D. Baetens

Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system

A complete mapping of immunoreactive sites for vitamin D-dependent calcium-binding protein (CaBP) was performed on serial sections from the rat central nervous system. CaBP immunoreactivity was found in the perikarya, dendrites and axons of some neurons from the limbic system, from many neurosecretory nuclei, from most sensory nuclei and from the cerebral and cerebellar cortex. In contrast, no CaBP antigenic sites were detectable in the motoneurons of the spinal cord and in those of the cranial nerve nuclei, nor in the neurons from the cerebellar nuclei. A quantitative evaluation revealed a great variability in the number of CaBP-immunoreactive neurons among different areas of the central nervous system. Positive cells represented less than 1% of the neurons in the frontal cortex, whereas 74% of the Purkinje cells from the cerebellar cortex showed immunoreactive staining for CaBP. In addition, 45% of the ependymal cells of the telencephalic ventricles were positive. These data show that CaBP is widely distributed in neurons and ependymal cells from the rat central nervous system although it is more concentrated in some specific areas.

Specific neurons in chick central nervous system stain with an antibody against chick intestinal vitamin D-dependent calcium-binding protein

A specific antiserum against intestinal vitamin D-dependent calcium-binding protein (CaBP) was used for a systematic immunohistochemical evaluation of immunoreactive sites in the central nervous system of chick. CaBP was observed in the perikarya, dendrites and axons of a specific population of neurons. It is concluded that CaBP represents a marker for selected neurons in the central nervous system of chick.

Synaptic remodelling in arcuate nucleus after injection of estradiol valerate in adult female rats

Adult female rats were injected with a single dose (20 mg/kg) of estradiol valerate (EV). The number of synapses was evaluated in thin sections of arcuate nucleus fixed 3, 8, 16 and 32 weeks after EV treatment and compared with the values obtained in the arcuate nucleus of uninjected proestrus control rats. By 8 weeks after EV treatment a significant (P less than 0.001) decrease was found in the number of axo-somatic and axo-dendritic synapses on dendritic shafts, but not in the number of axo-dendritic synapses on dendritic spines. However, by 32 weeks postinjection, the number of axo-somatic and axo-dendritic synapses had returned to control values. This transient decrease in the number of synapses was preceded by a massive appearance of neuronal degenerative images by 3 weeks after EV injection. These results are interpreted as reflecting a process of circuitry remodelling in the arcuate nucleus after a neuronal lesion induced by estrogen.

Alteration of islet cell populations in spontaneously diabetic mice.

Endocrine-cell populations in the islets of Langerhans of mutant mice with a severe hypoinsulinemic diabetes (obiob or dbldb on the C57BL/KsJ background) or with a mild hyperinsulinemic diabetes (ob/ob or db/db on the C57BL/6J background) were studied quantitatively by immunofluorescence and morphometry. In severely diabetic mice, islets presented a reduced proportion of insulin-containing cells buf increased glucagon-, somatostatin-, and pancreatic polypeptide (PP)-containing cells, as compared with islets of control (+/+) mice. An inverse change was observed in islets of mildly diabetic mice: islets were hypertrophie and composed mostly of insulin-containing cells, with decreased proportions of glucagon-, somatostatin-, and PP-containing cells. In both types of diabetic syndromes, the changes in cell populations induced a qualitative alteration of cellular interrelationships in the affected islets.

Reduced beta-cell glucose transporter in new onset diabetic BB rats.

Previous studies from our laboratories have suggested a defect in glucose transport in islets isolated from BB rats on the first day of overt diabetes. To quantitate by immunostaining the glucose transporter of beta-cells (GLUT-2) before and at the onset of autoimmune diabetes we employed an antibody to its COOH-terminal octapeptide. On the first day of overt diabetes, defined as the day the daily blood glucose first reached 200 mg/dl, the volume density ratio of GLUT-2-positive to insulin-positive beta-cells was only 0.48 +/- 0.06, compared to 0.91 +/- 0.02 in age-matched nondiabetic diabetes-resistant controls (P less than 0.001). In age-matched nondiabetic diabetes-prone rats, most of which would have become diabetic, the ratio was 0.85 +/- 0.02, also less than the controls (P less than 0.05). Protein A-gold labeling of GLUT-2 in beta-cells of day 1 diabetic rats revealed 2.17 +/- 0.16 gold particles per micrometer length of microvillar plasma membranes compared to 3.91 +/- 0.14 in controls (P less than 0.001) and 2.87 +/- 0.24 in the nondiabetic diabetes-prone rats (P less than 0.02). Reduction in GLUT-2 correlates temporally with and may contribute to the loss of glucose-stimulated insulin secretion that precedes profound beta-cell depletion of autoimmune diabetes.

A Study of Glucagonomas by Light and Electron Microscopy and Immunofluorescence

Five tumors associated with the complete glucagonoma syndrome, as well as a series of glucagon-cell adenomas from three patients without this syndrome, were investigated by light and electron microscopy and by immunofluorescence. All tumors associated with the syndrome were large, from 3 to 35 cm along the major axis, and three of them were proved to be malignant. No common histologic arrangement of tumor cells was apparent for the five neoplasms examined. Immunofluorescent staining for glucagon and glicentin was carried out: while most cells were negative, a varying number of scattered cells were positive with both antisera in all tumors except one; three tumors contained more glicentin- than glucagon-immunoreactive cells. Moreover, three tumors were multihormonal, with cells positive for pancreatic polypeptide and/or insulin. Ultrastruc-turally, the secretory granules of cells from these tumors were not typical of those found in A-cells from adult human islets. The glucagon-cell tumors from patients without the syndrome were benign, usually multiple, and were small, with diameters from 0.5 mm to 1 cm. In most cases, the cells from these neoplasms arranged in a characteristic pattern (ribbonlike or “gyriform”). In most tumors, the majority of cells showed both glucagon and glicentin immunofluorescence and the ultra-structural appearance of their secretory granules was similar to that of normal islet A-cells. From the morphologic point of view, therefore, cells from tumors not associated with the glucagonoma syndrome resemble normal glucagon cells more closely than those from tumors associated with the syndrome.

Sex differences and maturational changes in arcuate nucleus neuronal plasma membrane organization

Intramembrane particles (IMP) are believed to represent protein-containing structures in the membrane. Hypothalamic arcuate nucleus neuronal plasma membranes from male and female rats studied from birth to adulthood were quantitatively assessed for IMP number and size using freeze-fracture techniques. We found that newborn female rats have a significantly greater number of IMP than newborn males. There is also a progressive increase in the number of IMP during the first 20 days of postnatal life in both sexes. The rate of protein particle insertion favors females, maintaining the unequal protein particle content into adulthood with female rats having more IMP than males of the same age. The differences in IMP concentration are mainly due to greater numbers of small (less than 10 nm) particles in neuronal membranes from female rats. These data extend previous reports of sexual dimorphism in the arcuate nucleus to the level of plasma membrane organization.

Neurons with whorl bodies have increased numbers of synapses

The endoplasmic reticulum in some neurons of the rat hypothalamic arcuate nucleus forms concentric sheets of smooth cisternae which are known as whorl bodies. It has been reported that the number and size of these structures change under varying endocrine conditions. For example, the number of rat arcuate nucleus neurons containing whorl bodies increases after gonadectomy. In studying their relationship to other components of the cell, we found that neuron profiles which contain whorl bodies receive a significantly increased number of axosomatic presynaptic terminals (P less than 0.001). Whorl bodies may mark a subpopulation of endocrine sensitive cells which are characterized by a different pattern of connections and whose response to stimuli includes changes in endoplasmic reticulum organization.

Effects of climbing fiber destruction on large dendrite spines of purkinje cells

SummaryThe number and freeze-fracture membrane organization of spines on Purkinje cell dendrites were studied in rats after destruction, by intraperitoneal injection of 3-acetyl-pyridine (3-AP), of the climbing fiber afferents to the cerebellum. The results obtained show that: (1) there is a 1.6 fold increase in the total number of dendritic spines in the 3-AP-treated animals as compared to controls, but no change in the total number of spine synaptic profiles; (2) the spines from main dendrites in both control and 3-AP-treated animals have more than 1,000 IMP/μm2 of P-face membrane; and, (3) the spines formed on Purkinje cell dendrites in the absence of their presynaptic axons have the same P-face membrane organization (i.e. more than 1,000 IMP/μm2) as spines normally innervated by afferent climbing fibers. These data suggest that each type of Purkinje cell spine has a specific P-face membrane organization which does not appear to be influenced by presynaptic terminals.

Freeze-fracture cytochemistry of neuronal membranes: inhomogeneous distribution of filipin-sterol complexes in perikarya, dendrites and axons

Vibratome slices of cerebellar cortex fixed in a glutaraldehyde solution containing filipin, a sterol-specific probe, were freeze-fractured to study the distribution of filipin-sterol complexes in neuronal plasma membranes. These complexes appear as 25-30 nm protuberances or pits in the fracture face of plasma membranes, and their density was low in dendrites of Purkinje and granule cells. In contrast, the plasma membranes of neuronal perikarya showed an abundant filipin labeling, 4-6 times greater than in dendrites. Parallel fibers, the axons of granule cells also had significantly more filipin-sterol complexes than granule cell dendrites, but fewer than granule cell perikarya. The results reveal difference in the organization of specific regions of neuronal plasma membranes, which are also characterized by a different pattern of synaptic contacts.