Joseph G. Verbalis

c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems.

Expression of c-Fos, or other immediate early gene products, by individual neurons can be used as a marker of cell activation, making staining of these proteins an extremely useful technique for functional anatomical mapping of neuroendocrine systems. Because these proteins are located in the nucleus, identification of the phenotype of the activated neuron using substances located within the cytoplasm can be accomplished with standard double-labeling immunocytochemical techniques. Although it is clear that neurons have the capacity to express a number of immediate early gene products, what remains to be established is whether there is a different pattern of expression following various stimuli. In our studies, we focus primarily on expression of one immediate early gene product, the c-Fos protein. We also include some experiments using expression of other members of the Fos family and Jun proteins as markers for neuronal activation. Our studies describe uses of c-Fos expression in both parvocellular and magnocellular hypothalamic systems to address the following issues: (a) identification of neuroendocrine cells activated by specific treatments and conditions, (b) ascertainment of functional differences in subpopulations activated by specific stimuli, (c) evaluation of neuronal activity in complex areas containing multiple neuroendocrine systems, (d) identification of other brain areas activated in conjunction with neuroendocrine systems following specific stimuli, (e) analysis of connectivity of activated neuroendocrine systems with other parts of the brain, and (f) identification of stimuli that decrease neuronal activity. The neuroendocrine systems studied include those that secrete arginine vasopressin (AVP), oxytocin (OT), corticotropin-releasing hormone (CRH), luteinizing hormone-releasing hormone (LHRH), and dopamine (DA). The use of c-Fos expression has permitted functional neuroanatomical mapping of these systems in response to specific stimuli such as cholecystokinin (CCK), hyperosmolality, and volume depletion, or during various physiological states such as the proestrous ovulatory luteinizing hormone (LH) surge and lactation. Although the use of c-Fos as a marker of neuronal activation will continue to be an extremely powerful technique, future studies will also be directed at relating immediate early gene expression to changes in neuroendocrine gene expression. To this end, we have shown that both c-Fos and c-Jun are expressed in neuroendocrine neurons in response to a number of stimuli, setting the stage for potential regulatory drive to genes containing AP-1 binding sites.

Hyponatremia causes large sustained reductions in brain content of multiple organic osmolytes in rats

Brain adaptation to hypoosmolality is known to involve volume regulatory losses of both extracellular and intracellular electrolytes. We studied the effects of acute and chronic hypoosmolality on brain content of organic osmolytes as well as electrolytes in rats to ascertain the relative contributions of different brain solutes to the brain volume regulation that occurs under these conditions. Brains were dissected from rats after 2, 7 and 14 d of sustained hyponatremia induced by continuous infusion of 1-deamino-[8-D-arginine]-vasopressin (DDVAP) in combination with a liquid formula, along with control rats fed the same formula in the absence of DDAVP infusions. One half of each brain was analyzed for organic osmolyte contents and the other half for water and electrolyte contents. Brain Na+, K+ and Cl- and multiple organic osmolytes (glutamate, creatine, taurine, myo-inositol, glutamine and glycerophosphoryl-choline) decreased markedly by 2 d of hyponatremia, and brain electrolyte and most organic osmolyte contents then remained at these reduced levels throughout the duration of the hyponatremia. Although the absolute magnitude of the brain electrolyte losses was greater than the magnitude of the brain organic osmolyte losses, the organic osmolyte losses accounted for approximately 35% of the total measured brain solute losses during sustained hyponatremia. These results demonstrate that organic osmolytes constitute a significant proportion of the brain solute losses that take place during hyponatremia, and indicate that reductions in both organic osmolyte and electrolyte contents are necessary to accomplish brain volume regulation during adaptation to sustained hypoosmolality.

Cholecystokinin activates C-Fos expression in hypothalamic oxytocin and corticotropin-releasing hormone neurons

The effect of systemically‐administered Cholecystokinin octapeptide (CCK) on hypothalamic oxytocin, vasopressin, and corticotropin‐releasing hormone neurons was studied by analysis of c‐fos antigen expression in immunocytochemically‐characterized neurons in the supraoptic and paraventricular nuclei. CCK (100μg/kg intraperitoneally) caused a marked increase in nuclear c‐fos immunocytochemical staining, which peaked at 60 to 90 min after injection. C‐fos expression was found in most magnocellular oxytocin neurons in the supraoptic nucleus and in all magnocellular subdivisions of the paraventricular nucleus, but in no vasopressin neurons in either area. C‐fos expression was also found in several parvocellular subdivisions of the paraventricular nucleus: in oxytocin neurons within the medial and lateral, but not the dorsal, parvocellular subdivisions, and in corticotropin‐releasing hormone neurons in the medial parvocellular subdivision. Injection of lower doses of CCK showed that c‐fos expression closely paralleled the pattern of pituitary oxytocin secretion in response to CCK, with a threshold for activation at 1 μg/kg, near maximal responses by 10 μg/kg, and maximal responses by 100 μg/kg. These studies demonstrate that the pattern of c‐fos expression in hypothalamic magnocellular neurons following systemic CCK administration mirrors the neurosecretory response of these neurons, both with regard to specificity for the peptides secreted as well as intensity of secretion. They also demonstrate that systemic CCK administration activates c‐fos expression in parvocellular oxytocin and corticotropin‐releasing hormone neurons, and therefore likely causes secretion of oxytocin and corticotropin‐releasing hormone within the brain at the terminal fields of these neurons.

c-Fos Expression in Rat Brain and Brainstem Nuclei in Response to Treatments That Alter Food Intake and Gastric Motility.

Expression of the proto-oncogene protein c-Fos was evaluated immunocytochemically in individual brain cells as a marker of treatment-related neuronal activation following pharmacological and physiological treatments that are known to alter food intake and gastric motility in rats. c-Fos expression in response to each treatment was analyzed in the brainstem dorsal vagal complex, the limbic system, and the hypothalamus, representing the areas thought to be involved in coordinating the autonomic, behavioral, and neuroendocrine responses that occur during conditions of stimulated or inhibited food intake. Our results indicate that the patterns of brain c-Fos expression associated with treatments that inhibit food intake differ significantly from the patterns produced by treatments that potentiate food intake. Treatments that inhibited food intake (administration of the anorexigenic agents cholecystokinin, LiCl, and hypertonic saline, and food ingestion following fasting or insulin treatment) were associated with widespread stimulation of c-Fos expression in cells in the nucleus tractus solitarius (NTS), and to a more variable degree the area postrema (AP), but without significant activation of neurons in the dorsal motor nucleus of the vagus nerve (DMN). In contrast, treatments that potentiated food intake (food deprivation and insulin-induced hypoglycemia) were associated with marked stimulation of c-Fos expression in DMN neurons, but little or no activation in cells in the NTS or the AP. Pharmacological treatments that inhibited food intake and gastric motility also were associated with pronounced c-Fos expression in several forebrain areas, including the parvocellular and magnocellular subdivisions of the paraventricular nucleus of the hypothalamus (PVN), the central nucleus of the amygdala (CeA), and the bed nucleus of the stria terminalis (BNST). In contrast, more physiological inhibition of food intake resulting from spontaneous food ingestion did not cause significant activation of c-Fos expression in these forebrain regions, nor did treatments that stimulated food intake. Central administration of oxytocin, which also is known to inhibit food intake, was associated with a pattern of c-Fos activation analogous to that produced by spontaneous food ingestion after fasting (c-Fos expression in the NTS and AP, but without significant activation in the DMN or forebrain areas). Finally, acute gastric distension produced complex results, in that it was associated with activation of c-Fos expression in all areas of the brainstem (NTS, AP, and DMN), as well as in multiple forebrain areas (PVN, CeA, and BNST). Our results therefore demonstrate that specific patterns of brain c-Fos expression are associated with treatments that alter food intake in rats, and indicate that assessment of c-Fos immunoreactivity in different brain areas can identify common functional neuroanatomical networks that are activated by diverse treatments which nonetheless produce similar behavioral, autonomic, and neuroendocrine effects in animals.

Medullary c-Fos activation in rats after ingestion of a satiating meal

The distribution and chemical phenotypes of hindbrain neurons that are activated in rats after food ingestion were examined. Rats were anesthetized and perfused with fixative 30 min after the end of 1-h meals of an unrestricted or rationed amount of food, or after no meal. Brain sections were processed for localization of the immediate-early gene product c-Fos, a marker of stimulus-induced neural activation. Hindbrain c-Fos expression was low in rats that ate a rationed meal or no meal. Conversely, c-Fos was prominent in the medial nucleus of the solitary tract (NST) and area postrema in rats that ate to satiety. There was a significant positive correlation between postmortem weight of gastric contents and the proportion of NST catecholaminergic neurons expressing c-Fos. Cells in the ventrolateral medulla (VLM) were not activated in rats after food ingestion, in contrast with previous findings that stimulation of gastric vagal afferents with anorexigenic doses of cholecystokinin activates c-Fos expression in both NST and VLM catecholaminergic cells. These findings indicate that anatomically distinct subsets of hindbrain catecholaminergic neurons are activated in rats after food ingestion and that activation of these cells is quantitatively related to the magnitude of feeding-induced gastric distension.

Cholecystokinin induces c-fos expression in hypothalamic oxytocinergic neurons projecting to the dorsal vagal complex

Systemic administration of cholecystokinin (CCK) decreases gastric motility and stimulates pituitary secretion of oxytocin (OT). Although peripheral OT does not affect gastric function, increasing evidence suggests that central OT secretion acting within the dorsal vagal complex (DVC) can alter gastric motility. To evaluate whether systemically administered CCK is capable of activating oxytocinergic neurons projecting to the DVC, we utilized fluorogold retrograde labeling from the DVC in combination with c-fos and OT immunocytochemical staining to quantitatively analyze paraventricular nucleus (PVN) neurons of rats following injection of CCK at a dose known to cause maximal pituitary OT secretion (100 micrograms/kg i.p.). Our results showed that 2320 +/- 63 PVN neurons were retrogradely labeled from the DVC; 146 +/- 21 (6.3%) of these contained OT, and these cells were predominantly located in the medial parvocellular subdivision of the PVN. Of all retrogradely labeled cells, 671 +/- 112 (28.9%) expressed c-fos after CCK stimulation, and 68 +/- 14 of these (10.1%) contained OT. Approximately 50% of the OT-containing neurons retrogradely labeled from the DVC stained positively for c-fos. Many magnocellular OT neurons in the PVN that were not retrogradely labeled from the DVC also expressed c-fos after CCK stimulation. These results demonstrate that parvocellular OT neurons projecting to the DVC are co-activated along with magnocellular OT neurons projecting to the pituitary following administration of a large dose of CCK, and lend support to a possible functional role for OT as a central neurotransmitter that modulates vagal efferent traffic to the gastrointestinal tract.

Central inhibitory control of sodium appetite in rats: correlation with pituitary oxytocin secretion.

In the present experiments we examined neurohypophyseal hormone secretion in various models of sodium appetite in rats. Basal plasma levels of oxytocin were found to be low in sodium-deficient adrenalectomized rats and in intact animals treated daily with desoxycorticosterone acetate, both of which groups drank large amounts of NaCl solution, whereas basal plasma levels of arginine vasopressin were neither stimulated nor suppressed. Conversely, sodium appetite consistently was inhibited by treatments that stimulated pituitary oxytocin secretion. However, sodium appetite was not inhibited by administration of exogenous oxytocin, nor was it stimulated by administration of an oxytocin receptor antagonist. These and other results suggest that sodium appetite may be inhibited by activity in the supraoptic and/or paraventricular nuclei, the location of the neurons responsible for the synthesis of oxytocin, and can be stimulated only when activity in those neurons is reduced. Whatever the final neural pathway, our data support the hypothesis that the control of sodium appetite is governed by inhibitory as well as excitatory central mechanisms.

Rapid correction of hyponatremia produces differential effects on brain osmolyte and electrolyte reaccumulation in rats

Studies from these and other laboratories have shown that hyponatremia causes marked depletion of both electrolytes and organic osmolytes from the brain. The present studies evaluated brain reaccumulation of both classes of solute after correction of chronic hyponatremia. Hyponatremia was induced by subcutaneous infusions of 1-deamino-[8-D-arginine]-vasopressin (dDAVP) in rats fed a balanced liquid diet. After 14 days of sustained hyponatremia the dDAVP minipumps were removed causing rapid correction of plasma sodium concentrations from 104 +/- 1 mmol/l to 139 +/- 1 mmol/l in 24 h. Water and solute contents were measured in brain extracts both before and for 5 days after correction of the hyponatremia, and compared to values in normonatremic rats maintained on the same diet for 14 days. Our results demonstrate that electrolytes, particularly Na+ and Cl-, reaccumulate rapidly in the brain, resulting in a significant overshoot above normal control brain Na+ and Cl- contents as early as 24 h after correction. In contrast, organic osmolyte reaccumulation occurs more slowly, requiring 5 or more days for a return to normal control brain contents in most cases. A prominent exception to this pattern was glutamate, which also returned rapidly to normal brain contents within 24 h similar to the electrolytes. Quantitative analysis of brain water and solute contents after correction of hyponatremia indicated that the reaccumulation of electrolytes and organic osmolytes was sufficient to account for the changes in brain volume that occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Central Oxytocin Mediates Inhibition of Sodium Appetite by Naloxone in Hypovolemic Rats

Pituitary oxytocin (OT) secretion is inversely related to saline consumption in several experimental models of sodium appetite in rats. Because systemic OT administration does not inhibit sodium appetite, release of OT as a neurotransmitter within the brain, coincident with its secretion from the pituitary, may be related to inhibition of sodium ingestion. The present studies evaluated this possibility by increasing brain OT concentrations both exogenously and endogenously in rats with hypovolemia produced by subcutaneous administration of polyethylene glycol (PEG) solution. Intracerebroventricular (i.c.v.) administration of OT completely abolished intake of 0.5 M NaCl in PEG-treated hypovolemic rats, but did not significantly affect PEG-stimulated water intakes. Endogenous OT secretion was stimulated by systemic treatment with naloxone, which has been shown to increase peripheral and central OT levels. In both one-bottle (0.5 M NaCl) and two-bottle (water and 0.5 M NaCl) drinking tests, intraperitoneal naloxone completely abolished sodium appetite in association with markedly increased pituitary secretion of OT. This inhibition of sodium appetite could be prevented by i.c.v. pretreatment with a specific OT-receptor antagonist, although the antagonist by itself did not affect PEG-stimulated sodium intake. These findings therefore support previous reports which have found that sodium appetite in rats is inhibited by treatments that elicit pituitary release of OT, and provide more direct evidence that brain OT is causally involved in the inhibition of sodium appetite stimulated by such treatments in rats.

Capsaicin pretreatment attenuates multiple responses to cholecystokinin in rats

Systemic injection of the peptide hormone cholecystokinin (CCK) is known to inhibit food intake and gastric emptying, and to stimulate neurohypophyseal secretion of oxytocin (OT) in rats. Previous studies also have shown that surgical destruction of afferent fibers in the gastric vagus eliminates the inhibitory effects of CCK on food intake. The present experiments used capsaicin to destroy peripheral sensory fibers in rats, and confirmed the failure of CCK to inhibit food intake. Similarly, capsaicin pretreatment markedly attenuated the stimulatory effect of CCK on OT secretion and the inhibitory effect of CCK on gastric emptying in rats. These and other results suggest that in rats CCK acts on receptors located on afferent fibers in the gastric vagus and stimulates inhibition of gastric emptying predominantly via a vagovagal reflex arc through the brainstem.