Shanta E. Kapadia

Targeted Expression of Calcitonin Gene‐Related Peptide to Osteoblasts Increases Bone Density in Mice

The neuropeptide calcitonin gene‐related peptide (CGRP) is concentrated in fine sensory nerve endings innervating all tissues, including bone. CGRP inhibits osteoclasts, stimulates insulin‐like growth factor I and inhibits tumor necrosis factor alpha production by osteoblasts in vitro. To investigate the role of CGRP in bone in vivo, mice were engineered to express CGRP in osteoblasts by placing the human CGRP gene under the control of the rat osteocalcin promoter (Ost‐CGRP tg+ mice). Calvaria cultures from transgene positive (tg+), but not tg− mice, produced bioactive CGRP. Trabecular bone density and bone volume, determined by peripheral quantitative computed tomography and bone histomorphometry, respectively, were higher in tg+ than tg− littermates. This increase in bone volume was associated with an increased bone formation rate. Trabecular bone density decreased in tg+ mice as a result of ovariectomy, but remained higher than in sham tg− mice. Targeting CGRP to osteoblasts appears to favor the establishment of a higher trabecular bone mass in mice.

Immunohistochemical and electron microscopic demonstration of vascular innervation in the mammalian brainstem

Ultrastructural morphological relationship between microvessels and neural elements containing serotonin-like immunoreactivity, substance P and methionine-enkephalin like immunoreactivity were studied in the monkey and cat brain. A close relationship between serotonin-like immunoreactive dendritic processes, enkephalin-like immunoreactive profiles and capillary basement membrane was observed; they were usually separated by a double layer of glial membrane. More importantly, serotonin and substance P-like immunoreactive dendritic profiles are reported in close approximation with capillary basement membrane with no glial limitans at the site of contact. These intimate morphological associations may be the basis for neural regulation of tonicity, permeability and chemosensor function in the brain microvasculature.

Immunocytochemical and electron microscopic study of serotonin neuronal organization in the dorsal raphe nucleus of the monkey

Serotonin neurons in the dorsal raphe nucleus were identified using an antibody to a serotonin-bovine serum albumin conjugate and the peroxidase anti-peroxidase method. Nerve cell bodies showing serotonin-like immunoreactivity ranged in size from 15 to 22 micron in diameter; their dendrites were also immunoreactive. Immunostaining was present in the cytoplasmic matrix, outer membranes of mitochondria, rough endoplasmic reticulum, multivesicular bodies and dense-cored vesicles. Heavily immunoreactive axonal varicosities contained small round vesicles (18-35 nm) and larger dense-cored vesicles (50-90 nm). Both unmyelinated (0.2-0.5 micron) and myelinated (0.8-1.1 micron) serotonin-like immunoreactive axons were found, often interspersed within bundles of similar caliber unlabeled axons. Serotonin-like immunoreactive somata and dendrites were postsynaptic to numerous unlabeled terminals that contained either (a) clear round vesicles (18-25 nm) with many small dense-cored vesicles (30-50 nm), (b) clear round vesicles (18-25 nm) with large dense-cored vesicles (90-110 nm) or (c) clear round vesicles (18-25 nm) with or without flat vesicles. In addition pairs of unlabeled terminals formed crest synapses onto serotonin-like immunoreactive dendritic spines. This variety of unlabeled terminals making contact with serotonin-like immunoreactive elements suggests that several neuronal systems with possibly different transmitters may regulate serotonin raphe neurons. We occasionally observed serotonin-like immunoreactive dendrites and terminals in apposition to other serotonin-like immunoreactive dendrites with membrane specializations at the site of contact. This might represent a possible site for the self inhibition of serotoninergic neurons reported in physiological studies of the serotonin system in the dorsal raphe nucleus.

Populations of substance P, met-enkephalin and serotonin immunoreactive neurons in the interpeduncular nucleus of cat: cytoarchitectonics

The indirect antibody-peroxidase immunostaining method was used to study the distribution of substance P- (SPLI), Met-enkephalin- (MELI) and serotonin-like (5-HTLI) immunoreactivity within the interpeduncular nuclear complex (IPN) of the cat. Populations of immunoreactive cell bodies were found to have a distinct pattern of localization within the IPN. This distribution pattern is described as seen at 4 representative levels rostrocaudally through the IPN. SPLI cell bodies were mainly at rostral levels I, II and III especially in the median region and as a cluster of cells in the intermediate regions of the IPN. MELI cell bodies were seen at all 4 levels, confined mainly to the median and intermediate regions at levels I and II and also in the lateral regions at levels III and IV. There were few 5-HTLI cell bodies at the rostral levels I and II where they were confined to the dorsal zone of the median region, whereas at levels III and IV there were numerous cell bodies located throughout the intermediate region of the IPN. The immunoreactive cell bodies in the IPN may belong to both intrinsic and projection neurons. The distinctive neurochemical cytoarchitectonics of the IPN suggest that this region of the brain which lies along the course of major limbic pathways, may be a center of complex integrative mechanisms of neuronal activity.

Ultrastructure and localization of substance P and met-enkephalin immunoreactivity in the human fetal gastric antrum

SummaryThe ultrastructural localization and relations of substance P- and met-enkephalin-labeled neuronal structures were examined in the wall of the human gastric antrum during early fetal life. By 14–16 weeks of gestation, clearly discernable neural plexuses and a well developed external muscle coat were present. In the submucous coat, neural plexuses varied from immature forms consisting of 1–4 neurites partially enveloped by Schwann cell processes to more mature plexuses where neurons were completely enclosed by Schwann cell processes. Neuronal profiles with substance P- and met-enkephalin-like immunoreactivities were observed in the submucous plexus. In the myenteric plexus met-enkephalin-like immunoreactivity was seen within cell bodies and neurites. By contrast, although substance P-like immunoreactivity was observed in neurites in the myenteric plexus, no substance P-labeled somata could be identified. Unlabeled terminals were seen in contact with both unlabeled dendrites and met-enkephalinergic neurons. An increase in electron density was observed at the sites of contact. These structures probably represent early stages in the development of synaptic specializations. In addition, met-enkephalin-labeled varicosities were seen in apposition to smooth muscle cells of the circular muscle coat. This suggests that antral smooth muscle cells are directly innervated by met-enkephalin neurons.

Deafferentation-Induced Expression of GAP-43, NCAM, and NILE in the Adult Rat Dorsal Horn Following Pronase Injection of the Sciatic Nerve

The expression of growth-associated protein 43 (GAP-43), neural cell adhesion molecule (NCAM), and nerve-growth-factor-inducible large external glycoprotein (NILE) in the adult rat dorsal horn was examined at several survival times after unilateral pronase injection of the sciatic nerve. Pronase injection produces a permanent major loss of sciatic primary afferents in the dorsal horn, and there is a later sprouting of saphenous afferents into the sciatic territory. Small-diameter myelinated and nonmyelinated saphenous afferents sprout within the superficial dorsal horn, and larger, myelinated afferents sprout within the deep dorsal horn. In the present study, GAP-43 and NCAM immunoreactivity increased in the superficial dorsal horn by 10 days after injection. By 20 days, the increase spread into the deep dorsal horn; NCAM returned to normal after 1-2 months, but GAP-43 persisted up to 4 months. NILE immunoreactivity appeared in laminae I and II by 10 days and increased up to 30 days; by 2 months no NILE remained. NILE never spread into the deeper dorsal horn, regardless of survival time. These data suggest a correlation in the expression of both NCAM and NILE with the sprouting of fine-diameter sprouting afferents in laminae I and II, and of NCAM expression with the sprouting of larger-diameter afferents in the deep dorsal horn.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in substance P and calcitonin gene-related peptide binding in the dorsal horn of rat spinal cord following pronase-induced deafferentation

Quantitative receptor autoradiography was used to evaluate potential alterations in substance P (SP) and calcitonin gene-related peptide (CGRP) binding in the L4 spinal segment of rats following unilateral poisoning of the sciatic nerve with pronase. Ten days after pronase-induced deafferentation there was a significant increase in SP and CGRP binding in the superficial (I-II) and deeper (II-IV) laminae of the dorsal horn ipsilaterally. Densitometric measurements revealed a 50% return towards normal values for SP binding by 90 days postpronase injection in all laminae examined, while the density of CGRP binding showed a partial return towards normal values for laminae III-IV only. These differential responses may be indicative of the mechanisms underlying pronase-induced peripheral neuropathy.