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Growth/differentiation factor−15 (Gdf-15) is a member of the transforming growth factor-β (Tgf-β) superfamily and has been shown to be a potent neurotrophic factor for midbrain dopaminergic (DAergic) neurons both in vitro and in vivo. Gdf-15 has also been shown to be involved in inflammatory processes. The aim of this study was to identify the role of endogenous Gdf-15 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson’s disease (PD) by comparing Gdf-15 +/+ and Gdf-15 −/− mice. At 4 days and 14 days post-MPTP administration, both Gdf-15 +/+ and Gdf-15 −/− mice showed a similar decline in DAergic neuron numbers and in striatal dopamine (DA) levels. This was followed by a comparable restorative phase at 90 days and 120 days, indicating that the absence of Gdf-15 does not affect the susceptibility or the recovery capacity of the nigrostriatal system after MPTP administration. The MPTP-induced microglial and astrocytic response was not significantly altered between the two genotypes. However, pro-inflammatory and anti-inflammatory cytokine profiling revealed the differential expression of markers in Gdf-15 +/+ and Gdf-15 −/− mice after MPTP administration. Thus, the MPTP mouse model fails to uncover a major role of endogenous Gdf-15 in the protection of MPTP-lesioned nigrostriatal DAergic neurons, in contrast to its capacity to protect the 6-hydroxydopamine-intoxicated nigrostriatal system.

The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR-124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non-neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growth-factor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal cells.

The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood-brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor-2 (FGF-2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF-2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild-type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight-junction proteins and we observed no difference in the ultrastructure of the tight-junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight-junctions or for basic permeability properties and function of the blood-brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201-1212, 2016.

Growth/differentiation factor-15 (GDF-15) is a distant member of the transforming growth factor beta (TGF-β) superfamily. It is widely distributed in the nervous system, where it has been shown to play an important role in neuronal maintenance. The present study investigates the role of endogenous GDF-15 in sciatic nerve (SN) lesions using wild-type (WT) and GDF-15 knock-out (KO) mice. SN of 5-6-month-old mice were crushed or transected. Dorsal root ganglia (DRG) and nerve tissue were analyzed at different time points from 6 h to 9 weeks post-lesion. Both crush and transection induced GDF-15 mRNA and protein in the distal portion of the nerve, with a peak at day 7. DRG neuron death did not significantly differ between the genotypes; similarly, remyelination of regenerating axons was not affected by the genotype. Alternative macrophage activation and macrophage recruitment were more pronounced in the KO nerve. Protrusion speed of axons was similar in the two genotypes but WT axons showed better maturation, as indicated by larger caliber at 9 weeks. Furthermore, the regenerated WT nerve showed better performance in the electromyography test, indicating better functional recovery. We conclude that endogenous GDF-15 is beneficial for axon regeneration following SN crush.

The pan-neurotrophin receptor p75NTR is expressed in the adult brain in a discrete pattern. Although numerous studies have addressed its implications for hippocampal functions, the generated sets of data are surprisingly conflicting. We have therefore set out to re-investigate the impact of a deletion of the full-length p75NTR receptor on several parameters of the dentate gyrus (DG), including neurogenesis and hippocampus-related behavior by using p75NTR(ExIII) knockout mice. Moreover, we investigated further parameters of the DG (cholinergic innervation, dendritic spines). In addition, we analyzed on the morphological level the impact of aging by comparing adult and aged p75NTR(ExIII) mice and their age-matched littermates. Adult (4-6 months old), but not aged (20 months old), p75NTR(ExIII) knockout mice display an enhanced volume of the DG. However, adult neurogenesis within the adult DG was unaffected in both adult and aged p75NTR(ExIII) knockout mice. We could further demonstrate that the change in the volume of the DG was accompanied by an increased cholinergic innervation and increased spine densities of granule cells in adult, but not aged p75NTR deficient mice. These morphological changes in the adult p75NTR deficient mice were accompanied by specific alterations in their behavior, including altered behavior in the Morris water maze test, indicating impairments in spatial memory retention.

The neurotrophin receptor p75NTRhas been implicated in mediating neuronal apoptosis after injury to the CNS. Despite its frequent induction in pathologic states, there is limited understanding of the mechanisms that regulate p75NTRexpression after injury. Here, we show that after focal cerebral ischemiain vivoor oxygen–glucose deprivation in organotypic hippocampal slices or neurons, p75NTRis rapidly induced. A concomitant induction of proNGF, a ligand for p75NTR, is also observed. Induction of this ligand/receptor system is pathologically relevant, as a decrease in apoptosis, after oxygen–glucose deprivation, is observed in hippocampal neurons or slices after delivery of function-blocking antibodies to p75NTRor proNGF and inp75NTRandngfhaploinsufficient slices. Furthermore, a significant decrease in infarct volume was noted inp75NTR−/− mice compared with the wild type. We also investigated the regulatory mechanisms that lead to post-ischemic induction of p75NTR. We demonstrate that induction of p75NTRafter ischemic injury is independent of transcription but requires active translation. Basal levels of p75NTRin neurons are maintained in part by the expression of microRNA miR-592, and an inverse correlation is seen between miR-592 and p75NTRlevels in the adult brain. After cerebral ischemia, miR-592 levels fall, with a corresponding increase in p75NTRlevels. Importantly, overexpression of miR-592 in neurons decreases the level of ischemic injury-induced p75NTRand attenuates activation of pro-apoptotic signaling and cell death. These results identify miR-592 as a key regulator of p75NTRexpression and point to a potential therapeutic candidate to limit neuronal apoptosis after ischemic injury.

Neuroendocrine chromaffin cells exist in both intra- and extra-adrenal locations; the organ of Zuckerkandl (OZ) constitutes the largest accumulation of extra-adrenal chromaffin tissue in mammals. The OZ disappears postnatally by modes that are still enigmatic but can be maintained by treatment with glucocorticoids (GC). Whether the response to GC reflects a pharmacological or a physiological role of GC has not been clarified. Using mice with a conditional deletion of the GC-receptor (GR) gene restricted to cells expressing the dopamine β-hydroxylase (DBH) gene [GR(fl/fl) ; DBHCre abbreviated (GR(DBHCre) )], we now present the first evidence for a physiological role of GC signalling in the postnatal maintenance of the OZ: postnatal losses of OZ chromaffin cells in GR(DBHCre) mice are doubled compared to wild-type littermates. We find that postnatal cell loss in the OZ starts at birth and is accompanied by autophagy. Electron microscopy reveals autophagic vacuoles and autophagolysosomes in chromaffin cells. Autophagy in OZ extra-adrenal chromaffin cells is confirmed by showing accumulation of p62 protein, which occurs, when autophagy is blocked by deleting the Atg5 gene (Atg5(DBHCre) mice). Cathepsin-D, a lysosomal marker, is expressed in cells that surround chromaffin cells and are positive for the macrophage marker BM8. Macrophages are relatively more abundant in mice lacking the GR, indicating more robust elimination of degenerating chromaffin cells in GR(DBHCre) mice than in wild-type littermates. In summary, our results indicate that extra-adrenal chromaffin cells in the OZ show signs of autophagy, which accompany their postnatal numerical decline, a process that is controlled by GR signalling.

Fibroblast growth factor 2 (FGF-2) is an abundant growth factor in the brain and exerts multiple functions on neural cells ranging from cell division, cell fate determination to differentiation. However, many details of the molecular mechanisms underlying the diverse functions of FGF-2 are poorly understood. In a comparative microarray analysis of motor sensory cortex (MSC) tissue of adult knockout (FGF-2(-/-)) and control (FGF-2(+/+)) mice, we found a substantial number of regulated genes, which are implicated in cytoskeletal machinery dynamics. Specifically, we found a prominent downregulation of Arhgef6. Arhgef6 mRNA was significantly reduced in the FGF-2(-/-) cortex, and Arhgef6 protein virtually absent, while RhoA protein levels were massively increased and Cdc42 protein levels were reduced. Since Arhgef6 is localized to dendritic spines, we next analyzed dendritic spines of adult FGF2(-/-) and control mouse cortices. Spine densities were significantly increased, whereas mean length of spines on dendrites of layer V of MSC neurons in adult FGF-2(-/-) mice was significantly decreased as compared to respective controls. Furthermore, neurite length in dissociated cortical cultures from E18 FGF-2(-/-) mice was significantly reduced at DIV7 as compared to wildtype neurons. Despite the fact that altered neuronal morphology and alterations in dendritic spines were observed, FGF-2(-/-) mice behave relatively unsuspicious in several behavioral tasks. However, FGF-2(-/-) mice exhibited decreased thermal pain sensitivity in the hotplate-test.

The brain functions within a specialized environment tightly controlled by brain barrier mechanisms. Understanding the regulation of barrier formation is important for understanding brain development and may also lead to finding new ways to deliver pharmacotherapies to the brain; access of many potentially promising drugs is severely hindered by these barrier mechanisms. The cellular composition of the neurovascular unit of the blood-brain barrier proper and their effects on regulation of its function are beginning to be understood. One hallmark of the neurovascular unit in the adult is the astroglial foot processes that tightly surround cerebral blood vessels. However their role in barrier formation is still unclear. In this study we examined barrier function in newborn, juvenile and adult mice lacking fibroblast growth factor-2 (FGF-2), which has been shown to result in altered astroglial differentiation during development. We show that during development of FGF-2 deficient mice the astroglial contacts with cerebral blood vessels are delayed compared with wild-type animals. However, this delay did not result in changes to the permeability properties of the blood brain barrier as assessed by exclusion of either small or larger sized molecules at this interface. In addition cerebral vessels were positive for tight-junction proteins and we observed no difference in the ultrastructure of the tight-junctions. The results indicate that the direct contact of astroglia processes to cerebral blood vessels is not necessary for either the formation of the tight-junctions or for basic permeability properties and function of the blood-brain barrier. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1201-1212, 2016.

In conditions of increased erythropoiesis, expression of hepcidin, the master regulator of systemic iron homeostasis, is decreased to allow for the release of iron into the blood stream from duodenal enterocytes and macrophages. It has been suggested that hepcidin suppression is controlled by growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily of cytokines that is secreted from developing erythroblasts. In this study, we analyzed iron-related parameters in mice deficient for GDF15 under steady-state conditions and in response to increased erythropoietic activity induced by blood loss. We demonstrate that GDF15 suppresses the hepatic mRNA expression of some BMP/TGFβ target genes but not of hepcidin, and show that GDF15 is not required to balance iron homeostasis in response to blood loss.

The superfamily of transforming growth factor-βs (TGF-βs) constitutes one of the most versatile families of signaling molecules, being involved in a broad range of biological processes spanning from development, cell death and maintenance, to aging and disease. The field has made impressive progress during the past decade. Novel signaling pathways and contextual mechanisms have been discovered. New insights into the roles of TGF-βs in tumor initiation and progression have been achieved. Numerous associations of TGF-βs with various diseases have been newly discovered or elucidated in much more detail than before, including atherosclerosis, acute and chronic liver and kidney disease, osteoarthritis and neurodegenerative diseases. This Special Issue highlights several of the recent advances in the TGF-β field. The purpose of this collection of reviews is not to present a comprehensive overview of the current status of knowledge on TGF-βs. Rather, it is the guest editors’ concept to focus on recent progress seen from the perspective of dynamic cell performances, signaling and complex disease states. A brief overview of articles in this Special Issue

It is becoming increasingly clear that members of the transforming growth factor-β (TGF-β) family have roles in the central nervous system that extend beyond their well-established roles as neurotrophic and neuroprotective factors. Recent findings have indicated that the TGF-β signaling pathways are involved in the modulation of both excitatory and inhibitory synaptic transmission in the adult mammalian brain. In this review, we discuss how TGF-β, bone morphogenetic protein and activin signaling at central synapses modulate synaptic plasticity, cognition and affective behavior. We also discuss the implications of these findings for the molecular understanding and potential treatment of neuropsychiatric diseases, such as anxiety, depression and other neurological disorders.

Purpose Several members of the fibroblast growth factor (FGF) family have been shown to be dysregulated in individuals with major depression, and treatment with antidepressants has been reported to increase FGF-2 mRNA levels in the forebrain. Methods We have used the tail suspension test (TST), and olfactory bulbectomy (OBX), and FGF-2 deficient mice to investigate putative roles of FGF-2 as an antidepressant and mediator of antidepressive drug actions. Results FGF-2 applied intraventricularly generated antidepressant-like effects in the TST. FGF-2, similar to the antidepressant amitriptyline, attenuated neuron demise in the piriform cortex and posterolateral cortical nucleus of the amygdala following OBX. Moreover, OBX induced reduction in hippocampal neurogenesis could be ameliorated by subsequent treatment with either amitriptyline or FGF-2. Furthermore, FGF-2 was effective in reversing depressive-like behavior induced by OBX, monitored in the locomotor activity and the passive avoidance test. In bulbectomized FGF-2 deficient mice, treatment with amitriptyline protected neurons, but failed to reverse behavioral alterations. Conclusions Together, these results suggest that FGF-2 constitutes both a potential target for antidepressive treatments and an important growth factor in the cytokine network underlying the actions of antidepressive drugs. The results further suggest a requirement of endogenous FGF-2 for mediating behavioral, but not neuroprotective actions of amitriptyline.

Among the 23 members of the fibroblast growth factor (FGF) family, FGF-2 is the most abundant one in the central nervous system. Its impact on neural cells has been profoundly investigated by in vitro and in vivo studies as well as by gene knockout analyses during the past 2 decades. Key functions of FGF-2 in the nervous system include roles in neurogenesis, promotion of axonal growth, differentiation in development, and maintenance and plasticity in adulthood. From a clinical perspective, its prominent role for the maintenance of lesioned neurons (e.g., ischemia and following transection of fiber tracts) is of particular relevance. In the unlesioned brain, FGF-2 is involved in synaptic plasticity and processes attributed to learning and memory. The focus of this review is on the expression of FGF-2 and its receptors in the hippocampal formation and the physiological and pathophysiological roles of FGF-2 in this region during development and adulthood.

The LRRK2 gene was recently found to have multiple mutations that are causative for the most common inherited form of late onset Parkinson's disease. In the adult brain, LRRK2 mRNA is broadly expressed, also in regions other than the nigrostriatal system. In order to establish a basis for assessing more detailed functional implications of LRRK2 in development, we provide here an in-depth analysis of its mRNA expression patterns in neural and extra-neural tissues with a focus on murine embryonic development. LRRK2 mRNA is detectable at E8.5 in non-neural and at E10.5 in neural tissues. From E12.5 to E16.5, LRRK2 mRNA is prominently expressed throughout the neocortex and subsequently highly concentrated in ventricular and subventricular zones and cortical plate. In addition, developing cerebellar granule and Purkinje neurons and spinal cord neurons display robust LRRK2 expression. In non-neural tissues LRRK2 was highly expressed in limb interdigital zones, developing kidney glomeruli, and spermatogenetic cells. Together, our results suggest roles for LRRK2 in controlling proliferation, migration, and differentiation of neural cells as well as in morphogenesis of extra-neural tissues.

Extracellular signal-regulated kinase (ERK) is a versatile protein kinase that regulates many cellular functions. Growing evidence suggests that ERK1/2 plays a crucial role in promoting cell death in a variety of neuronal systems, including neurodegenerative diseases. It is believed that the magnitude and the duration of ERK1/2 activity determine its cellular function. In this review, we summarize recent evidence for a role of ERK1/2 in neuronal death. Furthermore, we discuss the mechanisms involved in ERK1/2 mediating neuronal death.

Periodic spontaneous activity represents an important attribute of the developing nervous system. The entorhinal cortex (EC) is a crucial component of the medial temporal lobe memory system. Yet, little is known about spontaneous activity in the immature EC. Here, we investigated spontaneous field potential (fp) activity and intrinsic firing patterns of medial EC layer III principal neurons in brain slices obtained from rats at the first two postnatal weeks. A fraction of immature layer III neurons spontaneously generated prolonged (2–20 s) voltage-dependent intrinsic bursting activity. Prolonged bursts were dependent on the extracellular concentration of Ca2+([Ca2+]o). Thus, reduction of [Ca2+]oincreased the fraction of neurons with prolonged bursting by inducing intrinsic bursts in regularly firing neurons. In 1 mm[Ca2+]o, the percentages of neurons showing prolonged bursts were 53%, 81%, and 29% at postnatal day 5 (P5)–P7, P8–P10, and P11–P13, respectively. Prolonged intrinsic bursting activity was blocked by buffering intracellular Ca2+with BAPTA, and by Cd2+, flufenamic acid (FFA), or TTX, and was suppressed by nifedipine and riluzole, suggesting that the Ca2+-sensitive nonspecific cationic current (ICAN) and the persistent Na+current (INap) underlie this effect. Indeed, a 0.2–1 s suprathreshold current step stimulus elicited a terminated plateau potential in these neurons. fp recordings at P5–P7 showed periodic spontaneous glutamate receptor-mediated events (sharp fp events or prolonged fp bursts) which were blocked by FFA. Slow-wave network oscillations become a dominant pattern at P11–P13. We conclude that prolonged intrinsic bursting activity is a characteristic feature of developing medial EC layer III neurons that might be involved in neuronal and network maturation.

Transforming growth factor-betas (TGF-betas) are among the most widespread and versatile cytokines. Here, we first provide a brief overview of their molecular biology, biochemistry, and signaling. We then review distribution and functions of the three mammalian TGF-beta isoforms, beta1, beta2, and beta3, and their receptors in the developing and adult nervous system. Roles of TGF-betas in the regulation of radial glia, astroglia, oligodendroglia, and microglia are addressed. Finally, we review the current state of knowledge concerning the roles of TGF-betas in controlling neuronal performances, including the regulation of proliferation of neuronal precursors, survival/death decisions, and neuronal differentiation.

Transforming growth factor-beta (TGF-beta) and glial-cell-line-derived neurotrophic factor (GDNF) have been shown to synergize in several paradigms of neuronal survival. We have previously shown that cerebellar granule neurons (CGN) degenerate in low potassium via ERK1/2 (extra-cellular-regulated kinase)-dependent plasma membrane (PM) damage and caspase-3-dependent DNA fragmentation. Here, we have investigated the putative synergistic function of GDNF and TGF-beta in CGN degeneration. GDNF alone prevents low-potassium-induced caspase-3 activation and DNA fragmentation but does not affect either low-potassium-induced ERK activation or PM damage. TGF-beta alone does not affect low-potassium-induced DNA fragmentation but potentiates low-potassium-induced PM damage. This effect of TGF-beta is independent of ERK1/2 activation but dependent on p38-MAPK (mitogen-activated protein kinase) activation. When co-applied with TGF-beta, GDNF paradoxically antagonizes TGF-beta-induced potentiation of PM damage by inhibiting TGF-beta-induced p38-MAPK activation. In addition, PI3K (phosphatidylinositol 3-kinase) inhibitors abolish the GDNF effect. This study thus demonstrates a differential mechanism of action of GDNF and TGF-beta on CGN degeneration. GDNF inhibits caspase-3-dependent DNA fragmentation but does not affect ERK-dependent PM damage. However, GDNF can attenuate TGF-beta-induced p38-MAPK-dependent PM damage via the PI3K pathway.

Newborn neurons elaborate an axon that undertakes a complicated journey to find its ultimate target in the brain or periphery. Although major progress in the study of this process has been made by analysis of dissociated neurons in vitro, one would like to observe and manipulate axonal outgrowth and pathfinding as it occurs in situ, as fasciculated nerves growing within the tissue itself. Here, we present a simple technique to do this, through cultivation of embryonic mouse slices expressing enhanced green fluorescent protein (EGFP) specifically in newborn neurons. This system allows for imaging of outgrowth of peripheral nerves into structures such as the developing limb. We demonstrate a reproduction of normal innervation patterns by spinal nerves derived from spinal cord motor neurons and sensory neurons of the dorsal root ganglia. The slices can be manipulated pharmacologically as well as genetically, by crossing the EGFP-expressing line with lines containing targeted mutations in genes of interest. Developmental Dynamics 236:3514–3523, 2007. © 2007 Wiley-Liss, Inc.

Analyses of mice carrying a deletion of the pan-neurotrophin receptor p75NTR have allowed identifying p75NTR as an important structural regulator of the hippocampus. Most of the previous analyses were done using p75NTR (ExIII) knockout mice which still express the short isoform of p75NTR. To scrutinize the role of p75NTR in the hippocampus, we analyzed adult and aged p75NTR (ExIV) knockout mice, in which both, the short and the full-length isoform are deleted. Deletion of these isoforms induced morphological alterations in the adult dentate gyrus (DG), leading to an increase in the thickness of the molecular and granular layer. Based on these observations, we next determined the morphological substrates that might contribute to this phenotype. The cholinergic innervation of the molecular and granular layer of the DG was found to be significantly increased in the knockout mice. Furthermore, adult neurogenesis in the DG was found to be significantly altered with increased numbers of doublecortin (DCX) positive cells and reduced numbers of apoptotic cells in p75NTR (ExIV) knockout mice. However, cell proliferation as measured by phosphohiston H3 (PH3) positive cell numbers was not affected. These morphological alterations (number of DCX-positive cells and increased cholinergic fiber densities) as well as reduced cell death in the DG are likely to contribute to the observed thickening of the granular layer in p75NTR (ExIV) knockout mice. In addition, Sholl-analysis of DCX-positive neurons revealed a higher dendritic complexity and could thus be a possible morphological correlate for the increased thickness of the molecular layer in p75NTR deficient animals. Our data clearly demonstrate that deletion of both, the short and the full-length isoform of p75NTR affects DG morphology, due to alterations of the cholinergic system and an imbalance between neurogenesis and programmed cell death within the subgranular zone.

Fibroblast growth factor 2 (FGF-2) was the first growth factor discovered that exerted prominent protective and regenerative effects in an animal model of Parkinson's disease, the MPTP-lesioned dopaminergic nigrostriatal system. To address the putative physiological relevance of endogenous FGF-2 for midbrain dopaminergic neurons, we have analysed densities of tyrosine hydroxylase (TH)positive cells in the substantia nigra (SN) and TH-positive fibers in the striatum and amygdala of adult FGF-2-deficient mice. We found that densities of TH-immunoreactive (ir) cells in the SN as well as densities of TH-ir fibers in the striatum and amygdala were unaltered as compared with wild-type littermates. There is evidence to suggest that growth factor deficits do not become apparent unless a system is challenged in a lesioning paradigm. We therefore tested the ability of the nigrostriatal system with respect to its ability to cope with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) intoxication. Treatment with 20 mg/kg MPTP on three consecutive days reduced dopamine levels in the striatum by about 80%. Densities of TH-positive neurons in the SN were reduced by 71%. However, both parameters did not significantly differ between FGF-2 (-/-) mice and wild-type littermates. Our results therefore suggest that FGF-2, despite its prominent pharmacological potency as a neurotrophic factor for the dopaminergic nigrostriatal system, is not crucial for maintaining its structural integrity and ability to cope with MPTP intoxication.

The diversification of neural-crest-derived sympathoadrenal (SA) progenitor cells into sympathetic neurons and neuroendocrine adrenal chromaffin cells was thought to be largely understood. In-vitro studies with isolated SA progenitor cells had suggested that chromaffin cell differentiation depends crucially on glucocorticoids provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor gene had revealed that adrenal chromaffin cells develop mostly normally in these mice. Alternative cues from the adrenal cortex that may promote chromaffin cell determination and differentiation have not been identified. We therefore investigated whether the chromaffin cell phenotype can develop in the absence of an adrenal cortex, using mice deficient for the nuclear orphan receptor steroidogenic factor-1 (SF1), which lack adrenal cortical cells and gonads. We show that in Sf1–/– mice typical chromaffin cells assemble correctly in the suprarenal region adjacent to the suprarenal sympathetic ganglion. The cells display most features of chromaffin cells, including the typical large chromaffin granules. Sf1–/–chromaffin cells are numerically reduced by about 50% compared with the wild type at embryonic day (E) 13.5 and E17.5. This phenotype is not accounted for by reduced survival or cell proliferation beyond E12.5. However, already at E12.5 the `adrenal' region in Sf1–/– mice is occupied by fewer PHOX2B+ and TH+ SA cells as well as SOX10+ neural crest cells. Our results suggest that cortical cues are not essential for determining chromaffin cell fate, but may be required for proper migration of SA progenitors to and/or colonization of the adrenal anlage.

Evidence is presented that the seminal vesicle of the bull is an important source of a protein which is very similar to nerve growth factor from mouse submaxillary gland. Its chemical and biological properties in vitro are compared with those of nerve growth factor from other sources.

Phox2B, a homeodomain transcription factor closely related to Phox2A, is expressed in peripheral and central noradrenergic neurons. In neural crest (NC) derivatives Phox2B is restricted to sympathetic and parasympathetic ganglia, enteric neurons, and adrenal and extraadrenal chromaffin cells. Similar to MASH-1, Phox2B has been implicated in synchronizing pan-neuronal and catecholaminergic phenotype-specific aspects of neurogenesis. The role of Phox2B for the differentiation of the neuroendocrine NC derivatives, the adrenal medullary chromaffin cells, has not been explored. We have previously reported that in MASH-1-deficient mice most chromaffin cells are arrested at the early neuroblast stage and lack catecholaminergic differentiation. We show now that in Phox2B knockout/lacZ knockin mice the maturation of presumptive chromaffin cells is arrested at an even earlier stage of development. The cells lack the catecholaminergic marker enzyme TH and fail to form a centrally located medulla. In contrast to MASH-1 (−/−) mice they do not express dHand, Phox2A, c-ret, neurofilament, neuron-specific tubulin, and NCAM and appear ultrastructurally more immature. Many of these cells die by apoptosis. Despite the complete lack of differentiation, few lacZ-positive adrenal cells can still be found at E16.5. We conclude that Phox2B regulates very early events in the differentiation of adrenal chromaffin cells distinct to steps, which essentially require MASH-1.

Growth differentiation factor-15/macrophage inhibitory cytokine-1 (GDF-15/MIC-1) is a new member of the transforming growth factor beta (TGF-beta) superfamily, which has most recently been found in activated macrophages (MPhi). We have now investigated GDF-15/MIC-1 in human MPhi after exposure to oxidized low-density lipoproteins (oxLDL) related mediators in vitro and in arteriosclerotic carotid arteries. Using RT-PCR and Western blotting a pronounced induction of GDF-15/MIC-1 expression by oxLDL, C6-ceramide, tumor necrosis factor (TNFalpha) and hydrogen peroxide (H2O2) was found in cultured human MPhi. In 11 human arteriosclerotic carotid arteries, immunohistochemical analyses supported by computer-assisted morphometry and regression analyses demonstrated a significant colocalization of GDF-15/MIC-1 immunoreactivity (IR) with oxLDL IR and manganese superoxide dismutase (MnSOD) IR in CD68 immunoreactive (ir) MPhi, which were also expressing AIF-IR (apoptosis-inducing factor), caspase-3-IR (CPP32), PARP-IR, c-Jun/AP-1-IR and p53-IR. Our data suggest that GDF-15/MIC-1 is inducible in human MPhi by oxLDL and its mediators in vitro and is supposed to contribute to oxidative stress dependent consequences in arteriosclerotic plaques, e.g. modulating apoptosis and inflammatory processes in activated MPhi.

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.

Andreas Oksche Coordinating Editor of Cell & Tissue Research from 1978 to 1995, and Co-Editor for the section “Neuroendocrinology” for many years, will celebrate his ninetieth birthday on 27 July 2016. We, the Editors, Springer Nature Publishing Group, and many friends and former colleagues congratulate him on this occasion and wish him health and vitality for the years to come. Andreas Oksche is known as an eminent figure in neuroendocrinology with a world-wide reputation, as a leading anatomist in Germany and beyond and as a prominent personality in national science policy. He was born in Riga, Latvia, and received his training in anatomy and neuroscience at the University of Marburg under the guidance of Professors Benninghoff and Niessing. His MD thesis on the “Organon frontale” of the grass frog established the foundation of his successful career in neuroendocrinology. Andreas Oksche’s Habilitation addressed secretory features of glia, with special reference to the subcommissural organ, thereby expanding the list of cells with a secretory capacity in the brain and anticipating the functions of astroglia, which have only recently been analysed in detail. Following postdoctoral work with Ernst and Berta Scharrer, two prominent pioneers in neuroendocrinology and hypothalamic-pituitary neurosecretion, at the Albert Einstein College of Medicine, he moved to the University of Kiel, where, in the late 1940s, Wolfgang Bargmann had established a centre for research on hypothalamic neurosecretion. The concept of neurons having a secretory identity had been initially met with profound scepticism. Proceeding from the hypothalamus to the pineal gland and circumventricular organs, Andreas Oksche’s contributions were essential to the broadening of the perception of the brain as having secretory capacities and of the neural–endocrine hybrid character of neurons. The discovery that such neuroendocrine cells could simultaneously act as photoreceptors and the elucidation of their fine structure was the culmination of the search for mechanisms underlying the regulation of Andreas Oksche (by courtesy of Dr. Alexander Oksche)

Multiple evidence suggests that fibroblast growth factors (FGFs), most prominently FGF-2, affect astroglial proliferation, maturation, and transition to a reactive phenotypein vitro, and after exogenous administration,in vivo. Whether this reflects a physiological role of endogenous FGF is unknown. Using FGF-2 and FGF-5 single- and double mutant mice we show now a region-specific reduction of glial fibrillary acidic protein (GFAP), but not of S100 in gray matter astrocytes. FGF-2 is apparently the major regulator of GFAP, because in mice deficient for FGF-2, GFAP is distinctly reduced in cortex and striatum, whereas in FGF-5-/-animals only a reduction in the midbrain tegmentum can be observed. In FGF-2-/-/FGF-5-/-double mutant animals, GFAP-immunoreactivity is reduced in all three brain regions. Cortical astrocytes cultured from FGF-2-/-/FGF-5-/-double mutant mice revealed reduced levels of GFAP, but not S100 as compared with wild-type littermates. This phenotype could be rescued by exogenous FGF-2 but not FGF-5 (10 ng/ml). Electron microscopy revealed reduced levels of intermediate filaments in perivascular astroglial endfeet. This defect was accompanied by enhanced permeability of the blood-brain barrier (BBB), as detected by albumin extravasation. Levels of the tight junction proteins Occludin and ZO-1 were reduced in blood vessels of FGF-2-/-/FGF-5-/-double mutant mice as compared with wild-type littermates. Our data support the notion that endogenous FGF-2 and FGF-5 regulate GFAP expression in a region-specific manner. The observed defect in astroglial differentiation is accompanied by a defect in BBB function arguing for an indirect or direct role of FGFs in the regulation of BBB permeabilityin vivo.

c-Ret encodes a receptor tyrosine kinase that is essential for normal development of the kidney as well as enteric and sympathetic neurons. Since sympathetic neurons and neuroendocrine chromaffin cells originate from a common progenitor cell, we have examined the relevance of c-Ret for the development of adrenal chromaffin cells by analyzing mouse mutants lacking c-Ret. Adrenal chromaffin cells express c-Ret mRNA at embryonic day (E) 12.5 and 13.5, yet levels of expression decline at later embryonic and postnatal ages. Adrenal medullae of c-Ret deficient mice show normal numbers of tyrosine hydroxylase (TH)-immunoreactive cells at E13.5 and at birth. Ultrastructurally, adrenal chromaffin cells of c-Ret−/− mice appear unaltered: chromaffin cells develop typical secretory chromaffin granules, the morphological hallmark of chromaffin cells, and synaptic terminals appear normal. However, adrenaline levels and numbers of chromaffin cells immunoreactive for the adrenaline synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) are reduced by about 30% in c-Ret-deficient mice arguing for a direct or indirect role of c-Ret in the regulation of PNMT. Thus, despite expression of c-Ret, adrenal chromaffin cells develop largely normal in mice lacking c-Ret. We therefore conclude that sympathetic neurons and neuroendocrine chromaffin cells profoundly differ in their requirement for c-Ret signaling during development.

Numbers of catecholaminergic neurons are known to decline with aging. Whether projections of these neurons to the forebrain are similarly affected is not known. High densities of tyrosine hydroxylase-immunoreactive (TH-ir) fibers are found in the hippocampal formation (CA1-3, dentate gyrus) and in the amygdala of normal adult mice. We report here that densities of TH-ir fibers in the amygdala and hippocampus in aged mice (21-26 months) decrease dramatically and in a subregion-specific fashion. There is a reduction of 35% in the dentate gyrus, while hippocampal regions CA1 through CA3 are almost entirely spared. In the amygdala the lateral, basolateral, basomedial, and central nucleus were affected, with fiber reduction ranging from 19% to 34%. These results indicate that the age-related decline of TH-ir catecholaminergic cell bodies in the substantia nigra and the ventral tegmental area induces substantial losses of TH-ir fibers in the amygdala and dentate gyrus, but not in other areas of the hippocampal formation. This suggests that region-specific factors may be implicated in the regulation of maintenance vs. degeneration of TH-ir fibers during aging.

Declines in memory function and behavioural dysfunction accompany normal ageing in mammals. However, the cellular and morphological basis of this decline remains largely unknown. It was assumed for a long time that cell losses in the hippocampus accompany ageing. However, recent stereological studies have questioned this finding. In addition, the effect of ageing is largely unknown in another key structure of the memory system, the amygdala. In the present study, we have estimated neuronal density and total neuronal numbers as well as density of fragments of degenerated axons in different hippocampal subfields and amygdaloid nuclei. Comparisons were made among aged (21-26 months old) mice and normal adult littermates (8 months old). No significant volume loss occurs in the hippocampus of aged mice. Small but insignificant reductions in total neuronal numbers were found in the hippocampus and in the amygdaloid nuclei. In contrast to the mild effects of ageing upon neuronal numbers, fragments of degenerated axons were increased in both hippocampus and amygdala of aged mice. These data suggest that ageing does not induce prominent cell loss in the hippocampus or amygdala, but leads to degeneration of axons that innervate these forebrain structures. Thus, mechanisms underlying age-related dysfunction depend on parameters other than neuronal numbers, at least in the hippocampal formation and the amygdala.

We have investigated basic fibroblast growth factor (FGF-2) localization in and release from isolated bovine adrenal chromaffin cells. In contrast to previous reports, we found no evidence of fibroblast growth factor (FGF) storage in catecholamine-containing chromaffin granules. Subcellular fractionation studies did not show enrichment of FGF-2 immunoreactivity in granules, and cholinergic stimulation failed to release FGF-2 into the medium. Our results suggest that adrenal chromaffin cells resemble other FGF-2-synthesizing cell types with respect to FGF storage and secretion.

Bovine chromaffin cells contain within their storage vesicles and release upon cholinergic stimulation a complex mixture of proteins and peptides. We present data suggesting that one of these proteins resembles transforming growth factor (TGF)-beta in terms of its biological activity. The assay used to assess the activity of TGF-beta is based on cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. The assay is highly specific in detecting TGF-beta 1, -beta 2, and -beta 3 but does not detect several cytokines and growth factors, such as fibroblast growth factor-2, transforming growth factor-alpha, platelet-derived growth factor-AB, insulin-like growth factor-1, or neurotrophin-3 or -4. Moreover, we show that this assay does not detect a wide range of TGF-beta superfamily members (activin A, bone morphogenetic protein-2, -4, -6, and -7, growth/differentiation factor-5, and glial cell line-derived neurotrophic factor). Chromaffin granules contain approximately 1 ng of TGF-beta/10 mg of protein. The biological activity elicited by the chromaffin granule component can be neutralized by using an antibody against TGF-beta 1/beta 2/beta 3. TGF-beta is releasable from cultured chromaffin cells stimulated with the cholinergic agonist carbachol (10(-5) M). These data suggest that TGF-beta is stored in chromaffin granules and can be released by exocytosis.

We have previously shown that the neurotrophic effect of glial cell line-derived neurotrophic factor (GDNF) in vitro and in vivo requires the presence of transforming growth factor (TGF)beta. Using primary neurons (chick E8 ciliary) we show that the combination of GDNF plus TGFbeta promotes survival, whereas the single factors do not. This cooperative effect is inhibited by blocking the extracellular signal-regulated kinase (ERK)/MAPK pathway, but not by interfering with the PI3 kinase signaling cascade. Although there is no functional GDNF signaling in the absence of TGFbeta, pretreatment with TGFbeta confers GDNF responsiveness to the cells. This is not due to upregulation of GDNF receptors mRNA and protein, but to TGFbeta-induced recruitment of the glycosyl-phosphatidylinositol-anchored GDNF receptor (GFR)alpha1 to the plasma membrane. This is supported by the fact that GDNF in the presence of a soluble GFRalpha1 can promote survival in the absence of TGFbeta. Our data suggest that TGFbeta is involved in GFRalpha1 membrane translocation, thereby permitting GDNF signaling and neurotrophic effects.

The developmental diversification of neural crest-derived sympathoadrenal (SA) progenitor cells into neuroendocrine adrenal chromaffin cells and sympathetic neurons has been thought to be largely understood. Based on two decades of in vitro studies with isolated SA progenitor and chromaffin cells, it was widely assumed that chromaffin cell development crucially depends on glucocorticoid hormones provided by adrenal cortical cells. However, analysis of mice lacking the glucocorticoid receptor has revealed that the chromaffin cell phenotype develops largely normally in these mice, except for the induction of the adrenaline synthesizing enzyme phenylethylamine N-methyl transferase. In a search for novel candidate genes that might be involved in triggering the sympathetic neuron/chromaffin cell decision, we have studied putative contributions of transforming growth factor (TGF)-alpha, BMP-4, and the transcription factor MASH-1, molecules with distinct expressions in SA progenitor cells, in their migratory pathways and final destinations. TGF-beta2 and -beta3 and BMP-4 are highly expressed in the wall of the dorsal aorta and in the adrenal anlagen during and after immigration of SA progenitors but expressed at much lower levels in sympathetic ganglia. We found that neutralizing antibodies against all three TGF-beta isoforms applied to the chorionic-allantoic membrane (CAM) of quail embryos interfere with proliferation of immigrated adrenal chromaffin cells but do not affect their specific neuroendocrine ultrastructural phenotype. Grafting of noggin-producing cells to the CAM, which scavenges BMPs, interferes with visceral arch and limb development but does not overtly affect the chromaffin phenotype. The transcription factor MASH-1 promotes early differentiation of SA progenitors. Mice deficient for MASH-1 lack sympathetic ganglia, whereas the adrenal medulla previously has been reported to be present. We show here that most adrenal medullary cells in MASH-1(-/-) mice identified by Phox2b immunoreactivity lack the catecholaminergic marker tyrosine hydroxylase. More surprisingly, most cells do not contain chromaffin granules and display a neuroblast-like ultrastructure and show strongly enhanced expression of c-RET comparable to that observed in sympathetic ganglia. Together, our data suggest that TGF-betas and BMP-4 do not seem to be essential for chromaffin cell differentiation. In contrast with previous reports, however, MASH-1 apparently plays a crucial role in chromaffin cell development.

FGF-2 is a potent neurotrophic factor for several populations of CNS neurons and has been shown to protect hippocampal neurons from glutamate-induced cell death in vitro and in vivo. Mechanisms underlying the neurotrophic and protective actions of FGF-2 have been resolved only in part. Using glutamate-treated cultured hippocampal neurons we show that FGF-2 shares its neuroprotective capacity with GDNF. Hippocampal neurons express glial-cell-line-derived neurotrophic factor (GDNF), its receptors c-Ret and the lipid-anchored GDNF family receptor-alpha1 (GFRalpha-1), and the FGF receptor 1 (FGFR I). Neutralizing antibodies to GDNF abolish the neuroprotective effect of FGF-2. In support of the notion that GDNF is required to permit the protective effects of FGF-2 we find that FGF-2 up-regulates GDNF and GFRalpha-1 in hippocampal neurons. Furthermore, FGF-2-induced GDNF causes enhanced phosphorylation of c-Ret and the signaling components Akt and Erk. A putative downstream target of FGF-2 and GDNF are bcl-2 gene family members, whose mRNAs are differentially up-regulated by the two factors. Together, these data suggest that GDNF is an important protective factor for glutamate-lesioned hippocampal neurons and an essential mediator of the neuroprotective actions of FGF-2.

The development of sympathetic neurons and chromaffin cells is differentially controlled at distinct stages by various extrinsic and intrinsic signals. Here we use conditional deletion of Dicer1 in neural crest cells and noradrenergic neuroblasts to identify stage specific functions in sympathoadrenal lineages. Conditional Dicer1 knockout in neural crest cells of Dicer1Wnt1Cre mice results in a rapid reduction in the size of developing sympathetic ganglia and adrenal medulla. In contrast, Dicer1 elimination in noradrenergic neuroblasts of Dicer1DbhiCre animals affects sympathetic neuron survival starting at late embryonic stages and chromaffin cells persist at least until postnatal week 1. A differential function of Dicer1 signaling for the development of embryonic noradrenergic and cholinergic sympathetic neurons is demonstrated by the selective increase in the expression of Tlx3 and the cholinergic marker genes VAChT and ChAT at E16.5. The number of Dbh, Th and TrkA expressing noradrenergic neurons is strongly decreased in Dicer1-deficient sympathetic ganglia at birth, whereas Tlx3+/ Ret+ cholinergic neurons cells are spared from cell death. The postnatal death of chromaffin cells is preceded by the loss of Ascl1, mir-375 and Pnmt and an increase in the markers Ret and NF-M, which suggests that Dicer1 is required for the maintenance of chromaffin cell differentiation and survival. Taken together, these findings demonstrate distinct stage and lineage specific functions of Dicer1 signaling in differentiation and survival of sympathetic neurons and adrenal chromaffin cells.

The activation of epidermal growth factor receptor (EGFR) affects multiple aspects of neural precursor behaviour, including proliferation and migration. Telencephalic precursors acquire EGF responsiveness and upregulate EGFR expression at late stages of development. The events regulating this process and its significance are still unclear. We here show that in the developing and postnatal hippocampus (HP), growth/differentiation factor (GDF) 15 and EGFR are co-expressed in primitive precursors as well as in more differentiated cells. We also provide evidence that GDF15 promotes responsiveness to EGF and EGFR expression in hippocampal precursors through a mechanism that requires active CXC chemokine receptor (CXCR) 4. Besides EGFR expression, GDF15 ablation also leads to decreased proliferation and migration. In particular, lack of GDF15 impairs both processes in the cornu ammonis (CA) 1 and only proliferation in the dentate gyrus (DG). Importantly, migration and proliferation in the mutant HP were altered only perinatally, when EGFR expression was also affected. These data suggest that GDF15 regulates migration and proliferation by promoting EGFR signalling in the perinatal HP and represent a first description of a functional role for GDF15 in the developing telencephalon.

Connexins (cx) constitute a family of transmembrane proteins that form gap junction channels allowing metabolic and electrical coupling of cellular networks. Initial studies on the expression of cx in the developing brain have suggested that cx may undergo dynamic changes and may possibly be implicated in synchronizing development and differentiation of neural progenitor cells and young neurons. We have investigated expression of cx26, cx32, cx43, and cx45 in the midbrain floor, where nigrostriatal dopaminergic neurons originate and differentiate. This neuron population is of major importance in regulating motor-functions. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed low levels of cx26-mRNA in the midbrain floor at E12, which gradually increased during pre- and postnatal development, reaching a maximum in the adult. Cx32-mRNA-levels reached a first peak at E16, and showed highest levels in adulthood. Cx43 was highly expressed at E12, decreased until E18, and subsequently increased again until adulthood. Cx45 mRNA was prominent at all developmental ages, but slightly decreased after the first postnatal week. Double-labeling for the dopaminergic neuronal marker tyrosine hydroxylase (TH), and cx-immunoreactivities (ir) evaluated by quantitative confocal laser microscopy revealed both distinct and similar developmental patterns for the individual cx investigated. Cx26 was highest at E14, decreased towards birth, and subsequently increased again reaching about 50% of the E14 level in the adult. Cx32-ir peaked at E16 and dropped to low levels after birth. Cx43-ir was highest at E12, decreased sharply at E14, reached its lowest levels at birth, but modestly increased again afterwards. Cx45-ir showed a biphasic pattern, with two prominent peaks at E12 and E18, followed by a massive postnatal decrease. Taken together, our results reveal that expression and ir of cx in the midbrain floor and dopaminergic neurons, respectively, follow cx-type specific patterns that temporally coincide with important steps of midbrain morphogenesis, as e.g. progenitor cell formation and migration (E12), early differentiation (E14-16), target encounter (E16-18) and postnatal functional maturation of the nigrostriatal system.

We and others have recently cloned a new member of the transforming growth factor-beta superfamily, growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF-15/MIC-1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF-15/MIC-1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF-15/MIC-1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF-15/MIC-1-producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Fluorescent microscopy revealed both intra- and extracellular GDF-15/MIC-1 ir. Up-regulation of GDF-15/MIC-1 in activated macrophages (Mstraight phi) is also supported by RT-PCR, ICC, and Western blot experiments showing pronounced induction of GDF-15/MIC-1 expression (mRNA and protein) in retinoic acid/phorbol ester-stimulated human M phi. Our data suggest that 1) GDF-15/MIC-1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme-epithelial interactions. Finally, GDF-15/MIC-1 may also act within the antiinflammatory cytokine network activated in CNS lesions.

Psychotic symptoms in different neuropsychiatric disorders are treated by neuroleptic drugs. Neuroleptics are known to block dopamine (DA) neurotransmission, however, cell types mediating their actions have not been determined. Recently, astrocytes have been demonstrated to express D1- and D2-DA receptors, whose activation leads to transient increases in intracellular calcium concentration. We show here that DA-sensitivity of cortical and striatal rat astroglial cultures, as monitored by calcium imaging, is reduced by a 12-h exposure to the atypical antipsychotic agents Clozapine (>1 nmol/liter), Olanzapine (>100 nmol/liter), and Risperidone (>1 nmol/liter), but not by classical neuroleptics Haloperidol and Sulpiride. These effects could not be reverted by the receptor-specific antagonists SCH23390, Sulpiride, L745 870, Ergotamine, and Propranolol. In addition, RT-PCR and Western blot analyses concerning the effects of Clozapine, Olanzapine, and Risperidone on DA receptor expression in cortical and striatal astroglial cells revealed no alterations in mRNAs and immunoreactive protein of D1- and D2-DA receptor subtypes. These results provide the first evidence that atypical but not classical neuroleptic drugs reduce astroglial DA-sensitivity, a mechanism that may be important for a better understanding of differences in effects and side effects between atypical and classical neuroleptic drugs.