A combined electron microscopic HRP and immunocytochemical study of the limbic projections to rat hypothalamic nuclei containing vasopressin and oxytocin neurons.

Light microscopic studies in our laboratory have indicated that the lateral septum, amygdala, and ventral subiculum project in a perinuclear fashion to the paraventricular (PVN), supraoptic (SON), and suprachiasmatic (SCN) nuclei (Oldfield et al., '82; Silverman and Oldfield, '84). In the present paper a combined anterograde HRP and immunocytochemical procedure has been used to determine the connectivity between these limbic efferents and peptide-containing processes emanating from the above mentioned hypothalamic nuclei. Synaptic associations were found to exist between efferents from (1) the septum and both vasopressin (VP)- and oxytocin (OX)-positive dendrites derived from cells in the PVN and SON, (2) the septum and VP dendrites dorsal to the SCN, (3) the ventral subiculum and both VP and OX dendrites arising from the PVN and SON, and (iv) the amygdala and VP dendrites from the PVN. These observations help clarify an apparent discrepancy between electrophysiological data, in which limbic efferents have been shown to influence the activity of VP and OX neurons in the PVN and SON, and anatomical evidence which indicates only a perinuclear innervation from these sites not encroaching on the hypothalamic nuclei themselves. In each case the synaptic connections are made on dendrites external to the nucleus: those lateral and ventrolateral to the PVN, dorsal to the SON, and dorsal or dorsolateral to the SCN.

Ultrastructural studies of vasopressin neurons of the paraventricular nucleus of the hypothalamus using a monoclonal antibody to vasopressin: analysis of synaptic input.

The ultrastructure of the vasopressin neurons of the paraventricular nucleus of the hypothalamus was studied by immunocytochemical techniques. Tissue antigen was detected in unembedded tissue sections using a monoclonal antibody that recognizes vasopressin but not oxytocin or vasotocin. At the light-microscopic level, reaction product was seen to fill the cytoplasm of the neuron cell body as well as large portions of the dendrite and axon. Immunoreactive spines were seen on both somatic and dendritic surfaces and their presence was confirmed at the ultrastructural level. In the light-microscope, axonal processes do not have spines and are thinner and more varicose than dendritic processes. At the electron-microscopic level, both axons and dendrites of the vasopressin cells are filled with reactive neurosecretory granules. The presence of large numbers of these organelles made it difficult to distinguish proximal dendrites from Herring bodies (axonal swellings). At the ultrastructural level, reaction product was also observed in the cytoplasm of all segments of the vasopressin cells. The presence of reaction product outside of membranous compartments is undoubtably due to disruption of membranes by detergent treatment or exposure to basic pH. However, the staining procedure used did allow us to examine the synaptic input to the vasopressin cells. All portions of the vasopressin neuron receive a diverse innervation. The somata have synapses on their surfaces and on spines. These axo-somatic terminals are primarily, but not exclusively, symmetrical and the presynaptic elements contain spherical or elongate vesicles. On the dendrites, terminals again were observed on the surface or on spines. these axo-dendritic synapses were usually asymmetrical. The presynaptic elements contained clear spherical, elongate or pleomorphic vesicles. Occasional varicosities with dense-core granules were seen to make en passant contacts with dendrites; these contacts did not have obvious membrane specializations. Input to vasopressin axons was studied both along the paraventricular-neurohypophysial tract and in the median eminence. Vasopressin axons receive a synaptic input (axo-axonic), predominately of the asymmetric variety with clear, spherical vesicles in the presynaptic element. These findings demonstrate that the vasopressin neurons of the paraventricular nucleus receive a diverse innervation.

Technique for the simultaneous ultrastructural demonstration of anterogradely transported horseradish peroxidase and an immunocytochemically identified neuropeptide.

In order to extend the information obtainable from ultrastructural studies of synaptic connectivity using either horseradish peroxidase tracing or immunocytochemistry alone, we have developed a method of combining these two procedures. Thus it has been possible to examine the characteristics of axon terminals of known origin forming synaptic contacts with cells of identified neuropeptide content.

Ultrastructural identification of noradrenergic nerve terminals and vasopressin-containing neurons of the paraventricular nucleus in the same thin section.

Since many peptidergic cell groups receive a diverse and complex monoaminergic innervation, we have developed a double-label procedure to visualize a peptide and a catecholamine in the same ultrathin section. Radiolabeled norepinephrine (NE) is applied locally and its reuptake into NE terminals is demonstrated by ultrastructural radioautography. Controls in this and other studies demonstrate that the NE labels only NE (and possibly epinephrine) terminals and not dopaminergic or serotonergic terminals. In the same tissue, vasopressin is localized by immunocytochemistry on unembedded sections that are subsequently embedded in epoxy resins for thin sectioning. The procedure as described here shows that NE terminals in the periventricular zone of the paraventricular nucleus of the hypothalamus innervate both vasopressin-positive and vasopressin-negative structures. This technique is useful in determining the chemical connectivity of the hypothalamus.

A monoclonal antibody to vasopressin: preparation, characterization, and application in immunocytochemistry.

The hypothalamo-neurohypophysial system, containing the hormones oxytocin (OT) and vasopressin (VP) and their associated carrier proteins, the neurophysins (NPS), has been the subject of extensive investigation for more than 40 years. This system has been reinvestigated during the last decade by application of immunocytochemical methods employing the rabbit antisera to the hormones and NPS. In this study we describe the preparation and characterization of a monoclonal antibody to VP and its application in immunohistochemistry. The antibody did not cross-react with OT or arginine vasotocin (AVT). Its antigenic determinants as characterized by absorption with various VP analogs included two aromatic amino acids: Phe in position 3, and to a lesser extent Tyr in 2. Tissue fixation with formaldehyde resulted in inadequate immunostaining as compared to glutaraldehyde, most likely due to interference with the aromatic amino acid determinants by the former fixative.

The noradrenergic innervation of vasopressin neurons in the paraventricular nucleus of the hypothalamus: an ultrastructural study using radioautography and immunocytochemistry.

Immunocytochemical and radioautographic procedures were combined at the ultrastructural level to study the noradrenergic synaptic input to vasopressin neurons in selected portions of the paraventricular nucleus of the hypothalamus (PVN) of the rat. Radioactive norepinephrine (NE) was infused into the lateral ventricle or applied topically to the region of the PVN. After appropriate survival times, brain tissues were processed for ultrastructural immunocytochemical demonstration of vasopressin using a monoclonal antibody. [3H]NE varicosities were detected by electron microscopic radioautography. In the periventricular zone of the PVN, radioactive varicosities were numerous accounting for 20-30% of all nerve terminals in this zones. These NE terminals primarily innervated dendritic processes of non-vasopressinergic neurons. Although an occasional axosomatic synapse was observed, input to vasopressin positive neurons was exclusively to their dendrites. In the lateral magnocellular sub-nucleus of the PVN (designed pvl2), noradrenergic terminals were fewer in number accounting for only 1-2% of the total. These terminals were found predominately but not exclusively making axodendritic synapses onto non-vasopressin processes. In both regions, many of the radiolabeled terminals had well-defined membrane appositions with their post-synaptic partners which included a synaptic cleft and post-synaptic density of varying thickness. In both the periventricular zone and the lateral magnocellular regions, noradrenergic varicosities were seen in close proximity to numerous blood vessels.

Supraoptic neurons in the rat hypothalamo-neurohypophysial explant: double-labeling with Lucifer Yellow injection and immunocytochemical identification of vasopressin- and neurophysin-containing neuroendocrine cells.

By combining intracellular electrophysiology with double-labeled intracellular dye-marking and immunocytochemical identification of the same magnocellular neuroendocrine cell, we studied supraoptic neurons in the rat hypothalamo-neurohypophysial explant in vitro. This report examines neurophysiological and light microscopical features of vasopressin- and neurophysin-immunoreactive, pituitary-projecting supraoptic neurons.

Corticotropin-releasing factor synapses within the paraventricular nucleus of the hypothalamus.

Corticotropin-releasing factor (CRF) regulates the release of adrenocorticotropin (ACTH) from the anterior pituitary and these neurosecretory neurons reside in the paraventricular nucleus of the hypothalamus (PVN). In addition to its role as an ACTH secretogogue, exogenously administered CRF can act centrally to modify sympathetic outflow, alter various stress-induced behaviors and modulate its own secretion. Some of these effects might be mediated by CRF acting synaptically within the PVN as the nucleus is known to play a major role in integration of autonomic function. The current ultrastructural immunocytochemical study was designed to examine the range of synaptic relationships that CRF terminals make within the PVN. CRF-positive synapses were numerous, particularly in the periventricular zone. The majority of terminals formed axo-dendritic synapses and of these over 85% were the Gray's type II (symmetrical) class. Axo-somatic terminals were also encountered and both parvicellular and magnocellular neurons were innervated. Once again most of the terminals were Gray's type II. Although an innervation of unidentified structures was the most common, CRF synapses onto CRF neurons and dendrites were observed. All CRF/CRF interactions had symmetrical membrane specializations. These studies indicate that CRF could play a prominent role in the modulation of both parvicellular and magnocellular neurons within the paraventricular nucleus, including modulation of its own neurosecretory activity.

Comparative distribution of vasopressin and oxytocin neurons in the rat brain using a double-label procedure.

The distribution of vasopressin (VP) and oxytocin (OT) neurons in the rat supraoptic (SON), paraventricular (PVN), and accessory magnocellular (AMN) nuclei was studied by localizing both peptides on the same section with a double immunocytochemical staining procedure employing specific monoclonal antibodies (MAB). This procedure allows us to visualize the distribution of one cell type relative to the other. In the rostral SON, VP cells lie dorsal and medial to the OT cells. Near the mid-point of the nucleus along its rostral-caudal length, there is a transition zone in which the two cell types are mixed. Proceeding caudalward, the relative locations of OT and VP cells are exchanged so that most of VP cells are located in the ventral and medial sector of the nucleus, whereas the OT cells are situated dorsal and lateral. However, there is no absolute segregation of the two types of cells anywhere in the nucleus. In the anterior part of the PVN a rostral group (rPVN) of cells composed of a medial portion and a lateral wing can be recognized. Nearly all of the cells in the rPVN are oxytocin-containing. The rPVN is separated from the next group, the middle PVN (mPVN), by a cell poor zone of about 100-150 micron. The mPVN contains both OT and VP neurons. As one proceeds caudally, the OT cells extend in the rostrocaudal direction from an anterior and ventromedial location, forming a shell around a core of VP neurons. In the most caudal PVN (cPVN), a triangular cell group characterized by fusiform cells with long-beaded processes can be distinguished from the more rounded cells of the remaining PVN. Many fusiform cells in the cPVN appear to send their axons to the posterior perifornical nucleus and the nucleus of the medial forebrain bundle. Other fusiform cells of the cPVN are oriented in a rostral-caudal plane and are situated more medially in this subdivision. The dendrites of these cells project into the mPVN while their posterior processes, most of which also appear to be dendrites, project caudally along a medial route.

Immunocytochemical studies of vasotocin and mesotocin in the hypothalamo-hypophysial system of the chicken.

The hypothalamo-hypophysial system of the adult chicken has been studied with a monoclonal antibody that cross-reacts with arginine vasotocin and mesotocin. We have used this antibody on thick (100 micrometers) sections in conjunction with a peroxidase-conjugated rabbit antimouse antibody that permits the visualization not only of entire perikarya, but also of long portions of their axons and dendrites. Our results confirm older concepts based on classical methods, but the more sensitive immunocytochemical method reveals that the system is more extensive than previously recognized. Immunostained neurons in the chicken are widely scattered in the hypothalamus. In the rostral preoptic region, there are three immunostained neuronal cell groups: a prominent closely packed group that extends along the ventromedial surface, a diffusely distributed lateral group, and an external group that surrounds the lateral aspect of the septomesencephalic tract. Caudally in the preoptic area and in the anterior hypothalamus, the same groups are present; but there are also conspicuous periventricular perikarya. Many of them have processes that project to the lumen of the third ventricle, as well as parallel axons that arch lateroventrally in the hypothalamus. In the midhypothalamic area, the periventricular perikarya and processes are particularly numerous at the level of the pallial commissure. The dorsal periventricular group located at the level of the dorsomedial anterior nucleus of the thalamus are the most caudal perikarya. They extend laterally in a wing-like formation. The immunostained axons from all of these perikarya form a compact hypothalamo-hypophysial tract as they run from the mid-hypothalamus to the median eminence and converge beneath the third ventricle. Axons branching from this tract innervate the zone externa of the anterior median eminence; another group of axons running in the fibrous layer of the zona interna proceeds to the neural lobe.

Immunocytochemical study of the development of vasotocin/mesotocin in the hypothalamo-hypophysial system of the chick embryo.

The hypothalamo-hypophysial system of the chick embryo has been studied with a monoclonal antibody which cross-reacts with arginine vasotocin and mesotocin, using thick (100 micron) sections in conjunction with a peroxidase-conjugated rabbit anti-mouse antibody. Although weakly stained perikarya occur occasionally in the tuberal region on embryonic days 6 and 7, the most consistent immunostaining of perikarya is found in the periventricular region of the caudal midhypothalamus at the level of the optic chiasm after embryonic day 8 1/2. Synthesis of peptides, therefore, takes place while the cells are close to their site of origin. Between embryonic days 9 and 10, beaded axons run along the anterior median eminence closely apposed to the adenohypophysis, thereby forming the anlage of the zona externa. The axons of the hypothalamo-neurohypophysial tract surround the neural lobe between embryonic days 11 and 12. The caudal to rostral wave of neuronal maturation that occurs during development appears to be due to a progressive differentiation of the periventricular zone, as well as the migration of perikarya. The early periventricular perikarya at embryonic day 8 1/2 send processes rostrally in a wing-shaped formation that extends both dorso- and ventrolaterally. From embryonic days 10 to 12, perikarya can be observed in the wing-like extensions, apparently migrating to rostral levels. The dorsolateral pathway gives rise at its midportion to the lateral cell group, whereas those perikarya migrating more laterally form the anlage of the external supraoptic nucleus. The ventrolateral wing-shaped extension of perikarya appears to be directed toward the ventral group and those lateral perikarya continuous with it. The location of mature neuronal cell groups is well established by embryonic day 17.

Vasopressinergic and oxytocinergic pathways in the central nervous system.

Recent data obtained by immunohistochemical and other anatomical tracing methods indicate that oxytocin and vasopressin pathways are much more complex and extensive than previously recognized. In addition to the classic magnocellular neurons that project from the supraoptic and paraventricular (PVN) nuclei to the posterior pituitary gland, generally smaller neurons in various parts of the PVN send vasopressin fibers to the portal capillary bed in the median eminence, or send oxytocin or vasopressin projections to other brain and spinal cord sites. In addition, vasopressin neurons are also found in the suprachiasmatic nucleus and may contribute to extrahypothalamic projection areas. Many of these axonal projections appear to form synapses with other neurons in forebrain, hindbrain, and spinal cord regions, which suggests roles for these peptides in neuronal communication. In brain stem and spinal cord, terminal fields include both parasympathetic and sympathetic regulatory centers. Oxytocin terminals are also found on large intracerebral arteries where the peptide may regulate cerebral blood flow.