Gloria E. Hoffman

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.

Induction of c-Fos immunoreactivity in the rat forebrain by conditioned and unconditioned aversive stimuli.

The protein product of the c-fos proto-oncogene was immunocytochemically localized in forebrain regions of adult male Lewis rats subjected to a physically aversive footshock stimulus or a Pavlovian-conditioned, non-aversive, auditory stimulus. Animals receiving the conditioned stimulus were first conditioned by repeatedly pairing electric footshock, the unconditioned stimulus (US), with an auditory cue, the conditioned stimulus (CS). These animals were later tested with the CS in the absence of the US, a procedure which, like footshock itself, suppresses immune function. In animals exposed to the conditioned or unconditioned stressor, c-Fos was strongly expressed in cells of the paraventricular nuclei (PVN) of the hypothalamus, some of which contain corticotropin-releasing hormone (CRH), and other forebrain areas directly associated with autonomic function, the ventral lateral septal nuclei (LSV), the medial amygdaloid nuclei (AME), the sensorimotor cortex, the basal ganglia and thalamic nuclei. Control animals exhibited very little or no c-Fos in the above areas. The identified forebrain nuclei can now be targeted for further study aimed at elucidating their role in stress-induced immune alteration.

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.

Activation of brainstem catecholaminergic neurons by conditioned and unconditioned aversive stimuli as revealed by c-Fos immunoreactivity

In an attempt to define areas of the brain that respond to stressors and influence immune function, we have previously identified stress-induced, c-Fos-immunoreactive areas of the diencephalon. We found that c-Fos was strongly expressed in cells of the paraventricular nuclei (some of which contain corticotropin-releasing hormone (CRH)) and other hypothalamic areas directly associated with autonomic function. To further characterize the presumptive pathways mediating stress-induced immune alterations, including the assessment of brainstem catecholaminergic neuron involvement, the induction of c-Fos immunoreactivity was examined in the brainstem of rats exposed to conditioned and unconditioned, immunomodulating stimuli. In response to electric footshock (the unconditioned stimulus (US)), c-Fos immunoreactivity was strongly induced in the noradrenergic neurons of the locus ceruleus (A6), the nucleus of the solitary tract (A2/C2), the ventral lateral medulla (A1/C1), A5, and A7, as well as in unidentified neurons of the dorsal and ventral subdivisions of the periaqueductal gray (PAG), and in the serotonergic neurons of the dorsal raphe nuclei. Conditioned animals re-exposed to the conditioned stimulus showed c-Fos induction in these same areas but to a lesser degree. Control animals exposed only to the conditioning stimulus (CS) (electronic tone) in the absence of the US, expressed very little, if any, c-Fos activity in the above loci except for a small degree of baseline expression in the PAG. These results further confirm the role of autonomic and endocrine pathways as mediators of the stress response and will help to more fully characterize the pathways of stress-induced immune alteration.

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.

c-Fos expression in brain in response to hypotension and hypertension in conscious rats

Hypotension- and hypertension-evoked expression of the protein product, Fos, of the immediate early gene c-fos was assessed throughout the rat brain as an approach for describing the neuronal populations that respond to alterations in arterial blood pressure. Conscious, chronically catheterized rats were treated with the vasoconstricting drug phenylephrine or the vasodilatating drug hydralazine to increase or decrease, respectively, arterial pressure by approx. 40 mm Hg for 90 min. Rats were then anesthetized, fixed by vascular perfusion, and sections representing the entire brain were processed for the immunocytochemical localization of Fos. In control rats treated with isotonic saline, few Fos-positive neurons were observed. In contrast, phenylephrine and hydralazine treatments resulted in different, yet reproducible, patterns of Fos expression in the brain, with hydralazine evoking Fos expression in more brain regions than phenylephrine. Brain regions containing Fos-positive neurons in rats treated with hydralazine included nucleus tractus solitarius, area postrema, caudal ventrolateral medulla, rostral ventrolateral medulla, bed nucleus of the stria terminalis, amygdala, paraventricular nucleus, supraoptic nucleus, subfornical organ and the Islands of Calleja. The nucleus tractus solitarius, paraventricular nucleus and the amygdala also contained Fos-positive neurons in phenylephrine-treated rats, although the number of Fos-positive neurons was always less than that noted in the hydralazine-treated rats and the location of Fos-positive neurons within these regions tended to differ between treatments. These results generally fit within an emerging understanding of brain circuitry underlying cardiovascular regulation.

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.

Detecting steroidal effects on immediate early gene expression in the hypothalamus

Abstract Immediate early genes are rapidly and transiently expressed within neurons following stimulation. While it is likely immediate early gene products alter gene expression in neurons, the products of the Immediate early genes also serve as excellent markers for Identifying activated neurons. The immunocytochemical localization of immediate early gene products enables the analysis of changes In activation of individual neurons in the brain in response to gonadal steroids or other stimuli. The technique presented in this article outlines the strategies for using immediate early gene proteins in the fos family as markers for neuronal activity and focuses principally on the examination of one chemically identified neuron population, the luteinizing hormone-releasing hormone system. Included are comparisons of the staining of brain tissue for c-fos and related antigens following use of different tissue fixatives. Because some of the c-fos antibodies also recognize other proteins in the fos family, we have presented data comparing staining patterns obtained with a number of available antisera. We also describe some of the approaches we use to analyze c-fos expression.

Central c-Fos expression in neonatal and adult rats after subcutaneous injection of hypertonic saline

Centrally-mediated responses to plasma hyperosmolality include compensatory drinking and pituitary secretion of vasopressin and oxytocin in both adult and neonatal rats. However, the anorexia that is produced by plasma hyperosmolality in adult rats is not evident in neonates, perhaps due to functional immaturity of osmoresponsive hindbrain circuits. To examine this possibility, the present study compared treatment-induced brain expression of the immediate-early gene product c-Fos as a marker of neural activation in adult and two-day-old rats after subcutaneous injection of 2 M NaCl (0.1 ml/10 g body weight). This treatment produced marked hypernatremia in adult and two-day-old rats without altering plasma volume. Several brain regions (including components of the lamina terminalis, the paraventricular and supraoptic nuclei of the hypothalamus, and the area postrema) were activated to express c-Fos similarly in adult and two-day-old rats after 2 M NaCl injection, consistent with previous reports implicating a subset of these regions in osmotically-stimulated drinking and neurohypophyseal secretion. In contrast, other areas of the brain that were activated to express c-Fos in adult rats after 2 M NaCl injection were not activated in neonates: these areas included the central nucleus of the amygdala, the parabrachial nucleus and catecholamine cell groups within the caudal medulla. This study demonstrates that certain brain regions that are osmoresponsive in adult rats (as defined by induced c-Fos expression) are not osmoresponsive in two-day-old rats. When considered in the context of known differences between the osmoregulatory capacities of adult and neonatal rats, our results are consistent with the idea that osmoresponsive forebrain centres are primarily involved in osmotically-stimulated compensatory drinking and neurohypophyseal secretion, whereas osmoresponsive regions of the hindbrain are important for concomitant inhibition of feeding and gastric emptying.