Anosmin-1 over-expression increases adult neurogenesis in the subventricular zone and neuroblast migration to the olfactory bulb.

New subventricular zone (SVZ)-derived neuroblasts that migrate via the rostral migratory stream are continuously added to the olfactory bulb (OB) of the adult rodent brain. Anosmin-1 (A1) is an extracellular matrix protein that binds to FGF receptor 1 (FGFR1) to exert its biological effects. When mutated as in Kallmann syndrome patients, A1 is associated with severe OB morphogenesis defects leading to anosmia and hypogonadotropic hypogonadism. Here, we show that A1 over-expression in adult mice strongly increases proliferation in the SVZ, mainly with symmetrical divisions, and produces substantial morphological changes in the normal SVZ architecture, where we also report the presence of FGFR1 in almost all SVZ cells. Interestingly, for the first time we show FGFR1 expression in the basal body of primary cilia in neural progenitor cells. Additionally, we have found that A1 over-expression also enhances neuroblast motility, mainly through FGFR1 activity. Together, these changes lead to a selective increase in several GABAergic interneuron populations in different OB layers. These specific alterations in the OB would be sufficient to disrupt the normal processing of sensory information and consequently alter olfactory memory. In summary, this work shows that FGFR1-mediated A1 activity plays a crucial role in the continuous remodelling of the adult OB.

Guanosine-induced synaptogenesis in the adult brain in vivo.

Astrocytes release factors like cholesterol, apoE, and pleiotropic molecules that influence synaptogenesis in the central nervous system. In vitro studies have shown that guanosine elicits the production and further release of these synaptogenic factors. To demonstrate that such astrocytic factors are synaptogenic in vivo, osmotic pumps were implanted in primary visual cortex (VC) of Sprague-Dawley rats to deliver guanosine. Simultaneous injection of dextran amine as an anterograde tracer at the same site where the osmotic pumps were implanted enabled the morphology of the fibers emerging from the VC to be visualized as well. The guanosine-treated efferent connections from these animals showed a significant increase in the number and size of synaptic boutons along the efferent fibers when compared with controls. A similar increase in the number and size of synaptic boutons was also detected when the cortico-cortical connection to the lateral secondary visual area was studied in more detail. The ensuing morphological changes to the synapses did not show a clear preference for any particular type or site of the axonal branches that integrates this cortical connection. Moreover, the distribution of boutons along the fibers was clearly stochastic according to their size. Thus, guanosine administration appears to open up the possibility of manipulating connections to compensate for total or partial denervation.

NMDA-receptor blockade enhances cell apoptosis in the developing retina of the postnatal rat.

During visual system development, programmed cell death occurs in order to facilitate the establishment of correct connections and synapses. During this period, glutamate plays a very important role as an excitatory neurotransmitter. With a view to evaluating if NMDA glutamate receptors participate in the regulation of apoptosis which occurs during the development of the rat retina, we subcutaneously injected the NMDA receptor antagonist MK-801 into rats at different stages of early postnatal development (P2 to P9). Ensuing cell death in the retina and superior colliculus was analyzed by using the Feulgen method. MK-801 administration had no effect on the survival of photoreceptor cells. In contrast, the presence of this antagonist induced a significant increase in the number of apoptotic cells in the neuroblastic layer (P7 and P8) and ganglion cell layer (P6-P8), as well as in the superior colliculus which receives afferent contacts from retinal ganglion cells during P7-P9. We conclude that during development, specific types of cells in the mammalian retina are critically dependent for their survival on glutamate stimulation through NMDA receptors. These findings thus throw fresh light on the mechanisms of development of the rat visual system by identifying NMDA glutamate receptors as participants in the regulation of apoptotic processes which occur during the initial stages of development.

Organization and origin of the connection from the inferior to the superior colliculi in the rat.

The inferior colliculus (IC) is the main ascending auditory relay station prior to the superior colliculus (SC). The morphology and origin of the connection from inferior to superior colliculus (I-SC) was analyzed both by anterograde and retrograde tracing. Irrespective of the subregion of the IC in which they originate, the terminal fields of these connections formed two main tiers in the SC. While the dorsal one primarily involved the stratum opticum and the stratum griseum intermediale, the ventral one innervated the deep strata, although some fibers did connect these tiers. While the dorsal tier occupied almost the whole extension of the SC, the ventral one was mostly confined to its caudomedial quadrant. The fiber density in these tiers decreased gradually in a rostral gradient and the terminal fields became denser as the anterograde tracer at the injection site was distributed more externally in the cortex of the IC. Retrograde tracing confirmed this result, although it did not reveal any topographic ordering for the I-SC pathway. Most presynaptic boutons of the I-SC terminal field were located either inside or close to the patches of acetylcholinesterase activity. Together with previous anatomical and physiological studies, our results indicate that the I-SC connection relays behaviorally relevant information for sensory-motor processing. Our observation that this pathway terminates in regions of the superior colliculus, where neurons involved in fear-like responses are located, reinforce previous suggestions of a role for the IC in generating motor stereotypes that occur during audiogenic seizures.

Expression pattern of calcitonin gene-related peptide in the superior colliculus during postnatal development: demonstration of its intrinsic nature and possible roles.

Calcitonin gene-related peptide (CGRP) is a widespread neuropeptide with multiple central and peripheral targets. In an analysis on the expression of this peptide throughout the rat brain during postnatal development, we observed a discrepancy between results obtained by immunohistochemistry and by in situ hybridization. In the superior colliculus (SC), only the immunohistochemical signal could be detected (Terrado et al. [1997] Neuroscience 80:951-970). Here we focus our attention on this structure because the temporal pattern of CGRP immunoreactivity observed in the SC suggested the participation of this peptide in the postnatal maturation of the SC. In the present study, we describe in detail the postnatal development of collicular CGRP-immunoreactive structures and their spatiotemporal relationship with cholinergic modules and definitively demonstrate the local expression of CGRP in the SC. CGRP-immunopositive axons and neurons were distributed within the most ventral part of superficial strata and in the intermediate strata of the SC, showing a peak in staining intensity and density at the end of the first postnatal week. At P14, CGRPergic terminal fibers are arranged in small, clearly defined patches in a complementary manner with respect to the cholinergic modules, which start forming at this stage. By using Western blot and RT-PCR analyses, and by means of injections of antisense oligonucleotides, both the presence of CGRP peptide in the SC and the local expression of alpha-CGRP transcripts in collicular neurons were demonstrated. A possible role of CGRP is discussed in the context of postnatal modular compartmentalization of collicular afferents.

Developmental roles of p73 in Cajal-Retzius cells and cortical patterning.

To examine the role of the p53 homolog p73 in brain development, we studied p73-/-, p73+/-, E2F1-/-, and reeler mutant mice. p73 in developing brain is expressed in Cajal-Retzius (CR) cells, the cortical hem, and the choroid plexus. p73-expressing CR cells are lost in p73-/- embryos, although Reelin is faintly expressed in the marginal zone. Ectopic neurons in the p73-/- preplate and cortical hem at embryonic day 12 implicate p73 in the early developmental program of the cortex; however, preplate partition and early cortical plate formation are not disturbed. Postnatal p73-/- mice show a mild hypoplasia of the rostral cortex and a severely disrupted architecture of the posterior telencephalon. In the developing p73-/- hippocampus, the most striking abnormality is the absence of the hippocampal fissure, suggesting a role of p73 in cortical folding. p73+/- mice have a less severe cortical phenotype; they display a dorsal shift of the entorhinal cortex and a reduced size of occipital and posterior temporal areas, which acquire entorhinal-like features such as Reelin-positive cells in layer II. CR cells appear unaffected by heterozygosity. We relate the malformations of the posterior pole in p73 mutant mice to alterations of p73 expression in the cortical hem and suggest that p73 forms part of an early signaling network that controls neocortical and archicortical regionalization. In mice deficient for the transcription factor E2F1, a main activator of the TAp73 (transactivating p73) isoform, we find a defect of the caudal cortical architecture resembling the p73+/- phenotype along with reduced TAp73 protein levels and propose that an E2F1-TAp73 dependent pathway is involved in cortical patterning.

Connection from the dorsal column nuclei to the superior colliculus in the rat: topographical organization and somatotopic specific plasticity in response to neonatal enucleation.

Somatosensory stimuli from the body to deep and intermediate strata of the superior colliculus (SC) are relayed from the dorsal column nuclei (DCN), gracile (GrN) and cuneate (CuN). Electrophysiological studies have shown that the somatosensory representation in SC is arranged into a map-like pattern. However, there is a lack of studies confirming a morphological correlate of such an organization. On the other hand, after neonatal enucleation in rodents, somatosensory inputs ascend from their normal termination territory in intermediate and deep collicular strata to invade the more dorsally located visual strata. However, the origin of these reactive afferents has not been specified. By using anterograde (biotinylated dextran amine 10,000; BDA) and retrograde (Fluoro-Gold; FG) tracers, we studied separately the connection from GrN and CuN to the intact and neonatally deafferented SC. GrN-collicular afferents were found to terminate mainly within the periphery of the caudomedial SC quadrant, whereas CuN-collicular fibers innervated primarily the lateral part of the rostrolateral and caudolateral collicular quadrants, in a way consistent with previously described functional data. Retrograde tracing experiments using FG injected in SC confirmed this topographical arrangement. Injections of BDA in GrN or CuN of neonatally enucleated rats showed that reactive fibers reaching superficial strata are only those CuN-collicular fibers innervating the caudolateral SC quadrant, where the forelimb is represented. The present results provide an anatomical substrate for the known somatotopic organization of tactile representation in SC and further reinforce the previous proposal that the plastic reorganization of DCN-collicular afferents following neonatal enucleation constitutes a functional compensatory response.

Quantifying presynaptic terminals at the light microscope level in intact and deafferented central nervous structures.

Quantification of presynaptic terminals often requires laborious techniques that involve tissue preparation for ultrastructural analysis. Modern preembedding immunohistochemical techniques provide a high morphological resolution at the light microscope level, thus allowing us to identify immunostained presynaptic boutons using specific antibodies. When absolute density of boutons (D(a)) is analysed for comparison between control and deafferented nervous tissue, quantification may be distorted due to tissue shrinkage that follows deafferentiation. The magnitude of this effect must be, therefore, estimated to correct quantitative data. Using the superior colliculus (SC) as a model, an easily applicable protocol to quantify the density of small size labelled particles in control and deafferented nervous tissue is described. This protocol was used to analyse the effect of neonatal and adult enucleation on the adult pattern of cholinergic input to the rat SC. Statistical treatment of data demonstrated that neonatal enucleation caused a drastic increase in bouton density in the visual collicular layers, stratum zonale (SZ) and stratum griseum superficiale (SGS). The same lesion carried out in adult animals caused an increase in the bouton density exclusively in the SZ.

Plastic reaction of the rat visual corticocollicular connection after contralateral retinal deafferentiation at the neonatal or adult stage: axonal growth versus reactive synaptogenesis.

The effects of neonatal or adult enucleation on the final adult pattern of the rat visual corticocollicular (C-Co) connection were studied using the anterograde tracer biotinylated dextranamine 10,000 (BDA) iontophoretically injected in the primary visual cortex. In control animals, column-shaped terminal fields limited to a small portion of the collicular surface were observed. Synaptic boutons were present in all superficial strata of the superior colliculus (SC), with the highest density in the ventral part of the stratum griseum superficiale (SGS). Neonatal enucleation caused a considerable expansion of the contralateral visual C-Co terminal fields, which occupied almost the entire collicular surface, suggesting that axonal sprouting had occurred. In addition, terminal boutons tended to localize more dorsally in these cases compared with controls. Following enucleation in adult animals, no changes were observed with respect to the extension of the terminal fields, although a plastic reaction leading to an increase in the bouton density in the stratum zonale (SZ) and upper SGS was found, reflecting a process of reactive synaptogenesis at these levels. These results show that both neonatal and adult visual C-Co fibers react in response to retinal ablation, although this reaction shows distinct characteristics. Molecular factors, such as growth-associated cytoskeletal proteins operating in the cortical origin, and extracellular matrix components and myelin-associated axonal growth inhibitors acting on the collicular target very likely account for these differences.

Rat retinal ganglion cells co-express brain derived neurotrophic factor (BDNF) and its receptor TrkB.

The expression of brain derived neurotrophic factor (BDNF) and its preferred receptor (TrkB) in rat retinal ganglion cells (RGCs) have been determined in the present study. To identify RGCs retrograde labelling was performed with fluorogold (FG). Subsequently, retinas were immunostained with antibodies to BDNF and TrkB. We found that all RGCs labelled with FG express both BDNF and its preferred receptor, TrkB. Moreover, displaced amacrine cells were also found to be immunolabelled by both antibodies. Thus BDNF/TrkB signalling in RGCs probably involves endogenous BDNF produced by the RGCs themselves.