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Notes: 1. The putative regulatory role of the lamina terminalis in the central control of salivation was investigated in the rat using the viral-tracing technique and Fos-immunohistochemistry.2. Neurons situated in the lamina terminalis, such as the vascular organ of the lamina terminalis (OVLT), median preoptic nucleus (MnPO) and subfornical organ (SFO), were retrogradely labelled after pseudorabies virus injections into the submandibular or sublingual gland.3. Viral tracing combined with glandular denervation showed that lamina terminalis structures sent efferents, in particular, to the parasympathetic side of submandibular gland innervation.4. Saliva lost under heat stress has severe implications for the body fluid economy of rats and a key to the understanding of the central regulation of heat-induced salivation may be the integrative role of the lamina terminalis processing thermoregulatory and osmoregulatory information.

Notes: In order to identify likely sites of action of insulin-like growth factor-I (IGF-I) in rat brain and pituitary gland, we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize and quantify its receptors in coronal and sagittal sections. A discrete and characteristic distribution of IGF-I receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in the olfactory bulb were typical for authentic IGF-I receptors and computer analysis indicated a single class of binding site with a dissociation constant (Kd) of 13 nM for the choroid plexus and 5.1 nM for the olfactory cortex.IGF-I receptor density was very high in the choroid plexus in ail ventricles, but the binding in other circumventricular organs was variable, with high levels in the median eminence and the sub-fornical organ, and low levels in the organum vasculosum of the lamina terminalis. Highest binding was seen in the glomerular layer of the olfactory bulb and its associated regions the taenia tecta and anteromedial olfactory nucleus. The preoptic and septal regions showed moderate binding, while the hypothalamus, with the exception of the median eminence, showed low IGF-I binding. The pituitary gland showed very high binding density in both anterior and posterior lobes, similar to the median eminence. The thalamus had high IGF-I binding density, while it was low in basal ganglia. In the limbic system the hippocampal CA2, CAS, CA4 layers showed high binding, with little in CA1, while binding was high also in the adjacent amygdala. Binding was low in the mid and hindbrain, with the exception of the geniculate bodies, and the sensory nucleus of the trigeminal nerve. Binding was high in the primary olfactory and endopyriform cortex and in specific superficial layers. Cerebellar binding was also high in the molecular layer. Fibre layers showed no binding.Comparison with insulin receptors revealed common distribution in the choroid plexus, paraventricular nucleus, cerebellum, entorhinal cortex and amygdala, with receptor density three- to five-fold higher for IGF-I than for insulin. In contrast, in the hippocampus, insulin binding was high in the CA1 field, and low in CA2, CA3, CA4 while for IGF-I binding the converse was seen. The arcuate nucleus showed prominent insulin labelling and minimal IGF-I binding, while the median eminence showed low insulin and high IGF-I binding. The hypothalamus was more widely labelled with insulin, while in the thalamus the converse was true. Olfactory bulb laminae were labelled with differing intensity by insulin and IGF-I. In common with insulin receptor distribution was the high density of IGF-I receptors over areas of extensive dendritic arborizations which receive rich synaptic inputs, in the cerebellum, hippocampus and olfactory bulb.We conclude that IGF-I receptors are widespread throughout rat brain and pituitary gland, with concentration in regions concerned with olfaction, autonomie and sensory processing, as well as in regulation of growth hormone release, via feedback at the median eminence and pituitary gland. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-I and its receptors.

Notes: Immunopositive angiotensin II nerve fibres and terminals are widely distributed throughout the rat brain, including areas of the brain with and without a blood-brain barrier. Ultrastructural examination indicates that in the circumventricular organs (areas which lack a blood-brain barrier), many angiotensin ll-positive nerve terminals are closely aligned with fenestrated blood vessels and do not have synaptic specializations. This appearance is in contrast to that of angiotensin II terminals in regions with a blood-brain barrier where there exists a more typical synaptic configuration. In both cases, angiotensin II is contained within large (100 to 125 nm) vesicles which coexist with smaller, lucent, non-immunoreactive vesicles. These observations suggest a possible duality of function such that angiotensin II in circumventricular organs may be secreted into the circulation, whereas angiotensin II in the remainder of the brain is more likely to be acting as a neurotransmitter or neuromodulator.

Notes: Insulin-like growth factor-ll (IGF-II) and its receptor, which is homologous with the mannose-6-phosphate (M6P) receptor, are found in high levels in adult rat and human brain, though their role remains unclear. In order to point to possible regional functions, we have mapped and quantified IGF-II/M6P receptors in sagittal sections of adult rat brain by in vitro autoradiography/computerized densitometry and immunohistochemistry. While in vitro autoradiography allowed mapping and quantitation, immunohistochemistry both confirmed mapping and allowed more detailed determination of cellular distribution of receptors. The two methods were generally in agreement with few areas of mismatching. By in vitro autoradiography, a discrete and characteristic distribution of IGF-II receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in arcuate nucleus and choroid plexus were typical for authentic IGF-II receptors with half maximal displacement at 1 nM cold IGF-II. IGF-II receptor density, estimated by in vitro autoradiography, was very high in circumventricular organs, especially the median eminence, which had the highest binding in the brain. In the remainder of the brain there was concordance between the distribution of receptors identified by the two techniques, with greatest densities in the olfactory bulb and olfactory pathways, the hippocampus and discrete regions of the cerebral cortex, cerebellum, hypothalamus, thalamus and brainstem. There were however, some notable mismatches. Autoradiographic binding was high to very high in the median eminence, arcuate nucleus, suprachiasmatic nucleus and anterodorsal thalamic nucleus, whereas these areas were only poorly immunostained. Conversely, the septum showed moderate autoradiographic binding, but very prominent immunostaining of neurons in its dorsolat-eral aspect. Using the immunohistochemical technique IGF-II receptors were localized to specific neuronal groups such as the mitral cells of the olfactory bulb, Purkinje cells of the cerebellum and neurons in the red nucleus. Fibre pathways were not labelled by either technique.We conclude that IGF-II/M6P receptors are widespread throughout rat brain, specifically in neurons and blood vessels, with a similar, but distinct distribution to IGF-I and insulin receptors. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-II/M6P and their receptors.

Notes: There is disagreement between laboratories on the presence and location of angiotensinogen immunostaining in neuronal cells. We examined this issue by using different antisera and histological procedures to stain for angiotensinogen in brains from normal, colchicine-treated and nephrectomized rats. Five different antisera from three laboratories were used to stain sections of paraffin-embedded tissue, frozen sections and Vibratome sections of cerebral cortex, thalamus, hypothalamus, brainstem and cerebellum. All five antisera and all three tissue treatments were effective in showing angiotensinogen staining in glial cells, with the most intense staining being achieved in Vibratome sections. All five antisera gave identical results. Neuronal staining was also found with all antisera but mostly in paraffin-embedded sections, with occasional light staining in frozen sections. No neuronal staining was observed in Vibratome sections. Neuronal staining was frequently perivascular, tended to have a more variable anatomical localization, and to occasionally lack bilateral symmetry in coronal sections. These results provide an explanation for the disagreement between laboratories on the presence and location of angiotensinogen immunostaining in neuronal cells. Taken together with the limited concordance between published sites of angiotensinogen and angiotensin II staining, and the recent demonstration by hybridization in situ of a specifically glial cell localization of angiotensinogen mRNA, our own results suggest a need for caution in the interpretation of neuronal staining with anti-angiotensinogen antisera.

Notes: Summary Axon plasma membranes (axolemma) were studied by freeze-fracture electron microscopy at stages prior to and during myelination in the optic nerves of neonatal rats. In unensheathed axons, intramembranous particles associated with the internal (P) and external (E) leaflets of the axolemma increased in number before reaching a plateau (approximately 600/μm2 in both leaflets) at about 9 days postnatally. In newly myelinated fibres, by contrast, the distribution of particles was asymmetrical; fewer particles (approximately 200/μm2) were found on the E-face and greater numbers (approximately 1400/μm2) were present on the P-face, distributions similar to those observed in mature myelinated fibres. Node-like aggregations of particles were not found in unensheathed pre-myelinated axons nor were they present in axons presumed to be ensheathed by glial cytoplasm but not yet myelinated, although nodal specializations could be easily identified in fibres with only a few turns of compact myelin. These observations show first that there is a redistribution of particles in the P- and E-faces of the internodal axolemma coincident with the onset of myelination and secondly, that nodal specializations (represented by the increased densities of E-face particles) appear after ensheathment but before the formation of compact myelin in fibres of the rat optic nerve.