Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat

Immuno-enzyme histochemical investigations showed that, in the magnocellular hypothalamo-hypophysial neurosecretory system of the rat, vasopressin and oxytocin are synthetized in separate neurons. Both the vasopressin neurons and the oxytocin neurons are present in both the supraoptic and the paraventricular nuclei in about the same number. Preferential location of the two kinds of rat neurosecretory neurons is not as obvious as in the bovine hypothalamus. Their perikarya do not show distinct morphological differences. The two kinds of neurosecretory perikarya are the origin of separate vasopressin-containing and oxytocin-containing axons respectively. In the neural lobe, the distribution of the two different types of axons is described.     

Oxytocinergic circuit from paraventricular and supraoptic nuclei to arcuate POMC neurons in hypothalamus

Intracerebroventricular injection of oxytocin (Oxt), a neuropeptide produced in hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), melanocortin-dependently suppresses feeding. However, the underlying neuronal pathway is unclear. This study aimed to determine whether Oxt regulates propiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) of the hypothalamus. Intra-ARC injection of Oxt decreased food intake. Oxt increased cytosolic Ca²⁺ in POMC neurons isolated from ARC. ARC POMC neurons expressed Oxt receptors and were contacted by Oxt terminals. Retrograde tracer study revealed the projection of PVN and SON Oxt neurons to ARC. These results demonstrate the novel oxytocinergic signaling from PVN/SON to ARC POMC, possibly regulating feeding.     

Expression of P2X6 receptors in the enteric nervous system of the rat gastrointestinal tract

Expression of P2X₄ and P2X₆ receptor subunits in the gastrointestinal tract of the rat was studied with double-labeling fluorescence immunohistochemistry. The results showed that P2X₆ receptors were expressed widely in the submucosal and myenteric plexuses. In the myenteric plexus, P2X₆ receptors were expressed mainly in large size neurons which resembled Dogiel type II neurons. These P2X₆ receptor-immunoreactive (ir) neurons also expressed calbindin 28K, calretinin and neuronal nuclei (NeuN), proteins that are markers of intrinsic sensory neurons. In the submucosal plexus, all the calbindin 28K, calretinin and NeuN-ir cells were immunoreactive for P2X₆ receptors. P2X₆ receptors do not form homomultimers, but rather heteromultimers with either P2X₂ or P2X₄ receptors. P2X₄ receptors were not expressed in neurons, but were expressed in macrophages of the rat gastrointestinal tract. These data indicate that P2X₆ receptors are mainly expressed on intrinsic sensory neurons and that ATP, via P2X₆ receptors probably in heteromeric combination with P2X₂ receptors, may be involved in regulating the physiological functions of these neurons.     

Expression of P2X5 receptors in the rat, cat, mouse and guinea pig dorsal root ganglion

P2X receptors are ATP-gated cationic channels composed of seven cloned subunits (P2X_{1 –7}). P2X₃ homomultimer and P2X_2/3 heteromultimer receptors expressed by primary afferent dorsal root ganglion (DRG) neurons are involved in pain processing. The aim of the study was to investigate the expression of the P2X₅ receptor subunit in DRG in different species including mouse, rat, cat and guinea pig. Immunohistochemistry showed that P2X₅ receptors exhibited low levels of immunostaining in rat DRG, but high levels in mouse and guinea pig. Only a few neurons were immunoreactive for P2X₅ receptors in cat. In mouse DRG, the P2X₅ receptor was expressed largely by medium-diameter neurons (42.9 %), less in small (29.3 %) and large (27.8 %) neurons. In contrast, in the guinea pig DRG, P2X₅ receptor expression was greatest in small-diameter (42.6 %), less in medium- (36.3 %) and large-diameter (21.1 %) neurons. Colocalization experiments revealed that, in mouse DRG, 65.5, 10.9 and 27.1 % of P2X₅ receptors were immunoreactive for NF-200, CGRP and calbindin, while only a few P2X₅-immunoreactive (IR) neurons were coexpressed with IB4 or with NOS. In guinea pig DRG, a total of 60.5 and 40.5 % of P2X₅-IR neurons were coexpressed with IB4 or with CGRP, while 20.3 and 24.5 % of P2X₅ receptors were coexpressed with NF-200 or with NOS. Only a few P2X₅-IR neurons were coexpressed with calbindin in guinea pig DRG. It will be of great interest to clarify the relative physiological and pathophysiological roles of P2X₅ receptors.     

The purinergic calcium channels P2X1,2,5,7 are down-regulated while P2X3,4,6 are up-regulated during apoptosis in the ageing rat prostate

Subtype-specific antibodies were used to measure purinergic (P2X) receptor expression in the rat prostate. In mature Wistar rats, apoptosis and expression of P2X₁, P2X₂, P2X₅ and P2X₇ subtypes were all significantly decreased compared with the levels found in immature rat prostates. Accompanying this age-related reduction in purinergic calcium channel expression was a reduction in epithelial and stromal calcium as well as the calcium-regulating hormone stanniocalcin. In contrast, expression of P2X₃, P2X₄ and P2X₆ increased with age. These results suggest that distinct changes in P2X subtype expression accompany apoptosis in the rat prostate.     

Regional expression of P2Y4 receptors in the rat central nervous system

P2Y receptors are G protein-coupled receptors composed of eight known subunits (P2Y_{1, 2, 4, 6, 11, 12, 13, 14}), which are involved in different functions in neural tissue. The present study investigates the expression pattern of P2Y₄ receptors in the rat central nervous system (CNS) using immunohistochemistry and in situ hybridization. The specificity of the immunostaining has been verified by preabsorption, Western blot, and combined use of immunohistochemistry and in situ hybridization. Neurons expressing P2Y₄ receptors were distributed widely in the rat CNS. Heavy P2Y₄ receptor immunostaining was observed in the magnocellular neuroendocrine neurons of the hypothalamus, red nucleus, pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, and dorsal motor vagus nucleus. Both neurons and astrocytes express P2Y₄ receptors. P2Y₄ receptor immunostaining signals were mainly confined to cell bodies and dendrites of neurons, suggesting that P2Y₄ receptors are mainly involved in regulating postsynaptic events. In the hypothalamus, all the vasopressin (VP) and oxytocin (OT) neurons and all the orexin A neurons were immunoreactive for P2Y₄ receptors. All the neurons expressing P2Y₄ receptors were found to express N-methyl-d-aspartate receptor 1 (NR1). These data suggest that purines and pyrimidines might be involved in regulation of the release of the neuropeptides VP, OT, and orexin in the rat hypothalamus via P2Y₄ receptors. Further, the physiological and pathophysiological functions of the neurons may operate through coupling between P2Y₄ receptors and NR1.     

Multiple signalling modalities mediated by dendritic exocytosis of oxytocin and vasopressin

The mammalian hypothalamic magnocellular neurons of the supraoptic and paraventricular nuclei are among the best understood of all peptidergic neurons. Through their anatomical features, vasopressin- and oxytocin-containing neurons have revealed many important aspects of dendritic functions. Here, we review our understanding of the mechanisms of somato-dendritic peptide release, and the effects of autocrine, paracrine and hormone-like signalling on neuronal networks and behaviour.     

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X₁–P2X₇) and seven heteromeric receptors (P2X_1/2, P2X_1/4, P2X_1/5, P2X_2/3, P2X_2/6, P2X_4/6, P2X_4/7) have been described. ATP treatment of Leydig cells leads to an increase in [Ca²⁺]_i and testosterone secretion, supporting the hypothesis that Ca²⁺ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X₂. In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X₂, P2X₄, P2X₆, and P2X₇ subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 μM ivermectin induced an increase (131.2 ± 5.9%) and 3 μM ivermectin a decrease (64.2 ± 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X₄ subunits. P2X₇ receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X_2/4/6, is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X₂ subunit.     

Ultrastructural localisation of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamo-neurohypophysial system

The distribution of the P2X₂ subtype of the purine receptor associated with the extracellular signalling activities of ATP was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were labelled with ExtrAvidin-horseradish peroxidase preembedding immunocytochemistry using a polyclonal antibody against a fragment of an intracellular domain of the receptor. Immunoreactivity to P2X₂ receptors was localised in: (i) paraventricular and supraoptic nuclei—in subpopulations of endocrine neurones, neurosecretory and non-neurosecretory axons and dendrites; and (ii) the neurohypophysis—in pituicytes and subpopulation of neurosecretory axons. In both the hypothalamic nuclei examined, labelled asymmetric axo-dendritic synapses were commonly observed. These synapses involved either P2X₂-labelled axon terminals (synaptic buttons) and unlabelled dendrites or labelled dendrites and unlabelled axon terminals. Axo-somatic synapses established by P2X₂-positive axons on P2X₂-positive endocrine cell bodies as well as on P2X₂-negative somata were also observed. The functional significance of these findings is discussed.     

Increased 5-HT3-mediated signalling in pelvic afferent neurons from mice deficient in P2X2 and/or P2X3 receptor subunits

Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT₃ receptors, respectively. We have investigated the changes in P2X and 5-HT₃-mediated signalling in pelvic afferent neurons in mice deficient in P2X₂ and/or P2X₃ subunits by whole-cell recording of L₆–S₂ dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X₃ ^−/− neurons, whereas sustained currents were absent in P2X₂ ^−/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X₂/P2X₃ ^Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT₃ receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X ₂ ^−/−, P2X₃ ^−/− and P2X₂/P2X₃ ^Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X₂/P2X₃ ^Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X₂/P2X₃ ^Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X₂/P2X₃ ^Dbl−/− preparations. These data suggest that P2X receptors containing the P2X₂ and/or P2X₃ subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X₂ or P2X₃ receptor genes. This effect is more pronounced when both subunits are absent.     

Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary neurons: role of P2 and NGFRp75 receptors

In this study, we monitored the direct expression of P2 receptors for extracellular ATP in cerebellar granule neurons undergoing metabolism impairment. Glucose deprivation for 30–60 min inhibited P2Y₁ receptor protein, only weakly modulated P2X₁, P2X₂ and P2X₃, and up‐regulated by about two‐fold P2X₄, P2X₇ and P2Y₄. The P2X/Y antagonist basilen blue, protecting cerebellar neurons from hypoglycemic cell death, maintained within basal levels only the expression of P2X₇ and P2Y₄ proteins, but not P2X₄ or P2Y₁. Glucose starvation transiently increased (up to three‐fold) the expression of NGFRp75 receptor protein and strongly stimulated the extracellular release of nerve growth factor (NGF; about 10‐fold). Exogenously added NGF then augmented hypoglycemic neuronal death by about 60%, increasing the percentage of Höechst‐positive nuclei (from approximately 62 to 95%), reducing lactate dehydrogenase (LDH) release (from about 50 to 14%) and significantly overstimulating the hypoglycemia‐induced expression of P2X₇ and P2Y₄. Conversely, extracellular ATP augmented hypoglycemic neuronal death by about 80%, reducing the number of Höechst‐positive nuclei (from approximately 62% to 14%), augmenting LDH outflow (by about 30%) and further increasing the hypoglycemia‐induced expression of NGFRp75. Our results indicate that P2 and NGFRp75 receptors are modulated during glucose starvation and that extracellular ATP and NGF drive features of, respectively, necrotic and apoptotic hypoglycemic cell death, aggravating the consequences of metabolism impairment in cerebellar primary neurons.     

Localisation of P2Y<Subscript>1</Subscript> and P2Y<Subscript>4</Subscript> receptors in dorsal root,nodose and trigeminal ganglia of the rat

The presence and distribution of P2Y (nucleotide) receptor subtypes in rat sensory neurons has been investigated. RT-PCR showed that P2Y₁, P2Y₂, P2Y₄ and P2Y₆ receptor mRNA is expressed in sensory ganglia [dorsal root ganglion (DRG), nodose ganglion (NG) and trigeminal ganglion (TG)]. The regional and cellular distribution of P2Y₁ and P2Y₄ receptor proteins in these ganglia was investigated using immunohistochemistry. P2Y₁ polyclonal antibodies stained over 80% of the sensory neurons, particularly the small-diameter (neurofilament-negative) neurons. The P2Y₄ receptor antibody stained more medium- and large- (neurofilament-positive) diameter neurons than small-diameter neurons. P2Y₁ and P2Y₄ receptor immunoreactivity (P2Y₁-IR and P2Y₄-IR) was often coexpressed with P2X₃ receptor immunoreactivity (P2X₃-IR) in subpopulations of neurons. Double immunohistochemistry showed that 73–84% of P2X₃ receptor-positive neurons also stained for the P2Y₁ receptor in DRG, TG and NG while only 25–35% also stained for the P2Y₄ receptor. Subpopulations of P2Y₁-IR neurons were coexpressed with NF200, CGRP and IB₄; most P2Y₄-IR neurons were coexpressed with NF200, while only a few neurons were coexpressed with CGRP (10–20%) or with IB₄ (1–2%). The results suggest that P2Y as well as P2X receptor subtypes contribute to purinergic signalling in sensory ganglia.     

Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

We investigated the expression of P2X₅, P2X₇, P2Y₁ and P2Y₂ receptor subtypes in adult human anagen hair follicles and in relation to markers of proliferation [proliferating cell nuclear antigen (PCNA) and Ki-67], keratinocyte differentiation (involucrin) and apoptosis (anticaspase-3). Using immunohistochemistry, we showed that P2X₅, P2Y₁ and P2Y₂ receptors were expressed in spatially distinct zones of the anagen hair follicle: P2Y₁ receptors in the outer root sheath and bulb, P2X₅ receptors in the inner and outer root sheaths and medulla and P2Y₂ receptors in living cells at the edge of the cortex/medulla. P2X₇ receptors were not expressed. Colocalisation experiments suggested different functional roles for these receptors: P2Y₁ receptors were associated with bulb and outer root sheath keratinocyte proliferation, P2X₅ receptors were associated with differentiation of cells of the medulla and inner root sheaths and P2Y₂ receptors were associated with early differentiated cells in the cortex/medulla that contribute to the formation of the hair shaft. The therapeutic potential of purinergic agonists and antagonists for controlling hair growth is discussed.     

Changes in the distribution of different subtypes of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the pregnant rat urinary bladder

Clusters of purinergic receptor subunits, about 1 μm diameter, are found on the smooth muscle cell membrane beneath junctional varicosities in the detrusor muscle of the rat urinary bladder. We have examined the extent of redistribution of the six different subunit clusters, P2X₁ to P2X₆, with respect to junctional varicosities during pregnancy, as it is known that the detrusor muscle undergoes changes in purinergic innervation during this period. Before pregnancy, clusters at junctional varicosities are principally composed of the subtypes P2X₁, P2X₂, P2X₃ and P2X₅. However this subtype distribution changes dramatically during pregnancy, such that by day 14 of pregnancy, the extent of P2X₁, P2X₂, P2X₃ and P2X₅ junctional clusters has decreased by more than 80% whereas the extent of P2X₄ and P2X₆ junctional clusters has increased by more than 80%. These changes were confirmed with Western blots for different subtypes. It is suggested that the changes in the purinergic innervation of the detrusor muscle during pregnancy reflect changes in the P2X subtypes found on the smooth muscle membrane beneath junctional varicosities.     

Involvement of P2X and P2Y receptors in microglial activation in vivo

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X_1,2,4,7 and P2Y_1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X₇ receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X₇ receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists α,βmeATP, ADPβS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPγS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X₇ receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X₇-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects.     

P2X (purinergic) receptor redistribution in rabbit aorta following injury to endothelial cells and cholesterol feeding

The redistribution of purinergic P2X receptor subunits (P2X₁ to P2X₇) within the rabbit aorta wall three weeks after endothelial balloon injury/cholesterol feeding was examined. P2X₁ receptor cluster density was elevated in the media following balloon injury/cholesterol feeding by about 30% and these clusters appeared on smooth muscle cells throughout the greatly expanded neointima but they did not change significantly on the endothelial cells following balloon injury. P2X₄ clusters were found in high density throughout the media and in very high density in the enlarged neointima following balloon injury, particularly on the endothelial cells where the density increased about 10-fold after balloon injury. P2X₅ clusters were found in high density in the media of normal aorta but with little change following balloon injury. P2X₃, P2X₆ and P2X₇ cluster density was low in normal aorta and remained unchanged following balloon injury. All receptor subunits were found on endothelial cells. It is suggested that the release of ATP from damaged endothelial cells and from smooth muscle cells sufficient to activate P2X₄ receptors may contribute to neointimal proliferation.     

Purinergic and glutamatergic interactions in the hypothalamic paraventricular nucleus modulate sympathetic outflow

P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5′-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ± 13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow.     

Purine receptors are required for DHA-mediated neuroprotection against oxygen and glucose deprivation in hippocampal slices

Docosahexaenoic acid (DHA) is important for central nervous system function during pathological states such as ischemia. DHA reduces neuronal injury in experimental brain ischemia; however, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on acute hippocampal slices subjected to experimental ischemia by transient oxygen and glucose deprivation (OGD) and re-oxygenation and the possible involvement of purinergic receptors as the mechanism underlying DHA-mediated neuroprotection. We observed that cellular viability reduction induced by experimental ischemia as well as cell damage and thiobarbituric acid reactive substances (TBARS) production induced by glutamate (10 mM) were prevented by hippocampal slices pretreated with DHA (5 μM). However, glutamate uptake reduction induced by OGD and re-oxygenation was not prevented by DHA. The beneficial effect of DHA against cellular viability reduction induced by OGD and re-oxygenation was blocked with PPADS (3 μM), a nonselective P2X_1–5 receptor antagonist as well as with a combination of TNP-APT (100 nM) plus brilliant blue (100 nM), which blocked P2X₁, P2X₃, P2X_2/3, and P2X₇ receptors, respectively. Moreover, adenosine receptors blockade with A₁ receptor antagonist DPCPX (100 nM) or with A_2B receptor antagonist alloxazine (100 nM) inhibited DHA-mediated neuroprotection. The addition of an A_2A receptor antagonist ZM241385 (50 nM), or A₃ receptor antagonist VUF5574 (1 μM) was ineffective. Taken together, our results indicated that neuroprotective actions of DHA may depend on P2X, A₁, and A_2B purinergic receptors activation. Our results reinforce the notion that dietary DHA may act as a local purinergic modulator in order to prevent neurodegenerative diseases.     

Immunocytochemical study of the hypothalamic magnocellular neurosecretory nuclei of the snake Natrix maura and the turtle Mauremys caspica

Summary An immunocytochemical study of the magnocellular neurosecretory nuclei was performed in the snake Natrix maura and the turtle Mauremys caspica by use of antisera against: (1) a mixture of both bovine neurophysins, (2) bovine oxytocin-neurophysin, (3) arginine vasotocin, and (4) mesotocin. Arginine vasotocin- and mesotocin-immunoreactivities were localized in individual neurons of the supraoptic and paraventricular nuclei, with a distinct pattern of distribution in both species. The same cells appeared to be stained by the anti-oxytocin-neurophysin and anti-mesotocin sera. The supraoptic nucleus can be subdivided into rostral medial and caudal portions. In N. maura, but not in M. caspica, neurophysin-immunoreactive neurons were found in the retrochiasmatic nucleus. No immunoreactive elements were seen in the suprachiasmatic nucleus of both species after the use of any of the antisera. A dorsolateral aggregation of neurophysin-containing cells, localized over the lateral forebrain bundle, was present in both species. Magnocellular and parvocellular neurophysin-immunoreactive neurons were present in the paraventricular nucleus of both species. In the turtle, the paraventricular neurons were arranged into four distinct layers parallel to the ependyma; these neurons were bipolar with the major axis perpendicular to the ventricle, and many of them projected processes toward the cerebrospinal-fluid compartment. In N. maura a group of large neurons of the paraventricular nucleus was found in a very lateral position. The posterior lobe of the hypophysis and the external zone of the median eminence contained arginine vasotocin- and mesotocin-immunoreactive nerve fibers. The lamina terminalis of both species was supplied with a dense bundle of fibers containing immunoreactive neurophysin. Neurophysin-immunore-active fibers were also present in the septum, some telencephalic regions, including the cortex and the olfactory tubercule, in the paraventricular organ, and the periventricular and periaqueductal gray of the brainstem.This work was partially supported by a Grant S-85-39 from the Direccion de Investigaciones, Universidad Austral de Chile to E.M. Rodriguez