Bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons.

Bone morphogenetic proteins (BMPs) induce autonomic neurogenesis in neural crest cultures and stimulate sympathetic neuron development when overexpressed in vivo. We demonstrate that inhibition of BMPs in the chick embryo bythe BMP antagonist Noggin prevents sympathetic neuron generation. In Noggin-treated embryos, the noradrenergic marker genes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), panneuronal neurofilament 160 (NF160) and SCG10 genes, and the transcriptional regulators Phox2b and Phox2a are not expressed in sympathetic ganglia. Whereas initial ganglion development is not affected, the expression of the basic helix-loop-helix transcription factor Cash-1 is strongly reduced. These results demonstrate that BMPs are essential for sympathetic neuron development and establish Cash-1 and Phox2 genes as downstream effectors of BMPs in this lineage.

Dynamics of core body temperature cycles in long-term measurements under real life conditions in women.

Studies under real life conditions become more and more relevant in chronobiological and chronomedical research. The present study aims to analyze one of the most prominent biological rhythms: the core body temperature (CBT) rhythm in the real world outside the laboratory. CBT was recorded continuously in 37 healthy women (age between 21 and 44 years, median 29 years) with a newly developed intravaginal temperature sensor for up to 102 days. Sleep logs were available from 23 participants. To quantify the daily dynamics of each individual CBT-curve, novel measurement parameters are introduced which permit the quantification of the phase and shape of the CBT rhythms as well as their relation to the sleep-wake cycle. In addition to the classical phase markers (i.e. nadir and peak), the daily curves were segmented into quartiles by introducing the t25/t50/t75-values which can be used as phase and shape markers. At variance to previous studies, a conspicuous day-to-day variation was shown not only for the time point of the peak, but also for the time point of the nadir. However, the t-values, particularly the t75-value were relatively closely locked to external time and thus represent more reliable phase markers than the nadir. The (variable) time point of the nadir determined the period length, phase and shape of the subsequent CBT cycle. If a nadir occurred close to the wake-up time, the following cycle was considerably shorter than 24 hours, while a nadir distant from the wake-up time was followed by a longer cycle. Thus, the period lengths of the daily CBT cycles of each individual were characterized by an "expand/contract" rhythm. The analyses of the novel phase markers (t25/t50/t75) of the CBT curves allowed to identify "early" and "late" participants who may differ in their phase-response curves with regard to the entraining effect of light. In addition, the novel phase markers mirrored the different social entrainment conditions on weekends and workdays.

Topologic and connectional analysis of the dorsal thalamus of Triturus alpestris (amphibia, urodela, salamandridae).

A morphological and connectional analysis was performed on the dorsal thalamus of the alpine newt, Triturus alpestris. We have used a graphic reconstruction technique for the evaluation of the connectional (HRP) data. On the basis of these reconstructions, we propose a subdivision of the salamandrid dorsal thalamus into subhabenular, anteroventral, and posterodorsal zones. Each of these zones is defined by its telencephalic projections ("ascending thalamofugal systems"). The posterodorsal zone projects to the striatum, the anteroventral zone to the pallium. The subhabenular zone projects to the subpallial telencephalon and to the tegmentum. This zonal subdivision allows a more detailed comparison of the salamandrid dorsal thalamic features with ranid dorsal thalamic structures. We compare our dorsal thalamic zones to the ones proposed by Herrick (J. Comp. Neurol. 62:239-261, '35, The Brain of the Tiger Salamander. Chicago: The University of Chicago Press, '48). Furthermore, using the same reconstructive technique, we undertook an analysis of the spatial relations of various inputs to the salamandrid dorsal thalamus ("thalamopetal systems"). Besides the well-known retinal inputs, we identified the tectum and the tegmentum as sources of inputs to the thalamus. We provide evidence that there is no extensive multi- or unimodal overlap of these thalamopetal systems.

Telencephalic connections in the Pacific hagfish (Eptatretus stouti), with special reference to the thalamopallial system.

The pallium of hagfishes (myxinoids) is unique: It consists of a superficial "cortical" mantle of gray matter which is subdivided into several layers and fields, but it is not clear whether or how these subdivisions can be compared to those of other craniates, i.e., lampreys and gnathostomes. The pallium of hagfishes receives extensive secondary olfactory projections (Wicht and Northcutt [1993] J. Comp. Neurol. 337:529-542), but there are no experimental data on its nonolfactory connections. We therefore investigated the pallial and dorsal thalamic connections of the Pacific hagfish. Injections of tracers into the pallium labeled many cells bilaterally in the olfactory bulbs. Other pallial afferents arise from the contralateral pallium, the dorsal thalamic nuclei, the preoptic region, and the posterior tubercular nuclei. Descending pallial efferents reach the preoptic region, the dorsal thalamus, and the mesencephalic tectum but not the motor or premotor centers of the brainstem. Injections of tracers into the dorsal thalamus confirmed the presence of reciprocal thalamopallial connections. In addition, these injections revealed that there is no "preferred" pallial target for the ascending thalamic fibers; instead, ascending thalamic and secondary olfactory projections overlap throughout the pallium. The mesencephalic tectum and tegmentum, which receive afferents from a variety of sensory sources, are interconnected with the dorsal thalamus; thus, ascending nonolfactory sensory information may reach myxinoid pallia via a tectal-thalamic-telencephalic route. A comparative analysis of pallial organization reveals that the subdivisions of the pallium in gnathostomes (i.e., medial, dorsal, and lateral pallia) cannot be recognized with certainty in hagfishes.

Secondary olfactory projections and pallial topography in the Pacific hagfish, Eptatretus stouti.

The extent of the secondary olfactory projections shows great variation among different groups of craniates. Gnathostomes typically display restricted secondary olfactory projections, whereas lampreys have more extensive projections. Any attempt to determine the phylogenetic polarity of these characters, that is, to decide which is primitive and which is derived, requires an investigation of the secondary olfactory system in the sister group of lampreys and gnathostomes, the hagfishes. Therefore the secondary olfactory projections of the Pacific hagfish, Eptatretus stouti, were traced with the use of horseradish peroxidase and the lipophilic fluorescent tracing compound DiI. The projections are bilateral and massive to all pallial areas and the septum, moderate to the striatum, and relatively weak to the preoptic and infundibular regions of the hypothalamus, reaching caudally to the diencephalic-mesencephalic boundary. Afferents to the olfactory bulb arise from the pallium, the preoptic area, and the ventral thalamus. We compare the secondary olfactory projections in hagfishes with those in lampreys and in gnathostomes, and we conclude that the presence of extensive secondary olfactory projections is a primitive character of craniate brains.

Distribution of gonadotropin-releasing hormone immunoreactivity in the brain of the Pacific hagfish, Eptatretus stouti (Craniata: Myxinoidea).

The distribution of gonadotropin-releasing hormone (GnRH)-like immunoreactivity in the brain of a myxinoid, the Pacific hagfish (Eptatretus stouti), was investigated via immunohistochemistry, including the use of six different antisera. In the diencephalon, immunoreactive cell bodies were found in two systems: the infundibular hypothalamus, a neuromodulatory nucleus with diffuse projections of varicose fibers to most areas of the brain, and a primarily preoptic system of putatively hypophysiotropic neurons that projects to the neurohypophysis. Some potential neurovascular and CSF contacts were also identified. These findings are consistent with those of similar studies in other craniates and suggest that a preoptic hypophysiotropic system may be present in all craniates. We therefore tentatively accept the homology of this system in hagfish and vertebrates. The homology of the distributed hypothalamic system is more dubious. It may be homologous to a caudal GnRH system of modulatory neurons found in many vertebrates. Antiserum PBL-49 displays a differential affinity for the two systems, indicating that the two systems differ in the amount or identity of the immunoreactive substance. We suggest that the two systems have distinct functions in hagfish. The primitive function of GnRH-like molecules in craniates may have thus been both neuromodulatory and hypophysiotropic. These findings also indicate that the brain-pituitary axis of hagfish is more similar to that of vertebrates than has been previously suggested.

A semiquantitative image-analytical method for the recording of dose-response curves in immunocytochemical preparations.

Knowledge about intracellular signal transduction cascades is largely based on investigations of cultured cells whose responses to different stimuli are typically quantified via RIA, ELISA, or immunoblots. These techniques, which require relatively large amounts of biological material, are performed with homogenized cells and therefore do not allow localization of the molecules under investigation. We describe a protocol for recording dose-response curves directly from immunocytochemical preparations using rat pinealocytes as a model system. The cells were exposed to beta-adrenergic stimuli inducing the phosphorylation of the transcription factor CREB (mediated by PKA), an increase in ICER protein levels, and synthesis and release of melatonin. Melatonin concentrations were determined by ELISA. cPKA, phosphorylated CREB, and ICER were demonstrated by immunocytochemistry and immunoblots. Dose-response curves were recorded by measuring the integrated density of the immunoreactive sites with an image analysis program. Dose-response curves from immunoblots and immunocytochemical preparations showed almost identical dynamics, validating the immunocytochemical approach, which minimizes the amount of biological material needed for such studies, allows combined quantification and localization of biomolecules, and may even be more sensitive than immunoblotting.

Distribution of myocardial beta-adrenoceptor subtypes and coupling to the adenylate cyclase in children with congenital heart disease and implications for treatment.

In congestive heart failure, down-regulation of myocardial beta-adrenoceptors (beta-AR) due to an elevated sympathetic tone is well known. In infancy and childhood, heart failure is usually related to congenital heart disease (CHD). Therefore, 71 samples of right atrial tissue of infants and children with CHD undergoing cardiac surgery were studied for beta-adrenoceptor density and distribution of the beta 1-/beta 2-AR subtypes. In 49 cases, the coupling of the beta-AR to the adenylate cyclase (AC) was examined. In a further study of 19 myocardial samples, AC was selectively stimulated with beta 1- or beta 2-AR whereas the other subtype was blocked by an antagonist. The following results were obtained: (1) Infants and children with severe acyanotic or cyanotic CHD had severely reduced beta-AR densities. (2) In most of the cases, the beta-AR down regulation is beta 1-subtype selective, but in critically ill newborns with congenital aortic valve stenosis or transposition of the great arteries, there is additional significant beta 2-AR down-regulation. In Fallot patients treated with the beta-antagonist propranolol, a significant increased beta-AR number compared with untreated Fallot patients was found. (3) beta-Adrenoceptor reduction in CHD is correlated with elevated noradrenaline plasma levels, thus proving a sympathetic dysregulation. (4) In CHD with moderate hemodynamic load, beta 2-AR coupling to AC was markedly more efficient than beta 1-AR coupling. The small number of myocardial beta 2-AR produced most of the cyclic adenosine monophosphate. (5) In severe acyanotic and cyanotic CHD, a partial decoupling of the beta 2-AR to the AC occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Two thalamo-telencephalic pathways in a urodele, Triturus alpestris.

Ascending thalamo-telencephalic projection systems have been investigated in an urodele, Triturus alpestris, using the horseradish peroxidase technique. Two separate dorsal thalamic projections onto the telencephalon have been identified; one arises from the posterior dorsal thalamus and terminates in the ipsilateral striatum, the other originates from anterior dorsal thalamic cells and reaches the medial pallium and a part of the dorsal pallium bilaterally. Both systems, which are spatially well segregated, might carry visual information to the telencephalon, as the posterior dorsal thalamus receives tectal, and the anterior dorsal thalamus direct retinal input. The urodele projection scheme as described here shows great similarities to the one described in anurans, although there are remarkable cytoarchitecture differences between the anuran and the urodele thalamus.

A possible homologue of the suprachiasmatic nucleus in the hypothalamus of lampreys (Lampetra fluviatilis L.).

This study in lampreys (Lampetra fluviatilis L.) is concerned with the phylogenetic age of the suprachiasmatic nucleus of the hypothalamus which in mammals receives input from the retinohypothalamic tract and acts as circadian pacemaker. Tracing experiments with the fluorescent dye DiI demonstrated a retinohypothalamic projection in lampreys which terminates in a distinct area of the hypothalamus corresponding to the infrachiasmatic part of the nucleus of the postoptic commissure. Immunocytochemical investigations showed vasotocinergic neurons and a relatively high concentration of neuropeptide Y-, substance P- and serotonin-immunoreactive nerve fibers in this region. These hodological and immunocytochemical data suggest that the infrachiasmatic part of the nucleus of the postoptic commissure of lampreys might be homologous to the suprachiasmatic nucleus of other craniates.

Afferent and efferent connections of the thalamic eminence in the axolotl, Ambystoma mexicanum.

Afferent and efferent connections of the thalamic eminence of the axolotl were determined using the fluorescent compound DiI as a tracer. The thalamic eminence is connected reciprocally with a number of telencephalic and diencephalic areas, particularly with the medial pallium, the amygdala and the preoptic region. Efferent connections are widespread throughout the ipsilateral diencephalon. These findings are discussed in relation to the homology of this nucleus, especially its homologue in agnathan brains.

Signal transduction in the rodent pineal organ. From the membrane to the nucleus.

The rodent pineal organ transduces a photoneural input into a hormonal output. This photoneuroendocrine transduction leads to highly elevated levels of the hormone melatonin at night-time which serves as a message for darkness. The melatonin rhythm depends on transcriptional, translational and posttranslational regulation of the arylalkylamine-N-acetyltransferase, the key enzyme of melatonin biosynthesis. These regulatory mechanisms are fundamentally linked to two second messenger systems, namely the cAMP- and the Ca(2+)-signal transduction pathways. Our data gained by molecular biology, immunohistochemistry and single-cell imaging demonstrate a time- and substance-specific activation of these signaling pathways and provide a framework for the understanding of the complex signal transduction cascades in the rodent pineal gland which in concert not only regulate the basic profile but also fine-tune the circadian rhythm in melatonin synthesis.

Observations on the development of the lateral line system in the Pacific hagfish (Eptatretus stouti, Myxinoidea).

We investigated the development of the lateral line system of the Pacific hagfish, Eptatretus stouti, using three-dimensional reconstructions from serial sections. Adult hagfishes possess a number of densely innervated skin grooves of unknown function, and these grooves do not contain typical lateral line receptors (i.e. neuromasts). However, three separate lateral line placodes appear to be present during development and these placodes give rise to groups of neuromast primordia. Unlike in other craniates, the neuromast primordia do not develop into neuromasts, but they apparently transform into the skin grooves of adults.

Early postembryonic neural development in the zebrafish: a 3-D reconstruction of forebrain proliferation zones shows their relation to prosomeres.

Based on a section-by-section analysis of the morphology (combined silver/Nissl stain) and of the distribution of proliferation zones (immunohistochemical detection of the proliferating cell nuclear antigen) in the zebrafish (Danio rerio) forebrain at 5 days postfertilization, we created a three-dimensional reconstruction of proliferation zones of that developmental stage. The resulting model visualizes the size, number, location and morphology of forebrain proliferation zones. The latter foreshadow closely adult neuroanatomical forebrain entities. Furthermore, the detailed distribution of proliferation zones in the posterior forebrain - but not in the more anterior secondary prosencephalon - supports a segmental prosomeric organization.

The brains of lampreys and hagfishes: characteristics, characters, and comparisons.

A comparison of the brains of lampreys and hagfishes is carried out in an attempt to reconstruct the anatomy of the brain of the last common ancestor of craniates: i.e., the morphotype of the craniate brain. This brain consisted of tel-, di-, mes-, and rhombencephalic divisions; the presence of a metencephalic/cerebellar division is questionable. All major sensory and motor systems (with the possible exception of the oculomotor system) that are typical of craniates were present in the morphotype. There were extensive bilateral secondary olfactory projections to the telencephalic pallium, as well as bilateral retinofugal projections to diencephalic, pretectal, and tectal targets. The rhombencephalon was subdivided into dorsal (viscero- and somatosensory) and ventral (branchiomotor) zones. The spinal cord projected to most rhomb- and mesencephalic areas; in turn, it received descending projections from the mes- and rhombencephalic reticular formation and from the octaval nuclei. The reconstruction of such a morphotype depends on the recognition of characters that are plesiomorphic for craniates, as determined by comparative, cladistic analysis. In many cases (gross morphological, topological and cytoarchitectural characters), such an analysis cannot be carried out, because characters that appear as discrete entities in one taxon are lacking or difficult to delineate from other characters in other taxa. The distribution of these characters in lampreys and hagfishes, and the developmental mechanisms that brought them about, offer a challenging problem in evolutionary neurobiology.

FMRFamide-like immunoreactivity in the brain of the Pacific hagfish, Eptatretus stouti (Myxinoidea).

The distribution of FMRFamide-like immunoreactivity was investigated in the brain of a myxinoid, the Pacific hagfish, Eptatretus stouti, by means of immunocytochemistry. In the forebrain, labelled cell bodies occurred in the infundibular nucleus of the hypothalamus and some closely adjacent nuclei. Labelled fibers formed a diffuse network in the forebrain, but there was no evidence for the presence of intracerebral ganglionic cells of the terminal nerve or a central projection of the terminal nerve. In the hindbrain, a group of labelled cells was found in the trigeminal sensory nucleus. A distinct terminal arborization occurred in the ventrally adjacent nucleus A of Kusunoki and around the nuclei of the branchial motor column. These findings suggest that FMRFamide may play a role in the central control of branchiomotor activity.

Afferent and efferent connections of the lateral and medial pallia of the silver lamprey.

Injections of the carbocyanine dye, DiI, into the lateral pallium of the silver lamprey reveal that this pallial region receives bilateral inputs from the olfactory bulbs, dorsomedial telencephalic neuropil, and the habenular nuclei, and ipsilateral inputs from the septum, preoptic area, medial pallium, thalamus, and, possibly, the striatum. The efferent projections of the lateral pallium form dorsal (olfacto-habenular tract of Heier) and ventral (olfacto-thalamic and hypothalamic tracts of Heier) bundles. The dorsal bundle terminates ipsilaterally in the dorsal pallium, medial pallium, habenular nuclei, and pretectum and contralaterally in the habenular nuclei and, possibly, the dorsal pallium. The ventral bundle terminates ipsilaterally in the septum, striatum, and preoptic areas and bilaterally within the hypothalamus. Injections of DiI into the medial pallium reveal bilateral inputs to this pallial formation from the olfactory bulbs, the dorsomedial telencephalic neuropil, septum, habenular nuclei, thalamic nuclei, preoptic area and hypothalamus, as well as ipsilateral inputs from the lateral pallium, dorsal isthmal grey and midbrain tegmentum. The efferent projections of the medial pallium form dorsal, ventral and descending bundles. The dorsal bundle terminates ipsilaterally in the dorsal and lateral pallia and in the olfactory bulb. The ventral bundle terminates ipsilaterally in the dorsal pallium and bilaterally within the lateral pallium and in preoptic and hypothalamic areas. The descending bundle terminates bilaterally in thalamic and hypothalamic areas and in the pretectum and optic tectum. These data support a number of earlier hypotheses concerning pallial homologues in lampreys and other vertebrates but suggest that the earlier hypothesis of an olfactory origin of the telencephalon of craniates should be rejected.

Retinofugal and retinopetal projections in the Pacific hagfish, Eptatretus stouti (Myxinoidea).

The retinofugal and retinopetal connections of the Pacific hagfish were determined using the fluorescent compound DiI as an in vitro tracer. The retinofugal connections were found to be bilateral and to reach preoptic, thalamic and pretectal, as well as tectal, targets. No distinct basal optic root was found. A retinopetal projection in hagfishes is described here for the first time. It originates from two cells groups in the rostral mesencephalic tegmentum. These retinal connections in hagfishes are compared to the organization of the visual system in the second Recent group of jawless fishes, the lampreys. Both visual systems are similar in the number and location of the targets of retinal fibers. Furthermore, there is a striking similarity in the retinopetal systems. Major differences are found in the organization of the optic tract. These observations allow the identification of some cell groups not previously recognized in myxinoid brains or interpreted differently. Finally, the findings are discussed in relation to visually guided behavior in hagfishes.

[Pineal body in vertebrates: a model for investigations of receptor and effector mechanisms of neuronal systems]. / Das Pinealorgan der Wirbeltiere: ein Modell für Untersuchungen von Rezeptor- und Effektormechanismen in neuronalen Systemen.

Cell and molecular biological investigations have greatly contributed to our understanding of receptor and effector mechanisms in sensory, neuronal, and endocrine cells. A fascinating aspect of this line of research is how such mechanisms have evolved and how they interact with each other. As shown in this contribution, the vertebrate pineal organ is an interesting model to study these problems, because it undergoes a conspicuous transformation during phylogeny, comprises two well-characterized receptor mechanisms (photoreception and adrenoreception), and acts upon its targets via neuronal and neuroendocrine signals.