The distribution of the calcium binding protein calretinin (protein 10) was examined in the rat forebrain by immunohistochemistry. The main and accessory olfactory bulbs had immunoreactive label in granule, periglomerular, and mitral cells. Positive fibers were noted in the external plexiform and granule cell layers, glomeruli, and in the molecular layer of the anterior olfactory nucleus. The cerebral cortex contained calretinin label in nonpyramidal bipolar cells. Cells in the substantia nigra compacta and ventral tegmental area were also calretinin positive as were nigrostriatal and mesolimbic projections (caudate-putamen, nucleus accumbens). In the hippocampus, interneurons were stained in all the subfields of the CA1-CA4 regions. In the thalamus, many positive cells were observed in the periventricular, reticular, lateral habenula, and reunions nuclei. Calretinin immunoreactive cells were particularly abundant in the lateral mamillary and septofimbrial nuclei. Several fiber tracts were also revealed, i.e., the lateral olfactory tract, mamillothalamic tract, fasciculus retroflexus, optic tract, and stria medullaris. These results demonstrate a distinct distribution of calretinin within cell bodies and fibers.

The localization of calretinin in the rat hindbrain was examined immunohistochemically with antiserum against calretinin purified from the guinea pig brain. Calretinin immunoreactivity was found within neuronal elements. The distribution of calretinin-immunoreactive cell bodies and fibers is presented in schematic drawings and summarized in a table. Major calretinin-immunoreactive neurons were found in the lateral and medial geniculate nuclei, substantia nigra, ventral tegmental area, interpeduncular nucleus, periaqueductal gray, mesencephalic trigeminal nucleus, superior and inferior colliculi, pontine nuclei, parabrachial nucleus, dorsal and laterodorsal tegmental nuclei, cochlear nuclei, vestibular nuclei, medullary reticular nuclei, nucleus of the solitary tract, area postrema, substantia gelatinosa of the spinal trigeminal nucleus, and cerebellum. These results show that distinct calretinin-immunoreactive neurons are widely distributed in the rat hindbrain.

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A calcium-binding protein (protein 10) having a molecular mass of 29 kDa and an isoelectric point of 5.3 was purified from guinea pig brain. The amino acid sequence of fragments from proteolytic digestion of protein 10 revealed an 86% sequence identity with a calcium-binding protein (calretinin) found in chicken retina. Polyclonal antibodies against protein 10 revealed a specific distribution ofthis protein within sensory neurons of auditory, visual, olfactory, nociceptive, and gustatory systems as well as other discrete neuronal circuits in rat and guinea pig brain, whereas no specific label was observed in any of several peripheral tissues examined.

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The predominantly neuronal, calcium-binding protein calretinin is highly expressed in the guinea pig auditory system. Within the ventral cochlear nucleus (VCN), calretinin-positive auditory nerve fibers terminate on many calretinin-containing bushy, octopus, and multipolar cells. The abundance of calretinin in the cochlear nucleus provides an ideal system for examining the effects of altered neuronal input on the expression of this calcium-binding protein. The present experiments examined the effects of unilateral cochlea ablation on calretinin immunoreactivity and mRNA levels in the VCN. Calretinin mRNA was labeled by in situ hybridization histochemistry using a radioactive oligonucleotide probe and was quantified by optical density measures on autoradiograms. Survival times of 1, 7, and 56 days postlesion were examined. The results revealed a consistent increase in calretinin mRNA in the rostral portion of the ipsilateral anterior VCN 1 day postlesion but no effect on calretinin mRNA in this region at 7 and 56 days postlesion. The intensity of immunohistochemical label was also increased at 1 and 7 days after surgery. In contrast, calretinin mRNA was not affected 1 day postlesion in the ipsilateral posterior VCN but was decreased at both 7 and 56 days postlesion. The decrease in calretinin mRNA in the posterior VCN at longer survival times was accompanied by decreased immunolabeling of fibers projecting from VCN cells to the superior olivary complex. These results suggest that calretinin gene expression is regulated in part by auditory nerve activity in some cochlear neurons but that additional factors related to the unique cellular milieu also control calretinin expression.

The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF‐hand calcium‐binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane‐associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.

The expression of the calcium-binding protein calretinin (CR) in the chick brainstem auditory nuclei angularis (NA), laminaris (NL), and magnocelularis (NM) was studied during normal development and after deafening by surgical removal of the otocyst (embryonic precursor of the inner ear) or columella (middle ear ossicle). CR mRNA was localized by in situ hybridization by using a radiolabeled oligonucleotide chick CR probe. CR immunoreactivity (CR-IR) was localized on adjacent tissue sections. CR mRNA signal in the auditory nuclei was expressed at comparable levels at embryonic day (E)9 and E11 and increased thereafter to reach the highest levels in posthatch chicks. CR-IR neurons were apparent in NM and NA at E11 and in NL by E13, and CR-IR increased in all three auditory nuclei thereafter. Neither unilateral nor bilateral otocyst removal caused detectable changes in the intensity of CR mRNA expression or CR-IR in the auditory nuclei at any of the several ages examined. Similarly, columella removal at posthatching day 2 or 3 failed to significantly affect CR mRNA or CR-IR levels at 3 hours, 1 day, or 3-4 days survival times. We conclude that cochlear nerve input is not necessary for expression of either calretinin mRNA or protein and that the profound decrease in sound-evoked activity caused by columella removal does not affect the maintenance of CR expression after hatching.