The use of quantitative immunocytochemistry (QICC) to measure calbindin D28k-like immunoreactivity in the rat brain

We have developed a method of quantitative immunocytochemistry using an iodinated second antibody to visualise the anatomical distribution of primary antibodies in tissue sections, by macroautoradiography. Computer-assisted densitometry was used to analyse the pattern of optical densities within autoradiograms. The amount of antigen present in tissue sections was then quantified by comparison with non-biological standards which were processed in parallel with the tissue sections. Using this technique we have measured calbindin like-immunoreactivity in 4 areas of rat brain and have found that the values obtained are similar to those obtained by radioimmunoassay. A similar approach can be used to quantify autoradiograms by comparison with antigen standards to measure amounts of radiolabelled immunoreactivity and determine concentrations of biologically active molecules in discrete brain areas.     

Immunohistochemical observation of growth-associated protein 43 (GAP-43) in the developing circumvallate papilla of the rat

The distribution and development of growth-associated protein 43 (GAP-43)-like immunoreactivity (-LI) in the rat circumvallate papilla (CVP) were compared to those of protein gene product 9.5 (PGP 9.5)-LI. In the adult, thick GAP-43-like immunoreactive (-IR) structures gathered densely in the subgemmal region. Some of these further penetrated the apical epithelium and trench wall epithelium. At least two types of GAP-43-IR structures were recognized; taste bud-related and non-gustatory GAP-43-IR neural elements. Immunoelectron microscopy revealed that GAP-43-LI was localized predominantly in the Schwann cells, and a few axons displayed GAP-43-LI in the lamina propria. In the trench epithelium, GAP-43-LI was detected in the cytoplasmic side of the axonal membrane. Some intragemmal GAP-43-IR axons made synaptic-like contacts with taste bud cells. At least four developmental stages were defined on the basis of the changes in distribution of GAP-43-LI. In stage I [embryonic day (E) 16–17] GAP-43-IR structures accumulated at the lamina propria just beneath the newly-formed circumvallate papilla. In stage II (E18–19) GAP-43-IR nerve fibers began to penetrate the apical epithelium. In stage III [E20-postnatal day (P) 0] GAP-43-IR nerve fibers first appeared in the trench wall epithelium. Penetration of GAP-IR nerve fibers occurred in the inner trench wall epithelium first, and then in the outer trench wall epithelium. In stage IV (P1-) the distribution of GAP-43-LI was similar to that observed in the adult; but the density of GAP-43-LI was much higher than in adults. PGP 9.5-LI showed a similar distribution pattern to that of GAP-43-LI, except for round-shaped cells in the apical epithelium at the late embryonic stages, and in taste bud cells and intralingual ganglionic cells which lacked GAP-43-LI. The similarities in distribution patterns of GAP-43-LI and PGP 9.5-LI during the development and mature circumvallate papilla suggest that GAP-43 may be a key neuronal molecule for induction and maintenance of the taste buds.     

Calbindin D28k and calretinin in oxytocin and vasopressin neurons of the rat supraoptic nucleus

The aim of the present study was to examine quantitatively whether two calcium-binding proteins, calbindin D28k and calretinin, are localized in oxytocin and vasopressin neurons of the supraoptic nucleus of the male rat. We used a triple-labeling immunofluorescence method with a confocal laser scanning microscope. Of the oxytocin-labeled cells, 70% were stained for both calbindin D28k and calretinin, 15% were stained for only calbindin D28k, 13% were stained for only calretinin, and 2% were stained for neither protein. Of the vasopressin-labeled cells, 73% were stained for neither calbindin D28k nor calretinin, 21% were stained for only calbindin D28k, 4% were stained for only calretinin, and 2% were stained for both proteins. Calbindin D28k and calretinin have been shown previously to contribute to calcium homeostasis by buffering [Ca2+]i. Therefore, these findings suggest that most of the oxytocin neurons may have a higher Ca(2+)-buffering capacity than most of the vasopressin neurons.     

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

Summary We 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.     

Observations on the ultrastructure of ganglion cells in the circumvallate papilla of rat and mouse

Ganglion cells in the circumvallate papilla of adult rodents are described as typical autonomic neurons. Some neurons are aggregated to form a discrete structure in the base of the papilla; others are scattered through the core, along the nerve bundles, and particularly near the dome. The term "circumvallate ganglion" is applied to the entire population. Satellite cells completely ensheathe each neuron. Preganglionic fibers, containing clear vesicles, synapse on the soma and stumpy dendrites of the neurons. Axons, containing dense-cored vesicles, are observed in close proximity to the neurons. However, these fibers do not establish true morphological synaptic contacts with the neurons. We have not observed serial or reciprocal synapses on or in the vicinity of the ganglion cells. The hypothesis that the axons of the circumvallate ganglion neurons act as parasympathetic vasodilators is indicated by the proximity of the two structures and by nerve terminations on the arteriole muscle cells. Direct modulation of taste transduction by these neurons is ruled out.     

Secretions of von Ebner's glands influence responses from taste buds in rat circumvallate papilla

The influence of secretions from von Ebner's lingual salivaryglands on gustatory function was studied in the rat. Neurophysiologicaltaste responses elicited by chemical stimulation of the circumvallatepapilla were recorded from the glossopharyngeal nerve whileinitiating salivary secretion in the same papilla. Salivarysecretion from von Ebner's glands significantly reduced tasteresponses to stimulation of the circumvallate papilla with variouschemicals. However, the magnitude of the reduction in responsediffered depending on the taste stimulus used. The reductionin response due to salivary secretion was blocked by prior administrationof the parasympathetic antagonist, atropine. These results demonstratea direct effect of salivary secretion on taste responses andillustrate the close relationship between taste function andthe secretion of von Ebner's glands.     

Influence of innervation on the levels of noradrenaline and serotonin in the circumvallate papilla of the rat

The levels and the distribution of monoamines within the rat circumvallate papilla have been studied. Noradrenaline was found in the connective tissue underlying the taste buds, whereas serotonin was located in the basal area of the gustatory epithelium but not inside the taste buds. Following denervation, noradrenaline levels decreased and serotonin levels increased. These results suggest that both neurotransmitters may have some mutual interaction in modulating transmission at the papilla.     

Calbindin D28k, parvalbumin, and calretinin immunoreactivity in the main and accessory olfactory bulbs of the gray short-tailed opossum, Monodelphis domestica

The vertebrate main and accessory olfactory bulbs (MOB and AOB) are the first synaptic sites in the olfactory pathways. The MOB is a cortical structure phylogenetically well conserved in its laminar structure and overall synaptic organization, while the AOB has significant species variation in size. In order to better understand signal processing in the two olfactory systems and the species differences, immunocytochemical staining and analysis were done of the neuronal expression patterns of the calcium-binding proteins calbindin D28k (CB), parvalbumin (PV), and calretinin (CR) in the MOB and AOB in a marsupial species, the gray short-tailed opossum, Monodelphis domestica. In the MOB, antibody to CB labeled periglomerular cells, superficial short axon cells / Van Gehuchten cells; antibody to PV labeled Van Gehuchten cells; and antibody to CR immunostained periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. In the AOB, CB immunoreactivity was detected in periglomerular cells and a subpopulation of granule cells; antibody to PV labeled the superficial short axon cells / Van Gehuchten cells and granule cells; and antibody to CR labeled a small number of periglomerular cells, superficial short axon cells / Van Gehuchten cells, and granule cells. These results showed that the patterns of CB, PV, and CR expression differ in the opossum main and accessory olfactory bulbs and differ from that in other animal species. These varying patterns of neuronal immunostaining may be related to the different functions of the main and accessory olfactory bulbs and to the differing signal processing features.     

Protein phosphorylation in developing and regenerating rat kidney

Renal cytosolic extracts from rats of different ages and mononephrectomized rats were incubated with gamma-[32P]ATP and analysed by high resolution two-dimensional electrophoresis and autoradiography. Extracts from new-born and young rats showed a great number of phosphorylated proteins migrating between the origin and Mr 52,000. Among these proteins, the group co-migrating with phosphorylase b (Mr 97,000) was particularly evident in new-born and days-old rats. In extracts from mature rats, other proteins of lower molecular weight, particularly those migrating between Mr 60,000 and 44,000, became intensely phosphorylated. The number and intensity of phosphorylated proteins from extracts of normal and nephrectomized rats, however, did not vary. Activity of cAMP-dependent protein kinase and [3H]cAMP binding was also modified during neonatal development but not in compensatory renal growth. Since cAMP-PK and protein phosphorylation are known to be regulated in response to hormonal stimulations, these results may provide good indications for the understanding of hormonal involvement in kidney growth.     

Calbindin immunoreactivity of enteric neurons in the guinea-pig ileum

Previous studies have identified Dogiel type II neurons with cell bodies in the myenteric plexus of guinea-pig ileum to be intrinsic primary afferent neurons. These neurons also have distinctive electrophysiological characteristics (they are AH neurons) and 82-84% are immunoreactive for calbindin. They are the only calbindin-immunoreactive neurons in the plexus. Neurons with analogous shape and electrophysiology are found in submucosal ganglia, but, with antibodies used in previous studies, they lack calbindin immunoreactivity. An antiserum that is more effective in revealing calbindin in the guinea-pig enteric nervous system has been reported recently. In the present work, we found that this antiserum reveals the same population that was previously identified in myenteric ganglia, and does not reveal any further population of myenteric nerve cells. In submucosal ganglia, 9-10% of nerve cells were calbindin immunoreactive with this antiserum. The submucosal neurons with calbindin immunoreactivity were also immunoreactive for choline acetyltransferase, but not for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). Small calbindin-immunoreactive neurons (average profile 130 microm2) were calretinin immunoreactive, whereas the large calbindin-immunoreactive neurons (average profile 330 microm2) had tachykinin (substance P) immunoreactivity. Calbindin immunoreactivity was seen in about 50% of the calretinin neurons and 40% of the tachykinin-immunoreactive submucosal neurons. It is concluded that, in the guinea-pig ileum, only one class of myenteric neuron, the AH/Dogiel type II neuron, is calbindin immunoreactive, but, in the submucosal ganglia, calbindin immunoreactivity occurs in cholinergic, calretinin-immunoreactive, secretomotor/vasodilator neurons and AH/Dogiel type II neurons.     

Protein phosphorylation in developing and regenerating rat kidney

Abstract. Renal cytosolic extracts from rats of different ages and mononephrectomized rats were incubated with γ-[³²P]ATP and analysed by high resolution two-dimensional electrophoresis and autoradiography. Extracts from new-born and young rats showed a great number of phosphorylated proteins migrating between the origin and Mr 52,000. Among these proteins, the group co-migrating with phosphorylase b (Mr 97,000) was particularly evident in new-born and days-old rats. In extracts from mature rats, other proteins of lower molecular weight, particularly those migrating between Mr 60,000 and 44,000, became intensely phosphorylated. The number and intensity of phosphorylated proteins from extracts of normal and nephrectomized rats, however, did not vary. Activity of cAMP-dependent protein kinase and [³H]cAMP binding was also modified during neonatal development but not in compensatory renal growth. Since cAMP-PK and protein phosphorylation are known to be regulated in response to hormonal stimulations, these results may provide good indications for the understanding of hormonal involvement in kidney growth.     

Calbindin D28k in mammalian intestinal absorptive cells: immunohistochemical evidence

Calbindin D28k and D9k are two cytosolic calcium-binding proteins abundant in intestinal absorptive cells which appear to play a role in calcium translocation. Until today, calbindin D28k was found in avian and reptilian absorptive cells but not in mammalian ones. We have described the presence of calbindin D28k-immunoreactivity in intestinal absorptive cells of pig and jerboa (Jaculus jaculus). Pig calbindin D28k-immunoreactive absorptive cells were prominent in duodenum, they were scattered along the villi and nearly absent in the crypts. Jerboa labelled absorptive cells were located along the colonic mucosal surface. No calbindin D28k could be detected in mouse, rat and goat absorptive cells. Topography of calbindin D28k absorptive cells was compared with calbindin D9k distribution. Our results confirmed the data of the literature showing a gradient of labelling increasing from the crypt to the top of the villus and no positive endocrine cell. Young (48 h old) pigs did not expressed calbindin D28k in absorptive cells although calbindin D9k was detected. Calbindin D28K was also observed in endocrine cells which were numerous in pig and goat duodenum and very rare in mouse and jerboa. Western blot experiments confirmed the presence of calbindin D28k in the adult pig intestine, in the jerboa colon and the absence of cross-reactivity between calbindin D28k antibody and calbindin D9k.     

The reversible effect of colchicine on the taste bud cells of the central circumvallate papilla in the rat

Summary It is believed that differentiation and maintenance of taste buds in vertebrates is dependent on the trophic function of their sensory nerve supply. In the present work colchicine was injected into the circumvallate papilla of the rat. This produced a reversible blockade of neuroplasmic transport and disappearance of taste buds. Colchicine inhibited the further differentiation of bud cells, but apparently did not change the life cycle of the cells present already at the time of injection. It is speculated that the neurotrophic factors in this particular cell system are effective to induce cell differentiation only.This work was supported by CAIT Grant No 1776     

Calbindin D28k is essentially located in the colonic part of the toad intestine

Summary— The distribution of calbindin D28k in the digestive system and the urinary bladder of the toad was investigated using immunohistochemistry and Western blotting. By analogy with mammals and birds, the protein was expected to be located preferentially in the duodenal part of the intestine. Interestingly, absorptive cells of the duodenum were totally devoid of calbindin D28k while the colon contained high amounts of the calcium-binding protein. This reversed polarity of calbindin D28k content in the toad intestine should obviously correspond to a different scheme of calcium absorption regulation between amphibians and higher vertebrates. Calbindin D28k containing neuroendocrine-like cells were found scattered in the proximal parts of the gut with a similar distribution to what has been described in rat and chick intestine. The oesophagus, the stomach, and the intrinsic nervous sytem of the intestine were negative. No significant amounts of the proteins were found in the urinary bladder, which is known to be a site of Ca²⁺ active transport.     

Calbindin D28k is essentially located in the colonic part of the toad intestine

The distribution of calbindin D28k in the digestive system and the urinary bladder of the toad was investigated using immunohistochemistry and Western blotting. By analogy with mammals and birds, the protein was expected to be located preferentially in the duodenal part of the intestine. Interestingly, absorptive cells of the duodenum were totally devoid of calbindin D28k while the colon contained high amounts of the calcium-binding protein. This reversed polarity of calbindin D28k content in the toad intestine should obviously correspond to a different scheme of calcium absorption regulation between amphibians and higher vertebrates. Calbindin D28k containing neuroendocrine-like cells were found scattered in the proximal parts of the gut with a similar distribution to what has been described in rat and chick intestine. The oesophagus, the stomach, and the intrinsic nervous sytem of the intestine were negative. No significant amounts of the proteins were found in the urinary bladder, which is known to be a site of Ca²⁺ active transport.     

Effect of testosterone on serotonin and noradrenaline concentrations and taste bud cell number of rat circumvallate papilla

Intramuscular administration of testosterone (T) to male orfemale rats produced a significant increase in the rate of developmentof the intermediate type of taste bud cells. T treatment produceda similar effect in rats previously submitted to the in-blockor selective removal of the main salivary glands. 5-Hydroxytryptamine(5-HT) and noradrenaline (NA) concentrations in the vallatepapilla of normal rats were sensitive to T. The concentrationof 5-HT decreased significantly and the concentration of NAincreased slightly, both with respect to the controls.     

Calbindin D-28k-immunoreactivity in rat muscle spindles during postnatal maturation and after denervation

Summary Calbindin D-28k-immunoreactivity has been demonstrated in some of the intrafusal muscle fibres and in the capsule of adult rat muscle spindles. In this study, the immunocytochemical localization of calbindin D-28k in the muscle spindles of triceps surae muscle was studied during postnatal maturation and after denervation. In young rats calbindin D-28k-immunoreactivity was seen in a few intrafusal fibres, first at the age of 4 days. At the 7th day, three calbindin D-28k-immunoreactive fibres and one unlabelled fibre were seen in most muscle spindles, as in adult rats. The spindle capsule and perineurial sheath of nerves were first seen to exhibit calbindin D-28k immunoreactivity at the age of 14 days, and thereafter the localization of calbinding D-28k-like immunoreactivity was similar to that in adult rats. After denervation, calbindin D-28k-immunoreactivity remained in intrafusal muscle fibres and the spindle capsule for a long period. After two months of denervation, calbindin D-28k immunoreactivity could still be seen in the spindle capsule, but the intrafusal fibres were not labelled.The innervation is known to have trophic effects on the intrafusal fibres. The present findings suggest that the expression of calbindin D-28k-immunoreactivity in maturating muscle spindles may be induced by the developing innervation. The decrease of calbindin D-28k-immunoreactivity in intrafusal fibres after denervation may be due to the loss of trophic factors released by the nerves.