Carlos Crespo

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.

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.

Calretinin- and parvalbumin-immunoreactive neurons in the rat main olfactory bulb do not express NADPH-diaphorase activity.

The presence of nitric oxide synthase (NOS) in neuronal elements expressing the calcium-binding proteins calretinin (CR) and parvalbumin (PV) was studied in the rat main olfactory bulb. CR and PV were detected by using immunocytochemistry and the nitric oxide (NO) -synthesizing cells were identified by means of the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) direct histochemical method. The possible coexistence of NADPH-diaphorase and each calcium-binding protein marker was determined by sequential histochemical-immunohistochemical double-labeling of the same sections. Specific neuronal populations were positive for these three markers. A subpopulation of olfactory fibers and olfactory glomeruli were positive for either NADPH-diaphorase or CR. In the most superficial layers, groups of juxtaglomerular cells, superficial short-axon cells and Van Gehuchten cells demonstrated staining for all three markers. In the deep regions, abundant granule cells were NADPH-diaphorase- and CR-positive and a few were PV-immunoreactive. Scarce deep short-axon cells demonstrated either CR-, PV-, or NADPH-diaphorase staining. Among all these labeled elements, no neuron expressing CR or PV colocalized NADPH-diaphorase staining. The present data contribute to a more detailed classification of the chemically- and morphologically-defined neuronal types in the rodent olfactory bulb. The neurochemical differences support the existence of physiologically distinct groups within morphologically homogeneous populations. Each of these groups would be involved in different modulatory mechanisms of the olfactory information. In addition, the absence of CR and PV in neuronal groups displaying NADPH-diaphorase, which moreover are calmodulin-negative, indicate that the regulation of NOS activity in calmodulin-negative neurons of the rat olfactory bulb is not mediated by CR or PV.

Calretinin-like immunoreactivity in the optic tectum of the tench (Tinca tinca L.)

The distribution of calretinin-like immunopositive cells and fibers in the optic tectum of the tench (Tinca tinca) was studied by using a polyclonal antibody and the avidin-biotin-peroxidase technique. A clear laminated pattern of calretinin-like immunoreactivity was observed. The stratum periventriculare demonstrated a large number of strongly labeled cells whereas in the strata album centrale and griseum centrale, and at the boundary between the strata griseum centrale and fibrosum et griseum superficiale, some scarce, weakly immunostained cells were observed. No immunoreactive cells were seen in the strata fibrosum et griseum superficiale, opticum and marginale. Cells belonging to neuronal types X and XIV, previously characterized using Golgi impregnation, were found to be calretinin-like immunoreactive. Most calretinin-like immunopositive fibers were found in the strata fibrosum et griseum superficiale and opticum with a distribution pattern similar to retinotectal axons in these layers. In agreement with previous biochemical studies, our data suggest that, by contrast to all other classes of vertebrates, instead of calretinin and calbindin D-28k, only one protein is present in teleosts. Nevertheless, the calretinin-like immunostaining pattern in the teleost optic tectum was more complex than that previously described for calbindin D-28k. When compared to the calretinin-immunostaining in the rat superior colliculus, it is evident the presence in both amniotes and anamniotes of calretinin-immunopositive retinotectal axons. However, the distribution patterns of intrinsic calretinin-immunoreactive cells were different.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide synthase activity in the olfactory bulb of anuran and urodele amphibians

Nitric oxide synthase activity was studied by means of NADPH-diaphorase activity and nitric oxide synthase immunoreactivity in the main and accessory olfactory bulbs of the frog Rana perezi and the newt Triturus marmoratus. In both species, NADPH-diaphorase staining was observed in all olfactory fibers. Vomeronasal fibers were NADPH-diaphorase-positive in Triturus but they were NADPH-diaphorase-negative in Rana. Nitric oxide synthase immunoreactivity was not observed in the primary afferents in any case. Granule cells were NADPH-diaphorase-positive and nitric oxide synthase-immunopositive in the main and accessory olfactory bulb of Rana, and in the main olfactory bulb of Triturus. The homogeneous NADPH-diaphorase staining of olfactory fibers is similar to what has been reported in teleosts, and it contrasts with the spatial segregation of NADPH-diaphorase-positive and -negative olfactory projections in rodents. These results confirm the interspecies variability of the NADPH-diaphorase/nitric oxide synthase distribution in the olfactory system of vertebrates.

Calretinin-, neurocalcin-, and parvalbumin-immunoreactive elements in the olfactory bulb of the hedgehog (Erinaceus europaeus).

The distribution pattern and morphology of calretinin‐, neurocalcin‐, and parvalbumin‐immunoreactive neurons were studied in the main and accessory olfactory bulbs of the hedgehog. The detection of these markers was carried out by using monoclonal or polyclonal antibodies and the avidin‐biotin‐immunoperoxidase method. Specific neuronal populations were positive for these calcium‐binding proteins in the hedgehog olfactory bulb, revealing both similarities to and differences from the data reported in the olfactory bulb of rodent species. The distribution pattern of each calcium‐binding protein studied in the accessory olfactory bulb was highly similar to that described in other macrosmatic species. However, in the main olfactory bulb , the markers analyzed were expressed in similar interneuronal populations as they are in the rodent olfactory bulb, whereas cell groups categorized as projecting neurons demonstrated striking differences in the expression of these calcium‐binding proteins. These results suggest that the expression of calcium‐binding proteins in a given brain region is not a constant feature among species despite a similar organization but that different factors could influence their expression. Thus, the accessory olfactory system involved in the processing of specific and similar olfactory cues among species demonstrates a more constant organization among species. By contrast, the functionally important role of the main olfactory system in the hedgehog is accompanied by a more complex organization, which is reflected in an increased diversity of calcium‐buffering systems. J. Comp. Neurol. 429:554–570, 2001.