Ultrastructural organization of actin filaments in neurosecretory axons of the rat

Summary The ultrastructural organization of actin filaments was studied in the neurohypophysial system of the rat after heavy meromyosin (HMM) labeling. This structural pattern is characterized by (1) a straight arrangement of the filaments parallel to the axonal axis in the proximal nondilated parts of axons, (2) a central location within axonal dilatations, and (3) a higher concentration within axonal endings where the filaments form a complex three-dimensional network. The relationships of the filaments to other axonal structures and organelles was further studied by use of electron microscopic stereoscopy. The actin filaments frequently appear anchored to the axolemma with either polar arrangements of the arrowhead decoration (i) at structurally undifferentiated sites, and (ii) more particularly within perivascular endings, at sites with electron-dense thickenings. In all axonal divisions actin filaments are also found to bind to filamentous material surrounding the microtubules and to various organelles. Within the terminal portions of the axons actin filaments exhibit close relationships to neurosecretory granules and to the numerous smooth microvesicles found in this region. Such preferential relationships are particularly observed both in axon terminals and in pituicytes, with coated vesicles frequently binding actin filaments. In water-deprived rats, the concentration of actin filaments is conspicuously increased along the axons and more clearly in the axonal swellings and endings, where they form a more complex and interconnected network. These data are discussed in the light of a possible involvement of contractile proteins in the mechanisms of axonal transport and terminal release of neurosecretory products.     

Anatomical and functional evidence for a stress-responsive,monoamine-accumulating area in the dorsomedial hypothalamus of adult rat brain

The dorsomedial hypothalamus (DMH) plays an important role in relaying information to neural pathways mediating neuroendocrine, autonomic, and behavioral responses to stress. Evidence suggests that the DMH is a structurally and functionally diverse integrative structure that contributes to both facilitation and inhibition of the hypothalamo-pituitary-adrenal axis, depending on the nature of the stimulus and the specific neural circuits involved. Previous studies have determined that stress or stress-related stimuli elevate tissue concentrations of serotonin (5-hydroxytryptamine; 5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine, and noradrenaline selectively within the DMH. In order to determine the specific region of the rat DMH involved, we used high-performance liquid chromatography with electrochemical detection to measure tissue concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline within five different subregions of the DMH in adult female Lewis and Fischer rats immediately or 4 h following a 30-min period of restraint stress. Compared to unrestrained control rats, restrained rats had elevated concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline immediately after a 30-min period of restraint and had elevated concentrations of 5-HT 4 h following the onset of a 30-min period of restraint stress. These effects were confined to a specific region that included medial portions of the dorsal hypothalamic area and dorsal ependymal, subependymal, and neuronal components of the periventricular nucleus. Furthermore, these effects were observed in Lewis rats, but not Fischer rats, two closely related rat strains with well-documented differences in neurochemical, neuroendocrine, autonomic, and behavioral responses to stress. These data provide support for the existence of a stress-responsive, amine-accumulating area in the DMH that may play an important role in the differential stress responsiveness of Lewis and Fischer rats.     

Functional topography of the dorsomedial hypothalamus

The dorsomedial hypothalamus (DMH) has been proposed to play key roles in both the defense reaction to acute stress and in the thermoregulatory response to cold. We reasoned that the autonomic/respiratory motor patterns of these responses would be mediated by at least partly distinct DMH neuron populations. To test this, we made simultaneous recordings of phrenic nerve and plantar cutaneous vasoconstrictor (CVC) activity in 14 vagotomized, ventilated, urethane-anesthetized rats. Microinjections of d,l-homocysteic acid (DLH; 15 nl, 50 mM) were used to cause localized, short-lasting (<1 min) activation of DMH neuron clusters. Cooling the rat's trunk skin by perfusing cold water through a water jacket-activated plantar CVC activity but depressed phrenic burst rate (cold-response pattern). The expected "stress/defense response" pattern would be phrenic activation, with increased blood pressure, heart rate, and possibly CVC activity. DLH microinjections into 76 sites within the DMH region never reduced phrenic activity. They frequently increased phrenic rate and/or plantar CVC activity, but the magnitudes of those two responses were not significantly correlated. Plantar CVC responses were evoked most strongly from the dorsal hypothalamic area and most dorsal part of the dorsomedial nucleus, whereas peak phrenic rate responses were evoked from more caudal sites; their relative magnitudes varied systematically with rostrocaudal position. Tachycardia correlated with plantar CVC responses but not phrenic rate. These findings indicate that localized activation of DMH neurons does not evoke full "cold-response" or stress/defense response patterns, but they demonstrate the existence of significant functional topography within the DMH region.     

Ultrastructural localization of the receptor for leptin in the rat hypothalamus

Ultrastructural localization of the leptin receptor in the rat hypothalamus was studied by immunocytochemistry. The antiserum against the leptin receptor which was used specifically recognized the carboxy terminal of the cytoplasmic domain. Intense leptin receptor immunoreactivity was detected in the arcuate, paraventricular, and ventromedial nuclei of the hypothalamus and in the lateral hypothalamic area. At the ultrastructural level, leptin receptor-like immunoreactivity appeared to be concentrated predominantly in perikarya and dendrites of these areas and strong immunolabeling for the leptin receptor was detected in the plasma membrane, rough endoplasmic reticulum, Golgi apparatus, and cytoplasmic matrix. This study provides the first detailed fine structure of leptin receptor-immunoreactive neurons in the rat hypothalamus. It may help to provide better understanding of the functions of leptin in the rat hypothalamus.     

Ultrastructural localization of radiolabelled L-dopa in the endocrine hypothalamus of the rat

Summary Light and electron microscopic autoradiography has been employed to define the neuroanatomical patterns of uptake and binding of radiolabelled L-dopa in the endocrine hypothalamus of the rat. A dorsomedial continuum of arcuate and periventricular neurons selectively sequester ³H L-dopa 20 min following its intraventricular infusion. By 40 and 60 min following the infusion labelling of neurons is minimal and supports the notion of rapid degradation. Other cell compartments such as tanycytes demonstrate uptake of ³H L-dopa. The ultrastructural localization and distribution of radiolabelled L-dopa (or its metabolites) in the rodent hypothalamus is discussed with respect to mechanisms and cell compartments involved in neuroendocrine regulatory processes.Supported by USPHS Program Project Grant NS-11642-04 (DES) and RR-05403Career Development Awardee RO4GM-70001     

Ultrastructural demonstration of proteoglycans in adult rat cornea

Proteoglycans in the adult rat cornea were demonstrated at the electron microscope level using two approaches: (a) staining with cuprolinic blue dye in the presence of 0.3 MgCl2, and (b) immunocytochemical localization of glycosaminoglycans with monoclonal antibodies and protein A-gold complexes. In the stroma two kinds of cuprolinic blue-induced filaments were morphologically differentiated and characterized according to their sensitivity to enzymatic degradations as keratan sulphate-rich and chondroitin-dermatan sulphate-rich proteoglycans respectively. Both types were mostly associated with collagen fibres, occupying the whole stroma except in certain areas whose significance is discussed. By immunocytochemistry, anterior and posterior regions of the stroma were found to be richer in chondroitin sulphate than the middle part, whereas keratan sulphate showed an homogeneous distribution throughout the stroma. Glycosaminoglycans were also detected in corneal basement membranes, epithelium and endothelium. The latter localizations are discussed in the light of what is known at present about the production of glycosaminoglycans by corneal cells.     

The dorsomedial hypothalamus: a new player in thermoregulation

Neurons in the dorsomedial hypothalamus (DMH) play key roles in physiological responses to exteroceptive ("emotional") stress in rats, including tachycardia. Tachycardia evoked from the DMH or seen in experimental stress in rats is blocked by microinjection of the GABA(A) receptor agonist muscimol into the rostral raphe pallidus (rRP), an important thermoregulatory site in the brain stem, where disinhibition elicits sympathetically mediated activation of brown adipose tissue (BAT) and cutaneous vasoconstriction in the tail. Disinhibition of neurons in the DMH also elevates core temperature in conscious rats and sympathetic activity to least significant difference interscapular BAT (IBAT) and IBAT temperature in anesthetized preparations. The latter effects are blocked by microinjection of muscimol into the rRP, while microinjection of muscimol into either the rRP or DMH suppresses increases in sympathetic nerve activity to IBAT, IBAT temperature, and core body temperature elicited either by microinjection of PGE(2) into the preoptic area (an experimental model for fever), or central administration of fentanyl. Neurons concentrated in the dorsal region of the DMH project directly to the rRP, a location corresponding to that of neurons trans-synaptically labeled from IBAT. Thus these neurons control nonshivering thermogenesis in rats, and their activation signals its recruitment in diverse experimental paradigms. Evidence also points to a role for neurons in the DMH in thermoregulatory cutaneous vasoconstriction, shivering, and endocrine adjustments. These directions provide intriguing avenues for future exploration that may expand our understanding of the DMH as an important hypothalamic site for the integration of autonomic, endocrine, and behavioral responses to diverse challenges.     

Inhibition of serotonin release by bombesin-like peptides in rat hypothalamus in vitro

We investigated the activity of bombesin (BN), neuromedin-C (NM-C) and neuromedin-B (NM-B) on serotonin (5-HT) release and reuptake in rat hypothalamus (HYP) in vitro. BN and NM-C but not NM-B (all 1 microM) decreased K+ evoked 3H-5-HT release from superfused HYP slices by 25%. Bacitracin (BCN, 2 micrograms/ml), a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K+ evoked 3H-5-HT release. Phosphoramidon (PAN, 10 microM) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K+ evoked 3H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 microM), enhanced both BN and NM-C inhibition of 3H-5-HT release. Bestatin (BST, 10 microM) had no effect on BN or NM-C inhibitory activity on 3H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of 3H-5-HT into HYP synaptosomes alone or in combination with any of the peptidase inhibitors, nor did these peptides alter the ability of fluoxetine to inhibit 3H-5-HT uptake. These data suggest: a) that BN-like peptides may alter neurotransmission in the HYP by acting presynaptically on the 5-HT release mechanism; b) a similarity in the structural requirements for the BN induced inhibition of 5-HT release and BN evoked thermoregulatory disturbances; and c) that peptidases may selectively augment or reduce pharmacologic activity of BN-like peptides upon CNS administration.     

Ultrastructural changes in the median eminence of the rat following deafferentation of the basal hypothalamus

Summary This report concerns a light and electron microscopic investigation of the median eminence and dorsal infundibular stem of the rat following surgical isolation (deafferentation). Using a modification of the Halász technique, the basal hypothalamus, including the arcuate nucleus and median eminence were surgically isolated from surrounding structures. Special attention was directed to the contact (external) zone of the median eminence and rostral infundibulum where tuberohypophyseal axons as well as ependymal cell processes abut upon the abluminal basement membrane of the portal perivascular space. The results of this study to date suggest that 9, 20, and 40 days following surgical isolation, there is a distinct increase in the population of tuberohypophyseal dense core vesicles. It is suggested that deafferentation abolishes inhibitory and excitatory input that serves to modify the cellular dynamics of tuberohypophyseal neurosecretory elements. Comments are also made on the presence of cistern-like structures in the lateral median eminence; the presence of vesicle-like inclusions in terminal ependymal processes is discussed in relationship to the role that ependyma may play in linking the third ventricle with the adenohypophysis.This research was supported by USPHS Grants NB 08171 and AM 10002. The authors are indebted to the excellent technical assistance provided by G. Krobisch Dudley. Further, the authors wish to express their thanks to Dr. Adolph Weindl for his valuable advice and criticism, and to Matilde Holzwarth for her helpful assistance.     

Changes due to age in the regenerating axons of the adult rat]

Muscle reinnervation after nerve crush was observed in rats at different ages with a combined technique that simultaneously demonstrates nerve endings and endplates. At early times of reinnervation the amount of sprouting was higher in older rats than in younger rats; according to this finding an enhanced number of polyinnervated endplates was found in older rats. A similar enhancement of sprouting and polyinnervation was observed during muscle reinnervation of vitamin E deficient rats, supporting the proposed analogy between vitamin E deficiency and aging.     

Ca2+-dependent secretion of serotonin by the rat hypothalamus during ontogenesis

A study was made of functional maturity of the terminal part of serotoninergic system of rat hypothalamus in perinatal period: the maturity was estimated by the ability to release serotonin. The release of specifically taken up serotonin (3H-5-OT) by the tissue of hypothalamus of 16-20-day-old rat fetuses, 8-9-day males and adult males was studied in the perfusion system. Spontaneous release of the labelled amine was recorded in the earliest studied period--on the 16th day of the prenatal period, but the response to K+ depolarization was absent at this time. For the first time the increase of the rate of 3H-5-OT release in response to depolarization was noted on the 17th day of development. In the absence of Ca2+ depolarizing stimulus produced no increase in the release of the labelled product. Similar results were obtained with perfusion of fetal hypothalamus on the 18t hand 20th days of development. In neonatal animals the release of 5-OT in response to depolarization was equal to that in adults. The data obtained point to a possible functioning of serotoninergic elements of hypothalamus in the perinatal period in rats.     

Evolvement of systems of serotonin uptake in rat hypothalamus during its development]

Kinetics of 3H serotonin accumulation into slices from hypothalamus have been compared in adult, puppy and foetus rat. In 15 days-old, as in adult rat, there are two components of 5 HT accumulation corresponding to the low and high affinity transport systems. For this latter, Km and Vmax values are much higher in adult than in 15 days old rat (in adult, Km=1,3 X 10(-7) and Vmax=0,33 X 10(-10); in 15 days old rat, Km=0,5 X 10(-7) and Vmax=0,125 X 10(-10)). On the opposite, in the 7 days old rat and in the 21 days old foetus, it is only possible to arbitrarely define one uptake system corresponding to the following apparent values: in the 7 days old rat, Km= 5 X 10(-7) and Vmax=2 X 10(-10), in the foetus, Km=0,2 X 10(-7) and Vmax=0,15 X 10(-10). These results showed important developmental differences in affinity of 3H serotonin to hypothalamus. The low and high affinity uptake systems existing in adult are only individualized in the 15 days old little rat.     

Effects of disinhibition of neurons in the dorsomedial hypothalamus on central respiratory drive

Neurons within the dorsomedial hypothalamus (DMH) play a critical role in subserving the cardiovascular and neuroendocrine response to psychological stress. An increase in respiratory activity is also a characteristic feature of the physiological response to psychological stress, but there have been few studies of the role of DMH neurons in regulating respiratory activity. In this study we determined the effects of activation of DMH neurons on respiratory activity (assessed by measuring phrenic nerve activity, PNA) and the relationship between evoked changes in respiratory activity and changes in sympathetic vasomotor activity in spontaneously breathing urethane-anesthetized rats. Microinjections of bicuculline (4-40 pmol in 20 nl) into the DMH evoked dose-dependent increases in PNA burst frequency and amplitude. These were accompanied by dose-dependent decreases in mean tracheal CO(2) levels, indicative of hyperventilation. In control experiments, microinjections of bicuculline into sites adjacent to the DMH evoked much smaller or no changes in PNA. In experiments where renal sympathetic nerve activity (RSNA) was also measured, cycle-triggered averaging revealed that RSNA under resting conditions was partly correlated with the PNA, but in response to DMH disinhibition there was no consistent change in the amplitude of the respiratory-related variations in RSNA. The results indicate that DMH neurons can exert a powerful stimulatory effect on respiratory activity, causing hyperventilation. This is not associated with an increase in the degree of coupling between PNA and RSNA, indicating that the DMH-evoked increase in RSNA is not a consequence of increased central respiratory drive.     

Ultrastructural study of experimental muscle degeneration and regeneration in the adult rat

Summary Methyl-bupivacaine is a local anaesthetic with a selective myotoxic action. A single subcutaneous injection of the drug into the hind leg of adult rats produces a uniform, complete and irreversible destruction of superficial layers of fibres in the underlying extensor digitorum longus muscle. The degeneration of muscle fibres is followed by phagocytosis and a rapid and complete regeneration.The first stage in the regeneration process is the appearance of presumptive myoblasts within the original basement membrane of the sarcolemmal tube. On the second day after injury aggregates of myoblasts are present and fusion is observed between the cells. The myotubes thus formed increase in size by fusing with additional myoblasts. Myotubes are also observed to fuse with one another. On the fifth day after injury the regeneration process has proceeded to the stage of early muscle fibres with fully differentiated myofibrils with typical sarcomere structures. By ten days only mature muscle fibres of about normal size are present and regeneration appears complete.In previously denervated and methyl-bupivacaine treated muscles the stages of regeneration are similar to those observed in innervated muscles, the only apparent difference being a slowing of cell differentiation and incomplete maturation.An electrophysiological study shows that the motor nerve at the third day after injury forms synaptic contacts with regenerating muscle cells. At that stage of myogenesis the myotubes are highly sensitive to applied acetylcholine.1 (1-n-butyl-DL-piperidine-2-carboxylic acid-2,6-dimethyl-anilide-hydrochloride); Marcaine®, manufactured by AB Bofors, Nobel-Pharma, Mölndal, Sweden.The study was carried out under the auspicies of The Czechoslovak Academy of Sciences and the Royal Academy of Sciences in Sweden.     

Growth preferences of adult rat retinal ganglion cell axons in retinotectal cocultures

We examined whether regenerating axons from adult rat ganglion cells are able to recognize their appropriate target region in vitro. Explants from adult rat retina were cocultured with embryonic sagittal midbrain slices in Matrigel®. The midbrain sections contained the superior colliculus, the main target for retinal ganglion cell axons in rats, and the inferior colliculus. We observed a statistically significant preference of both temporal and nasal retinal axons to grow toward their appropriate target region (anterior and posterior superior colliculus, respectively). No preferential growth of retinal ganglion cell axons was detected in controls, for which retinal explants were cultured on their own. When retinal ganglion cell axons were given a choice between superior colliculus and inferior colliculus, axons from nasal retina preferentially grew toward the posterior superior colliculus and avoided the inferior colliculus. In contrast, temporal axons in the same assay did not show preference for either of the colliculi. These findings suggest that regenerating axons from adult rat retina are able to recognize target-specific guidance cues released from embryonic midbrain targets in vitro. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 379–387, 1998