Jesús G. Briñón

Calbindin D-28k-positive neurons in the rat olfactory bulb

SummaryWe have studied the distribution of calbindin D-28k immunoreactivity in the rat olfactory bulb using specific monoclonal antibodies and the avidin-biotin-immunoperoxidase method. The largest number of positive neurons was located in the periglomerular layer. These neurons were identified as periglomerular cells; they have been described also by other authors as calbindin-positive elements. Close to these neurons, a second population of nerve cells was identified as superficial shortaxon neurons. The remaining layers showed a smaller number of stained elements. Other labeled neurons were located along the external border of the external plexiform layer; the scarce neurons marking its internal border were identified as van Gehuchten cells. No immunoreactive structures were found in the mitral cell layer, although we observed another population of immunostained short-axon cells at its internal border. Some reactive structures, identified by us as horizontal and vertical cells of Cajal, were located in the boundary zone between the internal plexiform layer and the granule layer. In the white matter, we found a neuronal type characterized by its large size and oriented arborization of varicose dendrites.

Calbindin D-28K and NADPH-diaphorase activity are localized in different populations of periglomerular cells in the rat olfactory bulb

Calbindin D-28k (CaBP) immunocytochemistry and NADPH-diaphorase (ND) histochemistry have been combined in the rat olfactory bulb by successive incubations of the same sections. The outer strata showed a similar neuronal staining pattern for both markers with positive periglomerular neurons (although the CaBP-stained periglomerular cells were six-fold more abundant than the ND-active ones) and larger neurons scattered in the glomerular and external plexiform layers. Both populations of periglomerular cells were distinct but they did not show specific morphological characteristics nor a predominant distribution around ND-positive and negative glomeruli. The colocalization study demonstrates that the larger ND and CaBP-stained juxtaglomerular cells, identified according to their size, location and processes branching patterns as two types of short axon cells (superficial short-axon and Van Gehuchten Cells) were also independent populations.

Coexpression of neurocalcin with other calcium‐binding proteins in the rat main olfactory bulb

The distribution patterns of four calcium‐binding proteins (CaBPs)—calbindin D‐28k (CB), calretinin (CR), neurocalcin (NC), and parvalbumin (PV)—in the rat main olfactory bulb were compared, and the degrees of colocalization of NC with the other CaBPs were determined by using double immunocytochemical techniques.

Subcellular localization of m2 muscarinic receptors in GABAergic interneurons of the olfactory bulb

We analysed the ultrastructural distribution of the m2 muscarinic receptor (m2R) in the rat olfactory bulb (OB) using immunohistochemical techniques and light and electron microscopy. m2R was differentially distributed within the cellular compartments of γ‐aminobutyric acid (GABA)ergic bulbar interneurons. It is located in the gemmules of granule cells and in the synaptic loci of the interneurons of the external plexiform layer, suggesting that m2R activation could modulate the release of GABA from these interneurons onto principal cells by a presynaptic mechanism. By contrast, the receptor appears in the somata and dendritic trunks of second‐order short‐axon interneurons located in the inframitral layers, suggesting that postsynaptic muscarinic activation in these cells could elicit the inhibition of granule cells, leading to a disinhibition of principal cells. We also detail the anatomical substrate for a new putative muscarinic modulation that has not been previously described, and that could influence the reception of sensory information within the olfactory glomeruli. m2R appears in a subset of GABAergic/dopaminergic juxtaglomerular cells innervated by olfactory axons but is absent in juxtaglomerular cells that do not receive sensory inputs. This finding suggests that m2R activation could modify, through dopaminergic local circuits, the strength of olfactory nerve inputs onto principal cells. Activation of the muscarinic receptor may modulate the olfactory information encoding within olfactory glomeruli and may facilitate the bulbar transmission to superior centres influencing the GABA release by presynaptic and postsynaptic mechanisms. Taken together, our data provide the neuroanatomical basis for a complex action of m2R at different levels in the mammalian OB.

Dasatinib as a Bone-Modifying Agent: Anabolic and Anti-Resorptive Effects

Background Bone loss, in malignant or non-malignant diseases, is caused by increased osteoclast resorption and/or reduced osteoblast bone formation, and is commonly associated with skeletal complications. Thus, there is a need to identify new agents capable of influencing bone remodeling. We aimed to further pre-clinically evaluate the effects of dasatinib (BMS-354825), a multitargeted tyrosine kinase inhibitor, on osteoblast and osteoclast differentiation and function. Methods For studies on osteoblasts, primary human bone marrow mensenchymal stem cells (hMSCs) together with the hMSC-TERT and the MG-63 cell lines were employed. Osteoclasts were generated from peripheral blood mononuclear cells (PBMC) of healthy volunteers. Skeletally-immature CD1 mice were used in the in vivo model. Results Dasatinib inhibited the platelet derived growth factor receptor-β (PDGFR-β), c-Src and c-Kit phosphorylation in hMSC-TERT and MG-63 cell lines, which was associated with decreased cell proliferation and activation of canonical Wnt signaling. Treatment of MSCs from healthy donors, but also from multiple myeloma patients with low doses of dasatinib (2–5 nM), promoted its osteogenic differentiation and matrix mineralization. The bone anabolic effect of dasatinib was also observed in vivo by targeting endogenous osteoprogenitors, as assessed by elevated serum levels of bone formation markers, and increased trabecular microarchitecture and number of osteoblast-like cells. By in vitro exposure of hemopoietic progenitors to a similar range of dasatinib concentrations (1–2 nM), novel biological sequelae relative to inhibition of osteoclast formation and resorptive function were identified, including F-actin ring disruption, reduced levels of c-Fos and of nuclear factor of activated T cells 1 (NFATc1) in the nucleus, together with lowered cathepsin K, αVβ3 integrin and CCR1 expression. Conclusions Low dasatinib concentrations show convergent bone anabolic and reduced bone resorption effects, which suggests its potential use for the treatment of bone diseases such as osteoporosis, osteolytic bone metastasis and myeloma bone disease.

Calretinin immunoreactivity in the developing olfactory system of the rainbow trout

The distribution of calretinin immunoreactivity in the developing olfactory system of the rainbow trout was studied by using an indirect immunocytochemical method. Calretinin immunoreactivity was firstly detected at 150 day-degrees in the olfactory placode, where labeled primordial cells were observed. At 250 day-degrees, precursor cells of the olfactory receptor neurons located in the olfactory pit were calretinin-immunoreactive. At 300 day-degrees, recognizable olfactory receptor neurons displayed calretinin immunoreactivity in the olfactory epithelium, and calretinin-immunopositive olfactory axons reached the presumptive olfactory bulb. After hatching (400 day-degrees) and during the subsequent development and maturation of the olfactory system, the number of calretinin-immunopositive olfactory receptor cells increased and distributed homogeneously throughout the olfactory epithelium. Accordingly, new positive olfactory fibers arrived to the olfactory bulb arborizing in olfactory glomeruli distributed in nine different terminal fields. Six days after hatching, calretinin-immunopositive interneurons within the olfactory bulb were also observed. The size and number of calretinin-immunoreactive interneurons increased from this stage to adulthood. The adult pattern demonstrated both similarities and differences with the distribution of calretinin immunoreactivity previously described in the olfactory system of mammals.

Calcium-binding proteins in the periglomerular region of typical and atypical olfactory glomeruli

The distribution of chemically identified neuronal populations was studied in the glomerular layer of the rat olfactory bulb using calcium-binding protein immunocytochemistry combined with acetylcholinesterase histochemistry. Four calcium-binding proteins (calbindin D-28k, parvalbumin, calretinin, and neurocalcin) were analyzed in the periglomerular region of two different glomerular subsets: typical and atypical glomeruli. Atypical glomeruli were clearly distinguishable from typical ones by their dense network of acetylcholinesterase-positive centrifugal fibers. Each calcium-binding protein studied showed a specific distribution pattern in the rat olfactory bulb. Calbindin D-28k-, calretinin-, and neurocalcin-immunoreactive neurons were specially abundant in the glomerular layer. These three calcium-binding proteins had their main expression in neuronal subpopulations directly involved in the glomerular circuitries of the rat olfactory bulb. Specific populations of periglomerular cells were stained for calbindin D-28k, parvalbumin, calretinin, or neurocalcin, whereas external tufted cells were only immunoreactive to neurocalcin. Both neuronal types, periglomerular cells and external tufted cells, were found in the periglomerular region of both glomerular subsets. Nevertheless, a homogeneous distribution of calbindin D-28k- or calretinin-immunopositive periglomerular cells were found between typical and atypical glomeruli, whereas the neurocalcin-immunostained external tufted cells were statistically more abundant in typical glomeruli than in atypical ones (P<0.001). These data suggest that some neuronal subpopulations are related with general properties of the glomerular physiology, and they have a homogeneous distribution in different subsets of glomeruli, whereas other chemically identified populations are related with a finer tuning of the olfactory processing, and they are segregately distributed in relation to particular glomerular subsets. In addition, this work adds new differences in the cellular composition of typical and atypical glomeruli.

Chemical organization of the macaque monkey olfactory bulb: II. Calretinin, calbindin D‐28k, parvalbumin, and neurocalcin immunoreactivity

The distribution and morphologic features of calcium‐binding protein‐ (calbindin D‐28k, calretinin, neurocalcin, and parvalbumin) immunoreactive elements were studied in the macaque monkey olfactory bulb by using specific antibodies and the avidin‐biotin‐immunoperoxidase method. A characteristic laminar pattern of stained elements was observed for each marker. Scarce superficial short‐axon cells and superficial stellate cells demonstrated calbindin D‐28k immunoreactivity in the outer layers, whereas a moderate number of calbindin D‐28k–immunoreactive granule cells and scarce deep short‐axon cells were observed in the inner layers. Calretinin‐staining demonstrated abundant periglomerular cells and granule cells and a scarce number of other interneuronal populations. Most neurocalcin‐immunopositive elements were external and medial tufted cells and periglomerular cells, although other scarcer interneuronal populations were also immunostained. A few superficial and deep short‐axon cells as well as small interneurons in the external plexiform layer were the only elements immunoreactive to parvalbumin. The distribution of the immunoreactive elements in the olfactory bulb of the macaque monkey showed a high similarity to that reported in the human, whereas it demonstrated a different and simpler pattern to what has been reported in the olfactory bulb of macrosmatic animals. It suggests more homogeneous calcium‐mediated cell responses after stimulation that could be correlated to the lower capability to modulate olfactory signals in microsmatic animals. In addition, these results indicate that experimental models in rodents do not provide an accurate estimation of calcium‐binding protein‐immunoreactive neuronal populations in the primate olfactory system. J. Comp. Neurol. 432:389–407, 2001.

Distribution of parvalbumin immunoreactivity in the brain of the tench (Tinca tinca L., 1758).

The distribution of parvalbumin (PV) immunoreactivity in the tench brain was examined by using the avidin‐biotin‐peroxidase immunocytochemical method. This protein was detected in neuronal populations throughout all main divisions of the tench brain. In the telencephalic hemispheres, PV‐immunopositive neurons were distributed in both the dorsal and ventral areas, being more abundant in the area ventralis telencephali, nucleus ventralis. In the diencephalon, the scarce distribution of PV‐containing cells followed a rostrocaudal gradient, and the most evident staining was observed in the nucleus periventricularis tuberculi posterioris and in a few nuclei of the area praetectalis. In the mesencephalon, abundant PV‐immunoreactive elements were found in the tectum opticum, torus semicircularis, and tegmentum. In the tectum opticum, PV‐immunoreactivity presented a laminar distribution. Three PV‐containing neuronal populations were described in the torus semicircularis, whereas in the tegmentum, the PV staining was mainly located in the nucleus tegmentalis rostralis and in the nucleus nervi oculomotorii. In the metencephalon, Purkinje cells were PV‐immunopositive in the valvula cerebelli, lobus caudalis cerebelli, and in the corpus cerebelli. In the myelencephalon, PV immunoreactivity was abundant in the nucleus lateralis valvulae, in the nucleus nervi trochlearis, nucleus nervi trigemini, nucleus nervi abducentis, nucleus nervi glossopharyngei, and in the formatio reticularis. Mauthner cells were also PV immunostained. By contrast to other vertebrate groups, only a restricted population of PV‐containing neurons was GABA‐immunoreactive in the tench, demonstrating that this calcium‐binding protein cannot be considered a marker for GABAergic elements in the teleost brain. This study demonstrates a low phylogenetic conservation of the distribution of PV comparing teleosts and tetrapods. J. Comp. Neurol. 413:549–571, 1999.

Chemical anatomy of the macaque monkey olfactory bulb: NADPH-diaphorase/nitric oxide synthase activity.

The distribution and the morphology of nicotinamide adenine dinucleotide phosphate (NADPH)‐diaphorase (ND)‐activeneuronal nitric oxide synthase (NOS)‐immunoreactive neuronsfibers were studied in the olfactory bulb of three species of primates, i.e., the cynomolgus macaque monkey (Macaca fascicularis), the Japanese macaque monkey (Macaca fuscata), and the pig‐tail macaque monkey (Macaca nemestrina). The ND staining was carried out by means of a direct histochemical method with β‐NADPH as cosubstratenitro blue tetrazolium as chromogen. The NOS immunostaining was carried out by using a polyclonal antibodythe avidin‐biotin peroxidase method. Similar results were found in the three species, where a distinct distribution pattern of ND/NOS‐stained neuronsfibers was observed. All olfactory fibers demonstrated ND‐positive labeling but they were NOS‐immunonegative. In the superficial modulatory area of the olfactory bulb, a few weakly ND‐NOS‐positive periglomerular cells, stellate cells,and darkly stained superficial short‐axon cells were observed. In the inframitral layers, granule cells, deep stellate cells, and deep short‐axon cells were distinguished. Short‐axon cells had oriented morphologiesspiny dendrites. Many thick, varicose ND/NOS‐stained fibers identified as centrifugal fibers were observed in the white matter, granule cell layer, internal plexiform layer, mitral cell layer, and external plexiform layer. This distribution of ND activityNOS immunoreactivity showed similarities to and differences from what has been reported in the olfactory bulb of macrosmatic mammals including rodents (rat, mouse, and hamster)insectivores (hedgehog). These data confirm that the complexity of the ND/NOS staining in the olfactory bulb of one species correlates with the importance of olfaction in the biology of such species. J. Comp. Neurol. 402:419–434, 1998.