Helmut Wicht

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.

The brainstem pathologies of Parkinson's disease and dementia with Lewy bodies.

Parkinsons disease (PD) and dementia with Lewy bodies (DLB) are among the human synucleinopathies, which show alpha‐synuclein immunoreactive neuronal and/or glial aggregations and progressive neuronal loss in selected brain regions (eg, substantia nigra, ventral tegmental area, pedunculopontine nucleus). Despite several studies about brainstem pathologies in PD and DLB, there is currently no detailed information available regarding the presence of alpha‐synuclein immunoreactive inclusions (i) in the cranial nerve, precerebellar, vestibular and oculomotor brainstem nuclei and (ii) in brainstem fiber tracts and oligodendroctyes. Therefore, we analyzed the inclusion pathologies in the brainstem nuclei (Lewy bodies, LB; Lewy neurites, LN; coiled bodies, CB) and fiber tracts (LN, CB) of PD and DLB patients. As reported in previous studies, LB and LN were most prevalent in the substantia nigra, ventral tegmental area, pedunculopontine and raphe nuclei, periaqueductal gray, locus coeruleus, parabrachial nuclei, reticular formation, prepositus hypoglossal, dorsal motor vagal and solitary nuclei. Additionally we were able to demonstrate LB and LN in all cranial nerve nuclei, premotor oculomotor, precerebellar and vestibular brainstem nuclei, as well as LN in all brainstem fiber tracts. CB were present in nearly all brainstem nuclei and brainstem fiber tracts containing LB and/or LN. These findings can contribute to a large variety of less well‐explained PD and DLB symptoms (eg, gait and postural instability, impaired balance and postural reflexes, falls, ingestive and oculomotor dysfunctions) and point to the occurrence of disturbances of intra‐axonal transport processes and transneuronal spread of the underlying pathological processes of PD and DLB along anatomical pathways.

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.

CREB phosphorylation and melatonin biosynthesis in the rat pineal gland: Involvement of cyclic AMP dependent protein kinase type II

Abstract: Phosphorylation of cyclic AMP response element binding protein (CREB) at amino acid serine 133 appears as an important link between the norepinephrine (NE)‐induced activation of second messenger systems and the stimulation of melatonin biosynthesis. Here we investigated in the rat pineal gland: 1) the type of protein kinase that mediates CREB phosphorylation; and 2) its impact on melatonin biosynthesis. Immunochemical or immunocytochemical demonstration of serine133‐phosphorylated cyclic AMP regulated element binding protein (pCREB) and radioimmunological detection of melatonin revealed that only cyclic AMP‐dependent protein kinase (PKA) inhibitors suppressed NE‐induced CREB phosphorylation and stimulation of melatonin biosynthesis, whereas inhibitors of cyclic GMP‐dependent protein kinase (PKG), mitogen‐activated protein kinase kinase, protein kinase C, or calcium‐calmodulin‐dependent protein kinase (CaMK) were ineffective. Investigations with cyclic AMP‐agonist pairs that selectively activate either PKA type I or II link NE‐induced CREB phosphorylation and stimulation of melatonin biosynthesis to the activation of PKA type II. Our data suggest that PKA type II plays an important role in the transcriptional control of melatonin biosynthesis in the rat pineal organ.

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 β-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.

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.

An immunocytochemical investigation of glial morphology in the Pacific hagfish: radial and astrocyte-like glia have the same phylogenetic age

SummaryThis study attempts to reconstruct the early phylogenetic history of macroglial cells among craniates. Since glia does not fossilize, such a reconstruction must be based on a cladistic comparison of glial characters in the Recent craniate taxa (hagfishes, lampreys, and gnathostomes); however, there are only few data on glial morphology and none on glial immunocytochemistry in hagfishes. Therefore, we investigated the presence and localization of various macroglia-specific epitopes in the brain and spinal cord of the Pacific hagfish,Eptatretus stouti (Myxinoidea) by means of immunocytochemistry. Antibodies directed against S100-pfotein and vimentin showed no cross reactivity. Antibodies directed against glial fibrillary acidic protein and glutamine synthetase labelled various glial structures. Glial fibrillary acidic protein-like immunoreactivity was observed in ependymal cells with radially oriented processes in some regions. However, throughout the entire CNS, labelling of non-ependymal cells and their processes prevailed. The processes of these cells made occasional vascular contacts and they also made contacts with neuronal perikarya. Glutamine synthetase-like immunoreactivity was also found in some processes with radial orientation and in ependymal cells; but the antibody stained mainly non-ependymal cells which gave rise to a felt-like meshwork of interdigitating fine and very fine processes penetrating the neuropil of the entire brain. Additionally, there was labelling in the walls of blood vessels and in processes enwrapping individual neurons. The occurrence of glial fibrillary acidic protein- and glutamine synthetase-like immunoreactivity in non-ependymal glial elements in the brain of hagfishes and the relative scarcity of labelling in radial glial elements necessitates a re-interpretation of the evolutionary history of glial cells. Non-ependymal macroglia with immunocytochemical and morphological characters resembling typical (mammalian) astrocytes appears to be as primitive as the various forms of radial ependymal glia.

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. J. Comp. Neurol. 395:245–260, 1998.

An Immunohistochemical Study of the Telencephalon and the Diencephalon in a Myxinoid Jawless Fish, the Pacific Hagfish, Eptatretus stouti

The forebrain of adult hagfishes (jawless craniates, Myxinoidea) displays a unique morphology. The forebrain is thick-walled and well-differentiated cytoarchitecturally but lacks a well-developed ventricular system, and there is a pronounced compression of the entire brain along the longitudinal axis. This combination of characters obscures the boundaries between the major subdivisions of the forebrain; thus, some elementary morphological issues, such as the location of the pallial-subpallial and the telencephalic-diencephalic boundaries, have remained a matter of dispute over the past 100 years. In an attempt to resolve some of these issues, we investigated the chemoarchitecture of the forebrain of the Pacific hagfish, Eptatretus stouti. Because a number of previous studies of other craniates, mainly gnathostomes, have shown that the spatial distribution of some neuroactive substances and enzymes mirrors the main subdivisions of the forebrain, we localized acetylcholinesterase, enkephalins, substance P, tyrosine hydroxylase, and alpha-melanocyte stimulating hormone by means of histochemistry. Surprisingly, our data show that there are very limited chemoarchitectural similarities shared by hagfishes and other craniates. Some striking similarities occur in the organization of the catecholaminergic systems. Hagfishes, as well as other craniates, possess catecholaminergic cell groups at the ventral junction of the mesencephalon and diencephalon that give rise to a catecholaminergic innervation of the basal forebrain. The distribution of all other substances examined is dissimilar to that found in other craniates. In particular, there are many neurons positive for acetylcholinesterase in the pallium; the subpallium contains relatively small amounts of neuroactive peptides; and the highest densities of structures positive for neuroactive peptides and acetylcholinesterase occur in the central prosencephalic nucleus. Therefore, a comparative chemoarchitectural analysis proved to be of limited value in revealing homologies among cell groups of hagfishes, lampreys, and gnathostomes. We conclude that some chemoarchitectural features that appear to be well conserved within gnathostomes, such as the histochemical differences between the pallium and subpallium, may result from evolutionary change early in craniate history.

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

SummaryThe 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 distinet 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.