Response of Substances Co-Expressed in Hypothalamic Magnocellular Neurons to Osmotic Challenges in Normal and Brattleboro Rats

Abstract

The intention of this review is to emphasize the current knowledge about the extent and importance of the substances co-localized with magnocellular arginine vasopressin (AVP) and oxytocin (OXY) as potential candidates for the gradual clarification of their actual role in the regulation of hydromineral homeostasis. Maintenance of the body hydromineral balance depends on the coordinated action of principal biologically active compounds, AVP and OXY, synthesized in the hypothalamic supraoptic and paraventricular nuclei. However, on the regulation of water–salt balance, other substances, co-localized with the principal neuropetides, participate. These can be classified as (1) peptides co-localized with AVP or OXY with unambiguous osmotic function, including angiotensin II, apelin, corticotropin releasing hormone, and galanin and (2) peptides co-localized with AVP or OXY with an unknown role in osmotic regulation, including cholecystokinin, chromogranin/secretogranin, dynorphin, endothelin-1, enkephalin, ferritin protein, interleukin 6, kininogen, neurokinin B, neuropeptide Y, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, TAFA5 protein, thyrotropin releasing hormone, tyrosine hydroxylase, and urocortin. In this brief review, also the responses of these substances to different hyperosmotic and hypoosmotic challenges are pointed out. Based on the literature data published recently, the functional implication of the majority of co-localized substances is still better understood in non-osmotic than osmotic functional circuits. Brattleboro strain of rats that does not express functional vasopressin was also included in this review. These animals suffer from chronic hypernatremia and hyperosmolality, accompanied by sustained increase in OXY mRNA in PVN and SON and OXY levels in plasma. They represent an important model of animals with constantly sustained osmolality, which in the future, will be utilizable for revealing the physiological importance of biologically active substances co-expressed with AVP and OXY, involved in the regulation of plasma osmolality.

General Assessment

Central Nervous System and Water–Salt Balance

Approximately 60% of body weight is formed by water stored in intracellular and extracellular body compartments. Constancy of water volume is essential for biochemical processes to maintain body fluid homeostasis. Loss of water activates appropriate sensory signals and pathways (humoral and neuronal), which relay information about the body hydromineral status to brain (Johnson and Thunhorst 1997).

Experimental studies have shown that several brain structures are involved in the regulation of water–salt balance, including the area postrema, nucleus of the solitary tract, caudal and rostral ventrolateral medulla, parabrachial nuclei, anteroventral third ventricle (median preoptic nucleus, organum vasculosum of the lamina terminalis OVLT, and subfornical organ SFO), amygdalae nuclei, bed nucleus of the stria terminalis, lateral, dorsomedial, and ventromedial hypothalamus, zona incerta, locus coeruleus, dorsal vagal nucleus, and intermediolateral spinal cord (Johnson and Thunhorst 1997). Signals from these structures are conveyed to the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei that are essential in the regulation of water–salt balance.

Brief Anatomy of SON and PVN

The hypothalamic SON is a paired structure constituted from two main populations of magnocellular cells, vasopressinergic and oxytocinergic, situated along the lateral sides of the optic chiasm (Armstrong 1995). These magnocellular neurons project via the internal zone of the median eminence to posterior pituitary (Smith and Armstrong 1990). Vasopressinergic neurons are present in the parvocellular PVN as well as in the suprachiasmatic nucleus, and OXY is also expressed in the dorsal cap of the PVN projecting to the brain stem; however, these areas are not involved in osmotic regulation.

The hypothalamic PVN is also a paired structure, occupying mainly the middle part of the hypothalamus (Armstrong 2004). Cytoarchitectonically, PVN represents a more complex structure than SON. It consists of magnocellular, parvocellular, and mediocellular neurons, projecting to different brain areas. Magnocellular neurons, via the internal zone of the median eminence, send projections to neurohypophysis (Wiegand and Price 1980). Axons from parvocellular neurons are directed to SFO, OVLT, and several other brain areas and via the external zone of the median eminence to anterior pituitary. Mediocellular neurons project caudally to the brain stem and spinal cord areas (Hoysoya and Matsushita 1979).

SON and PVN Principal Osmoregulatory Neuropeptides

Principal osmoregulatory neuropeptides produced by magnocellular cells of SON and PVN are AVP and OXY. Although AVP is involved in a wide spectrum of physiological functions, e.g., cardiovascular control, stimulation of adrenocorticotropic hormone (ACTH) release, influence of cognition, learning, and memory, it is mainly known as an antidiuretic hormone for its crucial role in osmoregulation. Increased plasma osmolality, decreased arterial pressure, and reduced cardiac filling are the most effective stimuli for AVP secretion via the neurohypophysis (Treschan and Peters 2006).

Different paradigms or osmotic stimulations show that AVP and AVP mRNA levels are in the SON and PVN under an imminent control of water balance. After exposing the rats to water deprivation or salt loading, plasma levels of AVP and AVP mRNA are approximately doubled in the PVN and SON magnocellular neurons (Gottlieb et al. 2006); however, the intensity of AVP immunoreactivity in PVN and SON cell bodies is decreased. In addition, cell and nuclear sizes of AVP neurons increase up to 170% compared to basal values. On the other hand, hypoosmolality produces a dramatic inhibition of AVP and OXY gene expression in the SON (Yue et al. 2006). Hypoosmolality/hyponatremia decreases AVP mRNA to approximately 10–20% and cell and nuclear sizes to 60% (Zhang et al. 2001). Under certain osmotic conditions, such as lactation and prolonged water deprivation (3 days), some population of magnocellular neurons are capable of AVP/OXY co-expression. However, this phenomenon recovers after terminating the physiological or experimental challenge (Telleria-Diaz et al. 2001). AVP synthesizing magnocellular neurons can also serve as osmoreceptors, responding to increased osmotic pressure in the extracellular environment by raising firing rate and AVP secretion into the general circulation (Leng et al. 1999).

The prominent role of AVP and other individual peptides produced in magnocellular neurons of SON and PVN has been studied in AVP deficient Brattleboro strain of rats. In contrast to normal rats, the homozygous diabetes insipidus (di/di) Brattleboro rats have deficiency in the ability to synthesize functional AVP (Valtin et al. 1962), inherited as an autosomal recessive trait occurring at a single gene locus (Saul et al. 1968; Vandesande and Dierickx 1976). The kidneys of these animals do not reabsorb water to general circulation, and therefore, these animals drink and urinate excessively, i.e., they suffer from an incessant polyuria and polydipsia. They can excrete hypotonic urine almost 70% of their body weight/day (Brimble et al. 1991). The distribution of AVP neurons in the hypothalamus of di/di rats is identical to wild-type rats; however, the expression of the mutant AVP precursor considerably alters the morphology of AVP cells. The perikaryal cross-cuts of AVP neurons indicate that these neurons are up to 90% greater than those of heterozygous strain. Increased size of nucleoli is a characteristic feature of their hypertrophy and it can be normalized by AVP substitution. Persisting osmotic stimulation of AVP and OXY neurons maintains their constant hypertrophy and hyperactivity (Ma and Morris 2002). Although faint Fos immunoreactivity, as an indicator of neuronal activity, has been found in the SON of di/di rats even under basal conditions, no differences in the distribution of Fos immunoreactivity after hypertonic saline injection were observed between the di/di and Long Evans control rats in SON and PVN. Fos expression after hypertonic saline injection was enhanced in many brain areas in both strains of rats (Guldenaar et al. 1992).

OXY producing magnocellular neurons are situated antero-dorsally in the SON (Hou Yu et al. 1986) and in several portions in the PVN (Swanson and Kuypers 1980). OXY is a hormone with a wide spectrum of central and peripheral effects (Kiss and Mikkelsen 2005). It is involved in many physiological processes connected with male and female reproductive system, cardiovascular regulation, modulation of neuroendocrine regulations, and establishment of complex social and bonding behaviors related to the reproduction and care of the offspring. Besides, OXY acts as a non-hypertensive natriuretic agent. In kidneys, likewise AVP, it takes control over the hydromineral excretion. Intraperitoneal injection of OXY causes dose-dependent increase in the urinary osmolality, natriuresis, and kaliuresis and evokes concomitant release of atrial natriuretic peptide (Haanwinckel et al. 1995). OXY release into the blood is initiated by stimuli such as suckling, parturition, hemorrhage, fever, pain, mating, hypovolemia, and hyperosmolality (Gimpl and Fahrenholz 2001). OXY effect is mediated by a single OXY receptor, which is differently expressed in various tissues, but OXY may also act on pituitary vasopressin V_1b receptors to stimulate ACTH secretion (Schlosser et al. 1994).

Osmotic stress, induced by the injection of hypertonic saline, causes a distinct increase in neuronal activity visualized as Fos immunoreactivity in the OXY producing neurons in the PVN and SON (Giovannelli et al. 1990). Prolonged salt loading and water deprivation, in a time-dependent manner, significantly increase OXY mRNA levels (da Silveira et al. 2007), but decrease OXY peptide levels in the PVN, SON (Meister et al. 1990b), and posterior pituitary and elevate its level in the plasma (Balment et al. 1980). This strongly indicates that increase in the turn-over of OXY in the hypothalamus and OXY levels in plasma is in positive correlation with increased osmolality status (Verbalis and Dohanics 1991). On the other hand, during chronic hypoosmolar conditions, likewise AVP in AVP neurons, OXY mRNA decreases to 10–20% compared to normal animals. Inadequate OXY secretion can occur after disruption of mechanosensitive gating of OXY cells (Bacova et al. 2006).

In di/di Brattleboro rats, suffering from chronic hypernatremia and hyperosmolality, increased OXY mRNA in the PVN and SON (Sherman et al. 1988) and OXY levels in plasma have been observed (Brimble et al. 1991). It has been shown that in the absence of functional AVP, OXY may serve as a weak AVP agonist and exhibit antidiuretic activity (Edwards and LaRochelle 1984).

Peptides Co-Localized with AVP or OXY with Unambiguous Osmotic Function (Table 1)

Table 1.

Different biologically active substances co-localized (+++++) or not co-localized (−−−−−) with oxytocin or vasopressin in the hypothalamic PVN and SON magnocellular perikarya

Neuropeptides colocalized with AVP or OXY in the SON and PVN	AVP	OXY
Agmatine	+++++	+++++
Angiotensin II	+++++	−−−−−
Apelin	+++++	+++++
Calbindin	+++++	+++++
Calretinin	−−−−−	+++++
CART	+++++	+++++
Corticotropin releasing hormone	+++++	+++++
Galanin	+++++	+++++
Cholecystokinin	−−−−−	+++++
Chromogranin/secretogranin II	+++++	−−−−−
Dynorphin	+++++	+++++
Endothelin-1	+++++	+++++
Leu-enkephalin	+++++	−−−−−
Met-enkephalin	+++++	+++++
Ferritin	+++++	−−−−−
Interleukin-6	+++++	−−−−−
Kininogen	−−−−−	+++++
T-kininogen	+++++	−−−−−
Leptin	+++++	+++++
NADPH-diaphorase	+++++	+++++
Nesfatine	+++++	+++++
Neuroendocrine polypeptide 7B2	+++++	+++++
Neurokinin B	+++++	−−−−−
Neuromedin U	+++++	+++++
Neuropeptide AF/FF	+++++	+++++
Neuropeptide B	+++++	−−−−−
Neuropeptide Y	+++++	+++++
NO synthase	+++++	+++++
Peptide histidine-isoleucine	+++++	−−−−−
Pituitary adenylate cyclase-activatig polypeptide	+++++	+++++
18 regulated endocrine specific protein	−−−−−	+++++
TAFA 5	+++++	+++++
Thyrotropin releasing hormone	+++++	+++++
Transforming growth factor beta	+++++	−−−−−
Tyrosine hydroxylase	+++++	−−−−−
Urocortin	+++++	+++++
Vasoactive intestinal peptide	+++++	−−−−−

Angiotensin II (ANGII) containing cell bodies were identified in several brain areas, including SON and magnocellular parts of the PVN (Lind et al. 1985a), where ANGII is co-localized with AVP (Imboden and Felix 1991). ANGII is associated with the regulation of the activity of gonadotropic hormone releasing hormone and pituitary hormones during the reproductive cycle and pregnancy. It can also act as a neurotransmitter and via AT₁ receptors potentiate the excitatory synaptic inputs into SON. During hypovolemia, ANGII induces thirst, increases blood pressure, sodium appetite, and excretion, and stimulates AVP, ACTH, and aldosterone secretions (Phillips 1987). Using push-pull cannulas to perfuse the PVN and radioimmunoassay to analyze the superfusate for immunoreactive angiotensins, 24 h of water deprivation resulted in an approximate five-fold increase in the angiotensin release rate, whereas 48-h deprivation produced a dramatic 492-fold increase in release (Harding et al. 1992).

The di/di Brattleboro rats have significantly decreased angiotensinogen levels in the hypothalamus, but they are elevated in the posterior pituitary (Hawkins and Printz 1983). The amount of ANGII immunopositive cells in the parvocellular subdivision of the PVN has been shown to be similar to that in normal rats; however, in magnocellular subdivision of the PVN, their number is decreased (Lind et al. 1985b). Decreased amount of ANGII leads to a decline in the basal secretion of aldosterone, which in di/di rats is three to four-fold lower in comparison with Long Evans rats (Laulin et al. 1988). The density of ANGII-binding sites in the anterior pituitary is higher in di/di compared to Long Evans rats and water deprivation further increases their number (Israel et al. 1986).

Apelin (APL) is widely expressed in the CNS, including the magnocellular neurons of the SON and PVN, where it is co-localized with both AVP and OXY (Brailoiu et al. 2002; Llorens-Cortés and Beaudet 2005). Apelin is involved in several physiological processes in the organism. It participates in the regulation of the cardiovascular system and immune responses, and it can act as an endocrine adipokine and co-receptor in the process of human immunodeficiency virus type1 infections (Falcao-Pires and Leite-Moreira 2005). Functional evidence supporting APL relation to fluid regulation and AVP regulation is supported by observations that i.c.v. administration of apelin decreases circulating plasma levels of AVP (−47%) (Reaux et al. 2001). In mice deprived of water, i.c.v. administration of apelin significantly reduced the water intake in the initial 30 min after re-exposure to drinking water and lowered circulating levels of AVP (−42.6%) (Reaux et al. 2001). AVP and APL are conversely regulated in the facilitation of systemic AVP release and suppression of diuresis (Llorens-Cortes and Moos 2008). Opposite effects of AVP and APL are likely to occur at the hypothalamic level through autocrine/paracrine modulation of the phasic electrical activity of AVP neurons. Both the number and intensity of magnocellular APL-immunoreactive cells were increased significantly after 24-h or 48-h dehydration, whereas the number and density of AVP-immunoreactive neurons significantly decreased. Dehydration-induced release of AVP from magnocellular hypothalamic neurons may be responsible for the increase in immunoreactive APL levels within the same neurons, and thus, the release of one peptide may block that of another peptide synthesis in the same cells (Reaux-Le Goazigo et al. 2004). There is no data available about the role of APL in magnocellular neurons in di/di Brattleboro rats.

Corticotropin releasing hormone (CRH) is expressed in neurons in several brain regions of the adult rat brain, but most prominently in the PVN parvocellular cells (Cummings et al. 1983). It is well known as a key mediator in endocrine (Chen et al. 2008a), autonomic (Aguilera 1998), behavioral (Koob and Bloom 1985), and immune (Baigent 2001) responses to stress. However, during osmotic stimulation, CRH can also regulate the activity of AVP and OXY neurons (Arima and Aguilera 2000).

Although the CRH levels in the PVN parvocellular neurons are also influenced (decreased) by osmotic stimuli, magnocellular neurones in the PVN and SON showed increased CRH-immunoreactivity that emerged only in a subset of magnocellular OXY neurons (Dohanics et al. 1990). I.c.v. administration of CRH increased OXY by four-fold but not AVP plasma levels. On the other hand, intravenous administration of CRH induced secretion of AVP and OXY. It seems that CRH may act directly or indirectly upon magnocellular neurons to increase OXY release (Bruhn et al. 1986). While CRH receptor-1mRNA was undetectable in the SON and PVN in control rats, its expression was significantly increased after i.p. hypertonic saline injection or after 12 days of salt loading (Arima and Aguilera 2000). On the other hand, expression of CRH receptor-2α was detectable even under basal conditions and was elevated only after chronic osmotic stimuli. Acute osmotic stimulation had no effect on its expression (Arima and Aguilera 2000; Young et al. 2007).

In the hypothalamus of di/di Brattleboro rats, basal content of CRH was similar to that of Long Evans ones (Kjaer et al. 1993). Only a minor elevation of CRH mRNA was observed in di/di rats, which however, had no effect on basal ACTH and corticosterone levels (Mlynarik et al. 2007).

Galanin (GAL) mRNA and galanin immunoreactivity have been demonstrated throughout the central nervous system, including the magnocellular neurons of the SON and PVN (Skofitsch and Jacobowitz 1986). It is mainly co-localized with AVP and to a lesser extent with OXY (Gaymann and Martin 1989). Galanin regulates numerous physiological actions, including wake-sleep states (Sherin et al. 1998), reproduction (Rossmanith et al. 1996), nociception (Liu and Hökfelt 2002), cognition (McDonald et al. 1998), and energy, and osmotic homeostasis (Crawley 1999). Water homeostasis is regulated by GAL at the level of the hypothalamus (Lang et al. 2007). It regulates the electrophysiological and secretory activities of the magnocellular AVP and OXY neurons and its effect depends upon the actual body fluid osmotic status. During dehydration, GAL via the GAL1 receptor (GAL-R1) inhibits AVP and OXY secretion into the systemic circulation, but during equilibrated water metabolism, it inhibits biosynthesis and axonal transport of only OXY and not AVP (Cisowska-Maciejewska and Ciosek 2005). Prolonged 2% NaCl intake or dehydration increases GAL mRNA levels and markedly decreases GAL protein immunoreactivity in the PVN and SON, and depletion of GAL immunoreactivity in the porterior pituitary has been also demonstrated (Meister et al. 1990a). Salt loading has been shown to increase the expression of GAL-R1 receptors that seems to be parallel with the functional and morphological changes in hypothalamic magnocellular neurons (Burazin et al. 2001).

AVP deficient di/di Brattleboro rats have elevated mRNA levels encoding preproGAL in the PVN and SON (Rökaeus et al. 1988) and also galanin mRNA in PVN (Beck and Max 2007). Galanin concentration in the median eminence of di/di rats did not differ from heterozygous Brattleboro or Sprague-Dawley rats. However, in the posterior pituitary of di/di rats, the level of GAL immunoreactivity was reduced by 75% (Koenig et al. 1989). These data suggest that production and secretion of GAL in this strain of rats is increased.

Peptides Co-Localized with AVP or OXY with Unknown Role in Osmotic Regulation

The following biologically active substances (organized in alphabetical order) co-localize in the magnocellular neurons with osmotically important molecules (AVP and OXY), but their role in osmoregulation, up to date, is unclear. There exists only a few data about their occurrence in Brattleboro rats.

Cholecystokinin (CCK) immunoreactivity has been demonstrated in OXY neurons of the SON (Vanderhaeghen et al. 1981; Liu and Ju 1994). Differences in the hypothalamic distribution of CCK have been described between male and female rats (Micevych et al. 1987). CCK exerts peripheral and central effects. In the pancreas, it increases the proliferation of insulin-producing beta cells and reduces insulin-induced hyperphagia. Elevated cholecystokinin levels decrease appetite and reduce intestinal inflammation caused by parasites and bacterial toxins. In the hypothalamus, CCK inhibits the expression of orexigenic peptides and prevents the stimulation of specialized neurons by ghrelin (Chandra and Liddle 2007). In untreated and salt loaded Sprague-Dawley rats, no CCK immunoreactive neurons were observed in the PVN or SON without colchicine pretreatment (Meister et al. 1990b). Prolonged administration of 2% sodium chloride, as drinking water, induced proportionally higher increase of CCK mRNA in the PVN than SON. Within the nerve fibers of the posterior pituitary of salt loaded rats, small depletion in CCK immunoreactivity was observed (Meister et al. 1990a). Experimental data showed that salt loading increases the mRNA of CCKA and CCKB receptors within the brain. Osmotically induced differential changes in cholecystokininA and cholecystokininB receptor messenger ribonucleic acids within the brain show that cholecystokinin receptors within the rodent hypothalamus are capable of plastic responses to chronic osmotic stress (Hinks et al. 1995).

In di/di Brattleboro rats, CCK immunopositive neurons have been described only after colchicine pretreatment of animals (Meister et al. 1990b). However, cholecystokinin octapeptide binding was greatly elevated in the paraventricular, supraoptic, and accessory nuclei of di/di Brattleboro rats compared to controls (Day et al. 1989), indicating that cholecystokinin receptors may be involved in the oxytocin release process.

Chromogranin/secretogranin (SgII), as shown by immunoelectron microscopy, is co-stored with vasopressin, and in salt loaded rats, the levels of mRNA for vasopressin, galanin, and secretogranin II are increased in the PVN and SON. Analogous changes were observed in di/di Brattleboro rats with the exception of the vasopressin message, which was decreased in these animals (Mahata et al. 1993). Measurements of changes in secretogranin II and chromogranin mRNA may be a useful tool for assessing both rapid and long-lasting increases and decreases in the neuronal activity. They also may reflect specific changes in neuronal secretory activity associated with transmitter/peptide release (Shen and Gundlach 1996). Because of its ability to aggregate in a low pH and high Ca²⁺environment, SgII has been implicated in sorting and packaging of peptide precursors into the secretory pathway (Huttner et al. 1991). SgII may also serve as a precursor of biologically active peptides with an extracellular function (Gerdes et al. 1988). Although after an osmotic challenge both AVP and OXY neurons are stimulated, SgII after salt loading and colchicine pretreatment was exclusively expressed in a subpopulation of AVP magnocellular neurons in the SON and PVN of Wistar rats (Ang et al. 1997). After 4 days of water deprivation, SgII mRNA significantly increased in the magnocellular neurons of SON and PVN (Mahata et al. 1992; Shen and Gundlach 1996). Since chronic stimulation of vasopressin neurons leads to a concomitant up-regulation of the biosynthesis of neuropeptides and secretogranin II, it seems that the secretogranin II message might be a useful general marker for identifying chronically stimulated neurons (Mahata et al. 1992).

Dynorphin (Dyn) is localized in AVP (Watson et al. 1982) and to a less extent in OXY magnocellular neurons of the PVN and SON (Meister et al. 1990b). Dynorphin is involved in a variety of normative physiologic functions, including antinociception, neuroendocrine signaling pathways, cardiovascular and temperature regulations, and response to stress (Smith and Lee 1988). It may be protective to neurons and oligodendroglia (Hauser et al. 2005). It takes part in the transmission of information concerned with the interaction of feeding and maintenance of energy balance (Baile et al. 1983), and regulation of fluid homeostasis (Bodnar and Klein 2005). In untreated or salt loaded rats, only a few Dyn-immunoreactive neurons are found in the PVN and SON, but their number markedly increases after colchicine pretreatment (Meister et al. 1990b). After prolonged salt loading, elevated Dyn mRNA levels in the PVN and SON and markedly depleted Dyn-like immunoreactivity have been found within the nerve fibers of the posterior pituitary, which suggest that high plasma osmolality increases Dyn release (Meister et al. 1990a).

Brain and pituitary content of immunoreactive Dyn in di/di Brattleboro rats under basal conditions is similar to that in di/+ and +/+ animals (Cox et al. 1980). Colchicine pretreatment elevates the amount of Dyn neurons in di/di rats (Meister et al. 1990b).

Endothelin-1 (ET-1) is colocalized with AVP and to a lesser extent with OXY in the magnocellular PVN and SON neurons. Double immunogold labeling revealed intragranular colocalization of ET-1 with AVP or OXY in the rat neural lobe (Nakamura et al. 1993). ET-1 is one of the most effective vasoconstrictive mediators (Namer et al. 2007). It enhances the rhythmicity and magnitude of contractions in the isolated rat uterine horns (Kozuka et al. 1989). It is involved in certain signaling pathways (Anita et al. 2006). It can decrease glomerular filtration rate and urine volume (Katoh et al. 1990). Elevated levels of ET-1 in circulation have predominantly excitatory effects on AVP and OXY neurons in the SON and PVN (Wall and Ferguson 1992). ET-1, by autocrine or paracrine way, may influence AVP or OXY release from the neural lobe (Nakamura et al. 1993). Dehydration causes a significant decrease in ET-1 immunoreactive content and number of neurosecretory granules in the neurohypophysis. However, in hemorrhaged rats, ET-1 immunoreactivity increased in the tissue nearly three-fold, whereas the ET-1 immunolabeling in the axon terminals was unchanged. Plasma concentration of immunoreactive ET-1 is unchanged following hemorrhage or dehydration, whereas immunoreactive AVP is remarkably increased. It suggests that ET-1 in the hypothalamo-hypophysial system may be involved in the local modulation of vasopressin secretion during hypovolemic and/or osmotic stress challenges (Uemura et al. 1994).

Enkephalins (ENK) are present in both PVN and SON structures. Although Methionin-ENK (Met-ENK) is localized within both OXY and AVP neurons, Leucine-ENK (Leu-ENK) is found only in AVP neurons in the SON and PVN (Martin and Voigt 1981). The ENK system represents a major modulatory system in the adaptation to stress (Drolet et al. 2001), plays a role in the processing of sensory information, or serves as neuroendocrine (Dores et al. 2002) and synaptic activity modulator (Snyder 2004). Met-ENK is involved in humoral and cell-mediated immune reactions (Jankovic and Radulovic 1992) and signaling pathways (Lukiw 2006). Although in untreated Sprague-Dawley rats no Leu-ENK or Met-ENK immunoreactive cell bodies were observed in the SON or PVN, the number of Leu-ENK neurons increased mainly in the SON after salt loading following colchicine treatment and hypophysectomy (Meister et al. 1990b). Prolonged salt loading caused a significant decrease of Met-ENK only in the PVN but not in the SON (Jessop et al. 1990). In the PVN and SON of di/di rats, ENK immunoreactive neurons were observed only after colchicine pretreatment (Meister et al. 1990b).

Ferritin protein is found almost in all AVP-immunoreactive neurons of the rat PVN and SON (Tokunaga et al. 1992). It is involved in the intracellular storage of iron, and it has been implicated in the pathogenesis of many diseases such as atherosclerosis, Parkinson’s disease, Alzheimer disease, and restless legs syndrome (You and Wang 2005). It can act as an immuno-suppressor (Recalcati et al. 2008) and as an oxygen free radical-mediated damage protectant (Orino et al. 2001). Water deprivation causes enlargement of the cell bodies and increases the number of AVP-ferritin containing neurons (Tokunaga et al. 1992). There are no data available about ferritin presence in di/di Brattleboro PVN and SON neurons.

Interleukin-6 (IL-6) is robustly expressed in AVP neurons of the SON and PVN. IL-6 is associated with inflammation, haematopoiesis, and immune responses (Hoene and Weigert 2008). It has a pathogenetic role in chronic heart failure (Chen et al. 2008b). It can act as an adipokine or myokine and can exert lipolytic effects and anti-obesity potential (Hoene and Weigert 2008). Dehydration up-regulates IL-6 levels in the SON cells, PVN, axons of the internal zone of the median eminence, and reduces its levels in the posterior pituitary. It has been suggested that IL-6 takes the same neurosecretory pathway as AVP and is secreted from the posterior pituitary following a physiological stimulus (Ghorbel et al. 2003). There are no data available about IL-6 in di/di Brattleboro rats.

Kininogen (HKg) and T-kininogen (TKg) were both detected in the magnocellular neurons of the PVN and SON. HKg immunostaining was detected mainly in OXY neurons, while TKg immunostaining appears to be restricted to AVP neurons. HKg can act as a precursor of bradykinin, but both of them are potent thiolprotease inhibitors that can modulate maturation processes of peptidic hormones, their inactivation, and catabolism. TKg stains in parallel with AVP during water deprivation and is undetectable in di/di Brattleboro rats (Richoux et al. 1991).

Neurokinin B (NKB), as revealed by dual fluorescence immunohistochemistry, is co-expressed with AVP in the PVN and SON neurons (Hatae et al. 2001a). NKB belongs to tachykinin peptide family. Tachykinins modulate blood pressure and heart rate (Walsh et al. 2006). NKB seems to be involved in nociception, neuroimmunomodulation, development of bronchial asthma, inflammatory bowel syndrome, and psychiatric disorders (Almeida et al. 2004). It can act as a neurotransmitter or neuromodulator (Chahl 2006). NKB, by paracrine/autocrine way, may regulate the AVP release via its NK-3 receptors (Hatae et al. 2001a). In the magnocellular neurons of the PVN and SON of intact rats, NKB and AVP show almost the same immunohistochemical reactivity. Water deprivation considerably increases NKB precursor peptide and mRNA in magnocellular neurons of PVN and SON but NKB immunoreactivity drastically decreases. These data indicate that the synthesis and release of NKB in the PVN and SON are enhanced by water deprivation in the same manner as AVP, and thus, NKB seems to be involved in the central control of body fluid balance (Hatae et al. 2001b). In di/di Brattleboro rats, there are no available data about NKB.

Neuropeptide Y (NPY) co-exists with AVP or OXY in magnocellular neurons of the PVN and SON (Larsen et al. 1994). It influences cardiovascular function (Carter et al. 1985), feeding, anxiety, depression, epilepsy, neuroprotection, neurogenesis (Xapelli et al. 2006), and regulation of water homeostasis. Under basal conditions, no NPY immunoreactivity was observed in the magnocellular SON and PVN neurons. However, increased NPY synthesis in these neurons appeared in response to elevated plasma osmolality (Larsen et al. 1992). NPY stimulates the release of AVP and OXY from the SON and PVN via activation of NPY Y1 receptors, whose expression in SON is increased during salt loading and water deprivation. NPY can support AVP/OXY secretion during prolonged osmotic challenge to reach fluid homeostasis (Urban et al. 2006). On the other hand, however, in the neurohypophysis, NPY is situated outside the blood-brain barrier. NPY inhibits/modulates the release of AVP (Larsen et al. 1992). Seven days of salt loading elevated NPY concentration in the median eminence and neurohypophysis (Hooi et al. 1989).

In di/di Brattleboro rats, NPY immunopositive neurons were observed in the PVN and SON only after intravenous administration of colchicine (Kagotani et al. 1990). NPY concentration in the neurohypophysis of di/di rats was found to be two-fold greater than in di/+ rats and four-fold greater than in normal Sprague-Dawley rats (Hooi et al. 1989).

Nitric oxide synthase (NOS) catalyzes the synthesis of the gaseous neuromessenger nitric oxide (NO) from l-arginine. The AVP and OXY neurons of the PVN and SON exhibit intense staining for NOS, and dense immunopositive terminals in the inner layer of the median eminence and neurohypophysis (Hatakeyama et al. 1996). Water deprivation markedly increases NOS mRNA in both the SON and PVN nuclei. It appears that NOS mRNA, NOS enzymatic activity, and also presumably NO production, are modulated by alterations in fluid homeostasis (O’Shea and Gundlach 1996; Yamauchi et al. 2007).

Expression of NOS gene in di/di Brattleboro rats in the magnocellular PVN and SON neurons is upregulated when compared to Wistar and heterozygous (di/+) rats (Yamamoto et al. 1997).

Vasoactive intestinal peptide (VIP) is colocalized with AVP in the magnocellular neurons of the PVN and SON. VIP reaches detectable levels only under certain experimental conditions, including hypophysectomy and salt loading. A large population of magnocellular VIP-immunoreactive neurons was described in the PVN and SON as well as in the accessory hypothalamic nuclei of the mink (Mustela vision), which were co-localized with vasopressin or oxytocin (Larsen and Mikkelsen 1992). However, less extensive VIP/AVP and VIP/OXY co-expressions were seen in rodents. Immunohistochemical localization of VIP in the circumventricular organs of the rat (Mikkelsen 1989) and hypothalamohypophysial system of the mongolian gerbil (Mikkelsen and Møller 1988) have been demonstrated.

VIP induces vasodilatation, relaxation of smooth muscle, exocrine gland secretion, secretion of different hormones (prolactin, growth hormone, oxytocin, vasopressin, and ovarial, and thyroid hormones) (Zudenigo and Lackovic 1989). I.c.v. infusion of VIP rises plasma OXY and AVP levels, which suggests that endogenous VIP is also a physiological regulator of OXY and AVP release in rats (Bardrum et al. 1988). In di/di Brattleboro rats, an additional colchicine pretreatment is needed for its detection (Ceccatelli et al. 1991).

Pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactivity has been found in magnocellular cells of the SON and PVN, which synthesize and release AVP or OXY (Gillard et al. 2006). PACAP is important in the control of several endocrine and homeostatic processes, such as anterior pituitary hormone release, insulin and glucagon secretion (Arimura 1998), and food intake (Nakata et al. 2004). The distribution of PACAP in the hypothalamus suggests a role in osmoregulation (Gillard et al. 2006). PACAP activates PVN and SON neurons via PACAP receptors and transcription of the AVP gene (Nomura et al. 1999). PACAP type I receptor gene was found to be moderately expressed in the whole PVN and SON (Nomura et al. 1996). PACAP might be a modulator of magnocellular neuron function during dehydration by influencing the local AVP release. In the SON of euhydrated control rats, PACAP and PACAP type I receptor immunoreactivities were modest and prolonged dehydration-induced their marked increase (Gillard et al. 2006). There are no data available about PACAP occurrence in di/di Brattleboro rats.

TAFA5 protein is located in the magnocellular AVP and OXY neurons of the PVN and SON (Paulsen et al. 2008). It belongs to a novel family of proteins with brain-specific expression and structural similarity to chemokines (de Stahl et al. 2005). Although both 48-h water deprivation and salt loading cause decline in TAFA 5mRNA in the PVN, only water deprivation lowers its level significantly, which may suggest that TAFA5 regulation is more sensitive to hypovolemia (Paulsen et al. 2008). There are no data available in di/di Brattleboro rats.

Thyrotropin releasing hormone (TRH) immunoreactive magnocellular cells were occasionally detected in the PVN and SON in both OXY and AVP neurons after colchicine pretreatment, hypophysectomy, or salt loading with colchicine pretreatment in Sprague-Dawley rats. It was not detected in untreated or salt loaded Sprague-Dawley rats or di/di rats even after colchicine pretreatment (Meister et al. 1990b). I.c.v. injected TRH significantly increases hypothalamic AVP and decreases OXY content in euhydrated and salt loaded rats (Ciosek and Stempniak 1995). I.p. injection of TRH decreased plasma AVP and diminished the nucleoli size of OXY cells. It may influence release and secretion processes in the PVN and SON AVP and OXY cells (Glazova and Krasnovskaia 1996).

Tyrosine hydroxylase (TH), a key enzyme for catecholamine synthesis (Panayotacopoulou et al. 1994; Weihe et al. 2006), is in the magnocellular neurons of PVN and SON co-expressed with AVP (Meister et al. 1990b). Salt loading markedly increases TH-like immunoreactivity in the SON and PVN, whereas AVP- and OXY-like immunoreactivities exhibit very weak or even undetectable signal in these nuclei (Meister et al. 1990b). During long-term osmotic stimulation, not only the number of TH-immunoreactive neurons but also the TH content are elevated in cell bodies and their axons. Thus, chronic stimulation of AVP neurons is associated with a series of adaptive reactions, the most important of which appears to be the expression of AVP and TH synthesis by neurons, which do not synthesize them under basal conditions (Kiss and Mezey 1986; Abramova et al. 2000).

In contrast to untreated Sprague-Dawley rats, where only single TH-immunopositive neurons were observed in the SON, di/di Brattleboro rats showed markedly increased TH immunoreactivity in the PVN and SON (Meister et al. 1990b). TH mRNA was also found to be increased by salt loading in the PVN and SON cells of di/di rats (Kiss and Mezey 1986; Young et al. 1987). Moreover, the presence of TH-immunopositive dendritic swellings after salt loading in the ventral part of SON reveals the high state of plasticity of these neurons in di/di rats. In aged di/di rats, a marked TH expression was even further potentiated by osmotic stimulation in AVP cells in the absence of AVP synthesis in the SON, indicating that TH expression is linked to the activation of AVP neurons but unrelated to AVP synthesis (Marsais et al. 2002). In addition, vasopressin treatment lowered TH immunoreactivity in the PVN and SON magnocellular neurons of di/di rats to normal levels indicating that extracellular factors, probably afferent inputs, regulate the level of TH immunoreactivity in magnocellular neurons (Kiss and Mezey 1986).

Urocortin, a member of CRF peptide family, is co-expressed with AVP and OXY in both PVN and SON magnocellular neurons (Imaki et al. 2001). Urocortin exerts physiological/pharmacological effects similar to those produced by CRH, including the stimulation of ACTH release from the anterior pituitary (Turnbull and Rivier 1997). It is an endogenous ligand for CRH receptor-2 and CRH receptor-1 types and it can bind with them with greater potency than CRH itself (Vaughan et al. 1995). I.c.v. injected urocortin significantly attenuates AVP release induced by hyperosmolality, which suggests that central urocortin may play an inhibitory role in AVP release during an osmotic challenge (Kakiya et al. 1998). Salt loading and water deprivation increase the number of urocortin-immunoreactive cells in the SON and PVN (Hara et al. 1997a, b). There are no data available in di/di Brattleboro rats.

Besides the above mentioned substances, there are many others co-localized with AVP or OXY (Table 1), including agmatine, calbindine and calretinine, cocaine and amphetamine regulated transcript (CART), NADPH-diaphorase, nesfatine, neuroendocrine polypeptide 7B2, neuromedin U, neuropeptide AF, neuropeptide FF, neuropeptide B, neurotensine, 18 regulated endocrine specific protein, transforming growth factor beta, etc., but there are no data available about their possible responsiveness to osmotic challenges.