Ultrastructural characteristics of vasopressin-containing neurons in the paraventricular nucleus of the hypothalamus

Summary Vasopressin-containing neurons, identified by immunocytochemistry, are located predominantly in the posterior magnocellular division of the paraventricular nucleus of the rat hypothalamus. By electron microscopy, the immunoreaction product is seen within the cell bodies and neuronal processes. In the perikarya and dendritic processes, the immunoreactive material is associated primarily with neurosecretory granules. Axonal processes, identified by their content of microtubules and accumulation of neurosecretory granules, show the immunoreaction product in association with both of these organelles. Afferent axo-dendritic, axo-somatic and putative axo-axonic synapses with immunostained vasopressinergic neurons can be identified. The presynaptic profiles do not contain immunoreactive material. This study contributes to the ultrastructural characterization of vasopressinergic neurons in the paraventricular nucleus and of their afferent synaptic input.Supported by NIH Grants HD-12956 and 2SO7RR05403     

The origin and ultrastructural characteristics of corticotropin-releasing factor (CRF)-immunoreactive nerve fibers in the posterior pituitary of the rat

Summary A fine network of corticotropin-releasing factor (CRF)-immunopositive fibers was found in the posterior lobe of the pituitary of the rat. The intermediate and distal lobes were free of CRF-immunoreactivity. Varicose, terminal-like axons were frequently observed around capillary vessels. Surgical isolation of the paraventricular nuclei resulted in a complete disappearance of CRF-immunoreactive fibers from the posterior lobe. CRF-immunopositive fibers show the general characteristics of peptidergic axons. These ultrastructural observations support the idea that CRF is secreted into capillary vessels.     

Corticotropin-releasing factor (CRF)-immunoreactive neurons in the mammillary body of the rat

Summary The presence and distribution of CRF-immunoreactive cells and nerve fibers were studied in the mammillary body of the rat, 12 days after placing various types of lesions within the hypothalamus. Anterior and anteriolateral cuts, placed in the midhypothalamus immediately behind the paraventricular nuclei resulted in an almost complete disappearance of CRF-immunoreactive fibers from the median eminence and simultaneous appearance of CRF-containing neurons in the mammillary body. Posterior or postero-lateral hypothalamic cuts carried out in front of the mammillary body caused the accumulation of CRF-immunoreactive material in neurons and neural processes located behind the cut-line. This type of intervention had no effect on the quantity of CRF fibers in the median eminence. A cut running through the central part of the mammillary body in the frontal plane resulted in appearance of CRF neurons only in the posterior half of the mammillary region. Placing a cut behind and over the mammillary body, CRF-immunoreactive neurons became detectable below the superior cut-line. No immunoreactive neurons were observed in the mammillary body when the frontal cut reached the base of the brain at the posterior border of the nucleus, leaving intact its anterior and superior connections. In all these cases when the mammillo-thalamic tract was transected, CRF neurons became detectable in the mammillary body.     

Ultrastructural alterations in skeletal muscle fibers of streptozotocin-diabetic rats

Summary The ultrastructure of fast-twitch-oxidative-glycolytic (FOG), fasttwitch-glycolytic (FG) and slow-twitch-oxidative (SO) fibers in plantaris and soleus muscles of normal and streptozotocin-diabetic rats was studied. In the diabetic animals, the mitochondria of FOG and SO fibers showed a loss of cristae and an increase in electron-dense granules. There was also an increased number of lipid droplets in close proximity to the mitochondria and the nuclei, and a separation of individual muscle nuclei to form satellite cells. Higher incidences of surface projections and sarcoplasmic splittings at the nuclear region were noticed in SO fibers. The FG fibers showed some disorientation of the T-tubular system. It is concluded that streptozotocin-diabetes has differential effects on the fine structure of the three fiber types of rat skeletal muscle.Supported by USPHS Grant AM 18280-04, Boston University Grant GRS-405-BI, and a grant-in-aid award from Sigma Xi Society     

Effect of Corticotropin Releasing Factor (CRF) Injected Into the Median Eminence on LH Secretion in Male Rats

We determined the dose-response relationship and examined the time-related effect of CRF (corticotropin releasing factor) injected directly into the Median Eminence (ME) on LH secretion in conscious intact and castrated male rats. Doses of 0.25, 0.75, 1, and 1.5 nmol CRF dissolved in 1 l of saline (or saline only in the controls) were injected into the ME and blood samples collected 30, 60, 90, and 120 min postinjection to determine by RIA serum LH. CRF at doses of 0.75, 1 and 1.5 nmol significantly decreased serum LH in castrated and intact animals. The lower dose of CRF did not decrease LH in the two groups studied. The results suggest that in males as in females, CRF inhibits by itself LH secretion, at least in part, by a central action in the ME; the inhibitory effect of CRF on LH is similar in castrated and intact males; the dose of 0.25 nmoles of CRF was ineffective in decreasing LH and finally that CRF at ME levels may participate in a variety of stress-related responses, including reproduction inhibition, through LH suppression.     

Ultrastructural immunocytochemical localization of luteinizing hormone-releasing hormone (LH-RH) in the median eminence of the guinea pig

Summary LH-RH was localized at the ultrastructural level in axons and nerve terminals of the median eminence of the male guinea pig. LH-RH positive neuronal profiles were most concentrated in the medial-dorsal aspect of the infundibular stalk and in the post-infundibular median eminence at the level immediately following separation of the stalk from the base of the brain. LH-RH containing axon profiles were most abundant in the palisade zone; nerve terminals in contact with the hypophysial portal vasculature were relatively rare. The hormone was present within granules that measured 900–1,200 Å in axons of the palisade zone and 400–800 Å in nerve terminals abutting on the portal plexus. The differently sized granules represent heterogeneous populations.Supported in part by U.S. Public Health Service grant HD-09636 from the National Institutes of Health and RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center Publication No. 15-031The authors wish to thank Dr. Sandy Sorrentino, Jr. for the gift of antiserum to LH-RH and Dr. Ludwig Sternberger for the peroxidase.antiperoxidase complex     

Ultrastructural characteristics of immunolabelled,corticotropin releasing factor (CRF)-synthesizing neurons in the rat brain

Summary The corticotropin releasing factor (CRF)-synthesizing perikarya and neural processes were detected at ultrastructural level in the hypothalamic paraventricular nucleus and in the median eminence of control and colchicine-pretreated rats. The unlabelled antibody peroxidase-antiperoxidase complex (PAP) immunohistochemical method was used in a pre-embedding manner, on thick, non-frozen sections. In CRF-perikarya, neurosecretory granules (80–120 nm in diameter), free ribosomes, and the rough endoplasmic reticulum were labelled. Unlabelled axon terminals formed asymmetric synapses on CRF-containing perikarya and dendrites. Immunolabelled axons terminated in the palisadic zone of the median eminence.     

Comparison of the effects of CRF and stress on levels of pituitary cyclic AMP and plasma ACTH in vivo

We have previously reported that acute stress increases levels of rat pituitary cyclic AMP in vivo. The present study was conducted to test the hypothesis that stress-induced increases in pituitary cyclic AMP in vivo were mediated via CRF. We compared the effects of various stressors with the effects of CRF or epinephrine administration on pituitary cyclic AMP and plasma ACTH responses in vivo. Stressors, epinephrine or CRF increased levels of pituitary cyclic AMP. Pituitary cyclic AMP response to either immobilization or CRF was much greater at light onset than at lights off in rats maintained on at 12 hr light: 12 hr dark lighting regimen. In rats with pituitary stalk transections, footshock did not increase levels of pituitary cyclic AMP, suggesting that some factor of central origin was required for this stress response. Exogenous CRF administration did increase levels of pituitary cyclic AMP in stalk-transected rats, while epinephrine increased levels in sham-operated but not in stalk-transected rats. Antisera to CRF markedly decreased pituitary cyclic AMP response to exogenous CRF administered 6 min following antisera and partially attenuated pituitary cyclic AMP response to forced running. Taken as a whole these data support a major role for CRF in the pituitary cyclic AMP response to stress.     

Ultrastructural localization of immunolabeled substance P and methionine-enkephalin-octapeptide in the surface layer of the dorsal horn of rat spinal cord

Summary A preembedding dual immunolabeling technique and electron microscopy were utilized to demonstrate the localization of immunoreactive substance P and methionine-enkephalin-octapeptide (Enk-8) in ultrathin sections of the surface layer (laminae I and II) of rat spinal dorsal horn. The immunoreaction of Enk-8 was visualized as goldtoned silver particles and that of substance P as diaminobenzidine reaction products. Axonal terminals with immunoreactive substance P, and also unlabeled axonal terminals, formed synaptic junctions with the perikarya and dendritic processes of Enk-8-containing neurons. Dendritic profiles immunolabeled for substance P were synaptically linked with unlabeled axons but not with Enk-8-positive ones. Furthermore, it was found that Enk-8 axons and substance P axons terminated synaptically in juxtaposition to one another on the same immunonegative dendrites. Among the Enk-8-containing neurons axonal profiles also appeared to be synaptically associated with immunoreactive Enk-8 dendritic processes.     

Ultrastructural immunocytochemical localization of neurophysin and vasopressin in the median eminence and posterior pituitary of the guinea pig

Summary With the use of tissue prepared by freeze-substitution and the unlabelled antibody enzyme technique, neurophysin and vasopressin were localized at the ultrastructural level in the posterior pituitary and median eminence of the guinea pig. In the posterior pituitary neurophysin was found in the large neurosecretory granules (1300–1500 Å) of axons, Herring bodies, and nerve terminals. In some of these axons immunoreactive neurophysin was found outside of granules in the axoplasm. By light microscopy neurophysin was found in both the zona interna and zona externa of the median eminence; this was confirmed by electron microscopy. In the zona interna as in the posterior pituitary, neurophysin was localized both inside and outside the large neurosecretory granules. In the zona externa, immunoreactive deposit was primarily located in granules with a diameter of 900–1100 Å in nerve terminals abutting on the primary portal plexus. The distribution of vasopressin paralleled that of neurophysin except that the hormone was rarely extragranular. These results demonstrate for the first time that both neurophysin and vasopressin are present in granules of axons that are in contact with the hypophysial portal vasculature.The authors wish to thank Dr. Alan Robinson for the gifts of antiserum to bovine neurophysin I and for purified bovine neurophysin I; Dr. Ludwig Sternberger for the peroxidase-anti-peroxidase complex; and Dr. Robert Utiger for antiserum to lysine vasopressinSupported in part by U.S. Public Health Service grant RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center publication No. 14-017.Recipient of NIH, NINDS Teacher-Investigator Award NS-1108.     

Immunocytochemical localization of growth hormone releasing factor (GHRF)-containing structures in the rat brain using anti-rat GHRF serum

The distribution of growth hormone releasing factor (GHRF) immunoreactive structures in the rat hypothalmus was studied after colchicine treatment with PAP immunocytochemistry in vibratome sections using an antiserum directed to rat hypothalamic GHRF. The majority of the GHRF-immunoreactive cell bodies were found in the arcuate nucleus, the medial perifornical region, and the ventral premammillary nuclei of the hypothalamus. Scattered cells were seen in the lateral basal hypothalamus, the medial and lateral portions of the ventromedial nucleus, and the dorsomedial and paraventricular nuclei. Immunoreactive fibers were observed in all the regions mentioned above. GHRF terminals were located in the central region of the median eminence. In addition, GHRF-immunoreactive neuronal processes were seen in the ventral region of the dorsomedial nucleus, the medial preoptic and suprachiasmatic regions, dorsal portion of the suprachiasmatic nucleus, bed nucleus of the stria terminals and the hypothalamic portion of the stria terminals. The localization of GHRF-immunoreactive terminals in the median eminence reinforces the view that GHRF plays a physiological role in the regulation of pituitary function. In addition, the localization of GHRF-immunoreactive structures in areas not usually considered to project to the median eminence suggest that GHRF may act as a neuromodulator or neurotransmitter.     

Corticotropin releasing factor-like immunoreactivity in sensory ganglia and capsaicin sensitive neurons of the rat central nervous system: colocalization with other neuropeptides

Immunohistochemistry and radioimmunoassay (RIA) revealed that corticotropin releasing factor (CRF)-like immunoreactivity was found to be colocalized with substance P (SP)-, somatostatin (SST)- and leu-enkephalin (LENK)-like immunoreactivity in the dorsal root- and trigeminal ganglia, the dorsal horn of the spinal cord (laminae I and II), the substantia gelatinosa, and at the lateral border of the spinal nucleus and in the tractus spinalis of the trigeminal nerve. These peptides were also located in fast blue labeled cells of the trigeminal ganglion following injection of the dye into the spinal trigeminal area. This indicates that there are possible sensory projections of these peptides into the spinal trigeminal area. Capsaicin treatment of neonatal rats resulted in a marked decrease in the density of CRF-, SP-, VIP- and CCK-containing neurons in the above mentioned hindbrain areas, whereas SST- and LENK-immunoreactivity were not changed. RIA revealed that, compared to controls, CRF, SP and VIP concentrations in these areas were decreased in rats pretreated with capsaicin, while SST levels were increased; CCK and LENK levels were unchanged. It is concluded that the primary afferent neurons of the nucleus and tractus spinalis of the trigeminal nerve are richly endowed with a number of peptides some of which are sensitive to capsaicin action. The close anatomical proximity of these peptide containing neurons suggests the possibility of a coexistance of one or more of these substances.     

Bipolarity of duodenal enterochromaffin cells in the rat

Summary Enterochromaffin cells of the rat duodenum have been studied immunocytochemically by use of a specific antiserum to serotonin. At the light-microscopic level serotonin immunoreactivity was observed in enterochromaffin cells located in the epithelium of the duodenal mucosa. Most of the serotonin-immunoreactive material was localized to the basal portion of the enterochromaffin cells, but small amounts of immunoreactive material were regularly observed in the apical portion. At the electron-microscopic level serotonin immunoreactivity in enterochromaffin cells was found to be concentrated over the dense cores of the cytoplasmic granules. The majority of these granules was located in the basal cytoplasm of the enterochromaffin cells, but serotonin-immunoreactive granules were also observed in the apical cytoplasm immediately beneath the microvilli. These observations indicate that duodenal enterochromaffin cells are bipolar and that they secrete serotonin both basally, to the circulation, and apically, to the gut lumen. Rat duodenal enterochromaffin cells thus appear to have an exocrine as well as an endocrine function.     

Evidence that an extrahypothalamic pituitary corticotropin-releasing hormone (CRH)/adrenocorticotropin (ACTH) system controls adrenal growth and secretion in rats

Within two weeks, hypophysectomy induced in rats a striking decrease in the level of circulating ACTH (the concentration of which was at the limit of sensitivity of our assay system), coupled with a net reduction in the plasma corticosterone concentration and an evident adrenal atrophy. Zona fasciculata, the main producer of glucocorticoids, was decreased in volume, due to a lowering in both the number and average volume of its parenchymal cells. Subcutaneous ACTH infusion (0.1 pmol·min^-1), administered during the last week following hypophysectomy, restored the normal blood level of ACTH and completely reversed all effects of hypophysectomy on the adrenals. Subcutaneous infusion for one week with -helical-CRH or corticotropin-inhibiting peptide (1 nmol·min^-1), which are competitive inhibitors of CRH and ACTH, evoked a further significant lowering of plasma corticosterone concentration and markedly enhanced adrenal atrophy in hypophysectomized rats. These findings strongly suggest that an extrahypothalamic pituitary CRH/ACTH system may be involved in the maintenance of the growth and steroidogenic secretory activity of the rat adrenal cortex.     

Ultrastructural demonstration of nerve endings containing a substance related to growth hormone-releasing factor in the guinea-pig paraventricular nucleus

Summary By means of a preembedding immuno-electronmicroscopic technique, a large number of nerve endings containing a substance related to human growth hormonereleasing factor (hGRF) have been demonstrated in the paraventricular nucleus of the guinea pig. They made synaptic contacts primarily with dendritic shafts: 80% of these contacts were symmetrical. The immunoprecipitate was located mainly in large granules and around small clear vesicles. These findings suggest that a peptide related to hGRF may play a role in neural communication in the paraventricular nucleus.     

Immunoreactive neuronal pathways of growth hormone-releasing hormone (GRH) in the brain and pituitary of the teleost Gadus morhua

Summary Using an antiserum directed against the C-terminus of hGRH(1–44)NH₂ and another recognizing the mid portion to C-terminal of hGRH(1–40)OH, we identify two immunocytochemically distinct GRH-immunoreactive systems in the brain of the codfish, Gadus morhua. The antiserum directed against GRF(1–44)NH₂ stains cell bodies exclusively in the rostral pars distalis. The other antiserum immunoreactive with GRF(1–40)OH reacts with a population of parvocellular and magnocellular neuronal cell bodies in the hypothalamus and with two major axonal pathways which project toward the median eminence and terminate primarily in the pars nervosa. These results indicate the presence of at least two forms of hGRH-like peptides in the teleost which may have different roles in the regulation of pituitary function.     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus

Summary The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethesone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer.These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feedback mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.Supported by NIH grant NS 19266 to WKP     

Iontophoretic mapping of corticotropin-releasing factor (CRF) sensitive neurons in the rat forebrain

Iontophoretic application of corticotropin-releasing factor (CRF) onto the membrane of individual brain neurons produced changes in the spontaneous occurrence of their extracellular action potentials. Neurons in the cortex and hypothalamus tended to be excited by the application of this 41-residue peptide, while those in the thalamus and lateral septal area were inhibited. In general, neurons excited by CRF were also inhibited by the local application of dopamine (DA) and morphine (MOR), while those which were inhibited by CRF were excited by DA and MOR. Glutamate excited the majority of cells tested independent of the other peptide responses. The results suggest that CRF activates several CNS regions with some specificity, and may be involved in neuronal modulation of pituitary as well as extrapituitary events.