Calbindin D28k expression in immunohistochemically identified Renshaw cells

Double immunofluorescence was utilized to determine whether Renshaw cells contain calbindin D28k immunoreactivity. Renshaw cells were identified by their characteristic expression patterns of gephyrin immunoreactivity in sections of rat and cat lumbar spinal cord. In the rat, all neurons classified as Renshaw cells (n = 487) also contained calbindin D28k-immunoreactivity, and all calbindin D28k-immunoreactive cells located in the ventral-most region of lamina VII expressed the characteristic gephyrin labeling and morphology of Renshaw cells. In the cat, fewer than half of the Renshaw cells (47%; n = 128) were double-labeled. In both species, occasional calbindin D28k-immunoreactive Renshaw cells were identified within motor nuclei in lamina IX. The distinctive immunolabeling of Renshaw cells allowed us to estimate that there are about 250 Renshaw cells in each ventral horn of the fourth lumbar segment of rat spinal cord, and about 750 cells per ventral horn in the L6 segment of the cat. We conclude that the functional properties of Renshaw cells, including their ability to fire action potentials at high rates, likely require specific homeostatic mechanisms including strong intracellular calcium buffering, the precise mechanisms of which may vary between species.     

Calbindin D28K regulation in precociously matured chick egg shell gland in vitro

Egg shell calcification in the hen uterus (egg shell gland, ESG) depends primarily on intestinal absorption of dietary Ca2+ as well as ESG Ca2+ transport into the shell. Intestinal Ca2+ absorption is linked to vitamin D-induced calbindin D28K (D28K) concentration. The ESG also contains D28K, and Ca2+ transport into the shell appears to be linked to D28K gene expression, but until this report, there was no direct proof that ESG D28K was or was not vitamin D-dependent. To address this issue, highly developed ESG from estradiol (E2)-injected, severely vitamin D-depleted chicks were cultured in serum-free medium with excellent viability. Addition of the vitamin D-hormone, 1,25(OH)2 vitamin D3 (1,25), to the culture medium increased ESG D28K levels as much as 70%. E2 alone had no effect, but E2 plus 1,25 further increased ESG D28K levels up to 160%. By contrast, progesterone (P4) prevented the 1,25-stimulated increase in D28K, while having no effect on basal D28K level. Of considerable interest, thapsigargin (THAPS), which increases intracellular Ca2+ concentration ([Ca2+]i) in many cell types, stimulated D28K synthesis in a concentration-dependent manner in the complete absence of 1,25 and independent of the [Ca2+] of the medium. These results are the first direct evidence that ESG D28K is under direct control of 1,25 and that both gonadal steroid hormones, E2 and P4, may be coregulators. Further, the effects of THAPS suggest that [Ca2+]i itself may also regulate D28K. This new in vitro model clearly represents a unique opportunity to study the regulation of the ESG calcium transport mechanism under stringently defined conditions.     

Quantitative examination of calcitonin gene-related peptide immunoreactive nerve fibres in the cat knee joint capsule

Aging is characterized by a decreased humoral and cell-mediated immunity to a large variety of exogenous antigens and by an increased propensity to autoantibody production, suggesting an age-related disregulation of the immune system. The decline in immune responsiveness to exogenous antigens has been attributed to thymus involution, consisting of a fall in the capacity to induce intrathymic T-cell growth and differentiation, and also to export mature T cells to the periphery. T-cell activation and secretion of soluble factors have been reported to change with aging, but, as with cytokines, the results are conflicting. We investigated the production of IL-2 and IFN-gamma (Th1 type) and IL-4 (Th2 type) cytokines by mitogen-activated spleen cells from young, adult and old mice and their regulation by the addition of a recombinant cytokine (IL-1 beta, IL-2, IL-3, IL-4, IL-12, IFN-gamma) at varying concentrations. The results indicate that cytokine production can be enhanced only when it is deficient, suggesting the possible use of recombinant cytokines as efficient immunomodulators in age-associated immune disorders.     

Effect of capsaicin treatment or inferior alveolar nerve resection on dentine formation and calcitonin gene-related peptide- and substance P-immunoreactive nerve fibres in rat molar pulp

The aim of this study was to investigate whether decreased sensory innervation induced by capsaicin treatment or axotomy of the inferior alveolar nerve has an effect upon dentine formation in the rat first molar. Dentine formation was visualized by intravital injection of Procion brilliant Red H8BS and denervation was verified immunohistochemically for the neuropeptides calcitonin gene-related peptide (CGRP) and substance P. The observation times were 6 weeks for the capsaicin-treated group and 11 days for the axotomized group. Capsaicin injections caused a consistent reduction in numbers of CGRP- and substance P-immunoreactive fibres in the pulps and a somewhat smaller reduction in the periodontal tissues. Unilateral axotomy of the inferior alveolar nerve induced an almost complete loss of immunoreactive fibres in the pulp and in the mesial gingiva of the first molar. Dentine formation at the mesial pulp horn and at the central pulp floor was significantly reduced in both groups compared to controls. The results suggest that sensory neuropeptides such as CGRP and substance P may play a part in dentine formation.     

Calbindin D-28k and monoamine oxidase A immunoreactive neurons in the nucleus basalis of Meynert in senile dementia of the Alzheimer type and Parkinson's disease

The lungs must be kept "dry" for efficient gas exchange. The mechanisms that contribute to clear alveoli from fetal lung fluid at birth are still present during adult life and allow recovery from alveolar flooding. It has recently been shown with the use of different approaches in vitro, as well as in vivo, that alveolar epithelium performs solute-coupled fluid transport. Fluid absorption from alveoli occurs chiefly as a result of active transepithelial Na+ transport. The mechanisms of Na+ transport have been partly elucidated; Na+ enters alveolar cells through apical Na+ channels and Na(+)-coupled solute transporters and is pumped out at the basolateral membrane by a Na(+)-K(+)-adenosinetriphosphatase (ATPase). Transepithelial Na+ transport and fluid absorption are stimulated by beta-adrenergic agonists, with adenosine 3',5'-cyclic monophosphate being the likely intracellular second messenger. K+ is probably secreted into alveoli because its concentration in the epithelial lining fluid is larger than expected for passive distribution. K+ channels have been described that, in conjunction with Na(+)-K(+)-ATP-ase, might provide pathways for active transport. Active proton secretion or bicarbonate absorption have been reported, which may explain the low pH of the alveolar epithelial lining fluid. It is probable that active solute transports are the main determinants of epithelial lining fluid depth and composition. A challenge for the future is to understand how this homeostasis is achieved.     

Distribution and origin of peptide-containing nerve fibres in the rat and human mammary gland

The structures in the mammary gland involved in milk ejection have been investigated with regard to their relation to different types of peptidergic nerve fibres and their origin. Lactating rats were studied with immunohistochemistry focusing on the nipple, the parenchyma, the mammary blood vessels and the mammary nerve. The human mammary gland was also analysed. In the mammary gland from rat and human, nerve endings in the subepidermis, around smooth muscle cells in the nipple, in the connective tissue surrounding lactiferous ducts and alveoli in the nipple and in the parenchyma of the mammary gland showed immunoreactivity for calcitonin gene-related peptide, substance P, vasoactive intestinal polypeptide, peptide histidine isoleucine, neuropeptide Y, galanin and tyrosine hydroxylase, whereas dynorphin-positive nerve fibres could not be detected. The mammary nerve contained calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y and tyrosine hydroxylase immunoreactivities; the adventitia of the mammary artery contained nerve fibres immunoreactive for neuropeptide Y and tyrosine hydroxylase, while vasoactive intestinal polypeptide-, peptide histidine isoleucine-, calcitonin gene-related peptide- and substance P-positive fibres were found in the tissue surrounding the artery. The wall of the mammary vein had nerve terminals immunoreactive for neuropeptide Y, tyrosine hydroxylase, calcitonin gene-related peptide and substance P. With the help of retrograde tracing using wheat germ agglutinin in combination with immunohistochemistry, projections of calcitonin gene-related peptide-immunoreactive cells in the dorsal root ganglia to the nipple were established. Neurons in the sympathetic stellate ganglion containing neuropeptide Y and tyrosine hydroxylase also projected to the mammary gland. Moreover retrogradely-labelled cells were found in the nodose ganglion, and they were vasoactive intestinal polypeptide-immunoreactive. These results demonstrate a rich distribution of different types of nerve fibres in structures of the mammary gland related to milk ejection. These nerve fibres and their peptides may be involved in the local control of milk ejection.     

Induction of enhanced postnatal expression of immunoreactive calbindin-D28k in rat forebrain by the calcium antagonist nimodipine

Male Copenhagen x Fisher F1 rats, transplanted with the androgen-sensitive Dunning R3327 PAP rat prostatic adenocarcinoma, were castrated when tumor volumes were approximately 1300 mm3. The rats were thereafter followed with measurements of tumor volume. Castration stopped tumor growth, but some of the tumors started to regrow after 7-36 weeks. These tumors relapsing from castration treatment were now considered to be androgen-insensitive. In this study, we defined relapse as the time when the tumor volume had increased to 200% of the volume at the time for castration. At this time, the rats were treated either with estradiol-17 beta (E2, 50 micrograms s.c. daily) or vehicle for 8 weeks. After this period, tumor morphology was examined. The tumors in the vehicle-treated group were heterogeneous, and both highly and more dedifferentiated parts were present. The tumor growth rate was correlated to the epithelial cell nuclear size and its variance, and to the mitotic index. In the E2-treated group, tumor growth rate was retarded throughout the treatment period, and dedifferentiated tumor areas were rare. Estrogen treatment resulted in a reduction of nuclear area and mitotic index, a changed nuclear shape, and an increased apoptotic index compared to that in vehicle-treated tumors. By castration, it is possible to induce an alteration of the androgen-sensitive Dunning R3327 PAP tumor phenotype to an androgen-insensitive tumor with an altered morphology. Estradiol-17 beta apparently inhibits not only the growth, but also postpones the castration-induced dedifferentiation of the tumor.     

Effects of pulsed Nd:YAG laser radiation on action potential conduction in nerve fibres inside teeth in vitro

OBJECTIVE: This study aimed to simulate the effects of lasing dentine on pulpal nerve function. METHODS: Rat spinal nerve roots were threaded through the prepared pulp canal of a 10 mm long tooth root segment which was mounted in a perspex bath. The protruding ends of the nerve were placed on platinum wire electrodes used to elicit and to record compound nerve action potentials (CAPs). Laser energy (average power = 0.3-3.0 W) was applied to the surface of the root segment using a pulsed Nd:YAG dental laser (dLase 300). RESULTS: With the laser probe tip placed in static contact with the tooth surface, the nerve CAP was irreversibly abolished within 60 s of lasing at 1.0-3.0 W power. When the laser tip was moved to and fro over the root surface in a scanning mode, similar levels of radiation produced less marked effects. In the latter mode, CAP attenuation increased with increasing power and duration of lasing. After 60 s lasing at 0.3 W, the CAP size was 95% (+/- 5, S.D.) of the prelasing controls value; with 2.0 W the CAP was reduced to 54% (+/- 33). The CAP recovered to 90% of control levels after lasing at powers up to 1.5 W, but reached only 72% of control values after lasing at 2.0 W power. CONCLUSIONS: Laser radiation applied to dentine caused a dose-dependent block of action potential conduction in nerve fibres in the underlying pulp chamber.     

Facilitation of adrenal sympathetic efferent nerve activity induced by mechanical stimulation of teeth in the rat

The effects of afferent signals from the periodontal mechanoreceptors and muscle spindles of jaw-closing muscles on adrenal nerve activity were examined using anesthetized rats. The adrenal nerve activity increased with pressure stimulation of the teeth and by biting a wooden stick. However, after denervation of the periodontal ligament, the facilitation due to the stick-biting was not observed. These results indicate that periodontal afferents facilitate adrenal nerve activity.     

Calbindin-D28K-immunoreactive cells and fibres in the human amygdaloid complex

The distribution of calbindin-D28k-immunoreactive cells and fibres in five human amygdalae was analysed from sections that had been stained immunohistochemically with a monoclonal antibody raised against calbindin-D28k. The highest density of calbindin-D28k-positive neurons were found in the anterior cortical, medial, posterior cortical and accessory basal nuclei, in the parvicellular division of the basal nucleus and in the amygdalohippocampal area. The lowest densities of immunopositive neurons were found in the paralaminar nucleus, in the periamygdaloid cortex (PAC1 and PACo) and in some of the intercalated nuclei. The deep nuclei (lateral, basal and accessory basal nuclei) contained a high density of calbindin-D28k-immunoreactive fibres and terminals. The cortical nuclei and the central nucleus were characterized by intense neuropil labelling. Morphologically, a large majority of the calbindin-D28k-immunoreactive neurons were aspiny or sparsely spiny and resembled inhibitory local circuit neurons. A small population of lightly-stained, pyramidal-shaped neurons was also observed. In most of the amygdaloid nuclei, calbindin-D28k-immunoreactive fibres travelled close to each other and formed bundles, which suggests that some of the immunostained neurons were double-bouquet cells. In the paralaminar nucleus, the calbindin-D28k-immunoreactive axons formed tortuous plexus (100-200 microns in diameter) that surrounded several unstained somata. This study provides baseline information on the morphology and distribution of calcium-binding protein-containing inhibitory cells and fibres immunoreactive for calbindin-D28k in the human amygdaloid complex. This information can be used in future studies on the pathogenesis of diseases known to damage the amygdala, such as Alzheimer's disease and temporal lobe epilepsy.     

Variation in nitric oxide synthase-immunoreactive nerve fibres with age and along the length of the vas deferens in the rat

The regional variation of nitric oxide synthase (NOS)-containing nerve fibres along the length of the vas deferens and cauda epididymidis was investigated in cross-sections of abdominal, intermediate and scrotal portions of the vas deferens as well as the cauda epididymidis of the rat. In adult rats, antibody to protein gene product 9.5 (protein found in all neurones) revealed very rich immunostaining in the muscle layers from all four areas studied. In the abdominal portion of the vas deferens NOS immunostaining revealed weak immunofluorescent nerve fibres just beneath the epithelium in the lamina propria extending into the innermost muscle layer where they became less dense. A similar pattern of NOS immunostaining was also seen in sections from the intermediate portion of the vas deferens. Fluorescent nerve fibres were not present in the outer muscle layers. Very few, if any, NOS-immunoreactive nerves were seen in scrotal portions of the vas deferens and cauda epididymidis. In tissue segments taken from aged rats the number of NOS-immunoreactive nerve fibres was increased in all segments, but were still more concentrated at the abdominal end of the male reproductive tract. In contrast, sections of vas deferens from immature rats showed no NOS immunostaining. These findings suggest that nitrergic nerves innervating the rat vas deferens may be involved in the initial relaxation of the abdominal portion of the vas deferens which allows for unidirectional sperm transport. The age-dependent increase in NOS-containing nerve fibres suggests that their development is also androgen-dependent.     

Localization of insulin-like immunoreactive neurons in the rat gracile nucleus

An insulin-like immunoreactivity (ILIR) was localized in the neuronal somata, dendrites and myelinated axons in the gracile nucleus of the male Wistar rat. In the neuronal somata, the reaction product was dispersed in the cell nucleus and cytoplasm. In the cell nucleus, the reaction product was scattered throughout the nucleoplasm, but not within the nucleolus. In the cytoplasm, the reaction product was evenly distributed, mainly in the vicinity of the cisternae of the rough endoplasmic reticulum. In labelled dendrites, the reaction product was closely associated with the parallel arrays of neurotubules and postsynaptic densities. Most of these labelled dendrites were postsynaptic to unlabelled axon terminals. A labelled dendrite often formed the central element of a synaptic glomerulus with several unlabelled axon terminals. Numerous labelled myelinated axons were also present in the neuropil. However, axon terminals appeared to be unlabelled. It is hypothesized that insulin-like substance(s) may be modulating nuclear activities as well as neurotransmission at the synapse in the gracile nucleus.     

Neurokinin A in rat renal afferent neurons and in nerve fibres within smooth muscle and epithelium of rat and guinea-pig renal pelvis

Neurokinin A-like immunoreactivity of dorsal root ganglion neurons innervating the kidney were studied with retrograde tracing of FluoroGold dye applied to the cut renal nerves. The proportions and sizes of renal afferent neurons with neurokinin A-like immunoreactivity were quantified in T9-L2 dorsal root ganglia from five rats. Of 240 renal afferent neuronal somata examined, 26 +/- 3% (S.E.M.) showed neurokinin A-like immunoreactivity. Compared with the overall size distribution of renal afferent neurons, those staining for neurokinin A were mostly small-sized neurons with a few medium-sized neurons. All somata with neurokinin A-like immunoreactivity were neurofilament-poor as judged by labelling with an anti-neurofilament antibody, RT97, and it is therefore likely that they had unmyelinated fibres. To examine the sites to which the renal afferent fibres with neurokinin A might project, sections of rat and guinea-pig kidney and upper ureter were examined. Fibres with neurokinin A-like immunoreactivity were found beneath and within the transitional epithelium lining the inner surface of the pelvis, and within the smooth muscle layer beneath the transitional epithelium. Epithelial innervation was found only in regions with underlying smooth muscle and loose connective tissue, and not in sites where the epithelium was closely applied to the renal parenchyma. The network of fibres was most dense towards the pelvo-uretic junction. Fibres with neurokinin A-like immunoreactivity were not seen beneath or within the cuboidal/columnar epithelium covering the papilla within the renal pelvis. Furthermore, only very few fibres with neurokinin A were observed penetrating the transitional epithelium of the upper ureter in both rat and guinea-pig. The distribution of fibres labelled with antibodies to substance P and calcitonin gene-related peptide in the renal pelvis was similar to that for fibres with neurokinin A-like immuno-reactivity, although a few fibres penetrated further into the fornices than fibres with neurokinin-A-like immunoreactivity. Thus, many afferent fibres in the renal pelvis may contain neurokinin A as well as substance P and calcitonin gene-related peptide. These fibres may be the source of the neurokinin A, substance P and calcitonin gene-related peptide which can be released by topical capsaicin treatment. In addition they may be the mechano- and chemo-receptive fibres in the renal pelvis that are known to play important roles in renal haemodynamics. The intra-epithelial position of some of these fibres in the epithelial layer suggests a possible chemosensory or osmosensory role.     

Mandibular molar teeth and the development of mastication in the miniature pig (Sus scrofa)

The only components of the orofacial complex that are fully developed as soon as they establish function are the occlusal surfaces of teeth. It is usually assumed that the occlusal surface of the molar teeth influences the orientation of the power stroke in spite of the fact that the data supporting this claim are lacking. The purpose of this longitudinal study was therefore to determine whether or not this hypothetical form-function relationship existed during development and whether or not it was related to growth. Serial dorsoventral and lateral radiography, and dorsoventral cineradiography were performed during natural feeding of 5 Hanford miniature pigs (Sus scrofa) of both genders between their 8th and 18th weeks. Sequences of power strokes were analyzed frame by frame and compared between sessions, and related to the position of the fourth primary mandibular molar (dm4). The changes in direction of the power stroke and in position of the dm4 relative to the midline were subsequently related to growth. The results of this study indicate that changes in orientation of the power stroke, though significant only on the balancing side, occur independently of the repositioning working and balancing side mandibular molars, as well as of skeletal growth. The null hypothesis that the mandibular primary molars influence the transverse orientation of the power stroke during growth is therefore refuted.     

Calcium binding by chick calretinin and rat calbindin D28k synthesised in bacteria

Hyperprostaglandin E-syndrome (HPS), a recently described variant of Bartter's syndrome (BS), resembles BS in a number of symptoms but is distinct from BS in others. Similar to BS, HPS is characterized by congenital hypokalemic alkalosis, hypertrophy of the juxtaglomerular apparatus, hyperreninemia, secondary hyperaldosteronism, normal blood pressure and renal diabetes insipidus. Other than BS, HPS is constantly associated with chronic hypercalciuria and nephrocalcinosis as well as both renal and systemic PGE2 overproduction. Correction of most of the symptoms in HPS is achieved by permanent inhibition of prostaglandin synthesis with indomethacin. Among the causes leading to HPS, a selective damage of the distal tubule in HPS has been suggested. Therefore, synthesis of Tamm-Horsfall protein (THP), a glycoprotein exclusively produced in the thick ascending limb of the loop of Henle, was measured by ELISA in the urine of seven infant HPS patients (aged 3 to 8 years). Patients were investigated both under constant indomethacin treatment and after a one week period without indomethacin. Nine healthy children (aged 5 months to 10 years) served as controls. In controls mean daily THP excretion was 54.2 +/- 13.9 (median 46.0) mg/24 hr/1.73 m2 whereas in HPS, THP levels were strongly diminished. During withdrawal of indomethacin treatment, mean THP level was 12.7 +/- 10.1 (median 7.2) mg/24 hr/1.73 m2 and 10.3 +/- 10.1 (median 3.5) mg/24 hr/1.73 m2 under indomethacin treatment, respectively. THP excretion values both without indomethacin and under indomethacin treatment were significantly different from controls (P < or = 0.005); however, there was no significant difference between the THP levels during or after cessation of indomethacin treatment. Creatinine clearance in HPS patients was 75.1 +/- 15.9 (median 76.2) ml/min/1.73 m2 without indomethacin and 81.9 +/- 15.1 (median 83.0) ml/min/1.73 m2 under indomethacin treatment. Control values were not obtained. Comparative measurements of THP excretion in six classical BS-patients (aged 3 months to 17 years) revealed normal THP values in two individuals and intermediate levels in the others: the mean level of six BS patients was 30.8 +/- 13.5 (median 25.0) mg/24 hr/1.73 m2 and was thus significantly higher than in HPS both with and without indomethacin treatment (P < or = 0.05). Immunohistochemistry in renal biopsies of three of the HPS patients showed a strong reduction of cortical tubular THP immunoreactivity in two cases and a less pronounced reduction in the third. In situ hybridization using a THP-riboprobe in these three biopsies revealed significantly reduced or absent THP-mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemosensitivity of nociceptive, mechanosensitive afferent nerve fibres in the guinea-pig ureter

The mechanosensitivity and chemosensitivity of afferent fibres were investigated in an in vitro preparation of the guinea-pig ureter. Electrophysiological recordings were obtained from 5 U-1 (low mechanical threshold, contraction-sensitive) and 74 U-2 units (high threshold). U-2 units had significant higher levels of spontaneous activity, lower conduction velocities, higher mechanical thresholds (U-1: 7 mmHg; U-2: 39 mmHg), less pronounced phasic responses and longer latencies in the response to distensions than the U-1 units. For chemical stimulation, guinea-pig urine (> 800 mosmol/L), bradykinin and capsaicin were applied intraluminally. The responses of U-1 units mainly corresponded to the contractions induced by the chemical stimulation. The vast majority of the U-2 units were excited by urine, bradykinin (threshold: 0.1-1 microM) and capsaicin (threshold: 0.03-0.3 microM). The responses to urine could be mimicked by high concentrations of potassium ions (> 200 mM), but not by an equiosmolar solution of NaCl, urea and mannitol. Chemical stimulation could also result in a transient sensitization of the U-2 units to mechanical stimuli. In the anaesthetized guinea-pig, pseudo-affective responses could be evoked by ureteric distension (threshold: 30-60 mmHg) and serosal application of capsaicin. Intraluminal application of urine in vivo did not evoke any reactions, suggesting that the responses of the U-2 units to urine might be due to an impaired barrier function of the urothelium in vitro. The data are in agreement with the hypothesis that U-2 units are visceral polymodal nociceptors. Since the U-1 units were also able to encode at least noxious mechanical stimuli, their involvement in visceral nociception cannot be excluded.