Luis Martínez-Millán

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.

Expression of the genes for α-type and β-type calcitonin gene-related peptide during postnatal rat brain development

Abstract In this study we have analysed the expression of the genes for both α -type and β -type calcitonin gene-related peptide (CGRP) during postnatal development of the rat brain and compared it with the expression of CGRP-like immunoreactivity. At birth both α -type and β -type CGRP messenger RNA were present in the parabrachial nucleus, inferior olive and motor nuclei (except for abducens nucleus), and only α -type CGRP messenger RNA in some posterior thalamic nuclei. As development advanced, new nuclei started to express either only α -CGRP gene (superior olive, parabigeminal, sagulum, and some hypothalamic and cranial thalamic nuclei) or both genes (abducens nucleus). In the inferior olive both genes were transiently expressed. β -CGRP messenger RNA disappeared by postnatal day 10 and α -CGRP messenger RNA by postnatal day 20. During the whole postnatal development β -CGRP gene expression predominated over that of α -CGRP in the trigeminal and eye motor nuclei, while in the remainder nuclei α -CGRP messenger RNA was either the predominant isoform or the sole one. CGRP-like immunoreactivity, which does not distinguish between α -type and β -type CGRP, was detected in those nuclei containing either α -CGRP messenger RNA or β -CGRP messenger RNA. However, no CGRP messenger RNA was detected in areas such as superior colliculus, lateral pontine nucleus, pars reticulata of the substantia nigra, perifornical area, or zona incerta in which CGRP-like immunoreactivity was prominent. CGRP-like immunoreactivity, but not CGRP messenger RNA, was also transiently detected by postnatal day 5 in some cells of the globus pallidus. In the adult brain, the levels of α - and β -CGRP messenger RNA as well as those of CGRP-like immunoreactivity were considerably reduced. This fact, similar to that of other growth- and development-associated factors, suggests a role for CGRP as a neuron-derived neurotrophic factor. The transient expression in neurons of the inferior olive, matching the period when climbing fibres and cerebellar cortex are developing, seems to support such an idea. The results of this study show that those nuclei expressing β -CGRP gene also express α -CGRP gene. However, there are a number of nuclei that only express α -CGRP gene. On the other hand, CGRP-like immunoreactivity is detected in some nuclei which express no CGRP messenger RNA. It suggests that such nuclei express any CGRP-related protein (identified by the antibodies against CGRP) or, if they really contain CGRP protein, this is produced from undetectable amounts (using our in situ hybridization histochemistry procedure) of CGRP messenger RNA or it comes from other nuclei that connect with them in which CGRP protein is synthesized and then transferred.

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. J. Comp. Neurol. 446:166–178, 2002.

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

Expression of the genes for α-type and β-type calcitonin gene-related peptide during rat embryogenesis

Abstract Throughout rat embryogenesis we analysed the expression patterns of the three mature transcripts generated from the two calcitonin gene-related peptide genes: calcitonin, α-calcitonin gene-related peptide, and β-calcitonin gene-related peptide messenger RNAs. In addition, we examined in parallel the distribution of calcitonin gene-related peptide and calcitonin immunoreactivity. Of the three transcripts, β-calcitonin gene-related peptide messenger RNA was first detected in sensory ganglia on embryonic day 14, and by embryonic day 15 was seen to a lesser degree in motor neurons and autonomic ganglia. Starting at embryonic day 16, however, the highest levels of β-calcitonin gene-related peptide messenger RNA were found in motor neurons rather than sensory ganglia. α-calcitonin gene-related peptide messenger RNA was first detected on embryonic day 16 in both sensory ganglia and motor neurons, but at lower levels than β-calcitonin gene-related peptide, particularly in the motor neurons of the spinal cord. By embryonic day 20, transcripts for α- and β-calcitonin gene-related peptide were expressed in distinct brain regions. High levels of α-calcitonin gene-related peptide messenger RNA were detected in hypoglossal, facial, and parabrachial nuclei, and moderate levels in the trigeminal motor and ambiguus nuclei. By contrast, β-calcitonin gene-related peptide messenger RNA was detected at low levels in hypoglossal, ambiguus, facial, and parabrachial nuclei, and at high levels in the trigeminal nucleus. In the oculomotor-trochlear nucleus, β-calcitonin gene-related peptide messenger RNA was the sole isotype expressed. Low levels of messenger RNA for both calcitonin gene-related peptide transcripts were appreciated in the inferior olive. Outside the nervous system, α-calcitonin gene-related peptide messenger RNA was weakly expressed in the thyroid gland and β-calcitonin gene-related peptide messenger RNA in the thymus. Throughout embryogenesis, calcitonin gene-related peptide immunoreactivity usually followed the expression of either α- or β-calcitonin gene-related peptide messenger RNA. Calcitonin messenger RNA and protein were detected only in the thyroid gland from embryonic day 18 onward. This work shows that of the three mature transcripts produced by the two calcitonin gene-related peptide genes, β-calcitonin gene-related peptide messenger RNA is the predominant transcript produced early in rat embryogenesis. However, by perinatal stages α-calcitonin gene-related peptide shows the highest expression in the brain and spinal cord. In autonomic ganglia, β-calcitonin gene-related peptide is either the sole or the predominant transcript. Unlike the chick embryo in which calcitonin messenger RNA is expressed early in the CNS, in rat it was only expressed outside the nervous system in the thyroid gland during the last days of embryogenesis.

Anatomical evidence for glutamate and/or aspartate as neurotransmitters in the geniculo-, claustro-, and cortico-cortical pathways to the cat striate cortex

Data obtained by using various experimental approaches suggest that in the mammalian brain, most neurons within the visual system projecting to the striate cortex employ excitatory amino acids as transmitters. In order to investigate further the neurotransmitter phenotype of the ipsilateral afferents to area 17 of the cat, we have injected D‐[3H]‐aspartate, a retrograde tracer which selectively reveals putative glutamatergic and/or aspartatergic pathways, into this area. Retrogradely labelled neurons were observed in the dorsal lateral geniculate nucleus, visual claustrum, cortical areas 18, 19, 21a, and in both posteromedial and posterolateral parts of the suprasylvian areas but not in other known thalamic afferents such as the lateral posterior‐pulvinar complex and the intralaminar nuclei. The distribution and localization of the labelled cells in all these regions were similar to that observed by using the non‐selective tracer horseradish peroxidase conjugated to wheat germ agglutinin, though the number of cells was higher with the latter. Our findings provide additional evidence for the presence of excitatory amino acids as neurotransmitters in the major afferents to the cat striate cortex.

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. J. Comp. Neurol. 499:716–731, 2006.

Ionotropic glutamate receptor subunits are differentially regulated in the motoneuronal pools of the rat hypoglossal nucleus in response to axotomy

Unilateral hypoglossal nerve axotomy was used as a model to analyse immunohistochemically the expression of the GluR1, GluR2, GluR3, and GluR4 glutamate receptor subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype and the NR1 subunit of the N-methyl-D-aspartate (NMDA) subtype in the different morphofunctional hypoglossal pools from 1 to 45 days postaxotomy. Following hypoglossal nerve axotomy, the percentage of motoneurons that were GluR1-immunopositive and the labeling intensity for this subunit was increased in some hypoglossal pools. Immunolabeling for the GluR2 subunit was undetectable. These results contrast with the unchanged pattern for these two subunits after sciatic nerve axotomy previously described. Image analysis showed a significant decrease in the intensity of immunohistochemical labeling for the GluR2/3 and GluR4 subunits in motoneurons, although most motoneurons were still immunopositive for these 2 subunits after axotomy. The intensity of immunolabeling for the NR1 subunit was slightly decreased postlesion, whereas the percentage of NR1-immunopositive motoneurons increased. Immunoreactivity returned to basal levels 45 days postlesion. These findings show that in axotomized hypoglossal motoneurons, i) AMPA and NMDA receptor subunits are still expressed, ii) the composition of the ionotropic glutamate receptor subunit pool is subjected to continuous changes during the regeneration process, iii) AMPA receptors, if functional, would have physiological properties different to those in intact motoneurons, and iv) the various AMPA receptor subunits are differentially regulated. The present results also suggest a faster recovery of basal levels of immunoreactivity for caudally localised groups of motoneurons which could reflect a caudo-rostral sequential functional revovery in the hypoglossal nucleus.

Sprouting of the visual corticocollicular terminal field after removal of contralateral retinal inputs in neonatal rabbits

Abstract The morphological changes occurring in the visual corticocollicular projection following removal of the contralateral retina (within the first 48 h of postnatal life) were studied using New Zealand rabbits. At 45–50 days after lesion, the corticocollicular terminal field was examined by anterograde transport of Phaseolus vulgaris leucoagglutinin, which was applied iontophoretically in the central region of the contralateral striate cortex. In contrast to normal intact rabbits of the same age, the corticocollicular terminal field was markedly enlarged in experimental animals. In the centre of the field we found abundant oblique fibres which sent out branches. These collateral fibres coursed over long distances, parallel to the pial surface, in the stratum zonale and in the upper part of the stratum griseum superficiale. The presence of these fibres, together with an increased density of synaptic boutons at more superficial levels of the sprouted terminal field, suggest that corticocollicular fibres tended to occupy territories left vacant when retinocollicular axons degenerated after enucleation. The high density and extensive distribution of these corticocollicular fibres may be due to the continued growth of the fibres, which occupy an extensive territory during the early postnatal stages and which, under normal circumstances are retracted during the process of postnatal maturation. Despite the expansion of the field occupied by corticocollicular synapses, its centre coincided topographically with the field centres in normal animals, indicating the existence of intrinsic positional cues that persisted after enucleation and determined the arrangement of visual cortical afferents. This model, which involves substantial changes in terminal field organization, should prove useful in elucidating the cellular and molecular processes underlying regeneration and plasticity in the visual system.

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. Anat Rec, 292:1968–1975, 2009.