An immunohistochemical study on the distribution of vasotocin neurons in the brain of two weakly electric fish, Gymnotus omarorum and Brachyhypopomus gauderio.

Hypothalamic nonapeptides (arginin vasotocin-vasopressin, oxytocin-isotocin) are known to modulate social behaviors across vertebrates. The neuroanatomical conservation of nonapeptide systems enables the use of novel vertebrate model species to identify general strategies of their functional mechanisms. We present a detailed immunohistochemical description of vasotocin (AVT) cell populations and their projections in two species of weakly electric fish with different social structure, Gymnotus omarorum and Brachyhypopomus gauderio. Strong behavioral, pharmacological, and electrophysiological evidence support that AVT modulation of electric behavior differs between the gregarious B. gauderio and the solitary G. omarorum. This functional diversity does not necessarily depend on anatomical differences of AVT neurons. To test this, we focus on interspecific comparisons of the AVT system in basal non-breeding males along the brain. G. omarorum and B. gauderio showed similar AVT somata sizes and comparable distributions of AVT somata and fibers. Interestingly, AVT fibers project to areas related to the control of social behavior and electromotor displays in both species. We found that no gross anatomical differences in the organization of the AVT system account for functional differences between species, which rather shall depend on the pattern of activation of neurons embedded in the same basic anatomical organization of the AVT system.

Post-hatching brain morphogenesis and cell proliferation in the pulse-type mormyrid Mormyrus rume proboscirostris.

The anatomical organization of African Mormyrids' brain is a clear example of departure from the average brain morphotype in teleosts, probably related to functional specialization associated to electrosensory processing and sensory-motor coordination. The brain of Mormyrids is characterized by a well-developed rhombencephalic electrosensory lobe interconnected with relatively large mesencephalic torus semicircularis and optic tectum, and a huge and complex cerebellum. This unique morphology might imply cell addition from extraventricular proliferation zones up to late developmental stages. Here we studied the ontogeny of these brain regions in Mormyrus rume proboscirostris from embryonic to adult stages by classical histological techniques and 3D reconstruction, and analyzed the spatial-temporal distribution of proliferating cells, using pulse type BrdU labeling. Brain morphogenesis and maturation progressed in rostral-caudal direction, from 4day old free embryos, through larvae, to juveniles whose brain almost attained adult morphological complexity. The change in the relative size of the telencephalon, and mesencephalic and rhombencephalic brain regions suggest a developmental shift in the relative importance of visual and electrosensory modalities. In free embryos, proliferating cells densely populated the lining of the ventricular system. During development, ventricular proliferating cells decreased in density and extension of distribution, constituting ventricular proliferation zones. The first recognizable one was found at the optic tectum of free embryos. Several extraventricular proliferation zones were found in the cerebellar divisions of larvae, persisting along life. Adult M. rume proboscirostris showed scarce ventricular but profuse cerebellar proliferation zones, particularly at the subpial layer of the valvula cerebelli, similar to lagomorphs. This might indicate that adult cerebellar proliferation is a conserved vertebrate feature.

Temporal patterns of tyrosine nitration in embryo heart development.

Tyrosine nitration is a biomarker for the production of peroxynitrite and other reactive nitrogen species. Nitrotyrosine immunoreactivity is present in many pathological conditions including several cardiac diseases. Because the events observed during heart failure may recapitulate some aspects of development, we tested whether nitrotyrosine is present during normal development of the rat embryo heart and its potential relationship in cardiac remodeling through apoptosis. Nitric oxide production is highly dynamic during development, but whether peroxynitrite and nitrotyrosine are formed during normal embryonic development has received little attention. Rat embryo hearts exhibited strong nitrotyrosine immunoreactivity in endocardial and myocardial cells of the atria and ventricles from E12 to E18. After E18, nitrotyrosine staining faded and disappeared entirely by birth. Tyrosine nitration in the myocardial tissue coincided with elevated protein expression of nitric oxide synthases (eNOS and iNOS). The immunoreactivity for these NOS isoforms remained elevated even after nitrotyrosine had disappeared. Tyrosine nitration did not correlate with cell death or proliferation of cardiac cells. Analysis of tryptic peptides by MALDI-TOF showed that nitration occurs in actin, myosin, and the mitochondrial ATP synthase α chain. These results suggest that reactive nitrogen species are not restricted to pathological conditions but may play a role during normal embryonic development.

Spatial domains of progenitor-like cells and functional complexity of a stem cell niche in the neonatal rat spinal cord.

During spinal cord development, progenitors in the neural tube are arranged within spatial domains that generate specific cell types. The ependyma of the postnatal spinal cord seems to retain cells with properties of the primitive neural stem cells, some of which are able to react to injury with active proliferation. However, the functional complexity and organization of this stem cell niche in mammals remains poorly understood. Here, we combined immunohistochemistry for cell-specific markers with patch-clamp recordings to test the hypothesis that the ependyma of the neonatal rat spinal cord contains progenitor-like cells functionally segregated within specific domains. Cells on the lateral aspects of the ependyma combined morphological and molecular traits of ependymocytes and radial glia (RG) expressing S100ß and vimentin, displayed passive membrane properties and were electrically coupled via Cx43. Cells contacting the ventral and dorsal poles expressed the neural stem cell markers nestin and/or vimentin, had the typical morphology of RG, and appeared uncoupled displaying various combinations of K(+) and Ca(2+) voltage-gated currents. Although progenitor-like cells were mitotically active around the entire ependyma, the proliferative capacity seemed higher on lateral domains. Our findings represent the first evidence that the ependyma of the rat harbors progenitor-like cells with heterogeneous electrophysiological phenotypes organized in spatial domains. The manipulation of specific functional properties in the heterogeneous population of progenitor-like cells contacting the ependyma may in future help to regulate their behavior and lineage potential, providing the cell types required for the endogenous repair of the injured spinal cord.

GABAergic signalling in a neurogenic niche of the turtle spinal cord.

The region that surrounds the central canal (CC) in the turtle spinal cord is a neurogenic niche immersed within already functional circuits, where radial glia expressing brain lipid binding protein (BLBP) behave as progenitors. The behaviour of both progenitors and neuroblasts within adult neurogenic niches must be regulated to maintain the functional stability of the host circuit. In the brain, GABA plays a major role in this kind of regulation but little is known about GABAergic signalling in neurogenic niches of the postnatal spinal cord. Here we explored the action of GABA around the CC of the turtle spinal cord by combining patch-clamp recordings of CC-contacting cells, immunohistochemistry for key components of GABAergic signalling and Ca(2+) imaging. Two potential sources of GABA appeared around the CC: GABAergic terminals and CC-contacting neurones. GABA depolarized BLBP(+) progenitors via GABA transporter-3 (GAT3) and/or GABA(A) receptors. In CC-contacting neurones, GABA(A) receptor activation generated responses ranging from excitation to inhibition. This functional heterogeneity appeared to originate from different ratios of activity of the Na(+)-K(+)-2Cl(-) co-transporter (NKCC1) and the K(+)-Cl(-) co-transporter (KCC2). In both progenitors and immature neurones, GABA induced an increase in intracellular Ca(2+) that required extracellular Ca(2+) and was blocked by the selective GABA(A) receptor antagonist gabazine. Our study shows that GABAergic signalling around the CC shares fundamental properties with those in the embryo and adult neurogenic niches, suggesting that GABA may be part of the mechanisms regulating the production and integration of neurones within operational spinal circuits in the turtle.

Enigmatic central canal contacting cells: immature neurons in "standby mode"?

The region that surrounds the central canal of the spinal cord derives from the neural tube and retains a substantial degree of plasticity. In turtles, this region is a neurogenic niche where newborn neurons coexist with precursors, a fact that may be related with the endogenous repair capabilities of low vertebrates. Immunohistochemical evidence suggests that the ependyma of the mammalian spinal cord may contain cells with similar properties, but their actual nature remains unsolved. Here, we combined immunohistochemistry for cell-specific markers with patch-clamp recordings to test the hypothesis that the ependyma of neonatal rats contains immature neurons similar to those in low vertebrates. We found that a subclass of cells expressed HuC/D neuronal proteins, doublecortin, and PSA-NCAM (polysialylated neural cell adhesion molecule) but did not express NeuN (anti-neuronal nuclei). These immature neurons displayed electrophysiological properties ranging from slow Ca(2+)-mediated responses to fast repetitive Na(+) spikes, suggesting different stages of maturation. These cells originated in the embryo, because we found colocalization of neuronal markers with 5-bromo-2'-deoxyuridine when injected during embryonic day 7-17 but not in postnatal day 0-5. Our findings represent the first evidence that the ependyma of the rat spinal cord contains cells with molecular and functional features similar to immature neurons in adult neurogenic niches. The fact that these cells retain the expression of molecules that participate in migration and neuronal differentiation raises the possibility that the ependyma of the rat spinal cord is a reservoir of immature neurons in "standby mode," which under some circumstances (e.g., injury) may complete their maturation to integrate spinal circuits.

Connexin 43 delimits functional domains of neurogenic precursors in the spinal cord.

The cells lining the central canal (CC) of the spinal cord derive from the ventral part of the neural tube and, in some vertebrates, are responsible for the functional recovery after spinal cord injury. The region that surrounds the CC in the turtle contains proliferating cells that seem to generate both glia and neurons. Understanding the biology of spinal progenitors with the potential to generate new neurons "in situ" is important for cell replacement therapies. Here, we aimed to identify and characterize precursor cells in the spinal cord of Trachemys dorbignyi. To evaluate the population of proliferating cells, 5-bromo-2'-deoxyuridine (BrdU) was injected every 4 h (50 microg/g, i.p.) during 24 h. We found BrdU(+) nuclei around the CC with a higher density in the lateral quadrants, in which whole-cell patch-clamp recordings showed extensive dye coupling of cells. Carbenoxolone (100 microM) increased the input resistance, suggesting strong gap junction coupling among precursors. The expression of brain lipid binding protein (a marker of a subtype of radial glia) and Pax6 matched the location of clusters, suggesting these cells belonged to a domain of neurogenic precursors. These domains were delimited by a high density of connexin 43 (Cx43) located on the endfeet of CC contacting cells. Our findings indicate that spinal precursors share basic properties with those in the embryo and neurogenic niches of the adult brain, and support a key role of functional clustering via Cx43 in spinal cord neurogenesis.

Cytological organization of the central gelatinosa in the turtle spinal cord.

This paper deals with the cytological organization of the central gelatinosa (CG) in the spinal cord of juvenile (2-12 months) turtles. We found two main cell classes in the CG: one with characteristics of immature neurons, the other identified as radial glia (RG). The cells surrounding the central canal formed radial conglomerates in such a way that the RG lamellae covered the immature neurons. We found three major subpopulations of RG that expressed S-100, glial fibrillary acidic protein, or both proteins. Electron microscopic images showed gap junctions interconnecting RG. As with the mammalian neuroepithelial cells, most CG cells displayed intrinsic polarity expressed by structural and molecular differences between the most apical and basal cell compartments. The apical zone was characterized by the occurrence of a single cilium associated with a conspicuous centrosomal complex. We found a prominent expression of the PCM-1 centrosomal protein concentrated close to the central canal lumen. In the particular case of RG, the peripheral end feet contacted the subpial basement membrane. We also found "transitional cell forms" difficult to classify by the usual imaging approaches. Functional clues obtained by patch-clamp recordings of CG cells defined some of them as already committed to follow the neuronal lineage, whereas others had properties of less mature or migrating cells. The CG appeared as a richly innervated region receiving terminal branches from nerve plexuses expressing gamma-aminobutyric acid, serotonin, and glutamate. The results presented here support our previous studies indicating that the CG is an extended neurogenic niche along the spinal cord of turtles.

Functional and molecular clues reveal precursor-like cells and immature neurones in the turtle spinal cord.

In lower vertebrates, some cells contacting the central canal (CC) retain the ability to proliferate, leading the reconstruction of the spinal cord after injury. A better understanding about the nature of these cells could contribute to the development of novel strategies for spinal cord repair. Here, by combining light and electron microscopy, immunocytochemistry and patch-clamp recordings, we provide evidence supporting the presence of precursor-like cells and immature neurones contacting the CC of juvenile turtles. A class of cells expressed the ependymal and glial cell marker S100 and displayed morphological and electrophysiological features of radial glia: relatively low input resistance, high resting potential, lack of active membrane properties and extensive dye-coupling. A second class of S100 reactive cells were characterized by a higher input resistance and outward rectification. Finally, some CC-contacting cells expressed HuC/D - a marker of immature neurones - and fired action potentials. The coexistence of cells with functional properties of precursor-like cells and immature neurones suggests that the region surrounding the CC is a site of active neurogenesis. It remains to be demonstrated by lineage analysis whether, as in the embryonic cerebral cortex, radial glia are the progenitor cells in the turtle spinal cord.

Environment temperature affects cell proliferation in the spinal cord and brain of juvenile turtles.

The spinal cords and brains--comprising dorsal cortex (DC), medial cortex (MC) and diencephalon (Dien)--of juvenile turtles acclimated to warm temperature [27-30 degrees C; warm-acclimated turtles (WATs)] revealed higher density values of bromodeoxyuridine-labeled cells (BrdU-LCs) than those acclimated to a cooler environment [5-14 degrees C; cold-acclimated turtles (CATs)]. Both populations were under the influence of the seasonal daily light-dark rhythms. Pronounced differences between WATs and CATs (independent t-test; confidence level, P<0.01) were found in the central area of the spinal gray matter and in the ependymal epithelium lining the brain ventricles. Forebrain regions (DC, MC and Dien) also revealed significant differences between WATs and CATs (independent t-test; confidence level, P<0.01-0.05). Unexplored biological clocks that may be affecting cell proliferation were equalized by performing paired experiments involving one WAT and one CAT. Both animals were injected on the same day at the same time and both were sacrificed 24 h later. These experiments confirmed that a warm environment increased cell proliferation in the CNS of turtles. Double- and triple-labeling experiments involving anti-BrdU antibody together with anti-glial protein antibodies revealed that temperature modulates not only cell populations expressing glial markers but also other cells that do not express them. As expected, in the case of short post-injection (BrdU) surviving time points, no cells were found colabeling for BrdU and NeuN (neuronal marker). The probable direct effect of temperature on the cell division rate should be analyzed together with potential indirect effects involving increased motor activity and increased food intake. The fate of the increased BrdU-LCs (death, permanence as progenitor cells or differentiation following neuronal or glial lines) remains a matter for further investigation. Results are discussed in the light of current opinions concerned with post-natal neurogenesis in vertebrates.

Neurogenesis and gliogenesis in the spinal cord of turtles.

A 5-bromo-3'-deoxyuridine (BrdU) pulse administered to juvenile turtles resulted in cell labeling throughout the gray matter (GM) and white matter (WM) of the spinal cord. One and twenty-four hours postinjection, larger densities of BrdU-labeled nuclei (LN) occurred within the GM, with a density peak localized in the central region (CR). Seven days later, density differences between GM and WM disappeared, accompanying a more uniform distribution of LN in the GM (absence of the central peak). Multiple injection experiments also showed similar evolution in the distribution of LN. Morphometric studies revealed that the size of LN had undergone time-related increments: Larger nuclei appeared at protracted fixation time points. Double-labeling experiments indicated that BrdU-labeled cells expressed neuroactive substances, such as gamma-aminobutyric acid (GABA), neuron-specific nuclear protein (NeuN), and the cytoplasmic early postmitotic neuronal marker (TUC-4). Other BrdU-labeled cells expressed the glial-specific protein (GFAP). GABA-BrdU, TUC-4-BrdU, and GFAP-BrdU double-labeled cells were recognized 6 days after the first BrdU injection. NeuN-BrdU double-labeled cells were found at 50 days postinjection. Three-dimensional transmission electron microscopy revealed the presence of synapses and typical kinocilia in putative immature nerve cells. Kinocilia were also found in putative immature glial cells. In consideration of the scattered distribution pattern of BrdU-labeled cells, in animals fixed 1 hour postinjection, the existence of a single proliferating center was discarded. The CR, including the ependymal epithelium, showed the highest density of LN.

A search for a mesencephalic periaqueductal gray-cochlear nucleus connection

Peroxidasa del rábano (HRP), introducida en la substancia grisperiacueductal mesencefálica (PAG) y en la region del complejo olivar superior lateral, demostró la existencia de vías indirectas desde estas zonas hacia el núcleo coclear (NC). No habiéndose demostrado vías directas, hemos propuesto conexiones indirectas a través del ya conocido sistema auditivo eferente. Los resultados obtenidos sugieren tres posibilidades: 1) La PAG está conectada al complejo olivar superior lateral donde existen neuronas cuyas fibras eferentes llegan hasta el NC; 2) Neuronas localizadas en la PAG dorsal demonstraron estar conectadas con el colículo inferior (IC). Se postula la posibilidad que estas fibras PAG-IC hagan sinapsis con neuronas conocidas cuyos axones van desde el IC hasta el NC; 3) Neuronas del cuerpo trapezoide, que comunican con el NC, están también conectadas hacia y desde la PAG. Un estudio electrofisiológico previo (1) ha demostrado cambios en la frecuencia y en la probabilidad de descarga de las neuronas de NC como consecuencia de la estimulación de la PAG. Se postuló además una acción, a través de encefalinas, de la PAG sobre el NC. Los resultados actuales apoyan, anatómicamente, las acciones funcionales de la PAG sobre el NC descritas

A search for a mesencephalic periaqueductal gray-cochlear nucleus connection

Peroxidasa del rábano (HRP), introducida en la substancia grisperiacueductal mesencefálica (PAG) y en la region del complejo olivar superior lateral, demostró la existencia de vías indirectas desde estas zonas hacia el núcleo coclear (NC). No habiéndose demostrado vías directas, hemos propuesto conexiones indirectas a través del ya conocido sistema auditivo eferente. Los resultados obtenidos sugieren tres posibilidades: 1) La PAG está conectada al complejo olivar superior lateral donde existen neuronas cuyas fibras eferentes llegan hasta el NC; 2) Neuronas localizadas en la PAG dorsal demonstraron estar conectadas con el colículo inferior (IC). Se postula la posibilidad que estas fibras PAG-IC hagan sinapsis con neuronas conocidas cuyos axones van desde el IC hasta el NC; 3) Neuronas del cuerpo trapezoide, que comunican con el NC, están también conectadas hacia y desde la PAG. Un estudio electrofisiológico previo (1) ha demostrado cambios en la frecuencia y en la probabilidad de descarga de las neuronas de NC como consecuencia de la estimulación de la PAG. Se postuló además una acción, a través de encefalinas, de la PAG sobre el NC. Los resultados actuales apoyan, anatómicamente, las acciones funcionales de la PAG sobre el NC descritas (AU)