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Odorant stimulation of receptor cells results in a calcium influx that activates the transduction pathway. The olfactory neurons extend axons to the olfactory bulb where they synapse onto mitral cells. Ca(2+)-acceptors also may participate in subsequent processing of olfactory information. The present study describes the distribution of calmodulin, calretinin, calbindin-D28k and neurocalcin during rat main olfactory bulb development. From postnatal day 1 (P1) we observed in the olfactory nerve layer a thin external bundle containing calbindin and calretinin whereas calmodulin was present in a large internal bundle. In tufted cells, neurocalcin immunoreactivity was detected at P10 and increased until P20. In mitral cells calmodulin was intensively immunoreactive at P1 but decreased during development to disappear at adulthood whereas calretinin was weakly labelled at P1 but raised in intensity until P20. In granule cells calbindin-D28k and calretinin were detected from P1. Giant neurons were positive for both calretinin and calbindin-D28k from postnatal day 20.

Odorant stimulation of receptor cells results in a calcium influx that activates the transduction pathway. Ca2+ acceptors, such as calmodulin, may mediate between the change in intracellular calcium and the conductance mechanism underlying the initial electrical event. Ca2+ acceptors also may participate in subsequent processing of olfactory information. The identification and characterization of these molecules, therefore, should provide important information about the complex signal transduction pathway involving calcium in olfaction as well as other sensory systems. The present study describes the distribution of six calcium-binding proteins in the rat main olfactory epithelium during postnatal development to determine when different Ca2+ acceptors can be detected and whether they segregate into different layers or portions of the epithelium. Calmodulin, calretinin, calbindin-D28k, neurocalcin, and recoverin were detected immunohistochemically in olfactory receptors but not in basal cells. S-100 immunoreactivity was restricted to glial cells primarily around the cribriform plate. During postnatal development (from P1 to P20), calmodulin, calretinin, calbindin-D28k, and neurocalcin formed a gradient of immunoreactivity descending from the central to the lateral areas in the nasal cavity, whereas recoverin was expressed only in sporadic, mature receptors in the proximal region of the mucosa. At P20, the immunoreactivity pattern for each calcium-binding protein was identical to the adult profile, indicating that the olfactory epithelium had reached maturity by this stage. Olfactory nerve fiber bundles displayed a differential staining pattern from P1 until adulthood for calbindin-D28k and calretinin (internal portions of bundles). Differential calmodulin immunoreactivity of olfactory nerves (large external portions of bundles) appeared at P10. The immunoreactivity of the nerve fiber bundles may reflect a further degree of organization relevant to odor discrimination.

The post-natal developmental expression in rat retina of four calcium-binding proteins belonging to the calmodulin-troponin-C family was investigated by immunohistochemistry using anti-calbindin-D28k, anti-calretinin, anti-hippocalcin and anti-neurocalcin polyclonal antibodies on paraffin sections from Wistar rat retinae aged from post-natal days 1 (P1), 5 (P5), 10 (P10), 20 (P20) to adulthood (8 weeks). Immunoblot using anti-hippocalcin and homogenates proteins from retina, cerebellar cortex, hippocampus and cerebellum was also performed. Hippocalcin immunoreactivity in adult rat retina was demonstrated by both immunohistochemistry and Western blot. During post-natal development, calbindin-D28k, calretinin and neurocalcin immunoreactivity were detected at P1 in ganglion cells, whereas hippocalcin immunoreactivity was seen later at P5 in this cell layer. In the amacrine cell layer, neurocalcin immunoreactivity was detected at P5 and hippocalcin at P10. Calbindin-D28k was labelling the immature horizontal cell, calretinin was detected in nearly all ganglion cells and in some amacrine cells since P1. These three calcium-binding proteins do not seem to play a role in synaptogenesis which takes place later. We confirmed that calbindin-D28k appeared to be a good marker for horizontal cells. The presence of hippocalcin, a myristoylated calcium-binding protein belonging to the recovering subfamily and previously localized in few brain areas has been detected for the first time in retina.

Calbindin-D28k, calretinin, and recoverin, three intracellular calcium-binding proteins belonging to the troponin C/calmodulin superfamily, were immunohistochemically localized in chick pineal during development [from embryonic day 16 (E16) to postnatal day 14 (P14)]. At E18, only calretinin immunoreactivity could be detected in nuclei from follicular pinealocytes. With development, calretinin immunoreactivity expanded from nucleus to cytoplasm, and calretinin immuno-positive cell number increased. At P14 almost al pinealocytes were calretinin positive. Calbindin-D28k immunoreactivity was not detected before E20. During development, many follicular and parafollicular pinealocytes became strongly calbindin-D28k positive, reaching a peak both in intensity and in number at P7; thereafter their number decreased. In addition to pinealocytes, neuron-like cells appeared calbindin-D28k positive at E20 and calretinin positive at P7. Recoverin, a myristoylated protein isolated from vertebrate photoreceptor and which might participate in the inactivation of the phototransduction cascade, was transiently expressed in follicular and parafollicular pinealocytes from P1 to P14 with a maximal expression at P7. This transitory expression may coincide with a transitory light sensitivity period in chick pinealocytes, before complete maturity of the pineal gland.

In pineal gland, melatonin is synthesized in pinealocytes. Pharmacological studies using calmodulin antagonists suggested that melatonin synthesis was regulated through calmodulin. However, immunohistochemical studies showed that calmodulin could only be detected in pineal glial cells, and not in pinealocytes. To further investigate this discrepancy, we have tried to detect calmodulin not seen by immunohistochemical methods. We have used rat and chicken pineal homogenate supernatants and Triton X-100-treated pellets denatured by sodium dodecyl sulfate, subjected to electrophoresis and immunoblotting using anti-calmodulin antibodies. Two different IgG (#465 and #860) purified from anti-calmodulin sera were used. In rat pineal homogenate supernatants, calmodulin could be detected by immunoblotting using both antibodies. Some calmodulin could also be detected in the Triton-treated pellet fractions, but no additional cross-reacting bands were detected. However, in both chicken pineal homogenate supernatants and Triton-extracted pellets, in addition to a calmodulin immunoreactive band, two other proteins with approximate molecular masses (M(r)) of 56 kDa and 60 kDa were detected using anti-calmodulin #465. For comparison, similar immunoblot experiments were performed for detection of calbindin-D28K and calretinin, two other calcium binding proteins expressed in different pineal cell populations. Interestingly, Triton extraction of chicken pineal pellets revealed additional bands cross-reacting with each antibody. Anti-calbindin-D28K cross-reacted strongly with a M(r) = 68 kDa protein and weakly with a M(r) = 56 kDa protein. Anti-calretinin cross-reacted strongly with a M(r) = 93 kDa protein and weakly with a M(r) = 56 kDa protein.

Calcium ions play a critical role in neural development. Insights into the ontogeny of Ca2+ homeostasis were gained by investigating the developmental expression of two E-F hand calcium-binding proteins. Calretinin and calbindin were monitored through their immunoreactivity in the developing chick cerebellum (from E6 to E20). Calbindin was detected from E13 and in Purkinje cells only. Intensity of labelling increased with Purkinje cell development. Calretinin presented a transitory immunoreactivity between E11 and E20 in the internal granular cell layer. This cell layer contains cells which will differentiate into Golgi and granular cells which are calretinin-negative in adult chick cerebellum. Calretinin immunoreactivity presented a peak (both in number of cells and in intensity) at E15 and fell dramatically after E20 while calbindin immunoreactivity was restricted to the Purkinje cells and increased with the development of these cells.

Profound morphological modifications occur during postnatal development of the rat pineal gland. We have immunohistochemically followed those events from postnatal day 1 to 20 by using three cytoarchitectonic markers (S-100, calbindin-D28k, and calretinin) that belong to the calmodulin/troponin C calcium-binding protein family. In the developing rat pineal, anticalbindin-D28k antibody labels three cell types: immature and mature astrocytes and perivascular type II pinealocytes. During development, calbindin-D28k positive cells migrate from the base of the pineal stalk into the superficial part of the pineal. Calbindin-D28k, usually used as a neuronal marker in the central nervous system, recognizes in rat pineal precursor astrocytes 5 days before S-100 and labels a subpopulation somewhat different from S-100 positive astrocytes. Calretinin immunoreactivity appeared in the postero-superior part of the pineal and was abundant until postnatal day 5, then its density dramatically felt to leave, after postnatal day 20, an occasional population of cells whose morphology is compatible with neuron-like cells.

Calmodulin, calbindin-D28k and calretinin are calcium-binding proteins largely distributed in the bipolar olfactory receptor cells. In the olfactory epithelium their distribution seemed to be random. Using immunohistochemistry we have examined their localization in rat olfactory axons extending to the olfactory bulb. Sections were analyzed both horizontally and vertically. Almost all fibers were immunoreactive for one of the three intracellular calcium-binding proteins whose distribution was not random among the bundles. Three different subclasses of fibers could be detected: calbindin-D28k and calretinin immunoreactivities were restricted to external fibers whereas calmodulin immunoreactivity was intense, abundant and largely distributed throughout the internal portion of the olfactory nerve. This additional degree of organization detected in the olfactory axons might play a role in odor discrimination.

Calmodulin distribution in the chicken pineal organ was investigated by immunohistochemistry. Calmodulin immunoreactivity was detected in ependymocytes in the follicular zone and in interstitial cells in the parafollicular zone. No calmodulin immunoreactivity was detected in pinealocytes. Lack of calmodulin immunoreactivity in pinealocytes raises questions about its proposed function in melatonin synthesis as suggested by pharmacological studies using calmodulin antagonists. The calmodulin distribution was comparable to that of S100, a glial cell marker. Two other markers, calbindin-D28k and calretinin, which in neuroanatomical studies give excellent cytoarchitectonic staining, in the chick pineal permitted the detection of two subclasses of pinealocytes. One was darkly stained by calbindin-D28k and rare. The other was very abundant and calretinin positive. In the parafollicular zone, calbindin-D28k and/or calretinin antibodies allowed us to visualize cells presenting a neuron-like morphology. Calretinin immunoreactivity was detected in nearly all pinealocytes in which hydroxy-indol-O-methyl transferase was also located. Comparison between the lack of calmodulin and the presence of calretinin, belonging to the same calcium-binding protein family, in chick pinealocytes raises the hypothesis about a possible role of calretinin in melatonin synthesis.