that capsaicin acts selectively on primary sensory neurons of the C-fiber type, it is suggested that corticotropin-releasing factor is also located in peripheral sensory neurons, representing a novel peptidergic neuronal system, possibly involved in the modulation or transmission of peripheral nociceptive impulses, which is different from the capsaicin-resistant hypothalamoinfundibular corticotropin-releasing factor system.

The distribution of corticotropin releasing factor (CRF)-like immunoreactivity in the rat brain has been demonstrated by immunohistochemistry and radioimmunoassay using 4 different antisera. Two antisera were directed against synthetic ovine CRF, two antisera were directed against synthetic rat/human CRF. Immunohistochemistry revealed that there are discrete regions where CRF immunoreactive cell bodies are seen with all 4 antisera (e.g., the paraventricular nucleus, the dorsolateral tegmental nucleus) whereas there are cells observed only with one rat CRF antiserum (e.g., in the cortex) or terminal fields observed only with ovine CRF antisera (e.g., the spinal trigeminal tract, the substantia gelatinosa, the spinal cord). Radioimmunoassay showed different cross reactivity of the antisera with synthetic ovine or rat/human CRF and sauvagine, however, there was no cross reactivity with a variety of other peptides. Tissue values of CRF obtained by RIA of micropunched brain nuclei with the 4 antisera were frequently dissimilar suggesting that different antisera recognize different substances. High performance liquid chromatography and radioimmunoassay of brain tissue samples revealed that there is more than one form of CRF-like immunoreactivity present. There is indirect evidence that there exists at least one peptide in the rat brain, prominent in the medulla and the spinal cord, which cross reacts with antisera directed to ovine CRF only.

The distribution of immunoreactive (ir) atrial natriuretic peptides (ANPs) in 47 microdissected brain and spinal cord regions of the rat was determined by radioimmunoassay. The highest concentrations of ir-ANPs exist in the paraventricular nucleus and median preoptic nucleus (580.9 and 558.0 fmol/mg protein, respectively). High concentrations of ir-ANP (greater than 300 fmol/mg protein) are present in the interpeduncular nucleus, preoptic and hypothalamic periventricular nuclei, median eminence and organum vasculosum of the lamina terminalis. Moderate concentrations of ir-ANPs (between 100 and 300 fmol/mg protein) are found in 16 brain regions such as the bed nucleus of the stria terminalis, nucleus of the diagonal band, most of the hypothalamic nuclei, central gray, locus coeruleus and parabrachial nuclei. Low levels of ir-ANPs (less than 100 fmol/mg protein) exist in 22 brain regions including cortical areas, amygdala, caudate nucleus, nucleus accumbens, hippocampus, supraoptic nucleus, subfornical organ, medial mammillary nucleus, substantia nigra, dorsal raphe nucleus, cerebellum, nucleus of the solitary tract and others. Cervical spinal cord and neurointermediate lobe of pituitary gland contain low levels of ir-ANPs.

In rats treated with capsaicin (CAP) as neonates, galanin-like (GA) immunoreactivity is markedly decreased in the trigeminal ganglion and the dorsal root ganglia as well as in the superficial layers of the dorsal spinal cord (laminae I and II), the substantia gelatinosa, the nucleus and tractus of the spinal trigeminal nerve and the nucleus commissuralis. Since CAP causes selective degeneration of primary sensory neurons of the C-fiber type and type B-cells of sensory ganglia, it is concluded that GA in CAP-sensitive primary sensory neurons represents a novel peptidergic system possibly involved in the transformation or modulation of peripheral nociceptive impulses. This system differs from the CAP-resistant GA-like neurons in other brain areas.

A close spatial relationship between serotonin-containing mast cells and substance P-containing nerves was shown by immunohistochemistry using a combination of antisera specific for serotonin and substance P. This supports earlier morphological results suggesting an innervation of mast cells and pharmacological studies which postulate an influence of substance P on the release of histamine from mast cells.

1. The release of histamine and serotonin by neuropeptides and capsaicin was measured in the isolated perfused rat hindquarter preparation. 2. Substance P and two antagonistic peptides, [d-Pro2, d-Phe7, d-Trp9]-SP and [d-Pro2, d-Trp7,9]-SP, released histamine, the SP(4-11) and SP(6-11) analogues did not. VIP and somatostatin released histamine and also serotonin. No amines were released by bombesin. Thus, all amine releasing peptides possessed at least two basic charges. However, the histamine releasing activity of the neuropeptides tested did not correlate with their reported ability to cause vasodilatation and plasma extravasation. 3. The SP(4-11) and SP(6-11) analogues which did not release histamine caused plasma extravasation. It is concluded that SP causes plasma extravasation by a direct action on blood vessels. 4. Capsaicin released only serotonin but no histamine either in untreated rats and such densensitized with capsaicin as neonates. 5. In rats desensitized with capsaicin 4 days prior to the experiment the substance P induced histamine release was as high as in untreated controls; it was, however, absent in rats desensitized with capsaicin as neonates. It is assumed that the sensitivity of mast cells to substance P is lost after degeneration of substance P containing primary sensory fibers.

Summary Serotonin (5-HT) was measured by HPLC with electrochemical detection in walnuts (Juglans regia) during ontogeny and germination. Whereas 5-HT levels in catkins were low and did not increase during maturation, they increased in seed buds. The embryo reached highest amounts of 5-HT mainly during the final development of the seeds. Extremely high concentrations of 5-HT (1.6 mg g-1) were observed in the seed coat. In the seeds the 5-HT content remained stable during their storage over a period of at least one year. The level of 5-HT in the cotyledons doubled during germination in two months. In the shoots the apical buds contained as much 5-HT as the seed coat whereas the 5-HT concentration in the roots was extremely low. Besides the possibility that 5-HT is not more than an end product of tryptophan metabolism its high concentration in seed coats and apical buds suggest a function in the defense against other plants or pests.

Galanin (GAL) binding sites in coronal sections of the rat brain were demonstrated using autoradiographic methods. Scatchard analysis of 125I-GAL binding to slide-mounted tissue sections revealed saturable binding to a single class of receptors with a Kd of approximately 0.2 nM. 125I-GAL binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the following areas: prefrontal cortex, the anterior nuclei of the olfactory bulb, several nuclei of the amygdaloid complex, the dorsal septal area, dorsal bed nucleus of the stria terminalis, the ventral pallidum, the internal medullary laminae of the thalamus, medial pretectal nucleus, nucleus of the medial optic tract, borderline area of the caudal spinal trigeminal nucleus adjacent to the spinal trigeminal tract, the substantia gelatinosa and the superficial layers of the dorsal spinal cord. Moderate binding was observed in the piriform, periamygdaloid, entorhinal, insular cortex and the subiculum, the nucleus accumbens, medial forebrain bundle, anterior hypothalamic, ventromedial, dorsal premamillary, lateral and periventricular thalamic nuclei, the subzona incerta, Forel's field H1 and H2, periventricular gray matter, medial and superficial gray strata of the superior colliculus, dorsal parts of the central gray, peripeduncular area, the interpeduncular nucleus, substantia nigra zona compacta, ventral tegmental area, the dorsal and ventral parabrachial and parvocellular reticular nuclei. The preponderance of GAL-binding in somatosensory as well as in limbic areas suggests a possible involvement of GAL in a variety of brain functions.

1. Distension of the proximal jejunum by increasing the intraluminal pressure for short time periods causes a reflex response in blood pressure of anaesthetized rats which correlates with the magnitude of distension. The blood pressure response consists of a short initial rise followed by a marked decrease for the time of distension. 2. The absence of the depressor response in capsaicin desensitized rats indicates its mediation by C fibre afferents. These afferents are located within the periarterial mesenteric nerves. The depressor response was also elicited by stimulation of these nerves and abolished by local application of percain or capsaicin onto the mesenteric stalk. Vagal afferents were not involved in this depressor response as shown by bilateral vagotomy or by afferent vagus stimulation. 3. The depressor response is absent in spinal rats. Therefore, the location of the reflex centre is assumed to be supraspinal. Because it is augmented by naloxone and abolished by morphine in a naloxone reversible way it is regarded as a nociceptive reflex response. 4. The efferent side of the depressor response is unknown; cholinergic and α-adrenergic activation were excluded. 5. The initial pressor response to intestinal distension or to afferent periaterial mesenteric nerve stimulation persists in capsaicin desensitized rats excluding the involvement of C fibre afferents and in spinal rats indicating that the reflex centre is within the spinal cord. It is not diminished by morphine and therefore not a nociceptive response. Its inhibition by phentolamine suggests an α-adrenergic spinal response to intestinal distension. In control rats the pressor response is greatly overlapped by the much more pronounced depressor response.

The immunocytochemical localization of atrial natriuretic factor (ANF), galanin (GAL), and calcitonin gene-related peptide (CGRP) in specific subnuclei of the interpeduncular nucleus (IPN) was determined by immunocytochemistry in rats with and without intraventricular colchicine injection. ANF-positive processes were present within the ovoid regions of the rostral subnucleus, the dorsal lateral subnuclei, and were densely concentrated along the medial aspects of the lateral subnuclei in the caudal half of the IPN. GAL-positive processes were concentrated within the lateral subnuclei, in a narrow band extending over the central and intermediate subnuclei, and within the central subnuclei. GAL-positive cell bodies were present in a narrow band ventral to the rostral subnucleus, and in the ventrolateral corners of the caudal IPN. CGRP-positive processes were primarily localized within the dorsal lateral subnuclei and dorsal aspects of the lateral subnuclei. The presence of ANF, GAL and CGRP peptides within the IPN in patterns similar to previously described localizations of substance P, vasoactive intestinal peptide, serotonin and Leu-enkephalin provides a morphologic basis for modulation of complex physiological actions yet to be elucidated.

1. The basic peptide substance P causes histamine release from peritoneal mast cells of the rat in vitro whereas the closely related neutral peptides eledoisin and physalaemin do not. 2. Infusion of substance P (7.4 nmol min-1), but not of eledoisin (8.4 nmol min-1) or physalaemin (7.9 nmol min-1), into the rat hindquarter preparation caused a more than 4-fold increase of the histamine content in the venous outflow. The outflow of 5-HT remained unchanged under infusion of all three peptides. 3. No histamine depletion in the skin of the rat hind paw was observed following antidromic stimulation of the saphenous nerve or cutaneous application of mustard oil. Infusion of substance P (7.4 nmol min-1) caused a 47% depletion of histamine in the paw skin although only a small proportion of the infused substance P seemed to enter the tissue from the blood vessels. 4. The results further substantiate the view that substance P upon release from peripheral nerve endings induces release of histamine from cutaneous mast cells, a mechanism which contributes to neurogenic vasodilatation and plasma extravasation.

Frenkelia glareoli in Clethrionomys glareolus (exper.), demonstration of circulating antibodies against cyst homogenate using gel diffusion methods

The corticotropin releasing factor-like immunoreactivity (CRF-LI) of discrete areas of rat brain were measured following reserpine (2 mg/kg i.p. for 3 days) or chronic desmethylimipramine (DMI) (20 mg/kg i.p. for 14 days) treatment. Reserpine caused a 40% and 36% reduction in the (CRF-LI) of the median eminence (ME) and posterior pituitary respectively, while DMI caused a 61% rise in CRF-LI of the posterior pituitary only. The results support the role of monoaminergic regulation of CRF release from the ME and further suggest an interaction between monoaminergic and CRF neurons in the posterior pituitary.

Corticotropin releasing factor (CRF)-like immunoreactive neurons have been identified in the rat retina by immunohistochemical methods using antisera directed against ovine and rat CRF. CRF-like immunoreactivity was observed in both amacrine and ganglion cells which projected fine varicose processes to the inner plexiform layer of the retina. It is suggested that CRF may play a role in retinal function.