A phase Ib dose-escalation study of the MEK inhibitor trametinib in combination with the PI3K/mTOR inhibitor GSK2126458 in patients with advanced solid tumors.

Introduction This Phase Ib trial investigated the safety, tolerability, and recommended phase 2 dose for the pan-PI3K/mTOR inhibitor, GSK2126458 (GSK458), and trametinib combination when administered to patients with advanced solid tumors. Patients and Methods Patients with advanced solid tumors received escalating doses of GSK458 (once or twice daily, and continuous or intermittent) and trametinib following a zone-based 3 + 3 design to determine the maximum tolerated dose (MTD). Assessments included monitoring for adverse events and response, and evaluating pharmacokinetic (PK) measures. Archival tissue and circulating free DNA samples were collected to assess biomarkers of response in the PI3K and RAS pathways. Results 57 patients were enrolled onto the continuous dosing cohort and 12 patients onto an intermittent BID dosing cohort. Two MTDs were established for the continuous daily dosing: 2 mg of GSK458 with 1.0 mg of trametinib or 1.0 mg of GSK458 with 1.5 mg of trametinib; no MTD was determined in the intermittent dosing cohort. The most frequent adverse events were rash (74 %) and diarrhea (61 %). Dose interruptions due to adverse events occurred in 42 % of patients. No significant PK interaction was observed. One patient achieved partial response and 12 patients had stable disease >16 weeks. Mutations in RAS/RAF/PI3K were detected in 70 % of patients, but no pattern emerged between response and mutational status. Conclusion GSK458 plus trametinib is poorly tolerated, due to skin and GI-related toxicities. Responses were minimal, despite enrichment for PI3K/RAS pathway driven tumors, which may be due to overlapping toxicities precluding sufficient dose exposure.

Integrative population pharmacokinetic and pharmacodynamic dose finding approach of the new camptothecin compound namitecan (ST1968).

AIMS: Namitecan is a new camptothecan compound undergoing early clinical development. This study was initiated to build an integrated pharmacokinetic (PK) and pharmacodynamic (PD) population model of namitecan to guide future clinical development. METHODS: Plasma concentration-time data, neutrophils and thrombocytes were pooled from two phase 1 studies in 90 patients with advanced solid tumours, receiving namitecan as a 2 h infusion on days 1 and 8 every 3 weeks (D1,8) (n = 34), once every 3 weeks (D1) (n = 29) and on 3 consecutive days (D1-3) (n = 27). A linear three compartment PK model was coupled to a semiphysiological PD-model for neutrophils and thrombocytes. Data simulations were used to interrogate various dosing regimens and give dosing recommendations. RESULTS: Clearance was estimated to be 0.15 l h(-1), with a long terminal half-life of 48 h. Body surface area was not associated with clearance, supporting flat-dosing of namitecan. A significant and clinically relevant association was found between namitecan area under the concentration-time curve (AUC) and the percentage drop of neutrophils (r(2) = 0.51, P < 10(-4)) or thrombocytes (r(2) = 0.49, P < 10(-4)). With a target for haematological dose-limiting toxicity of <20%, the recommended dose was defined as 12.5 mg for the D1,8 regimen, 23 mg for the once every 3 week regimen and 7 mg for the D1-3 regimen. CONCLUSION: This is the first integrated population PK-PD analysis of the new hydrophilic topoisomerase I inhibitor namitecan, that is currently undergoing early clinical development. A distinct relationship was found between drug exposure and haematological toxicity, supporting flat-dosing once every 3 weeks as the most adequate dosing regimen.

Phase I study of olaparib in combination with liposomal doxorubicin in patients with advanced solid tumours.

BACKGROUND: Olaparib, an oral PARP inhibitor, has shown antitumour activity as monotherapy in patients with germline BRCA1/2 (gBRCA)-mutated breast and ovarian cancer. This study evaluated olaparib capsules in combination with liposomal doxorubicin (PLD) in patients with advanced solid tumours (NCT00819221). METHODS: Patients received 28-day cycles of olaparib, continuously (days 1-28) or intermittently (days 1-7), plus PLD (40 mg m(-2), day 1); seven olaparib dose cohorts (50-400 mg bid) were explored to determine the recommended dose. Assessments included safety, pharmacokinetics, pharmacodynamics and preliminary efficacy (objective response rate (ORR)). RESULTS: Of 44 patients treated (ovarian, n=28; breast, n=13; other/unknown, n=3), two experienced dose-limiting toxicities (grade 3 stomatitis and fatal pneumonia/pneumonitis (200 mg per 28-day cycle); grade 4 thrombocytopenia (400 mg per 7-day cycle)). The maximum tolerated dose was not reached using continuous olaparib 400 mg bid plus PLD. Grade ≥3 and serious AEs were reported for 27 (61%) and 12 (27%) patients, respectively. No major pharmacokinetic interference was observed between olaparib and PLD. The ORR was 33% (n=14 out of 42; complete response, n=3). A total of 13 responders had ovarian cancer: 10 were platinum-sensitive, 11 had a gBRCA mutation. CONCLUSIONS: Continuous/intermittent olaparib (up to 400 mg bid) combined with PLD (40 mg m(-2)) was generally tolerated and showed evidence of antitumour activity in ovarian cancer.

Seminars in clinical pharmacology: an introduction to MET inhibitors for the medical oncologist.

MET is a tyrosine kinase receptor for hepatocyte growth factor (HGF), primarily expressed on epithelial cells; the activation of MET induces several biological responses relevant for the development and growth of many human cancers. Several human malignancies present altered expression of MET and this is usually associated with poor prognosis and aggressive phenotype. The majority of MET inhibitors in clinical development target directly the receptor through the use of monoclonal antibodies (MAbs) or through small molecule inhibitors of MET kinase activity; small molecule inhibitors are very potent but less specific than MAbs. MET inhibitors are of great clinical interest because of the extensive crosstalk of the HGF/MET axis with many other signaling pathways, including growth factor-dependent pathways (like PI3K/AKT/mTOR,RAS/RAF/ERK) and vascular endothelial growth factor (VEGF) axis. In preclinical studies, the treatment with MET inhibitors could prevent or reverse resistance to inhibitors of growth factor-dependent signaling; this hypothesis is currently tested in phase III trials with anti-epidermal growth factor receptor (EGFR) inhibitors in non-small-cell lung cancer (NSCLC). Based on preclinical and preliminary clinical results, a rational strategy for the clinical development of MET antagonists should include a selection of the tumors with MET overexpression, the identification of prognostic/predictive biomarkers, the evaluation of combinations with anti-VEGF compounds.

Integration and sensory experience-dependent survival of newly-generated neurons in the accessory olfactory bulb of female mice.

Newborn neurons generated by proliferative progenitors in the adult subventricular zone (SVZ) integrate into the olfactory bulb circuitry of mammals. Survival of these newly-formed cells is regulated by the olfactory input. The presence of new neurons in the accessory olfactory bulb (AOB) has already been demonstrated in some mammalian species, albeit their neurochemical profile and functional integration into AOB circuits are still to be investigated. To unravel whether the mouse AOB represents a site of adult constitutive neurogenesis and whether this process can be modulated by extrinsic factors, we have used multiple in vivo approaches. These included fate mapping of bromodeoxyuridine-labelled cells, lineage tracing of SVZ-derived enhanced green fluorescent protein-positive engrafted cells and neurogenesis quantification in the AOB, in both sexes, as well as in females alone after exposure to male-soiled bedding or its derived volatiles. Here, we show that a subpopulation of SVZ-derived neuroblasts acquires proper neurochemical profiles of mature AOB interneurons. Moreover, 3D reconstruction of long-term survived engrafted neuroblasts in the AOB confirms these cells show features of fully integrated neurons. Finally, exposure to male-soiled bedding, but not to its volatile compounds, significantly increases the number of new neurons in the AOB, but not in the main olfactory bulb of female mice. These data show SVZ-derived neuroblasts differentiate into new functionally integrated neurons in the AOB of young and adult mice. Survival of these cells seems to be regulated by an experience-specific mechanism mediated by pheromones.

Clinical and pharmacological phase I evaluation of Exherin (ADH-1), a selective anti-N-cadherin peptide in patients with N-cadherin-expressing solid tumours.

BACKGROUND: Upregulation of N-cadherin promotes dysregulated cell growth, motility, invasiveness, plus maintenance of vascular stability and is associated with cancer progression in several human tumour types. N-cadherin is expressed also on tumour cells and the anti-N-cadherin cyclic pentapeptide ADH-1, tested in the present study, can exert a direct antitumour effect. PATIENTS AND METHODS: Adult patients with advanced solid malignancies expressing N-cadherin on tumour biopsies carried out in the previous 12 months received escalating i.v. doses of ADH-1 given weekly (initially for 3 of 4 weeks, then every week). Plasma pharmacokinetics (PK) was studied at cycle 1. Blood flow changes were assessed after first dosing in all patients treated in the initial regimen. RESULTS: In all, 129 patients were screened, 65 (50%) were N-cadherin positive, and 30 were enrolled. The doses ranged from 150 to 2400 mg/m(2); no maximum tolerated dose was reached. Treatment was well tolerated with asthenia as the most frequent adverse event. Two patients with ovarian cancer showed prolonged disease stabilisation while one patient with fallopian tube carcinoma achieved a mixed response. PK was linear in the range of doses tested. CONCLUSION: ADH-1 is the first anti-N-cadherin compound tested in humans. In N-cadherin-positive patients, ADH-1 showed an acceptable toxicity profile, linear PK and hints of antitumour activity in gynaecological cancers.

Developmental regulation of metabotropic glutamate receptor 1 splice variants in olfactory bulb mitral cells.

Alternative splicing of the metabotropic glutamate receptor 1 (mGluR1) receptor gene generates two major receptor isoforms, mGluR1a and mGluR1b, differing in intracellular function and distribution. However, little is known on the expression profiles of these variants during development. We examined the mRNA expression profile of mGluR1a/b in microdissected layers and acutely isolated mitral cells in the developing mouse olfactory bulb. This analysis showed that the two mGluR1 variants are differentially regulated within each bulb layer. During the first postnatal week, the mGluR1a isoform replaces GluR1b in the microdissected mitral cell layer (MCL) and in isolated identified mitral cells, coinciding with a developmental epoch of mitral cell dendritic reorganization. Although mGluR1a mRNA is expressed at high levels in both the adult external plexiform layer (EPL) and MCL, Western blotting analysis reveals a marked reduction of the mGluR1a protein in the MCL, where mitral cell bodies are located, and strong labeling in the EPL, which contains mitral cell dendrites. This suggests that there is increased dendritic trafficking efficiency of the receptor in adult. The temporal and spatial shift in mGluR1b/a expression suggests distinct roles of the mGluR1 isoforms, with mGluR1b potentially involved in the early mitral cell maturation and mGluR1a in dendritic and synapse function.

Maternal deprivation and early handling affect density of calcium binding protein-containing neurons in selected brain regions and emotional behavior in periadolescent rats.

Adverse early life experiences can induce neurochemical changes that may underlie modifications in hypothalamic-pituitary-adrenal axis responsiveness, emotionality and cognition. Here, we investigated the expression of the calcium binding proteins (CBPs) calretinin, calbindin and parvalbumin, which identify subpopulations of GABAergic neurons and serve important functional roles by buffering intracellular calcium levels, following brief (early handling) and long (maternal deprivation) periods of maternal separation, as compared with non-handled controls. CBP-expressing neurons were analyzed in brain regions related to stress and anxiety. Emotionality was assessed in parallel using the social interaction test. Analyses were carried out at periadolescence, an important phase for the development of brain areas involved in stress responses. Our results indicate that density of CBP-immunoreactive neurons decreases in the paraventricular region of deprived rats but increases in the hippocampus and lateral amygdala of both early-handled and deprived rats when compared with controls. Emotionality is reduced in both early-handled and deprived animals. In conclusion, early handling and deprivation led to neurochemical and behavioral changes linked to stress-sensitive brain regions. These data suggest that the effects of early experiences on CBP containing neurons might contribute to the functional changes of neuronal circuits involved in emotional response.

Carnosine-related dipeptides in the mammalian brain.

Carnosine and structurally related dipeptides are a group of histidine-containing molecules widely distributed in vertebrate organisms and particularly abundant in muscle and nervous tissue. Although many theories have been proposed, the biological function(s) of these compounds in the nervous system remains enigmatic. The purpose of this article is to review the distribution of carnosine-related dipeptides in the mammalian brain, with particular reference to some cell populations wherein these molecules have been demonstrated to occur very recently. The high expression of carnosine in the mammalian olfactory receptor neurons led to infer that this dipeptide could play a role as a neurotransmitter/modulator in olfaction. This prediction, which has not yet been fully demonstrated, does not explain the localization of carnosine-related dipeptides in other cell types, such as glial and ependymal cells. A recent demonstration of high carnosine-like immunoreactivity in the subependymal layer of rodents, an area of the forebrain which shares with the olfactory neuroepithelium the occurrence of continuous neurogenesis during adulthood, supports the hypothesis that carnosine-related dipeptides could be implicated in some forms of structural plasticity. However, the particular distribution of these molecules in the subependymal layer, along with their expression in glial/ependymal cell populations, suggests that they are not directly linked to cell migration or cell renewal. In the absence of a unified theory about the role of carnosine-related dipeptides in the nervous system, some common features shared by different cell populations of the mammalian brain which contain these molecules are discussed.

Corneal transplantation activity over 7 years: changing trends for indications, patient demographics and surgical techniques from the Corneal Transplant Epidemiological Study (CORTES).

This study aims to examine evolving indications and changing trends for corneal transplantation in Italy. Corneal transplantations performed with donor tissues distributed by the Veneto Eye Bank Foundation between 2002 and 2008 were prospectively evaluated. Of the 13,173 keratoplasties performed on 11,337 patients, 10,742 (81.5%) were penetrating (PK), 1644 (12.5%) were anterior lamellar (ALK), and 787 (6.0%) were endothelial (EK). Keratoconus (42.5%), regraft (18.9%), and pseudophakic bullous keratopathy (PBK, 11.9%) were the leading indications for PK, with keratoconus (69.6%) and regraft (6.5%) showing higher indications for ALK, whereas pseudophakic bullous keratopathy (50.1%) and regraft (18.7%) were the major indications for EK. There was an overall decrease observed in corneal grafting for keratoconus (P = .0048) and an increase for PBK (P = .0653) and regrafting (P = .0137). These indications differed by age and gender. The number of keratoplasties over 7 years was stable (P = .2394), although the annual number of PKs declined by 34.0% (P = .0250), ALKs began to rise from 2005 (P = .0600), whereas EKs showed a huge growth, with their number tripling in 2007 and further doubling in 2008 (P = .0004). Leading indications for keratoplasty showed similar data that have been reported elsewhere for Western countries over the past few decades, albeit with a higher percentage of keratoconus. However, the overall number of keratoplasties for keratoconus was in decline, whereas regraft keratopathy and PKs increased due to the application of the newer surgical techniques for corneal grafting. This highlights an important shift in managing corneal diseases toward the application of selective and more conservative surgeries and changes in indications in corneal transplantation.

Endothelin stimulates steroid secretion by frog adrenal gland in vitro: evidence for the involvement of prostaglandins and extracellular calcium in the mechanism of actin of endothelin.

Endothelin (ET-1) is a pleiotropic regulatory peptide which exerts multiple endocrine actions on the cardiovascular system. In the present study, we have investigated the possible role of ET-1 in the regulation of adrenocortical cells using perifused frog interrenal (adrenal) slices. Graded doses of ET-1 from 10(-11)-10(-9) M stimulated both corticosterone and aldosterone production in a dose-dependent manner. Repeated 20-min pulses of ET-1 (10(-9) M), given at the frequency of one pulse per 90 min, resulted in a marked reduction of the secretory response after the second pulse. Prolonged administration (3 h) of ET-1 induced a rapid increase in corticosterone and aldosterone output, followed by a gradual decline of corticosteroid secretion. Perifusion of frog adrenal tissue with ET-1 (10(-9) M) caused a significant increase in the release of prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha, the stable metabolite of the prostacyclin PGI2. The enhancement of PG biosynthesis preceded by 10 min the peak of corticosteroids. When repeated pulses of ET-1 were administered, a significant diminution of the production of PGE2 and 6-keto-PGF1 alpha was observed after the second pulse. The cyclooxygenase inhibitor indomethacin (5 microM) totally suppressed the stimulatory effect of ET-1 on corticosterone and aldosterone secretion; in contrast, indomethacin did not affect ACTH-evoked corticosteroid secretion. Perifusion of adrenal slices with a calcium-free solution or addition of cobalt (4 mM) induced total inhibition of the stimulatory effect of ET-1. These results demonstrate that ET-1 is a potent stimulator of corticosterone and aldosterone secretion from frog adrenal gland in vitro. Our data show that repeated or prolonged administration of ET-1 induces a rapid desensitization phenomenon. The data also indicate that ET-1-evoked corticosteroid secretion is mediated by an increase in PG biosynthesis and requires the presence of extracellular calcium.

Hepatocyte growth factor/scatter factor facilitates migration of GN-11 immortalized LHRH neurons.

The molecular cues regulating the migratory process of LHRH neurons from the olfactory placode into the brain are not well known, but gradients of chemotropic and chemorepellent factors secreted by the targets are likely to play a key role in guidance mechanisms. Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine inducing cell migration. It is involved in a variety of developmental processes through interaction with its receptor c-Met. Here we show that c-Met-antibody labels LHRH migrating neurons in the olfactory mesenchyme of E12 mouse and analyze the potential chemotropic effect of HGF/SF on two immortalized LHRH cell lines, GT1-7 and GN11, isolated from tumors developed in the hypothalamus and in the olfactory bulb, respectively. By RT-PCR analysis, Western blotting, and immunocytochemistry, we provide evidence for a high level of c-Met expression in GN11, but not in GT1-7, cells. In addition, HGF/SF treatment promotes specific migratory activity of GN11 cells, as demonstrated by collagen gel assay, time-lapse video microscopy, and Boyden's chamber experiments. Such promotion is inhibited by the neutralizing antibody. The data reported here represent the first direct evidence of a chemotactic effect of HGF/SF on immortalized LHRH neurons.

Localization, identification, and action of calcitonin gene-related peptide in the frog adrenal gland.

The localization of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the adrenal gland of the frog, Rana ridibunda, was examined by the indirect immunofluorescence technique. Using an antiserum directed against rat alpha-CGRP, the presence of a network of positive fibers was observed in the adrenal parenchyma. The immunoreactive material has been characterized by HPLC analysis combined with RIA quantification. The elution profile revealed the existence of a single form of CGRP exhibiting the same retention time as synthetic frog CGRP. The possible involvement of CGRP in the regulation of corticosteroid secretion was studied in vitro using a perifusion system for frog adrenal slices. Graded doses of frog CGRP (from 3 x 10(-9) to 3 x 10(-6) M) increased corticosterone and aldosterone secretion in a dose-dependent manner (ED50, 4.1 x 10(-8) M). Several other forms of CGRP, i.e. rat alpha-CGRP and beta-CGRP, and human alpha-CGRP and beta-CGRP, were also capable of enhancing steroid output, but frog CGRP was the most effective stimulator of steroidogenesis. Repeated administration of rat alpha CGRP induced a reproducible stimulation of corticosteroid secretion without any tachyphylaxis. Prolonged infusion of the peptide (3 h) caused a rapid increase in corticosteroid release, followed by a gradual decline of steroid secretion, suggesting the occurrence of a desensitization phenomenon. Rat alpha-CGRP also gave rise to a significant increase in corticosteroid release from acutely dispersed adrenal cells. These results show the presence of CGRP in fibers innervating the frog adrenal gland. The data also demonstrate that synthetic CGRP exerts a direct stimulatory effect on corticosteroid secretion. Taken together, these findings suggest that CGRP, released by nerve fibers in the adrenal tissue, can locally regulate corticosteroid secretion.

Ranakinin, a naturally occurring tachykinin, stimulates phospholipase C activity in the frog adrenal gland.

We have previously shown that the frog adrenal gland is innervated by a dense network of fibers containing ranakinin, one of the endogenous tachykinins in the amphibian Rana ridibunda, and we have found that ranakinin stimulates in vitro corticosteroid secretion by frog adrenal tissue. To elucidate the mechanism of action of ranakinin on the frog adrenal gland, we investigated the effect of ranakinin on cAMP formation and polyphosphoinositide metabolism. Incubation of frog adrenal explants with various tachykinins, including ranakinin, substance P, neurokinin A, or neurokinin B, did not produce any significant modification of cAMP concentrations. In contrast, ranakinin induced a time- and dose-dependent stimulation of inositol phosphate formation with a concomitant decrease in membrane polyphosphoinositides. Pretreatment of the tissue slices with the phospholipase C inhibitor U-73122 or with pertussis toxin completely abolished the stimulatory effect of ranakinin on inositol phosphate formation. Prolonged administration of U-73122 to perifused frog adrenal explants markedly attenuated the ranakinin-evoked stimulation of corticosterone and aldosterone secretion. Taken together, these data indicate that in the frog adrenal gland, ranakinin has no effect on the adenylyl cyclase system, but enhances polyphosphoinositide hydrolysis. The stimulatory action of ranakinin on inositol phosphate formation and corticosteroid secretion is mediated through activation of a phospholipase C positively coupled to a pertussis toxin-sensitive G protein.

Carnosine-like immunoreactivity in the central nervous system of rats during postnatal development.

In the nervous system of adult rodents, the aminoacylhistidine dipeptides (carnosine and/or homocarnosine) have been shown to be expressed in three main populations of cells: the mature olfactory receptor neurons, a subset of glial cells, and the neuroblasts of the rostral migratory stream. The current study analyzed the distribution of these dipeptides during postnatal development within the rat brain and spinal cord focusing on their pattern of appearance in the glial cells. Double staining methods using antibodies against carnosine and some markers specific for immature (vimentin) and mature (glial fibrillary acidic protein and Rip) glial cell types were used. Glial immunostaining for the aminoacylhistidine dipeptides appears starting from postnatal day 6 and reaches the final distribution in 3-week-old animals. The occurrence of carnosine-like immunoreactivity in astrocytes lags behind that in oligodendrocytes suggesting that, as previously demonstrated by in vitro studies, oligodendrocytes are also able to synthesize carnosine and/or homocarnosine in vivo. Furthermore, the spatiotemporal patterns observed support the hypothesis that the production of these dipeptides coincides with the final stages of glia differentiation. In addition, a strong carnosine-like immunoreactivity is transiently seen in a small population of cells localized in the hypothalamus and in the subfornical organ from birth to postnatal day 21. In these cells, carnosine-like immunoreactivity was not colocalized with any of the glial specific markers used. Moreover, no evidence for colocalization of carnosine and gonadotropin-releasing hormone (GnRH) has been observed.

Distribution of neuropeptide Y-like immunoreactivity in the brain of the lizard Gallotia galloti.

The distribution of neuropeptide Y (NPY)-like immunoreactivity was studied in the brain of the lizard Gallotia galloti, in order to gain insight into the comparative topography of this peptide. Antisera against both NPY and its C-terminal flanking peptide (C-PON) were used, demonstrating a general coexistence of both peptides, as described in other vertebrates. Most NPY-like immunoreactive (NPY-LI) cell bodies were observed in the telencephalon, specifically in various olfactory structures, all cortices, septum, basal ganglia (except for the globus pallidus), the nucleus of the diagonal band of Broca, the amygdaloid complex, and the bed nucleus of the anterior commissure. NPY-LI cells were also seen in the preoptic and hypothalamic regions and the dorsal thalamus (mainly in the perirotundal belt), as well as in the mesencephalic tegmentum (in the ventral tegmental area, the substantia nigra, and the retrorubral area). NPY-LI fibers and terminals were widely distributed in the brain. All visual and auditory neuropiles were densely innervated. Specially dense plexuses were seen in the nucleus accumbens, the ventral pallidum, the suprachiasmatic and ventromedial hypothalamic nuclei, the nucleus medialis thalami, the left habenula, and the central nucleus of the torus semicircularis. Our analysis shows that the distribution of NPY-like immunoreactivity in the forebrain of Gallotia largely resembles that of other vertebrates, whereas differences are mainly observed in the brainstem. The widespread distribution of NPY in the lizard brain suggests several modulatory functional roles, either in local-circuit systems of the forebrain, or in various limbic, neuroendocrine, and sensory pathways.

Immunohistochemical investigation of gamma-aminobutyric acid ontogeny and transient expression in the central nervous system of Xenopus laevis tadpoles.

The ontogeny of the gamma-aminobutyric acid (GABA)-positive neurons in the brain of Xenopus laevis tadpoles was investigated by means of immunohistochemistry, using specific antibodies both against GABA and its biosynthetic enzyme, glutamate decarboxylase (GAD). The results obtained with the two antisera were comparable. The GABA system differentiates very early during development. At stages 35/36, numerous GABA-positive neurons were seen throughout the prosencephalon and formed two main bilateral clusters within the lateral walls of the forebrain that ran caudally toward the hindbrain. Other GABA-immunolabeled cell bodies, together with a conspicuous network of GABAergic fibers, were seen in the posterior hypothalamus. In the spinal cord, the lateral marginal zone was GABA-positive, as were Rohon-Beard neurons, interneurons, and Kolmer-Agdhur cells. A very rich GABA innervation was observed in the pars intermedia of the pituitary. At stage 50, plentiful immunopositive neurons and fibers were found in the telencephalic hemispheres, the diencephalon, and the mesencephalon (optic tectum and tegmentum). By stage 54, the number of GABA-immunoreactive neurons in the posterior hypothalamus had decreased, so that, at stage 58, there were very few GABA-labeled cell bodies in the dorsolateral walls of the infundibulum, despite a strong GABAergic innervation within the median eminence and the pars intermedia. From stage 58 to stage 66, the distribution pattern was very similar to that described in the adult X. laevis and in other amphibian species. These results point to transient GABA expression within the hypothalamus, possibly related to either 1) a naturally occurring cell death or 2) a phenotypic switch.

Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain of the crested newt.

The newt brain represents a simplified model for the increasingly complex vertebrate neuronal organization. The localization of neuropeptide Y-like (NPY-like) containing neurons in the brain of Triturus cristatus was studied by means of indirect immunofluorescence, peroxidase-antiperoxidase, and avidin-biotin techniques using a highly specific antiserum. NPY-like positive cell bodies were observed in several areas, most notably in the telencephalon (primordium hippocampi and amygdaloid complex), the preoptic and suprachiasmatic areas, the hypothalamus, the dorsal thalamus, the tegmentum, and the rhombencephalon (laterolateral grey column and raphe area). Nerve fibres were particularly abundant in the pallium, striatum, septum, amygdaloid, preoptic neuropils, and pars intercalaris diencephali. Bundles of NPY-immunoreactive fibres also were visualized in the dorsal thalamus and in the posterior hypothalamus. The pars intermedia lacked any NPY-like positive fibres. Neuronal processes also were found in the tectum mesencephali and in the body of the cerebellum. A prominent NPY-like fibre network was observed in the octavolateralis. Concentrations of NPY measured by means of a specific radioimmunoassay were threefold higher in the hypothalamus (15.2 +/- 1.3 ng/mg proteins) than in the rhombencephalon (4.9 +/- 0.3) and the mesencephalon (4.3 +/- 0.2). The concentration found in the telencephalon was 2.1 +/- 0.3 ng/mg proteins. Sephadex G-50 gel chromatography of whole brain extracts indicated the presence of high molecular weight forms of NPY-like material in addition to the authentic peptide. Both amphibian and mammalian NPY peptides had an apparent molecular weight of 4,000 daltons, as evidenced by immunoblotting analysis. High-performance liquid chromatography demonstrated, however, that the newt peptide was slightly less hydrophobic than porcine NPY. The present findings indicate that NPY-immunoreactive neurons are widely distributed in the brain of urodeles. Our data indicate that the NPY molecule has been relatively well preserved during evolution.

Organization of tyrosine-hydroxylase immunopositive neurons in the brain of the crested newt, Triturus cristatus carnifex.

The localization of neurons, fibers, and terminals containing tyrosine hydroxylase (TH)-like immunoreactivity was studied in the brain of the crested newt by using an antiserum to rat phaeochromocytoma tyrosine hydroxylase. Immunoreactive cells and fibers were found in the spinal cord, the medulla oblongata (lateral periventricular areas), and the acousticolateral area. In the tegmentum mesencephali, two bilateral clusters of labelled cells were localized in the ventrolateral periventricular gray extending toward the caudal hypothalamus. In the hypothalamic tuberal lobes, the TH-like reactive neurons, frequently of CSF-contacting type, lined the dorsal wall of the lateral infundibular recesses. A thick network of TH-like reactive nerve fibers and terminals was observed in the perivascular zone of the median eminence and in the adenohypophysial pars intermedia. A number of labelled cell bodies were also found in the dorsal thalamus (pars intercalaris diencephali), the paraventricular organ, and the ventral wall of the preoptic recess. In the telencephalon, immunoreactive innervation was identified in the striatum, together with immunopositive cell bodies in the olfactory bulbs. The pattern of organization of TH-immunoreactive systems in the newt showed, except for some peculiarities (e.g., the labelled cell bodies in dorsal thalamus), close similarities to the arrangement typical of mammals.

Immunohistochemical localization of gonadotropin-releasing-hormone-associated peptide in the brain of the frog.

Gonadotropin-releasing-hormone (GnRH)-associated peptide (GnRH-AP) is a 56 amino acid neuropeptide derived from the GnRH prohormone. GnRH-AP corresponds to the C-terminal fragment flanking the GnRH peptide. Using an antiserum raised against human GnRH-AP [1-56], or against human GnRH, we have investigated the neuronal systems containing either peptide in the central nervous system of the frog Rana ridibunda by immunohistological techniques. A main group of GnRH-AP-containing perikarya was found in a dorsoventral orientation of the supra anterior preoptic area (SAPA) just in front of the preoptic recess. Fibers originating from these perikarya projected rostrally toward the medial septal nucleus and the diagonal band of Broca. A network of GnRH-AP-immunoreactive (ir) fibers runs caudally from the SAPA toward the ventral hypothalamus. A high density of GnRH-AP-ir terminals was found in the median eminence. A few positive fibers were detected in the neural lobe of the pituitary, particularly in the region bordering the pars intermedia. Labelling of consecutive sections by either GnRH-AP or GnRH antibodies showed that GnRH and GnRH-AP-like irs were contained in the same cells of the SAPA. The double-staining technique with electrophoretic elution confirmed the colocalization of GnRH and GnRH-AP within the same neurons. Such a coexistence indicates that frog GnRH originates from a high molecular weight precursor which is closely related to rat pro-GnRH. The relative preservation of the C-terminal sequence of the pro-GnRH during evolution suggests that GnRH-AP may possess intrinsic biological activity. The high density of GnRH-AP-containing neurons projecting through the external zone of the median eminence would support the concept that GnRH-AP is involved in the modulation of pituitary hormone secretion.