Regeneration of neurosecretory axons into various types of intrahypothalamic grafts is promoted by the absence of blood brain barrier: fine structural analysis.

Isogenous grafts of neural lobe and optic nerve and autologous grafts of sciatic nerve were placed into contact with the intrahypothalamically transected hypothalamo-neurohypophysial tract, and their fine structural characteristics examined at various time periods thereafter. The vascular bed of neural lobe grafts is composed primarily of fenestrated capillaries, that are permeable to blood-borne HRP throughout the entire experimental period. The microvasculature of sciatic nerve grafts consists of continuous, as well as fenestrated capillaries, which are similarly permeable to HRP. Fenestrated capillaries and HRP leakage in optic nerve grafts are observed at 10 days, but only in grafts located ventrally in the hypothalamus at 30 days. Neurosecretory axon regeneration is seen only in grafts or adjacent hypothalamus where the blood-brain barrier is breached. Regenerating axons are closely associated with the specific glial cells of the respective graft. Based on these observations, we conclude that blood-borne factors are necessary to initiate and sustain regeneration of transected neurosecretory axons, and that such regeneration occurs only in the presence of glial cells.

Structure of the subfornical organ: a review.

In this review, the light microscopic and fine structural characteristics of neurons, axons, dendrites, glial cells, and capillaries and their topography within the subfornical organ are summarized, with an emphasis on recent findings. Structure-function relationships are discussed whenever possible and put into perspective in a concluding section.

Regeneration of neurosecretory axons into various types of intrahypothalamic graft is promoted by the absence of the blood-brain barrier: a neurophysin-immunohistochemical and horseradish peroxidase-histochemical study.

In order to test the hypothesis that neurosecretory axon regeneration occurs only in the presence of specific vascular, perivascular, and glial microenvironments, isografts of neural lobe and optic nerve and autografts of sciatic nerve were transplanted into the hypothalamo-neurohypophysial tract at the lateral retrochiasmatic area of adult male rats. The integrity of the blood-brain barrier (BBB) to intravenously administered horseradish peroxidase (HRP), the regenerative process of neurosecretory axons, and functional recovery from lesion-induced diabetes insipidus were analyzed at 18 hr, 36 hr, 10 days, 30 days, and 80 days postsurgery. Neurophysin-positive axons invaded all grafts, as well as perivascular spaces of the adjacent hypothalamus. Wherever neurosecretory axon regeneration occurred, the BBB was breached. Reestablishment of the BBB was paralleled by a decrease in both density and staining intensity of regenerated neurophysin-positive axons. These observations illustrate that neurosecretory axon regeneration is tributary of the absence of BBB. It is speculated that blood-borne factors, provided when the BBB is breached, initiate and sustain neurosecretory axon regeneration. In addition, products of glial elements may enhance or complement the above stimulatory processes.

Chronic intermittent salt loading enhances functional recovery from polydipsia and survival of vasopressinergic cells in the hypothalamic supraoptic nucleus following transection of the hypophysial stalk.

Hypophysial stalk-transected (ST) and sham-operated animals were subjected to a chronic intermittent salt loading regimen (CISL) for 14 days beginning 1 day post surgery (dps). Animals were sacrificed at 15 and 36 dps. Three days after the termination of CISL, water consumption in ST + CISL animals decreased to the same level as that of sham-operated animals, while that of ST + water animals was maintained at a significantly higher level. The number of the surviving vasopressinergic neurons in the supraoptic nuclei of the ST + CISL group was significantly higher than that of ST + water group. CISL induced vasopressinergic axonal sprouting into the external zone of the median eminence, and formation of subependymal perivascular plexus. While CISL also enhanced regeneration of oxytocinergic axons into the external zone, it did not, however, have any effect on the number of oxytocinergic neurons surviving axotomy.

Intrahypothalamically transected neurosecretory axons do not regenerate in the absence of glial cells.

Fifteen days after transection of the hypothalamo-neurohypophysial tract at the lateral retrochiasmatic hypothalamic area, neurosecretory axons had vigorously regenerated into transplants of explanted hypophysial neural lobe, to a lesser extent into sciatic nerve transplants, and least into optic nerve transplants. Regenerating axons were always closely associated with the specific glial cells of these grafts. When these glial cells were killed by cryotreatment prior to transplantation, neurosecretory axons did not regenerate into the abundant extracellular matrix of the transplants, including persisting basal lamina tubes in neural lobe and sciatic nerve grafts. The presence of viable glial cells is a prerequisite for neurosecretory axon regeneration.

Regeneration of neural lobe-like neurovascular contact regions in explanted neural lobes placed in the hypothalamo-neurohypophysial tract in the lateral retrochiasmatic area.

Neural lobes that had been explanted 30 days earlier were transplanted into retrochiasmatic lesions of the hypothalamo-neurohypophysial tract 15 days before being observed by electron microscopy and neurophysin immunohistochemistry. Neurovascular contact regions consisting of microvascular networks surrounded by neurophysin-immunoreactive terminals developed in 86% of the grafted explants. The fine structure of such regions resembled that of the neural lobe, with palisades of neurosecretory axon terminals abutting the basal laminae associated with the microvessels and plexuses of neurosecretory axons occupying the spaces within the vascular network. Both continuous and fenestrated blood vessels were present. Lamellopodia from glial cells partially ensheathed both axons and terminals, and sometimes separated the terminals from the perivascular basal lamina. Profiles in which neurosecretory granulated vesicles were depleted and many microvesicles were present were interpreted as terminals from which hormones had been released. No regeneration occurred into explants that had been cryotreated to kill their pituicytes and other cells before transplantation. These observations demonstrate that neurosecretory axons served in the hypothalamus can regenerate to form a new neural lobe-like structure when an appropriate microenvironment is available, and that neural lobes explanted 21 days earlier retain the elements required to supply that microenvironment. They also provide evidence that viable pituicytes are essential for regeneration of neurosecretory axons and terminals into transplanted explants.

Development of neural lobe-like neurovascular contact regions after intrahypothalamic transection of the hypothalamo-neurohypophysial tract.

Fifteen days after bilateral transection of the hypothalamo-neurohypophysial tract at the level of the lateral retrochiasmatic area, neurovascular contact regions had developed proximal to 66% of the lesions. Contact regions developed in every case when neural lobe explants were placed into the lesions, and near approximately half of the lesions into which small pieces of sciatic or optic nerve were transplanted. Neurovascular contact regions were characterized by microvascular networks surrounded by dense neurophysin-immunoreactive plexuses. At the fine structural level, the organization of such regions resembled that of the neural lobe, with the single exception that capillaries were not fenestrated. Numerous neurosecretory axons were present, and palisades of neurosecretory axon terminals abutted perivascular basal laminae. Lamellopodia from glial cells partially ensheathed regenerating neurosecretory axons and often lay between terminals and the perivascular basal lamina. Terminals with many microvesicles and few neurosecretory granulated vesicles provided morphological evidence of hormone release.

Fine structural changes in explants of the neural lobe of the rat hypophysis.

Abstract To obtain a purified population of pituicytes, pieces of rat neural lobes were dissected free of the pars intermedia, incubated in a variety of media, and fixed at regular intervals between 7 and 56 days of incubation. Many neurosecretory axons survived for up to 21 days without any apparent signs of degeneration. Most axons, however, degenerated and were progressively phagocytosed and subsequently eliminated by pituicytes and microglial cells. Lysed axons that were not eliminated, persisted as dense bodies or paracrystalline inclusions. After 30 days of culture, cluster-forming pituicytes predominated the explants. Pituicytes underwent morphologic changes such as medium-dependent decrease or increase of lipid inclusions, Golgi activation, process extension and interdigitation, formation of gap junctions and bundles of intermediate filaments. At the explant surface in contact with the culture medium, pituicytes differentiated into an epithelial layer of ciliated and microvilli-bearing cells linked by junctional complexes. Long-term neural lobe explants are a relatively pure source of viable pituicytes and should be useful for further studies on the functional significance of these cells.

Magnocellular neurosecretory axon regeneration into rat intrahypothalamic optic nerve allografts.

To test the working hypothesis that neurosecretory neuronal regeneration is largely dependent on microenvironmental conditions at the lesion site, intact or predegenerated optic nerves were allografted intrahypothalamically into the hypothalamo-neurohypophysial tract. Neurosecretory axons regenerated consistently into all grafts. Early regeneration proceeded without glial cell association. At later stages of regeneration, however, neurosecretory axons were associated consistently with astrocytes that, within perivascular spaces, were surrounded by a basal lamina. Axons in contact with that basal lamina had the characteristics of terminals, suggesting functional recovery. It is postulated that it is the initial absence of a blood-brain barrier at the graft site that provides a microenvironment similar to that in the neural lobe and that induces neurosecretory axon regeneration.

An immunohistochemical and fine-structural analysis of peptidergic hypothalamic neurosecretory axon regeneration into the leptomeninges of the rat.

Regeneration of severed hypothalamic peptidergic neurosecretory axons into the ventral pia-arachnoid was observed in rats at the light microscopic and fine-structural levels. A temporal increase occurred in the number of neurophysin-positive axons regenerating into the leptomeninges for distances up to 3.3 mm by 40 days post-lesioning. A consistent pattern of parallel, meshed and clustered axons, occurring either singly or in bundles, was present within the connective tissue, while plexus and bundles were observed in association with leptomeningeal blood vessels. Axons were characterized by preterminal and terminal dilatations. Neurosecretory granulated vesicles occurred throughout axons. The presence of microvesicles at contact points with basal lamina suggests the possibility of hormone release. Most axons were arranged as fascicles associated closely with basal lamina-bounded support cells whose thin lamellar processes wrapped single axons or fascicles of axons. We conclude, therefore, that cellular and intercellular leptomeningeal microenvironments support and sustain the growth and regeneration of transected neurosecretory axons.

Immunohistochemical investigation of the magnocellular peptidergic hypothalamo-neurohypophysial system of the rat chronically stimulated by long-term administration of hypertonic saline.

The hypothalamic supraoptic (SON) and paraventricular nuclei (PVN), median eminences (ME) and neural lobes (NL) of normally hydrated control rats (group 1), and of rats drinking 2% NaCl for 7 (group 2), 30 (group 3) or 90 days (group 4) were investigated using immunohistochemistry for neurophysins (NP), arginine vasopressin (AVP) or oxytocin (OXY). Animals from the 3 experimental groups showed equivalent decreased levels of immunoreactive NP in the SON and PVN, but the greatest decrease was in the SON. Dendrites of SON and PVN neurons became loaded progressively with immunoreactive NP, AVP and OXY as salt loading proceeded. In rats of group 2, axons leaving the SON and PVN showed a marked depletion of immunoreactive material. The latter was found mainly at the periphery of widely spaced axonal swellings, clearly contrasting with the small and narrowly spaced beads of the neurosecretory axons of control rats. In rats of groups 3 and 4, axons leaving the SON and PVN resembled those of control rats. In the ME of the animals in all experimental groups, the same degree of decrease of immunoreactive NP was observed. In rats of group 3, bundles of axons containing immunoreactive AVP and OXY frequently projected through the ependymal lining of the ME into the third ventricle. In the NL of all experimental animals, a marked decrease occurred in the amount of immunoreactive NP, AVP and OXY. The decrease of immunoreactive AVP, however, was more pronounced in rats of group 2 than in those of groups 3 and 4. The NL of rats in group 4 were approximately 80% larger than those of control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of colchicine on the hypothalamo-neurohypophysial system of chronically salt-loaded rats.

The effect of colchicine on the hypothalamo-neurohypophysial system of rats drinking 2% NaCl for 4, 6, or 90 days was investigated. Colchicine was injected at time 0 for the 4-day group and 48 h before sacrifice for the 6- and 90-day groups. Each experimental group was divided into two subgroups that were injected with 7 micrograms colchicine into either the cisterna magna or the lateral cerebral ventricle. The hypothalamo-neurohypophysial system was investigated immunocytochemically using an antiserum against both neurophysins (NPs) as primary antibody. The arginine vasopressin content of the neural lobe (NL) of rats salt loaded for 4 days and injected with colchicine on days 0 and 3 was determined by radioimmunoassay. The amount of immunoreactive NPs in the supraoptic nucleus, the paraventricular nucleus, and in the NL was decreased in salt-loaded rats. In salt-loaded rats injected with colchicine into the lateral ventricle, a substantially larger amount of immunoreactive NPs was present in these three areas. However, when colchicine was injected into the cisterna magna, only the supraoptic nucleus appeared loaded with immunoreactive NPs, while NPs were depleted from the paraventricular nucleus. In the NL of rats salt loaded for 90 days immunoreactive NPs were diminished markedly, and axon swellings (Herring bodies) had virtually disappeared. Colchicine treatment of these rats caused an increase of immunoreactive NPs in the NL approaching control values and a reappearance of a large number of axon swellings. Results similar to those obtained with immunohistochemistry for NPs were obtained when determining the arginine vasopressin content of the NL by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Frequency of involvement of pox virions in lesions of bovine dermatophilosis.

By combined electron microscopical and serological methods the rate of involvement of pox virions within lesions of bovine natural dermatophilosis was determined. Scabby lesions of 40 (77%) out of a total of 52 cases were positive for the virions, while sera of all infected animals which reacted positively for pox viral antibodies (LSD) was significantly higher (P less than 0.001) in comparison to those of healthy-appearing animals. The virions in all cases had a uniform oval to cylindrical morphology with a mean measurement of 110 X 280 nm. A synergistic interplay involving the initiation of the scabby lesions by the virions and their subsequent trapping within keratin deposits, followed by their release through the keratolytic activity of Dermatophilus congolensis appear to have obtained in cases.

Fine structural characteristics of neurophysin-positive perivascular plexus that develop in the rat hypothalamus following interruption of the hypothalamo-neurohypophysial tract.

Transection of neurosecretory axons of the hypothalamo-neurohypophysial tract within the hypothalamus by stereotactic grafts of various tissues or knife cuts induced the development of neurophysin-positive plexus around arterioles, venules and capillaries in the vicinity of these grafts or cuts. These plexus ranged from single axons to densely woven networks and tended to increase progressively with time after experimental intervention. At the fine structural level, typical neurosecretory axon profiles were either abutting the perivascular connective tissue space or located within it. They were usually accompanied by astrocyte processes or microglial cells. Many of these axons had extensive contact with the surrounding basal lamina at which point clusters of microvesicles reminiscent of axon terminals in the neural lobe were present.

Neurosecretory axon regeneration into intrahypothalamic neural lobe allografts: neurophysin immunohistochemistry and fine structure.

Neural lobe allografts placed stereotactically into the hypothalamo-neurohypophysial tract between the paraventricular and supraoptic nuclei were investigated between 5 and 70 days post-transplantation (dpt). They contained temporally increasing numbers of neurophysin-positive axons. At the fine structural level, endogenous neurosecretory axons had virtually disappeared from successful (vascularized) grafts by 5 dpt. At this time, single host neurosecretory axons and especially numerous growth cones were associated with pituicyte processes and/or scalloped basal lamina scaffolds. Axon terminals containing neurosecretory granulated vesicles and microvesicles were present only occasionally at 5 dpt but became much more numerous subsequently. These terminals were associated with pituicytes and abutted the parenchymal basal lamina of pericapillary connective tissue spaces. In addition, beginning at 10 dpt, neurolemmocyte-like cells were associated with neurosecretory axons. At 70 dpt, the fine structural characteristics of grafted neural lobes were virtually indistinguishable from those of intact controls, except for the presence of occasional areas of more extensive connective tissue, nonfenestrated capillaries and neurolemmocyte-like cells.

Xylazine-ketamine-induced anesthesia in rats and its antagonism by yohimbine.

A combination of xylazine and ketamine was used to anesthetize 60 male rats, and then yohimbine was given to evaluate its reversing effect on xylazine-ketamine-induced anesthesia. In experiment A, xylazine (21 mg/kg of body weight) and ketamine (45 mg/kg) were admixed and administered IM to 12 Sprague-Dawley rats. Anesthesia lasted approximately 70 minutes. The xylazine-ketamine combination also induced polyuria, bradycardia, and bradypnea. When yohimbine (2.1 mg/kg) was given intraperitoneally 20 minutes after the xylazine-ketamine injection, the rats regained consciousness and righting reflexes within approximately 10 minutes. Yohimbine also reversed the bradycardia and bradypnea and appeared to reduce the polyuria induced by the xylazine-ketamine combination. In experiment B, xylazine (15.4 mg/kg) and ketamine (33 mg/kg) were admixed and given IM to 48 Holtzman rats. The combination induced surgical anesthesia for at least 30 minutes, during which a surgical procedure involving grafting a section of the sciatic nerve into the hypothalamus was performed. In rats in which yohimbine (1 mg/kg) was given intraperitoneally 45 to 60 minutes after xylazine-ketamine administration (before natural recovery from the anesthesia), the righting reflex was apparent in less than 10 minutes.

Peptidergic neurosecretory axons regenerate into sciatic nerve autografts in the rat hypothalamus.

Pieces of intact or degenerated sciatic nerves autografted into contact with transected neurosecretory axons within the hypothalamus were invaded by neurophysin-positive axons. With increasing time after grafting, increasing numbers of axons were present in both types of grafts, but grafts of degenerated sciatic nerves always contained more axons. At the fine-structural level typical neurosecretory as well as nonneurosecretory axons were usually associated with basal lamina-enclosed neurolemmocyte processes; occasional axons occurred among collagen fibrils or within basal lamina scaffolds. Profiles with the fine structural characteristics of axon terminals were present by 20 days after transplantation.

Fine structural organization of the subfornical organ. A concise review.

This review of the subfornical organ, with special emphasis on the rat, summarizes the fine structural characteristics of the capillaries, the access route for blood-borne substances, the ependyma through which cerebrospinal fluid-borne substances penetrate the organ, neuronal perikarya, and types of synapses and axons, together with a brief discussion of the principal as yet unresolved problems.

Ultrastructural visualization of Na+-channel associated [125I]alpha-scorpion toxin binding sites on fetal mouse nerve cells in culture.

Purified neurotoxin II from the scorpion Androctonus australis Hector (alpha-ScTx) has previously been shown to bind specifically to the voltage-sensitive Na+ channels of excitable cells. Recent studies, using high specific activity 125I-labeled alpha-ScTx, demonstrated specific binding to neuronal cells derived from fetal mouse brains. In the present study, 125I-labeled alpha-ScTx was used to localize the voltage-sensitive Na+ channels in cultured fetal mouse brain cells. By quantitative electron microscope autoradiography we demonstrate that specific alpha-ScTx binding sites are selectively located at the plasma membrane. Estimates of their density revealed that neurites at 13 days in vitro carry at least 6 X more specific alpha-ScTx sites than cell body membrane.

Biosynthesis and axoplasmic transport of neurophysins in the hypothalamo-neurohypophysial system of Rana pipiens.

Biosynthesis, axoplasmic transport, and storage of neurophysin in the amphibian (Rana pipiens) magnocellular peptidergic neurosecretory system were studied, and the results were compared with those reported in mammals. After injection of [35S]cysteine into the preoptic recess, light microscopic autoradiography provides evidence that neurons of the preoptic nucleus (PON) synthesize cysteine-rich proteins. The time course of appearance of these [35S]cysteine-labeled proteins in different regions of the hypothalamo-neurohypophysial system was studied by slab gel autoradiography. [35S]Cysteine-labeled proteins were found in the PON less than 1 hr postinjection, whereas a major labeled protein, tentatively identified as the neurophysin, first appeared in the infundibulum and neural lobe 4 hr after the injection. In addition, the labeled neurophysin persisted in the neural lobe throughout the entire observation period of 5 days. The minimum transport rate for neurophysin was calculated as 0.9 mm/hr (22 mm/day) at 25 degrees C. Two different neurophysins (with isoelectric points (pI) 4.9 +/- 0.1, 4.6 +/- 0.1, and Mr = 23,000, 20,100) may be resolved from the neural lobe extracts by isoelectric focusing and SDS-polyacrylamide gel electrophoresis, respectively. In addition to the neurophysin peaks, two radioactive peaks with pI 5.2 and 5.8 may be detected in the preoptic nucleus and the infundibulum as early as 30 min after [35S]cysteine injection. Preliminary conversion studies suggest a putative precursor role for the pI 5.2 protein. The results indicate that in the amphibian peptidergic neurosecretory system, the synthesis of cysteine-rich neurophysin by the preoptic neurons, the transport through the infundibulum, and the storage in the neural lobe proceed similarily to their mammalian counterparts.