Hypothalamic NPY, AGRP, and POMC mRNA responses to leptin and refeeding in mice.

Food deprivation (FD) increases hypothalamic neuropeptide Y (NPY) and agouti-related protein (AGRP) mRNA levels and decreases proopiomelanocortin (POMC) mRNA levels; refeeding restores these levels. We determined the time course of changes in hypothalamic NPY, AGRP, and POMC mRNA levels on refeeding after 24 h FD in C57BL mice by in situ hybridization. After 24 h deprivation, mice were refed with either chow or a palatable mash containing no calories or were injected with murine leptin (100 microg) without food. Mice were perfused 2 or 6 h after treatment. Food deprivation increased hypothalamic NPY mRNA (108 +/- 6%) and AGRP mRNA (78 +/- 7%) and decreased hypothalamic POMC mRNA (-15 +/- 1%). Refeeding for 6 h, but not 2 h, was sufficient to reduce (but not restore) NPY mRNA, did not affect AGRP mRNA, and restored POMC mRNA levels to ad libitum control levels. Intake of the noncaloric mash had no effect on mRNA levels, and leptin administration after deprivation (at a dose sufficient to reduce refeeding in FD mice) was not sufficient to affect mRNA levels. These results suggest that gradual postabsorptive events subsequent to refeeding are required for the restoration of peptide mRNA to baseline levels after food deprivation in mice.

Persistence of meal-entrained circadian rhythms following area postrema lesions in the rat.

Rats anticipate daily meals with increased approaches to a feeder and an increase in core body temperature. Food-anticipatory activity (FAA) is thought to be under the control of a feeding-entrained circadian oscillator (FEO). Ibotenic acid and electrolytic lesions in the region of the parabrachial nuclei (PBN) in the rat severely disrupt FAA (feeder approaches) and temperature rhythms. The PBN receive dense input from the area postrema (AP), which lacks a blood-brain barrier and thus has access to humoral factors in the systemic circulation. The present study assesses development and maintenance of FAA in rats with cautery lesions of the AP. The results demonstrate that AP lesions do not alter FAA. This experiment does not support the hypothesis that the AP in the caudal brainstem detects and relays circulating signals from the periphery that trigger FAA.

Dynamics of c-fos and ICER mRNA expression in rat forebrain following lithium chloride injection.

Lithium is commonly used as a treatment for affective disorders in humans and as a toxin to produce conditioned taste aversions in rats. LiCl administration in rats has been correlated with activation of c-fos and cAMP-mediated gene transcription in many brain regions; however, little is known about the timing or duration of gene activation. We hypothesized that c-fos gene transcription is rapidly stimulated by LiCl, followed later by the expression of the inducible cAMP early repressor (ICER) transcription factor, a negative modulator of cAMP-mediated gene transcription. By in situ hybridization, we analyzed the timecourse of c-fos and ICER mRNA expression in the central nucleus of the amygdala (CeA), the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus (SON) at seven time points (0, 0.3, 1, 3, 6, 9 and 12 h) after intraperitoneal LiCl injection (0.15 M, 12 ml/kg, 76 mg/kg). Expression of c-fos mRNA peaked between 20 min and 1 h and returned to baseline by 3 h in the CeA, PVN and SON. ICER mRNA was detected in these regions at 20 min, peaked at 1-3 h and returned to nearly baseline 9 h following LiCl injection. The time lag between c-fos mRNA expression and ICER mRNA expression within the same regions is consistent with ICER terminating c-fos gene transcription. However, no refractory period was detected for restimulation of c-fos transcription by a second injection of LiCl during the period of peak ICER mRNA expression, suggesting the involvement of other transcriptional modulators.

Impaired conditioned taste aversion learning in spinophilin knockout mice.

Plasticity in dendritic spines may underlie learning and memory. Spinophilin, a protein enriched in dendritic spines, has the properties of a scaffolding protein and is believed to regulate actin cytoskeletal dynamics affecting dendritic spine morphology. It also binds protein phosphatase-1 (PP-1), an enzyme that regulates dendritic spine physiology. In this study, we tested the role of spinophilin in conditioned taste aversion learning (CTA) using transgenic spinophilin knockout mice. CTA is a form of associative learning in which an animal rejects a food that has been paired previously with a toxic effect (e.g., a sucrose solution paired with a malaise-inducing injection of lithium chloride). Acquisition and extinction of CTA was tested in spinophilin knockout and wild-type mice using taste solutions (sucrose or sodium chloride) or flavors (Kool-Aid) paired with moderate or high doses of LiCl (0.15 M, 20 or 40 mL/kg). When sucrose or NaCl solutions were paired with a moderate dose of LiCl, spinophilin knockout mice were unable to learn a CTA. At the higher dose, knockout mice acquired a CTA but extinguished more rapidly than wild-type mice. A more salient flavor stimulus (taste plus odor) revealed similar CTA learning at both doses of LiCl in both knockouts and wild types. Sensory processing in the knockouts appeared normal because knockout mice and wild-type mice expressed identical unconditioned taste preferences in two-bottle tests, and identical lying-on-belly responses to acute LiCl. We conclude that spinophilin is a candidate molecule required for normal CTA learning.

Subdiaphragmatic vagotomy induces NADPH diaphorase in the rat dorsal motor nucleus of the vagus.

Axotomy of the vagal motor neurons by cervical vagotomy induces NADPH diaphorase staining due to increased nitric oxide synthase expression in both the rat dorsal motor nucleus and nucleus ambiguous; furthermore, cerical vagotomy leads to cell death of the dorsal motor nucleus cells. Subdiaphragmatic vagotomy axotomizes the vagal motor cells further from the brainstem than cervical vagotomy, and cuts the fibers running only to the abdominal viscera. Here we report that subdiaphragmatic vagotomy is sufficient to induce NADPH diaphorase staining in the dorsal motor nucleus but does not induce staining in the nucleus ambiguus. Because the neurons of the dorsal motor nucleus do not undergo cell death after subdiaphragmatic vagotomy and are able to re-enervate the gut, the increased nitric oxide synthase expression after distal axotomy may be related more to regeneration than degeneration.

MK801 increases feeding and decreases drinking in nondeprived, freely feeding rats.

The noncompetitive NMDA receptor antagonist MK801 has been reported to increase food intake in rats during scheduled test meals of palatable foods or after food deprivation, but not in nondeprived rats given rodent chow. To determine if MK801 has an effect on spontaneous meals, MK801 (100 microg/kg) was administered 15 min prior to dark onset to nondeprived rats maintained on powdered rodent chow, and spontaneous food and water access was measured. MK801 increased the length of the first meal and the amount of time spent feeding within the meal. Conversely, MK801 decreased the length and size of the first drinking bout. MK801 did not alter the latency to the first meal or drinking bout, nor the intervals between successive meals or bouts. The effects of MK801 on feeding and drinking bouts were partially confirmed by measuring total chow and water intake over the first 2 h of the dark period. Thus, acute MK801 can significantly alter spontaneous chow feeding and drinking in nondeprived rats when administered prior to dark onset.

The effect of food deprivation and experimental diabetes on orexin and NPY mRNA levels.

Although exogenous orexin can induce feeding, reports of increased orexin gene expression after caloric manipulations have been inconsistent. We hypothesized that orexin gene expression is increased only by extreme negative energy balance challenges. We measured hypothalamic orexin and NPY mRNA by in situ hybridization and orexin-A immunoreactivity in rats after food deprivation, streptozotocin-induced diabetes, and combined deprivation and diabetes. Neither food deprivation, nor diabetes, nor the combination affected orexin mRNA levels, although orexin-A immunoreactivity was increased by diabetes. NPY mRNA levels were increased by either treatment. These results suggest that increased orexin gene expression is not a consistent correlate of negative energy balance challenges.

Molecular neurobiology of ingestive behavior.

The concepts and tools of molecular biology may be applied to almost any component of the animal involved in ingestion, but two categories of model system are particularly relevant for molecular analysis: homeostatic regulation of neuropeptide expression in the hypothalamus and neuronal plasticity underlying persistent changes in ingestive behavior. Molecular approaches to these models are reviewed, focusing on our strategy for analyzing conditioned taste aversion learning. Three questions must be answered: Where do the long-term changes occur within the distributed neural network that mediates feeding? This answer reveals the site of neuronal restructuring mediated by gene expression. When does the transition occur from short-term expression to long-term persistence of the change in behavior? This transition reveals the critical time of gene expression. What genes are expressed during the change in behavior? The expression of thousands of genes in discrete subpopulations of cells is likely to be required during critical periods of neuronal restructuring. The identification of these genes is a general challenge for molecular neurobiology. The analysis of ingestive behavior can profit from molecular tools, but ingestion also provides informative models that elucidate the principles of time- and neuron-specific gene expression mediating complex behaviors.

Spontaneous meal patterns in female rats with and without access to running wheels.

Rats display strong behavioral rhythms during the ovarian cycle. During estrus, food intake is minimal due to a decrease in meal size, and locomotor activity is maximal. To investigate how activity influences feeding patterns across the ovarian cycle, we used a computerized system to monitor spontaneous meal patterns in intact, cycling female rats with and without access to running wheels. We found that running wheel access decreased dark meal frequency, increased dark meal size, and increased 24-h water intake during each phase of the ovarian cycle. In contrast, body weight, 24-h food intake, and the ovarian rhythms of reduced food intake, meal size, and body weight during estrus were not affected by running wheel access. In particular, the reduction in food intake during estrus was due to a selective reduction in dark meal size, not dark meal frequency, and this occurred independent of wheel access. These data indicate that estrus-related changes in spontaneous meal patterns and locomotor activity are independently controlled and that the reduction in food intake during estrus involves a selective change in the neurobiological controls of meal size.

c-Fos induction in visceral and vestibular nuclei of the rat brain stem by a 9.4 T magnetic field.

Recently, it has been shown that rats placed in a 9.4T static magnetic field for 30 min after drinking a glucose-saccharin solution develop a conditioned taste aversion (CTA) to glucose-saccharin. We sought to identify brain stem regions that are activated by the 9.4 T magnetic field exposure using c-Fos immunohistochemistry. Rats were restrained in a 9.4 T magnet for 30 min; sham-exposed rats were restrained but not exposed to the magnetic field. The magnetic field induced significantly more c-Fos-positive cells than sham treatment in the solitary tract, parabrachial, medial vestibular, prepositus, and supragenualis nuclei. These results suggest that magnetic field exposure causes neural activation in visceral and vestibular nuclei that may promote CTA learning.

Neuropeptide Y overexpression in the preweanling Zucker (fa/fa) rat.

Hypothalamic preproNPY overexpression in the Zucker fatty (fa/fa) rat was examined. In situ hybridization was used to determine the relative level of preproNPY mRNA in the arcuate nucleus of +/+, +/fa, and fa/fa pups aged postnatal day 2 (P2), 5, 9, 12, or 25. The relative optical density (ROD) of probe hybridization in the arcuate, the area of hybridization (A), and the product of ROD x A (a measure of total arcuate preproNPY mRNA hybridization) were measured. Values were normalized to the mean +/fa value within each litter. Initial analysis showed that preproNPY mRNA hybridization (ROD x A) in fa/fa pups was significantly higher than +/fa and +/+ pups on P9, 12, and 25, and significantly higher than +/fa on P5. No significant difference between lean (+/+ and +/fa) genotypes, however, were observed at any age tested. Values from the lean genotypes were, therefore, pooled, and data were normalized to the mean value of lean animals for analysis. This analysis revealed that preproNPY mRNA hybridization in fa/fa pups was higher than lean littermates as early as P2.

Gastrin-releasing peptide suppresses independent but not intraoral intake.

Independent and intraoral intake tests have been used to separate the effects of various substances on the appetitive and consummatory phases of ingestive behavior. This study compared the ability of gastrin-releasing peptide1-27 (GRP) to suppress intraoral intake of nutrient solutions versus independent intake of the same solutions from a bottle. In a series of experiments, adult male Sprague Dawley rats implanted with anterior sublingual chronic intraoral catheters were injected intraperitoneally with saline control or 28 microg/kg GRP before 20-min intraoral and 20-min one-bottle intake tests of a sucrose (0.1 M) and milk solution (1.2 kcal/ml). GRP potently reduced independent intake of both sucrose and milk from a bottle but had no significant effect on intraoral intake of either solution. From these results, we conclude that GRP affects appetitive-related aspects of the feeding process to reduce food intake.

Rapid, labile, and protein synthesis-independent short-term memory in conditioned taste aversion.

Short-term memory is a rapid, labile, and protein-synthesis-independent phase of memory. The existence of short-term memory in conditioned taste aversion (CTA) learning has not been demonstrated formally. To determine the earliest time at which a CTA is expressed, we measured intraoral intake of sucrose at 15 min, 1 hr, 6 hr, or 48 h after contingent pairing of an intraoral infusion of 5% sucrose (6.6 ml over 6 min) and toxic lithium chloride injection (76 mg/kg). Rats were implanted with intraoral catheters to allow presentation of taste solutions at arbitrary times. Intraoral intake was measured under conditions of long-delay, single-trial learning typical of CTA. Rats decreased intraoral intake of sucrose at 15 min after contingent pairing of sucrose and LiCl, but not after noncontingent LiCl or sucrose. Thus CTA learning can be expressed rapidly. To determine if short-term CTA memory is labile and decays in the absence of long-term memory, we measured intraoral intake of sucrose after pairing sucrose with low doses of LiCl. Rats received an intraoral infusion of 5% sucrose (6 ml/6 min); 30 min later LiCl was injected at three different doses (19, 38, or 76 mg/kg). A second intraoral infusion of sucrose was administered 15 min, 1 hr, 3 hr, 4.5 hr, 6 hr, or 48 hr later. The formation of long-term CTA memory was dependent on the dose of LiCl paired with sucrose during acquisition. Low doses of LiCl induced a CTA that decayed within 6 hr after pairing. Central administration of the protein synthesis inhibitor cycloheximide prior to LiCl injection blocked long-term CTA expression at 6 and 48 hr, but not short-term CTA expression at 1 hr. Thus, short-term memory for CTA learning exists that is acquired rapidly and independent of protein synthesis, but labile in the absence of long-term memory formation.

Ontogeny of neuropeptide Y expression in response to deprivation in lean Zucker rat pups.

Hypothalamic neuropeptide Y (NPY) activity is believed to play an important role in the response to food deprivation in adult rats. Little is known, however, about the role of the hypothalamic NPY system in the control of food intake in the preweanling rat. To address this issue, we examined the effect of deprivation on arcuate nucleus preproNPY expression in lean Zucker rat pups, using in situ hybridization. PreproNPY expression within the arcuate nucleus was localized to cells in the medial portion. Twenty-four hours of food, water, and maternal deprivation significantly increased the relative abundance of preproNPY mRNA in pups on postnatal day (P) 2, P9, P12, and P15 by 14-31%. This response, however, was not observed on P5. The absence of an effect on P5 and the magnitude of the response at the other ages tested were not correlated with the amount of weight lost during deprivation.

Neuropeptide Y mRNA and serotonin innervation in the arcuate nucleus of anorexia mutant mice.

The anorexia (anx) mutation causes reduced food intake in preweanling mice, resulting in death from starvation within 3-4 weeks. In wild-type rodents, starvation induces increased neuropeptide Y (NPY) mRNA levels in the arcuate nucleus that promotes compensatory hyperphagia. Despite severely decreased body weight and food intake at 3-weeks age, anx/anx mice do not show elevated NPY mRNA levels in the hypothalamic arcuate nucleus compared to wild-type/heterozygous littermates. The NPY mRNA levels can be upregulated in normal mice at this chronological age, because 24-h food deprivation increased arcuate NPY mRNA in wild-type littermates. The unresponsiveness of NPY expression in the arcuate of anx/anx mice was paralleled by serotonergic hyperinnervation of the arcuate nucleus, comparable to the serotonergic hyperinnervation previously reported in the rest of the anx/anx brain. This result is consistent with the hypothesis that wasting disorders are accompanied by disregulation of NPY mRNA expression in the arcuate nucleus, and suggests that reduced food intake, the primary behavioral phenotype of the anx/anx mouse, may be the result of altered hypothalamic mechanisms that normally regulate feeding.

Differential expression of monoamine oxidase A, serotonin transporter, tyrosine hydroxylase and norepinephrine transporter mRNA by anorexia mutation and food deprivation.

The Anorexia (anx) mutation causes reduced food intake in preweanling mice, resulting in death from starvation within 3-4 weeks. We have found serotonin (5HT) hyperinnervation in the anx brain; altered noradrenergic (NE) innervation may also mediate eating disorders. We examined the expression of synthetic or catabolic monoamine enzyme genes in brainstem nuclei: serotonin transporter (5HTT) and monoamine oxidase A (MAOA) in the raphe nuclei (RN), and MAOA, norepinephrine transporter (NET), and tyrosine hydroxylase (TH) in the locus ceruleus (LC). We compared 3-week old anx with control and 24-h food-deprived wildtype littermates using in situ hybridization to measure mRNA levels by quantitative autoradiography. The anx mutation was correlated with decreased MAOA mRNA in the LC (but not RN), decreased 5HTT mRNA in the RN, and a trend towards lower NET mRNA in the LC. Food deprivation decreased MAOA mRNA in the LC (but not RN), increased TH mRNA in the LC, and did not alter NET or 5HTT mRNA levels. Thus, the effect of the anx mutation on MAOA expression in the LC paralleled the effect of food-deprivation, but the anx mutation and food-deprivation had differential effects on the expression of TH, NET, and 5HTT genes. Decreased 5HTT expression in the anx RN is consistent with upregulation of serotonergic neurotransmission that may accompany 5HT hyperinnervation. Central NE levels or innervation may be altered in anx mice by decreased expression of NET and MAOA and a lack of TH upregulation induced by food deprivation as in wild-type mice.

c-fos-like immunoreactivity in the subfornical organ and nucleus of the solitary tract following salt intake by sodium-depleted rats.

Acute sodium depletion by furosemide induces a robust salt appetite in the rat which is satiated rapidly by ingestion of sodium chloride (salt) solutions. To identify neuronal populations activated by sodium depletion and by salt intake, we quantified c-fos-like immunoreactivity (c-FLI) in the subfornical organ (SFO) and nucleus of the solitary tract (NTS) after sodium depletion and at time intervals from 30 min to 12 h after 1 h of access to 0.3 M NaCl. Rats drank 10+/-1.6 mL over 1 h, with most of the intake occurring by 30 min. Increased numbers of c-FLI-positive cells were observed in the SFO 24 h after sodium depletion; c-FLI remained elevated for 90 min after 0.3 M NaCl intake and then declined until the number of c-FLI-positive cells at 12 h was not significantly different from mock-depleted levels. Sodium depletion alone did not significantly elevate c-FLI in the NTS, but the number of c-FLI-positive nuclei in the NTS was significantly increased after 0.3 M NaCl intake. The cellular location and temporal pattern of c-FLI expression are consistent with activation of neural circuitry sensitive to humoral, gustatory, and postingestive stimuli accompanying sodium depletion and 0.3 M NaCl ingestion. c-FLI in the SFO and NTS may serve as quantifiable markers in the central nervous system of the state of sodium depletion and of ingestive (orosensory and gastrointestinal) sensory stimulation, respectively.

Intracerebroventricular angiotensin II increases intraoral intake of water in rats.

Ad lib and intraoral intake tests can separate the effects of drugs on the appetitive and consummatory phases of ingestive behavior. Central angiotensin II increases ad lib intake from water bottles, but its effect on intraoral intake has not been examined. Rats with both lateral intracerebroventricular (i.c.v.) cannulas and intraoral catheters were given angiotensin II (100 ng/5 microliters i.c.v.) followed by a 10-min intraoral infusion of water. Angiotensin II increased intraoral intake and increased ad lib water intake from bottles after the intraoral test. Thus angiotensin II increases water intake during both appetitive and consummatory phases of drinking.

High lick rate is maintained throughout spontaneous liquid meals in freely feeding rats.

To investigate the microstructure of spontaneous meals in freely feeding rats, 16 adult male Sprague Dawley rats were housed individually in custom-designed lickometer cages and maintained on a milk diet. Licks were recorded over 23 h at millisecond accuracy via a computer-controlled lickometer. Analysis of lick data revealed an average of about 12 discrete meals/day occurring mainly during the dark phase. The most striking feature of both dark and light meals was the maintenance of a high initial rate of licking until an abrupt decline at the end of the meal. This pattern of licking is very different from the exponential decay of lick rate reported in scheduled test meals of palatable solutions. Thus, the microstructure of licking for meals is affected in an apparently fundamental way by whether a meal is scheduled or spontaneous, suggesting a basic difference in the underlying physiologic controls.