Noradrenergic system of the zebra finch brain: Immunocytochemical study of dopamine-β-hydroxylase

Oscine birds are among the few animal groups that have vocal learning, and their brains contain a specialized system for song learning and production. We describe here the immunocytochemical distribution of dopamine-β-hydroxylase (DBH), a noradrenergic marker, in the brain of an oscine, the zebra finch (Taeniopygia guttata). DBH-positive cells were seen in the locus coeruleus, the nucleus subcoeruleus ventralis, the nucleus of the solitary tract, and the caudolateral medulla. Immunoreactive fibers and varicosities had a much wider brain distribution. They were particularly abundant in the hippocampus, septum, hypothalamus, area ventralis of Tsai, and substantia nigra, where they formed dense pericellular arrangements. Significant immunoreactivity was observed in auditory nuclei, including the nucleus mesencephalicus lateralis pars dorsalis, the thalamic nucleus ovoidalis, field L, the shelf of the high vocal center (HVC), and the cup of the nucleus robustus archistriatalis (RA), as well as in song control nuclei, including the HVC, RA, the lateral magnocellular nucleus of the anterior neostriatum, and the dorsomedial nucleus (DM) of the intercollicular complex. Except for the DM, DBH immunoreactivity within song nuclei was comparable to that of surrounding tissues. Conspicuously negative were the lobus paraolfactorius, including song nucleus area X, and the paleostriatum. Our results are in agreement with previous studies of the noradrenergic system performed in nonoscines. More importantly, they provide direct evidence for a noradrenergic innervation of auditory and song control nuclei involved in song perception and production, supporting the notion that noradrenaline is involved in vocal communication and learning in oscines. J. Comp. Neurol. 400:207–228, 1998. © 1998 Wiley-Liss, Inc.     

Prenatal development of the rodent rostral migratory stream

The aim of this study was to elucidate the embryological origins of the unique neuronal progenitor cells that form the rostral migratory stream (RMS), the path traversed by cells from the anterior part of the forebrain subventricular zone (SVZa) en route to the olfactory bulb. To determine when and where cells constituting the RMS initially exhibit their characteristic neuronal phenotype and high mitotic capacity, we analyzed the cells of the rat forebrain between embryonic day 14 (E14) and postnatal day 2 (P2). At E14, cells with a neuronal phenotype were observed within the ventricular zone in close proximity to the mantle layer of the future olfactory bulb. By E15, cells expressing neuronal markers are also PSA-NCAM immunoreactive and become aligned in chains of similarly oriented cells, a hallmark of the postnatal RMS. The cells that form chains organize into a patch that enlarges in the anterior-posterior and medial-lateral dimensions from E16 to E22 (birth). In comparing the forebrain cytoarchitecture to the pattern of cell type-specific staining, the patch constitutes only the central part of the proximal RMS. Early during development, the region of the RMS surrounding the patch expresses low levels of PSA-NCAM and neuron-specific markers. The proliferative activity of cells forming the patch vs. nonpatch regions of the RMS was analyzed following a short bromodeoxyuridine (BrdU) exposure. Between E15 and E22, the patch can be recognized by the mitotic activity of its cells; the cells of the patch incorporate less BrdU than the nonpatch portion of the RMS. The time course of appearance of cells forming the RMS indicates that the RMS arises in advance and independently of the cortical SVZ. Although the patch and the nonpatch regions of the embryonic RMS appear to merge postnatally, the two regions may originate separately under the influence of distinct intrinsic and extrinsic factors.     

Expression of the ecto-ATPase NTPDase2 in the germinal zones of the developing and adult rat brain

In the adult nervous system, multipotential stem cells of the subventricular zone of the lateral ventricles generate neuron precursors (type-A cells) that migrate via the rostral migratory stream to the olfactory bulb where they differentiate into neurons. The migrating neuroblasts are surrounded by a sheath of astrocytes (type-B cells). Using immunostaining, in situ hybridization and enzyme histochemistry, we demonstrate that the ecto-ATPase nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is expressed in the subventricular zone and the rostral migratory stream of the adult rat brain. This enzyme hydrolyses extracellular nucleoside triphosphates to the respective nucleoside diphosphates and is thought to directly modulate ATP receptor-mediated cell communication. Double labelling for the astrocyte intermediate filament protein GFAP and the glial glutamate transporter GLAST identifies the NTPDase2-positive cells as type-B cells. During development the enzyme protein is first detected at E18, long before expression of the astrocyte marker GFAP. It gradually becomes expressed along the ventricular and subventricular zone of the brain, followed by complete retraction to the adult expression pattern at P21. NTPDase2 is transiently expressed in the outer molecular layer of the dentate gyrus and within the cerebellar white matter and is associated with select microvessels, tanycytes of the third ventricle, and subpial astrocytes of the adult brain. Our results suggest that NTPDase2 can serve as a novel marker for specifying subsets of cells during in vivo and in vitro studies of neural development and raise the possibility that ATP-mediated signalling pathways play a role in neural development and differentiation.     

Neurogenesis within the juvenile zebra finch telencephalic ventricular zone: a map of proliferative activity

Localized regions of increased cellular proliferation within the ventricular zone (VZ) of juvenile male songbirds may contain progenitor cells that give rise to song-control neurons and, thereby, contribute to the construction of brain areas important for song learning. The purpose of this study was to examine levels of cell division throughout the telencephalic VZ of juvenile birds. A single pulse of [(3)H]thymidine was administered to 30-day male and female zebra finches, and the birds were killed 2 hours later. The VZ was divided into segments throughout the entire anterior-posterior and dorsal-ventral neuraxes, and levels of thymidine labeling were measured within each subdivision. By subdividing the VZ into segments, we were able to construct a "map" of proliferation throughout the telencephalic VZ, thereby allowing us to compare levels of mitotic activity within corresponding locations of the VZ between males and females. Our map revealed two major findings: (1) proliferation in both juvenile males and females was spatially differentiated throughout the VZ, suggesting that mitotic activity is differentially regulated across the neuraxis; (2) sex differences in proliferation were present in 30-day-old birds, but were highly restricted. The most robust sexual dimorphism occurred within the ventral aspect of the VZ at rostral levels of the song-control nucleus Area X, with males demonstrating an increased number of dividing cells compared with females. This result raises the possibility that Area X neurons in males are derived from committed progenitors within the adjacent VZ in close proximity to this nucleus.     

Drebrin E regulates neuroblast proliferation and chain migration in the adult brain

F‐actin‐binding protein drebrin has two major isoforms: drebrin A and drebrin E. Drebrin A is the major isoform in the adult brain and is highly concentrated in dendritic spines, regulating spine morphology and synaptic plasticity. Conversely, drebrin E is the major isoform in the embryonic brain and regulates neuronal morphological differentiation, but it is also expressed in neurogenic regions of the adult brain. The subventricular zone (SVZ) is one of the brain regions where adult neurogenesis occurs. Neuroblasts migrate to the olfactory bulb (OB) and integrate into existing neuronal networks, after which drebrin expression changes from E to A, suggesting that drebrin E plays a specific role in neuroblasts in the adult brain. Therefore, to understand the role of drebrin E in the adult brain, we immunohistochemically analyzed adult neurogenesis using drebrin‐null‐mutant (DXKO) mice. In DXKO mice, the number of neuroblasts and cell proliferation decreased, although cell death remained unchanged. These results suggest that drebrin E regulates cell proliferation in the adult SVZ. Surprisingly, the decreased number of neuroblasts in the SVZ did not result in less neurons in the OB. This was because the survival rate of newly generated neurons in the OB increased in DXKO mice. Additionally, when neuroblasts reached the OB, the change in the migratory pathway from tangential to radial was partly disturbed in DXKO mice. These results suggest that drebrin E is involved in a chain migration of neuroblasts.     

mDll1 and mDll3 expression in the developing mouse brain: role in the establishment of the early cortex

The Delta/Notch signalling system is involved in several developmental processes. During fly neurogenesis, Delta expression defines the fate of neuronal precursors and inhibits neighboring Notch-expressing cells from acquiring a neural fate, a process known as lateral inhibition. In vertebrates, recent evidence demonstrates that Notch activation can positively determine cell fate and affect neuronal process extension. Nevertheless, Delta-like expression patterns during brain development are relatively unknown. Using a transgenic mouse, which expresses LacZ under the mDll1 promoter, we show by immunofluorescence that in the developing telencephalon mDll1 is expressed in undifferentiated cells in close contact with radial glial cells. Based on in situ hybridization data on mDll1 and mDll3 mRNA expression and on the immunohistochemical detection of beta-galactosidase in the Dll1-lacZ transgenic mouse, we suggest that mDll1 and mDll3 are involved in the establishment of the early cortical plate and that mDll1-expressing cells are in close contact with radial glial cells, thereby modulating the latter population, which is known to express Notch1. Furthermore, we suggest that the decrease in mDll1 mRNA found toward the end of gestation could be related, first, to the slowing of neurogenesis and, second, to the differentiation of the radial glial cell population into astrocytes.     

Identification, localisation and functional implication of 26RFa orthologue peptide in the brain of zebra finch (Taeniopygia guttata)

Several neuropeptides with the C-terminal Arg-Phe-NH(2) (RFa) sequence have been identified in the hypothalamus of a variety of vertebrates. The present study was conducted to isolate novel RFa peptides from the zebra finch brain. Peptides were isolated by immunoaffinity purification using an antibody that recognises avian RFa peptides. The isolated peptide consisted of 25 amino acids with RFa at its C-terminus. The sequence was SGTLGNLAEEINGYNRRKGGFTFRFa. Alignment of the peptide with vertebrate 26RFa has revealed that the identified peptide is the zebra finch 26RFa. We also cloned the precursor cDNA encoding this peptide. Synteny analysis of the gene showed a high conservation of this gene among vertebrates. In addition, we cloned the cDNA encoding a putative 26RFa receptor, G protein-coupled receptor 103 (GPR103) in the zebra finch brain. GPR103 cDNA encoded a 432 amino acid protein that has seven transmembrane domains. In situ hybridisation analysis in the brain showed that the expression of 26RFa mRNA is confined to the anterior-medial hypothalamic area, ventromedial nucleus of the hypothalamus and the lateral hypothalamic area, the brain regions that are involved in the regulation of feeding behaviour, whereas GPR103 mRNA is distributed throughout the brain in addition to the hypothalamic nuclei. When administered centrally in free-feeding male zebra finches, 26RFa increased food intake 24 h after injection without body mass change. Diencephalic GPR103 mRNA expression was up-regulated by fasting for 10 h. Our data suggest that the hypothalamic 26RFa-its receptor system plays an important role in the central control of food intake and energy homeostasis in the zebra finch.     

Development of intersecting CNS fiber tracts: the corpus callosum and its perforating fiber pathway

What are the mechanisms acting during development at points of intersection of central nervous system fiber tracts which influence the direction taken by a population of growing axons? In order to address this question, the ontogeny of the intersecting rostral corpus callosum and its perforating fiber pathway (PF), and the microenvironment through which these fiber systems grow, were examined in a series of mouse embryos and early postnates. Our results show that the perforating fibers are identifiable in silverstained sections between embryonic days (E) 15 and 16, at least 1 day prior to the initial appearance of the callosal projection. Soon after the PF can be identified, a dense accumulation of subventricular cells surrounds the PF at a point just ventral to the location where the callosum and PF will intersect (i.e., at the corticoseptal boundary). Callosal axons, which are present at the point of intersection beginning on E17, do not join the perforating fibers, nor do they appear to penetrate the underlying population of subventricular cells. Instead, the callosal fibers turn across the PF and enter the contralateral cerebral hemisphere. Thus, the intersection of the callosal and perforating fiber systems during development may be related both to the sequential development of each pathway and to the altered nonneuronal environment at the point of intersection.     

Nitric oxide and gamma-aminobutyric acid as regulators of neurogenesis in the brain of adult mammals: Models of seizure activity

New neurons are known to be generated in the brain of adult mammals throughout their entire life in the area of the lateral ventricles and the subgranular zone of the dentate gyrus. The regulatory mechanisms of neurogenesis are complex and poorly understood. Numerous studies performed during the last decade have shown that the intensity of generation of new cells in the germinative regions of the brain is significantly influenced by various environmental factors. Pronounced changes in neurogenesis were also found in the models of various pathologies of the central nervous system (such as neurodegeneration, brain ischemia, and epilepsy). This review is focused on the regulation of neurogenesis in the brain of adult mammals in the course of experimental epilepsy. The involvement of nitric oxide and gamma-aminobutyric acid in the regulation of the proliferation and differentiation of brain cells during seizure activity is discussed.     

Cytoarchitectonic organization and morphology of cells of the field L complex in male zebra finches (Taenopygia guttata).

The organization of the field L complex, a thalamorecipient auditory region in the telencephalon of birds, was examined in Nissl and Golgi preparations of male zebra finches (Taenopygia guttata). The field L complex comprises five cytoarchitectonic subdivisions: L1, L2a, L2b, L3, and L, although the border between L and L2b is not distinct. L2a is a plate extending dorsocaudally from the dorsal medullary lamina in the caudal neostriatum. L1 lies on the anterodorsal border and L3 lies on the posteroventral border of L2a. L, the area designated "field L" by Rose (J. Psychol. Neurol., 1914, 2:278-352), forms the medial and posterior borders of the field L complex. L2b is a thick band that forms the dorsal and dorsolateral boundary of the field L complex and is continuous with L medially. Nucleus interface (NIf) is a nucleus that lies between L2a and L1 near the lateral edge of the complex. The four types of Golgi stained neurons that occur in the zebra finch field L complex correspond to those described for the European starling (Sturnus vulgaris). Additionally, type 3 neurons are subdivided into "unoriented" neurons with spherical dendritic fields and "oriented" neurons with bipolar dendritic fields. NIf contains a distinct class of neurons that have large somata with both thick and thin spiny dendrites. The distribution of Golgi cell types between the subdivisions of the field L complex corresponds to the morphology of cells seen in Nissl material. Type 3 oriented cells are found almost exclusively within L2a. L3 has significantly greater numbers of the largest cells (type 1) and significantly smaller numbers of the smallest cells (type 4) than does L1. There are no significant differences in the distribution of Golgi stained cells between L2b and L.     

Sex differences in cell proliferation and glucocorticoid responsiveness in the zebra finch brain

Neural proliferation is a conserved property of the adult vertebrate brain. In mammals, stress reduces hippocampal neuronal proliferation and the effect is stronger in males than in females. We tested the effects of glucocorticoids on ventricular zone cell proliferation in adult zebra finches where neurons are produced that migrate to and incorporate within the neural circuits controlling song learning and performance. Adult male zebra finches sing and have an enlarged song circuitry; females do not sing and the song circuit is poorly developed. Freshly prepared slices from adult males and females containing the lateral ventricles were incubated with the mitotic marker BrdU with or without steroid treatments. BrdU-labeled cells were revealed immunocytochemically and all labeled cells within the ventricular zone were counted. We identified significantly higher rates of proliferation along the ventricular zone of males than in females. Moreover, acute administration of corticosterone significantly reduced proliferation in males with no effects in females. This effect in males was replicated by RU-486, which appears to act as an agonist of the glucocorticoid receptor in the songbird brain. The corticosterone effect was reversed by Thiram, which disrupts corticosterone action on the glucocorticoid receptor. Sex differences in proliferation and responses to stress hormones may contribute to the sexually dimorphic and seasonal growth of the neural song system of songbirds.     

Expression of trophinin and bystin identifies distinct cell types in the germinal zones of adult rat brain

In the adult brain, the subventricular zone (SVZ) is one of the regions where active neurogenesis occurs. Relatively few specific markers are available to distinguish different types of cells in the SVZ and rostral migratory stream (RMS) of adult brain. Here, we showed that trophinin and bystin, both of which are required for early embryo implantation during development, were expressed in the SVZ and RMS of the adult rat brain, but not in the brain of embryos and early postnatal animals. Trophinin-expressing cells were immunopositive for both Ki-67 and nestin in the SVZ. Some of the trophinin-positive cells did not express either the type A cell marker polysialylated weakly adhesive form of the neural cell adhesion molecule (PSA-NCAM) or the type B cell marker glial fibrillary acidic protein (GFAP). Double-label immunohistochemistry revealed that bystin-positive cells co-expressed GFAP, Ki-67 and nestin, but not PSA-NCAM, suggesting that they are likely type B cells. Intracerebroventricular infusion of cytosine-beta-d-arabiofuranoside (Ara-C) eliminated trophinin-positive cells in the SVZ. Following its depletion, however, the remaining bystin-positive cells continued to divide and generate actively dividing trophinin-positive cells that were negative for PSA-NCAM, leading to reconstruction of SVZ network. These characteristics indicate that this subset of trophinin-positive cells in the SVZ is type C cells. Conversely in the RMS, trophinin co-localized with nestin and PSA-NCAM, suggesting that it is expressed in neuroblasts. Cultured neural precursor cells derived from the adult SVZ also expressed both trophinin and bystin. These findings provide insight into the molecular basis of adult neurogenesis in the SVZ and RMS.     

Generation recruitment and death of brain cells throughout the life cycle of Sorex shrews (Lipotyphla)

Young shrews of the genus Sorex that are born in early summer reduce their body size before wintering, including a reduction of brain weight of 10–30%. In the spring they mature sexually, double their body weight and regain about half of the loss in brain weight. To investigate the mechanisms of brain weight oscillations we studied the rate of cell death and generation in the brain during the whole life cycle of the common shrew ( Sorex araneus ) and pygmy shrew ( S. minutus ). After weaning, shrews generate new brain cells in only two mammalian neurogenic zones and approximately 80% of these develop into neurones. The increase of the shrew brain weight in the spring did not depend on recruitment of new cells. Moreover, adult Sorex shrews did not generate new cells in the dentate gyri. Injections of 5-HT1A receptor agonists in the adult shrews induced neurogenesis in their dentate gyri, showing the presence of dormant progenitor cells. Generation of new neurones in the subventricular zone of the lateral ventricles and their recruitment to olfactory bulbs continued throughout life. TUNEL labelling showed that the rate of cell death in all brain structures, including the proliferation zones and olfactory bulb, was very low throughout life. We conclude that neither cell death nor recruitment significantly contributes to seasonal oscillations and the net loss of brain weight in the Sorex shrews. With the exception of dentate gyrus and olfactory bulb, cellular populations of brain structures are stable throughout the life cycle of these shrews.     

Organization of the zebra finch song control system: II. Functional organization of outputs from nucleus Robustus archistriatalis.

The organization of projections from nucleus robustus archistriatalis (RA) was mapped by a combination of anatomical tracer techniques. After injections of retrograde tracers in the syringeal part of the hypoglossal motor nucleus (nXIIts), labelled cells were seen in ipsilateral RA, in agreement with previous work. However, a shallow band in the dorsal and dorsocaudal part of RA did not contain labelled cells. Cells in this "cap" area were labelled following tracer injections in the dorsomedial nucleus of midbrain ICo (DM), the other known target of RA projections. The topography of outputs to nXIIts was further examined by making small injections of retrograde tracer into physiologically identified control zones for individual syringeal muscles in nXIIts. The distribution of labelled cells in RA revealed bands of cells that cross RA in approximately horizontal layers and project to different parts of nXIIts. This topography was confirmed with the anterograde tracer PHA-L. Thus RA contains two functional subdivisions, one related to midbrain centers for vocalization and the other directly controlling syringeal motorneurons. The latter area can be further divided into zones that preferentially engage particular syringeal muscles.     

Review: Adult neurogenesis and its role in neuropsychiatric disease,brain repair and normal brain function

Neural stem/progenitor cells (NSPCs) in the mammalian brain retain the ability to generate new neurones throughout life in discrete brain regions, through a process called adult neurogenesis. Adult neurogenesis, a dramatic form of adult brain circuitry plasticity, has been implicated in physiological brain function and appears to be of pivotal importance for certain forms of learning and memory. In addition, failing or altered neurogenesis has been associated with a variety of brain diseases such as major depression, epilepsy and age‐related cognitive decline. Here we review recent advances in our understanding of the basic biology underlying the neurogenic process in the adult brain, focusing on mechanisms that regulate quiescence, proliferation and differentiation of NSPCs. In addition, we discuss how neurogenesis influences normal brain function, and in particular its role in memory formation, as well as its contribution to neuropsychiatric diseases. Finally, we evaluate the potential of targeting endogenous NSPCs for brain repair.     

Anatomy of the brain neurogenic zones revisited: fractones and the fibroblast/macrophage network

Cytogenesis in adult peripheral organs, and in all organs during development, occurs nearby basal laminae (BL) overlying connective tissue. Paradoxically, cytogenesis in the adult brain occurs primarily in the subependymal layer (SEL), a zone where no particular organization of BL and connective tissue has been described. We have reinvestigated the anatomy of the area considered the most neurogenic in the adult brain, the SEL of the lateral ventricle, in zones adjacent to the caudate putamen, corpus callosum, and lateral septal nucleus. Here, we report structural (confocal microscopy using laminin as a marker) and ultrastructural evidence for highly organized extravascular BL, unique to the SEL. The extravascular BL, termed fractones because of their fractal organization, were regularly arranged along the SEL and consisted of stems terminating in bulbs immediately underneath the ependyma. Fractones contacted local blood vessels by means of their stems. An individual fractone engulfed in its folds numerous processes of astrocytes, ependymocytes, microglial cells, and precursor cell types. The attachment site (base) of stems to blood vessels was extensively folded, overlying large perivascular macrophages that belong to a fibroblast/macrophage network coursing in the perivascular layer and through the meninges. In addition, collagen-1, which is associated with BL and growth factors during developmental morphogenetic inductions, was immunodetected in the SEL and particularly regionalized within fractones. Because macrophages and fibroblasts produce cytokines and growth factors that may concentrate in and exert their effect from the BL, we suggest that the structure described is implicated in adult neurogenesis, gliogenesis, and angiogenesis.     

The presence of an audience modulates responses to familiar call stimuli in the male zebra finch forebrain

The ability to recognize familiar individuals is crucial for establishing social relationships. The zebra finch, a highly social songbird species that forms lifelong pair bonds, uses a vocalization, the distance call, to identify its mate. However, in males, this ability depends on social conditions, requiring the presence of an audience. To evaluate whether the presence of bystanders modulates the auditory processing underlying recognition abilities, we assessed, by using a lightweight telemetry system, whether electrophysiological responses driven by familiar and unfamiliar female calls in a high‐level auditory area [the caudomedial nidopallium (NCM)] were modulated by the presence of conspecific males. Males had experienced the call of their mate for several months and the call of a familiar female for several days. When they were exposed to female calls in the presence of two male conspecifics, NCM neurons showed greater responses to the playback of familiar female calls, including the mate's call, than to unfamiliar ones. In contrast, no such discrimination was observed in males when they were alone or when call‐evoked responses were collected under anaesthesia. Together, these results suggest that NCM neuronal activity is profoundly influenced by social conditions, providing new evidence that the properties of NCM neurons are not simply determined by the acoustic structure of auditory stimuli. They also show that neurons in the NCM form part of a network that can be shaped by experience and that probably plays an important role in the emergence of communication sound recognition.     

Evidence for the spontaneous production but massive programmed cell death of new neurons in the subcallosal zone of the postnatal mouse brain

In the last 10 years, many studies have reported that neural stem/progenitor cells spontaneously produce new neurons in a subset of adult brain regions, including the hippocampus, olfactory bulb (OB), cerebral cortex, substantia nigra, hypothalamus, white matter and amygdala in several mammalian species. Although adult neurogenesis in the hippocampus and OB has been clearly documented, its occurrence in other brain regions is controversial. In the present study, we identified a marked accumulation of new neurons in the subcallosal zone (SCZ) of Bax-knockout mice in which programmed cell death (PCD) of adult-generated hippocampal and OB neurons has been shown to be completely prevented. By contrast, in the SCZ of wild-type (WT) mice, only a few immature (but no mature) newly generated neurons were observed, suggesting that virtually all postnatally generated immature neurons in the SCZ were eliminated by Bax-dependent PCD. Treatment of 2-month-old WT mice with a caspase inhibitor, or with the neurotrophic factor brain-derived neurotrophic factor, promoted the survival of adult-generated neurons, suggesting that it is the absence of sufficient neurotrophic signaling in WT SCZ that triggers the Bax-dependent, apoptotic PCD of newly generated SCZ neurons. Furthermore, following focal traumatic brain injury to the posterior brain, SCZ neurogenesis in WT mice was increased, and a subset of these newly generated neurons migrated toward the injury site. These data indicate that the adult SCZ maintains a neurogenic potential that could contribute to recovery in the brain in response to the injury-induced upregulation of neurotrophic signaling.