Neurofilament proteins form an annular superstructure in guinea-pig type I vestibular hair cells

Neurofilaments, the neuron-specific intermediate filaments, are composed of three immunochemically distinct subunits: NF-L, NF-M and NF-H that can be either phosphorylated or unphosphorylated. In mammals, the distribution of these subunits has been described in vestibular ganglion neurons, but there are no reports on the presence of neurofilaments in vestibular hair cells. We investigated, by immunocytochemistry, neurofilaments in vestibular hair cells from rat and guinea-pig using antibodies against the three subunits and to dephosphorylated NF-H (clone SMI 32, recognizes also NF-M on immunoblots), on Vibratome sections of the vestibular end-organs and on isolated hair cells. Various immunostaining protocols were used, as appropriate for the method of observation: laser scanning confocal microscopy (immunofluorescence) and transmission electron microscopy (immunoperoxidase, pre-embedding technique). In rat and guinea-pig cristae and utricles, neurofilament immunoreactivity was observed in axons inside and below the sensory epithelia. In guinea-pig, in addition to this staining, intensely immunoreactive annular structures were found in the basal regions of hair cells. These rings were detected with anti-NF-L, -NF-M and -dephosphorylated NF-H/M antibodies, but not with anti-phosphorylation-independent NF-H. Ring-containing hair cells were present in all regions of the sensory epithelia but were more abundant in the peripheral areas. All levels of observation (Vibratome and thin sections, and isolated hair cells) showed that only the guinea-pig type I hair cells contained a neurofilament ring. High-resolution observations showed that the ring was located below the nucleus, often close to smooth endoplasmic reticulum and the cell membrane.     

Developmental expression of alpha 9 acetylcholine receptor mRNA in the rat cochlea and vestibular inner ear

Expression of alpha9 acetylcholine receptor (AChR) mRNA was studied by in situ hybridization in the rat adult and developing cochlea and vestibular inner ear. Alpha9 AChR mRNA was first observed in cochlear hair cells (HCs) at embryonic day 18 (E18), increased markedly after birth, stayed high until postnatal day 10 (P10), and decreased to substantially lower adult levels by P14. High levels of alpha9 AChR mRNA expression were also noted in the developing nonneuronal structures of the inner sulcus, chondrocytes, and/or osteoblasts in the cochlear capsule and interscalar laminae. Both developing and adult bone marrow cells also expressed intense alpha9 AChR mRNA. In the vestibular system, alpha9 AChR mRNA was first observed in HCs at E16 in all sensory epithelia, increased to its highest levels by P0-P4, then decreased slightly to reach adult levels by P10. The results are consistent with the alpha9 AChR subserving efferent neurotransmission to both cochlear and vestibular HCs. The observation of alpha9 AChR mRNA in cochlear HCs 2 weeks prior to functional onset in the cochlea further suggests that expression of this gene is not related to HC activity. The observation of substantial nonneuronal expression of alpha9 AChR mRNA suggests that this receptor also has functions separate from its role in neurotransmission.     

Detection of nerve growth factor mRNA in rodent salivary glands with digoxigenin- and 33P-labeled oligonucleotides: effects of castration and sympathectomy

Nerve growth factor (NGF) is a protein highly expressed in the male mouse submandibular gland. We have applied a non-radioactive in situ hybridization method using digoxigenin-labeled NGF oligonucleotides, and have found the highest amounts of NGF mRNA in the secretory striated ducts of the male mouse submandibular gland. Scattered strongly positive cells were found in male mouse sublingual glands. Weakly labeled cells were seen in female mouse and in male rat submandibular gland striated duct cells. Using 33P as an alternative to 32P and 35S, we demonstrated a 1.3 KB NGF mRNA in salivary glands of male mice by Northern blot hybridization. Using 33P we detected NGF mRNA in male mouse submandibular glands by in situ hybridization but with a signal that, compared with the non-radioactive method, had a very low resolution. Castration of male mice almost abolished both the 1.3 KB NGF mRNA seen with Northern blots and the NGF mRNA labeling in submandibular glands 4 weeks after the operation, whereas levels were increased 6 hr and 2 days after sympathectomy. We conclude that hybridization with digoxigenin-labeled NGF oligonucleotides is a good tool to study the expression and regulation of NGF mRNA in male mouse submandibular glands.     

Analysis of insulin-like growth factors (IGF)-I, and -II, type II IGF receptor and IGF-binding protein-2 mRNA and peptide levels in normal and nephrectomized rat kidney

Immunocytochemistry, in situ hybridization, and radioimmunoassay were employed to examine the cellular distribution of mRNAs and proteins for IGF-I, II, IGF-II/M6P receptor, IGFBP2 as well as the levels of IGF-I and II in normal and unilaterally nephrectomized (Nx) adult rat kidneys. A similar distribution of immunoreactive IGF-I, and -II as well as IGF-II/M6P receptor was found in the principal cells of the cortical collecting duct and in all cells of the inner medullary collecting duct. In addition, immunostainable IGF-I and IGF-II/M6P receptor were noted in some inner medullary loops of Henle, while IGFBP2 was seen in the collecting ducts and loops of Henle of the inner medullar and the renal vasculature of all animals. By comparison, in situ hybridization revealed IGF-I mRNA only in the medullary thick ascending limbs while IGF-II mRNA was localized to the wall of the renal microvasculature in all kidneys. IGFBP2 mRNA was localized to the renal corpuscle and to inner medullary interstitial cells of all kidneys. These data suggest that renal IGF-I and IGFBP2 are synthesized at upstream sites along the nephron and then transported downstream for interaction with IGF receptors. Following nephrectomy, the renal levels of IGF-I peptide and mRNA were elevated at both 5 and 33 days post-nephrectomy, supporting a potential functional role for IGF-I in stimulating the structural and functional recovery in compensatory hypertrophy.     

Neuregulins and erbB receptors at neuromuscular junctions and at agrin-induced postsynaptic-like apparatus in skeletal muscle

This study has characterized the repertoire of the anion exchanger (AE) family members expressed within the guinea pig organ of Corti, the auditory neuroepithelia. Both AE2 and AE3 cDNAs were present, but AE1 cDNA was not detected. The more abundant AE2 was sequenced and its expression characterized in the cochlea. The 3888 base pairs (bp) AE2 sequence, compiled from multiple clones, includes 150 bp of upstream non-coding sequence and 3717 bp of open reading frame encoding a protein of 1238 amino acids. Immunoblot of cochlear homogenate revealed a single AE2-immunoreactive band of Mr 180 kDa. In situ hybridization and immunohistochemical analysis localized AE2 expression to several tissues and cell types within the guinea pig inner ear, including superior half of the spiral ligament and within the interdental cells lining the spiral limbus. However, AE2 was not clearly detected in the outer hair cells (OHC) of the organ of Corti by either immunohistochemistry or in situ hybridization. The results of these studies imply a physiologic role of AE2 in the cochlear homeostasis, but do not support its role as a potential 'motor protein' in mediating the in vitro-observed voltage-gated, ATP-independent OHC motility.     

Histone H3 messenger RNA in situ hybridization for identifying proliferating cells in formalin-fixed rat gastric mucosa

To devise a more sensitive method for identifying proliferative cells in routinely formalin-fixed, paraffin-embedded tissues, we applied an in situ hybridization (ISH) technique for the detection of histone H3 mRNA in rat gastric mucosa and amplified the signal by a silver intensification method. ISH was performed using a Fluorescein-labelled, single-stranded DNA probe for the human histone H3 gene. To determine the optimal conditions for detecting H3 mRNA in rat gastric mucosa, we tested the effect of changing conditions, such as fixation time and digestion time, by a proteinase before hybridization. Next, the proliferation indices obtained using H3 ISH were compared with those obtained using bromodeoxyuridine (BrdU) immunohistochemistry. In normal rat gastric mucosa, H3 ISH- and BrdU-positive cells were confined to the neck region of both fundic and pyloric mucosa. The two labelling indices were almost the same. In all the serial sections studied, H3 ISH-positive cells were almost always BrdU-positive too. Taken together, these results indicate that the H3 ISH technique is useful for the evaluation of proliferative activity in gastric epithelial cells by virtue of its detection of S-phase cells.     

Detection by a solid phase independent enzyme immunoassay of monoclonal anti-idiotypic antibodies against monoclonal antibodies neutralizing Semliki Forest virus and encephalomyocarditis virus

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) is the rate limiting step in the mevalonate pathway that produces isoprenoids and cholesterol. Inhibitors of HMG-CoA reductase are teratogenic in vivo and induce neural tube defects in rat embryo culture, effects which appear unrelated to cholesterol deficiency. This study is the first to localize HMG-CoA reductase mRNA by in situ hybridization (ISH). Expression of reductase mRNA was examined in post-implantation rat embryos, and for control purposes in rat liver and UT-1 cells, using a digoxigenin-11 (dig-11) labelled cRNA probe. Eighteen-day fetal liver showed heavy but patchy hybridization, and adult rat liver showed strong hybridization only on some periportal hepatocytes, which was absent in livers of fasted animals. UT-1 cells stimulated to overexpress HMG-CoA reductase mRNA were strongly positive with the same probe. Control hybridizations with sense strand RNA probe, or with cRNA probe on pre-RNased tissue were negative. Strong hybridization signal for HMG-CoA reductase mRNA was observed in all tissues of the post-implantation rat embryo, from egg cylinder to 30 somite stages (7 to 12 days). Heavy signal was noted in primitive ectoderm and neural tube. The wide embryonic and extraembryonic distribution and abundance of HMG-CoA reductase mRNA may reflect developmental requirements for products of the mevalonate pathway, e.g., isoprenoids for post-translational farnesylation of p21ras.     

Localization of dopamine D2 receptor in rat spinal cord identified with immunocytochemistry and in situ hybridization

In the present study the distribution of dopamine D2 receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D2 receptor and in situ hybridization (ISH) using a [35S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D2 receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D2 receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D2 receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D2 receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.     

Vagal and spinal afferent innervation of the rat esophagus: a combined retrograde tracing and immunocytochemical study with special emphasis on calcium-binding proteins

Vagal afferent neurons contain a variety of neurochemical markers and neuroactive substances, most of which are present also in dorsal root ganglion cells. To test for the suitability of the calcium-binding protein calretinin as a specific marker for vagal afferent fibers in the periphery, immunocytochemistry for this protein was combined with retrograde tracing. Nerve fibers in the rat esophagus, as well as vagal and spinal sensory neurons innervating the esophagus, were investigated for co-localization of calretinin with calbindin, calcitonin gene-related peptide, and NADPH diaphorase. The results indicated that calretinin immunocytochemistry demonstrates neuronal structures known as vagal afferent from other studies, in particular intraganglionic laminar endings. A few enteric neurons whose distribution was unrelated to intraganglionic laminar endings also stained for calretinin. Strikingly, calretinin immunoreactivity was absent from spinal afferent neurons innervating the rat esophagus. In intraganglionic laminar endings and nodose ganglion cells calretinin was highly co-localized with calbindin but not with calcitonin gene-related peptide. On the other hand, calbindin was also found in spinal afferents to the esophagus where it was co-localized with calcitonin gene-related peptide. Vagal afferent neurons innervating the esophagus were never positive for NADPH diaphorase. Thus, calretinin appears to be a more specific marker for vagal afferent structures in the esophagus than calbindin, which is expressed by both vagal and spinal sensory neurons. Calretinin immunocytochemistry may be utilized as a valuable tool for investigations of subpopulations of vagal afferents in certain viscera.     

Localization of corticotropin-releasing factor in primary and secondary lymphoid organs of the rat

Cells of the immune system produce a variety of neuropeptides or peptide hormones, either constitutively or upon induction, and possess specific neuropeptide receptors that display ligand-receptor interactions similar to those described in the central nervous system (CNS). These findings suggest that specific subsets of lymphoid cells can produce and respond to peptides previously thought to be principally neural mediators. Recently, corticotropin releasing factor (CRF) mRNA was detected in the rat thymus and spleen, although the cells that synthesize CRF were not identified. We examined the localization of CRF and its mRNA in the rat spleen, thymus, and mesenteric lymph nodes using immunocytochemistry (ICC) and in situ hybridization (ISH), respectively. Immunoreactive CRF was present in cells in the marginal zone and red pulp of the spleen, in connective tissue septa and the subcapsular region of the thymus, and in the medullary cords and sinuses of the mesenteric lymph nodes. Dual ICC/ISH for CRF and its mRNA, respectively, demonstrated CRF mRNA over CRF-immunoreactive cells, suggesting CRF synthesis. Double-label ICC for CRF and markers for specific immunocyte subsets suggest that CRF+ cells in the spleen and thymus are macrophages. CRF+ cells in primary and secondary lymphoid organs reside in compartments that are innervated by sympathetic nerves, and some cells appears to be contacted by noradrenergic sympathetic nerve fibers, suggesting that CRF release may be influenced by the sympathetic nervous system, as it is in the hypothalamo-pituitary-adrenal axis. The presence of CRF in organs of the immune system suggests that this neuropeptide may modulate immune functions after paracrine release.     

5-HT receptors in mammalian brain: receptor autoradiography and in situ hybridization studies of new ligands and newly identified receptors

In recent years the family of mammalian serotonin receptors has grown to 14 different subtypes, characterized by pharmacological or molecular biological techniques. In parallel, new ligand molecules have been developed for their study. However, selective ligands are not yet available to study every one of them. In addition the degree of selectivity of ligands, hitherto regarded as specific for a particular receptor subtype has been called in question by their affinities for newly discovered receptors. Consequently, a re-evaluation of past ligand receptor autoradiography work is necessary in view of the redefined receptor profiles of these ligands, and the introduction of newly developed ligands. A further difficulty for the characterization of these receptors is the absence of selective antagonist ligands which, for some of the subtypes, have become available only recently. In an attempt to overcome these difficulties we have combined in situ hybridization histochemistry and receptor ligand autoradiography to study the regional and cellular localization of several serotonin receptors in the rodent brain. In addition, for some receptors, we have expanded these studies to primates, including humans. We have found that the distribution of 5-HT1A receptors in monkey brain, labelled with the agonist 3H-8-OH-DPAT and the antagonist 3H-WAY 100635 was very similar at the levels examined, and corresponded well with that observed for the cells containing mRNA coding for this receptor, confirming the somatodendritic localization of 5-HT1A receptors in monkey brain. The labelling conditions to visualize 5-HT1F receptors in guinea pig brain, namely 3H-sumatriptan in the presence of 10(-8) M 5-CT to block 5-HT1D receptors, are suitable for visualizing this receptor, since the results agreed with those observed by in situ hybridization. By using 3H-ketanserin and 3H-mesulergine in parallel with in situ hybridization using the corresponding oligonucleotides, we were able to show that these ligands label respectively 5-HT2A and 5-HT2C binding sites in monkey brain. 5-HT4 receptors were localized in the brain of several species including humans by using 125I-SB 207710. In situ hybridization experiments performed in guinea pig confirmed that 5-HT4 receptors are localized on the terminals of the striatopallidal and striatonigral projections. 5-HT7 binding sites were labelled in rat and guinea pig brains by incubating with 3H-5-CT in the presence of 100 microM WAY 100135 and 250 microM GR 127935; the distribution obtained in both species agreed, in general, with that of the corresponding mRNA coding for them. These results are an illustration of the understanding of our current knowledge of the chemical neuroanatomy of the mammalian 5-HT system.     

Cholinergic, but not the rod pathway-related glycinergic (All), amacrine cells contain calretinin in the rat retina

Double-label immunocytochemistry was carried out on cryostat sections of rat retina to test for the presence of calretinin in cholinergic starburst and the rod pathway-related glycinergic (All) amacrine cells. All cholinergic cells contained calretinin, but calretinin-immunoreactive cells were much more numerous in both the inner nuclear and ganglion cell layers than the cholinergic cells. Glycinergic All amacrine cells have been found to contain calretinin in cat, monkey and rabbit retinas. Since All amacrine cells in rat can be selectively labeled with antibodies against parvalbumin, in a second experiment we attempted to colocalize these proteins. We found that calretinin- and parvalbumin-immunoreactive neurons belonged to distinct amacrine cell populations permitting the conclusion that, in the rat retina, All amacrine cells do not contain calretinin. The results indicate that even those amacrine cells of the mammalian retina that are highly conserved with respect to morphology and transmitter content, may differ with respect to other neurochemical characteristics, such as their calcium-binding proteins.     

AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus

Recent studies suggest a functional diversity of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)- and calretinin (CR)-positive cells, which represent different populations of non-principal cells. To this end, non-radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double-immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV-containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR-A and GluR-D mRNAs, moderate levels of GluR-C and low levels of GluR-B mRNAs in all the examined regions of hippocampus. The vast majority of CR-containing cells have a much lower expression of GluR-A, -C and -D mRNA than PV-positive neurons, although similarly featuring low levels of GluR-B mRNA. Only a subpopulation of CR-containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR-A and -D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR-B subunit expression, PV- and CR-containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.     

Evidence for differential regulation of calcium by outer versus inner hair cells: plasma membrane Ca-ATPase gene expression

The expression of mRNA encoding plasma membrane calcium ATPase (PMCA) subunit isoforms (1-4) and splice variants was examined in the adult and developing rat cochlea by PCR and in situ hybridization. High levels of PMCA mRNA expression were observed in the neurons of the spiral ganglion, and in hair cells. Spiral ganglion neurons expressed PMCA 1-3 beginning in embryonic development, reaching high levels shortly after birth, and continuing into adulthood. Inner hair cells expressed PMCA 1 at moderate levels from birth to the time of onset of cochlear function on postnatal day 12, and strongly from then until adulthood. Outer hair cells expressed PMCA 2 at high levels from shortly after birth through adulthood. The data suggest that the calcium clearance requirements of inner and outer hair cells are distinct. PMCA 2 is the isoform with the highest affinity for calmodulin, and has also been associated with high levels of inositol triphosphate. Its presence in outer hair cells suggests that regulation of the enzyme by calmodulin may be particularly important for this hair cell type. It further suggests that inositol phosphate may play a unique role in the outer hair cell.     

Distribution of I, II and III subtypes of voltage-sensitive Na+ channel mRNA in the rat brain

We examined the expression of the I, II and III subtypes of voltage-sensitive Na+ channel mRNA in the rat brain using in situ hybridization histochemistry with oligonucleotide probes. The distribution of cells with strongly positive signals was characteristic for each subtype. Synthesis of each subtype of Na+ channel protein may be regulated by differential mRNA expression.     

Hair cell formation in cultures of dissociated cells from the vestibular sensory epithelium of the bullfrog

HYPOTHESIS: Bullfrog vestibular hair cells are capable of regenerating in vitro. BACKGROUND: Recent studies have established that sensory organs in the inner ear of vertebrates continue to produce hair cells after birth. However, the mechanisms responsible for the regulation of this process are not well understood. The current study reports the development of a novel method for the culture of dispersed cells from the bullfrog inner ear. METHODS: New hair cell formation in this in vitro preparation was shown by sequential photomicroscopy. Studies with the selective marker for mitotic activity 5-bromo-2-deoxyuridine (BrdU) were done to estimate the level of cell proliferation and to quantify postmitotic hair cell formation. Finally, confirmation of cell type was obtained by scanning electron microscopy and by the use of specific markers for hair cells. RESULTS: Once the optimal culture conditions were established in the initial experiments, the formation of new hair cells was directly visualized in all unstained live cultures and fixed preparations without exception. Asymmetric division of progenitor cells, with subsequent differentiation of one of the daughter cells into new hair cells, also was documented by photomicroscopy. Approximately 12% of the cells were labeled with BrdU, of which 6% were hair cells, showing that new hair cell formation was subsequent to mitotic division in vitro. The identity of newly formed hair cells was verified as follows: 1) morphologically by scanning electron microscopy; 2) by positive labeling with phalloidin-rhodamine, a marker for actin; and 3) by positive calmodulin immunocytochemistry. CONCLUSIONS: This study reports the development of an in vitro culture preparation in which undifferentiated epithelial cells proliferate to become new hair cells. Evidence is provided of division of hair cell progenitors and subsequent differentiation of the daughter cells as one of the mechanisms involved in new hair cell formation in the culture preparation. This newly developed cell culture technique provides a powerful tool for further study of the process of hair cell formation in the vestibular end organ.     

Family diagnosis in psychological disorders of children and adolescents

The distribution of choline acetyltransferase messenger RNA (mRNA) among efferent vestibular neurons in the chinchilla was investigated. mRNA coding for choline acetyltransferase, the enzyme that synthesizes acetylcholine, was used as a marker for the cholinergic system. In order to retrogradely label the efferent vestibular neurons, Fluoro-gold was injected through the oval window into the inner ear of anesthetized young male chinchillas (6 to 12 months old). The animals were anesthetized and perfused through the heart 2 days post injection with 4% paraformaldehyde in phosphate buffer. Retrogradely labeled efferent vestibular neurons were mapped in brainstem sections prior to processing for in situ hybridization histochemistry using radiolabeled ribonucleic acid probes complementary to the 3' end of the choline acetyltransferase mRNA. At the levels of the ascending facial nerve and the genu of the facial nerve, we found that approximately 90% of the Fluoro-gold labeled cells in group E1 contained choline acetyltransferase mRNA. All of the group E2 cells that were labeled with Fluoro-gold were found to be cholinergic (contain choline acetyltransferase mRNA). Finally, 60% of the Fluoro-gold-labeled cells in the caudal pontine reticular nucleus contained choline acetyltransferase mRNA.