Target-specific projections of amygdala somatostatin-expressing neurons to the hypothalamus and brainstem.

Somatostatin neurons in the central nucleus of the amygdala (CeA/Sst) can be parsed into subpopulations that project either to the nucleus of the solitary tract (NST) or parabrachial nucleus (PBN). We have shown recently that inhibition of CeA/Sst-to-NST neurons increased the ingestion of a normally aversive taste stimulus, quinine HCl (QHCl). Because the CeA innervates other forebrain areas such as the lateral hypothalamus (LH) that also sends axonal projections to the NST, the effects on QHCl intake could be, in part, the result of CeA modulation of LH-to-NST neurons. To address these issues, the present study investigated whether CeA/Sst-to-NST neurons are distinct from CeA/Sst-to-LH neurons. For comparison purposes, additional experiments assessed divergent innervation of the LH by CeA/Sst-to-PBN neurons. In Sst-cre mice, two different retrograde transported flox viruses were injected into the NST and the ipsilateral LH or PBN and ipsilateral LH. The results showed that 90% or more of retrograde-labeled CeA/Sst neurons project either to the LH, NST, or PBN. Separate populations of CeA/Sst neurons projecting to these different regions suggest a highly heterogeneous population in terms of synaptic target and likely function.

Distinct Populations of Amygdala Somatostatin-Expressing Neurons Project to the Nucleus of the Solitary Tract and Parabrachial Nucleus.

Rostral forebrain structures, such as the central nucleus of the amygdala (CeA), send projections to the nucleus of the solitary tract (NST) and the parabrachial nucleus (PBN) that modulate taste-elicited responses. However, the proportion of forebrain-induced excitatory and inhibitory effects often differs when taste cell recording changes from the NST to the PBN. The present study investigated whether this descending influence might originate from a shared or distinct population of neurons marked by expression of somatostatin (Sst). In Sst-reporter mice, the retrograde tracers' cholera toxin subunit B AlexaFluor-488 and -647 conjugates were injected into the taste-responsive regions of the NST and the ipsilateral PBN. In Sst-cre mice, the cre-dependent retrograde tracers' enhanced yellow fluorescent protein Herpes Simplex Virus (HSV) and mCherry fluorescent protein HSV were injected into the NST and the ipsilateral PBN. The results showed that ~40% of CeA-to-PBN neurons expressed Sst compared with ~ 23% of CeA-to-NST neurons. For both the CeA Sst-positive and -negative populations, the vast majority projected to the NST or PBN but not both nuclei. Thus, a subset of CeA-to-NST and CeA-to-PBN neurons are marked by Sst expression and are largely distinct from one another. Separate populations of CeA/Sst neurons projecting to the NST and PBN suggest that differential modulation of taste processing might, in part, rely on differences in local brainstem/forebrain synaptic connections.

Gene Network Analysis in Amygdala following Taste Aversion Learning in Rats.

Conditioned taste aversion (CTA) is an adaptive behavior that benefits survival of animals including humans and also serves as a powerful model to study the neural mechanisms of learning. Memory formation is a necessary component of CTA learning and involves neural processing and regulation of gene expression in the amygdala. Many studies have been focused on the identification of intracellular signaling cascades involved in CTA, but not late responsive genes underlying the long-lasting behavioral plasticity. In this study, we explored in silico experiments to identify persistent changes in gene expression associated with CTA in rats. We used oligonucleotide microarrays to identify 248 genes in the amygdala regulated by CTA. Pathway Studio and IPA software analyses showed that the differentially expressed genes in the amygdala fall in diverse functional categories such as behavior, psychological disorders, nervous system development and function, and cell-to-cell signaling. Conditioned taste aversion is a complex behavioral trait which involves association of visceral and taste inputs, consolidation of taste and visceral information, memory formation, retrieval of stored information, and extinction phase. In silico analysis of differentially expressed genes is therefore necessary to manipulate specific phase/stage of CTA to understand the molecular insight.

Conditioned taste aversion dependent regulation of amygdala gene expression.

The present experiments investigated gene expression in the amygdala following contingent taste/LiCl treatment that supports development of conditioned taste aversion (CTA). The use of whole genome chips and stringent data set filtering led to the identification of 168 genes regulated by CTA compared to non-contingent LiCl treatment that does not support CTA learning. Seventy-six of these genes were eligible for network analysis. Such analysis identified "behavior" as the top biological function, which was represented by 15 of the 76 genes. These genes included several neuropeptides, G protein-coupled receptors, ion channels, kinases, and phosphatases. Subsequent qRT-PCR analyses confirmed changes in mRNA expression for 5 of 7 selected genes. We were able to demonstrate directionally consistent changes in protein level for 3 of these genes; insulin 1, oxytocin, and major histocompatibility complex class I-C. Behavioral analyses demonstrated that blockade of central insulin receptors produced a weaker CTA that was less resistant to extinction. Together, these results support the notion that we have identified downstream genes in the amygdala that contribute to CTA learning.

Terminal field specificity of forebrain efferent axons to the pontine parabrachial nucleus and medullary reticular formation.

The pontine parabrachial nucleus (PBN) and medullary reticular formation (RF) are hindbrain regions that, respectively, process sensory input and coordinate motor output related to ingestive behavior. Neural processing in each hindbrain site is subject to modulation originating from several forebrain structures including the insular gustatory cortex (IC), bed nucleus of the stria terminalis (BNST), central nucleus of the amygdala (CeA), and lateral hypothalamus (LH). The present study combined electrophysiology and retrograde tracing techniques to determine the extent of overlap between neurons within the IC, BNST, CeA and LH that target both the PBN and RF. One fluorescent retrograde tracer, red (RFB) or green (GFB) latex microbeads, was injected into the gustatory PBN under electrophysiological guidance and a different retrograde tracer, GFB or fluorogold (FG), into the ipsilateral RF using the location of gustatory NST as a point of reference. Brain tissue containing each forebrain region was sectioned, scanned using a confocal microscope, and scored for the number of single and double labeled neurons. Neurons innervating the RF only, the PBN only, or both the medullary RF and PBN were observed, largely intermingled, in each forebrain region. The CeA contained the largest number of cells retrogradely labeled after tracer injection into either hindbrain region. For each forebrain area except the IC, the origin of descending input to the RF and PBN was almost entirely ipsilateral. Axons from a small percentage of hindbrain projecting forebrain neurons targeted both the PBN and RF. Target specific and non-specific inputs from a variety of forebrain nuclei to the hindbrain likely reflect functional specialization in the control of ingestive behaviors.

Tumor necrosis factor-α regulates distinct molecular pathways and gene networks in cultured skeletal muscle cells.

BACKGROUND: Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-α (TNF-α) is one of the most important muscle-wasting cytokine, elevated levels of which cause significant muscular abnormalities. However, the underpinning molecular mechanisms by which TNF-α causes skeletal muscle wasting are less well-understood. METHODOLOGY/PRINCIPAL FINDINGS: We have used microarray, quantitative real-time PCR (QRT-PCR), Western blot, and bioinformatics tools to study the effects of TNF-α on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line). Microarray analyses of C2C12 myotubes treated with TNF-α (10 ng/ml) for 18h showed differential expression of a number of genes involved in distinct molecular pathways. The genes involved in nuclear factor-kappa B (NF-kappaB) signaling, 26s proteasome pathway, Notch1 signaling, and chemokine networks are the most important ones affected by TNF-α. The expression of some of the genes in microarray dataset showed good correlation in independent QRT-PCR and Western blot assays. Analysis of TNF-treated myotubes showed that TNF-α augments the activity of both canonical and alternative NF-κB signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-α affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-ß signaling. Furthermore, TNF-α was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes. CONCLUSIONS: TNF-α regulates the expression of multiple genes involved in various toxic pathways which may be responsible for TNF-induced muscle loss in catabolic conditions. Our study suggests that TNF-α activates both canonical and alternative NF-κB signaling pathways in a time-dependent manner in skeletal muscle cells. The study provides novel insight into the mechanisms of action of TNF-α in skeletal muscle cells.

Amygdalofugal influence on processing of taste information in the nucleus of the solitary tract of the rat.

Previous studies have shown that corticofugal input to the first central synapse of the ascending gustatory system, the nucleus of the solitary tract (NST), can alter the way taste information is processed. Activity in other forebrain structures, such as the central nucleus of the amygdala (CeA), similarly influence activation of NST taste cells, although the effects of amygdalofugal input on neural coding of taste information is not well understood. The present study examined responses of 110 NST neurons to 15 taste stimuli before, during, and after electrical stimulation of the CeA in rats. The taste stimuli consisted of different concentrations of NaCl (0.03, 0.1, 0.3 M), sucrose (0.1, 0.3, 1.0 M), citric acid (0.005, 0.01 M), quinine HCl (0.003, 0.03 M), and 0.03 M MSG, 0.1 M KCl, as well as 0.1 M NaCl, 0.01 M citric acid, and 0.03 M MSG mixed with 10 muM amiloride. In 66% of NST cells sampled (73/110) response rates to the majority of effective taste stimuli were either inhibited or augmented. Nevertheless, the magnitude of effect across stimuli was often differential, which provides a neurophysiological mechanism to alter neural coding. Subsequent analysis of across-unit patterns showed that amygdalofugal input plays a role in shaping spatial patterns of activation and could potentially influence the perceptual similarity and/or discrimination of gustatory stimuli by altering this feature of neural coding.

Genomic profiling of messenger RNAs and microRNAs reveals potential mechanisms of TWEAK-induced skeletal muscle wasting in mice.

BACKGROUND: Skeletal muscle wasting is a devastating complication of several physiological and pathophysiological conditions. Inflammatory cytokines play an important role in the loss of skeletal muscle mass in various chronic diseases. We have recently reported that proinflammatory cytokine TWEAK is a major muscle-wasting cytokine. Emerging evidence suggests that gene expression is regulated not only at transcriptional level but also at post-transcriptional level through the expression of specific non-coding microRNAs (miRs) which can affect the stability and/or translation of target mRNA. However, the role of miRs in skeletal muscle wasting is unknown. METHODOLOGY/PRINCIPAL FINDINGS: To understand the mechanism of action of TWEAK in skeletal muscle, we performed mRNA and miRs expression profile of control and TWEAK-treated myotubes. TWEAK increased the expression of a number of genes involved in inflammatory response and fibrosis and reduced the expression of few cytoskeletal gene (e.g. Myh4, Ankrd2, and TCap) and metabolic enzymes (e.g. Pgam2). Low density miR array demonstrated that TWEAK inhibits the expression of several miRs including muscle-specific miR-1-1, miR-1-2, miR-133a, miR-133b and miR-206. The expression of a few miRs including miR-146a and miR-455 was found to be significantly increased in response to TWEAK treatment. Ingenuity pathway analysis showed that several genes affected by TWEAK are known/putative targets of miRs. Our cDNA microarray data are consistent with miRs profiling. The levels of specific mRNAs and miRs were also found to be similarly regulated in atrophying skeletal muscle of transgenic mice (Tg) mice expressing TWEAK. CONCLUSIONS/SIGNIFICANCE: Our results suggest that TWEAK affects the expression of several genes and microRNAs involved in inflammatory response, fibrosis, extracellular matrix remodeling, and proteolytic degradation which might be responsible for TWEAK-induced skeletal muscle loss.

Terminal field specificity of forebrain efferent axons to brainstem gustatory nuclei.

Rostral forebrain structures like the gustatory cortex (GC), bed nucleus of the stria terminalis (BNST), central nucleus of the amygdala (CeA), and lateral hypothalamus (LH) send projections to the nucleus of solitary tract (NST) and the parabrachial nucleus (PBN) that modulate taste-elicited responses. However, the proportion of forebrain-induced excitatory and inhibitory effects often differs when taste cell recording changes from the NST to the PBN. The present study investigated whether this descending influence originates from a shared or distinct population of forebrain neurons. Under electrophysiological guidance, the retrograde tracers fast blue (FB) and fluorogold (FG) or green (GFB) and red (RFB) fluorescent latex microbeads were injected iontophoretically or by pressure pulses (10 ms at 20 psi) into the taste-responsive regions of the NST and the ipsilateral PBN in six rats. Seven days later, the animals were euthanized and tissue sections containing the LH, CeA, BNST, and GC were processed for co-localization of FB and FG or GFB and RFB. The results showed that the CeA is the major source of input to the NST (82.3+/-7.6 cells/section) and the PBN (76.7+/-11.5), compared to the BNST (31.8+/-4.5; 37.0+/-4.8), the LH (35.0+/-5.4; 33.6+/-5.7), and the GC (27.5+/-4.0; 29.0+/-4.6). Of the total number of retrogradely labeled cells, the incidence of tracer co-localization was 17+/-3% in the GC, 17+/-2% in the CeA, 15+/-3% in the BNST and 16+/-1% in the LH. Thus, irrespective of forebrain source the majority of descending input to the gustatory NST and PBN originates from distinct neuronal populations. This arrangement provides an anatomical substrate for differential modulation of taste processing in the first and second central relays of the ascending gustatory system.

Cracking taste codes by tapping into sensory neuron impulse traffic.

Insights into the biological basis for mammalian taste quality coding began with electrophysiological recordings from "taste" nerves and this technique continues to produce essential information today. Chorda tympani (geniculate ganglion) neurons, which are particularly involved in taste quality discrimination, are specialists or generalists. Specialists respond to stimuli characterized by a single taste quality as defined by behavioral cross-generalization in conditioned taste tests. Generalists respond to electrolytes that elicit multiple aversive qualities. Na(+)-salt (N) specialists in rodents and sweet-stimulus (S) specialists in multiple orders of mammals are well characterized. Specialists are associated with species' nutritional needs and their activation is known to be malleable by internal physiological conditions and contaminated external caloric sources. S specialists, associated with the heterodimeric G-protein coupled receptor T1R, and N specialists, associated with the epithelial sodium channel ENaC, are consistent with labeled line coding from taste bud to afferent neuron. Yet, S-specialist neurons and behavior are less specific than T1R2-3 in encompassing glutamate and E generalist neurons are much less specific than a candidate, PDK TRP channel, sour receptor in encompassing salts and bitter stimuli. Specialist labeled lines for nutrients and generalist patterns for aversive electrolytes may be transmitting taste information to the brain side by side. However, specific roles of generalists in taste quality coding may be resolved by selecting stimuli and stimulus levels found in natural situations. T2Rs, participating in reflexes via the glossopharynygeal nerve, became highly diversified in mammalian phylogenesis as they evolved to deal with dangerous substances within specific environmental niches. Establishing the information afferent neurons traffic to the brain about natural taste stimuli imbedded in dynamic complex mixtures will ultimately "crack taste codes."

Gustatory hedonic value: potential function for forebrain control of brainstem taste processing.

Among well-nourished populations, eating beyond homeostatic needs when presented with caloric-dense palatable food evidences the assertion that an increasing proportion of consumption is driven by pleasure, not just by the need for calories. This presents a major health crisis because the affective component of foods constitutes a behavioral risk factor that promotes over consumption [Sorensen, L.B., Moller, P., Flint, A., Martens, M., Raben, A., 2003. Effect of sensory perception of foods on appetite and food intake: a review of studies on humans. Int. J. Obes. Relat. Metab. Disord. 27, 1152-1166; Yeomans, M.R., Blundell, J.E., Leshem, M., 2004. Palatability: response to nutritional need or need-free stimulation of appetite? Br. J. Nutr. 92 (Suppl. 1), S3-S14]. Overweight or obese individuals have an increased risk of developing hypertension, stroke, heart disease, chronic musculoskeletal problems, type-2 diabetes, and certain types of cancers [Hill, J.O., Catenacci, V., Wyatt, H.R., 2005. Obesity: overview of an epidemic. Psychiatr. Clin. N. Am. 28, 1-23, vii]. The etiology of obesity is complex involving genetic, metabolic, and behavioral factors, but ultimately results from long-term energy imbalance. Evidence indicates that learned and some forms of unlearned control of ingestive behavior driven by palatability (i.e. hedonic value) are critically dependent on reciprocal interactions between brainstem gustatory nuclei and the ventral forebrain. This review discusses the current understanding of centrifugal control of taste processing in subcortical gustatory nuclei and the potential role of such modulation in hedonic responding.

Expression of Fos during sham sucrose intake in rats with central gustatory lesions.

For humans and rodents, ingesting sucrose is rewarding. This experiment tested the prediction that the neural activity produced by sapid sucrose reaches reward systems via projections from the pons through the limbic system. Gastric cannulas drained ingested fluid before absorption. For 10 days, the rats alternated an hour of this sham ingestion between sucrose and water. On the final test day, half of them sham drank water and the other half 0.6 M sucrose. Thirty minutes later, the rats were killed and their brains immunohistochemically stained for Fos. The groups consisted of controls and rats with excitotoxic lesions in the gustatory thalamus (TTA), the medial (gustatory) parabrachial nucleus (PBN), or the lateral (visceral afferent) parabrachial nucleus. In controls, compared with water, sham ingesting sucrose produced significantly more Fos-positive neurons in the nucleus of the solitary tract, PBN, TTA, and gustatory cortex (GC). In the ventral forebrain, sucrose sham licking increased Fos in the bed nucleus of the stria terminalis, central nucleus of amygdala, and the shell of nucleus accumbens. Thalamic lesions blocked the sucrose effect in GC but not in the ventral forebrain. After lateral PBN lesions, the Fos distributions produced by distilled H(2)O or sucrose intake did not differ from controls. Bilateral medial PBN damage, however, eliminated the sucrose-induced Fos increase not only in the TTA and GC but also in the ventral forebrain. Thus ventral forebrain areas associated with affective responses appear to be activated directly by PBN gustatory neurons rather than via the thalamocortical taste system.

Forebrain neurons that project to the gustatory parabrachial nucleus in rat lack glutamic acid decarboxylase.

Evidence suggests that GABA might mediate the inhibitory influence of centrifugal inputs on taste-evoked responses in the parabrachial nucleus (PBN). Previous studies show that activation of the gustatory cortex (GC), bed nucleus of the stria terminalis (BNST), central nucleus of the amygdala (CeA), and lateral hypothalamus (LH) inhibits PBN taste responses, GABAergic neurons are present in these forebrain regions, and GABA reduces the input resistance of PBN neurons. The present study investigated the expression of glutamic acid decarboxylase immunoreactivity (GAD_67 ir) in GC, BNST, CeA, and LH neurons that project to the PBN in rats. After anesthesia (50 mg/kg ip Nembutal), injections of the retrograde tracer Fluorogold (FG) were made in the physiologically defined gustatory PBN. Brain tissue containing the above forebrain structures was processed and examined for FG and GAD_67 ir. Similar to previous studies, each forebrain site contained retrogradely labeled neurons. Our results suggest further that the major source of input to the PBN taste region is the CeA (608 total cells) followed by GC (257 cells), LH (106 cells), and BNST (92 cells). This suggests a differential contribution to centrifugal control of PBN taste processing. We further show that despite the presence of GAD_67 neurons in each forebrain area, colocalization was extremely rare, occurring only in 3 out of 1,063 FG-labeled cells. If we assume that the influence of centrifugal input is mediated by direct projections to the gustatory region of the PBN, then GABAergic forebrain neurons apparently are not part of this descending pathway.

Central gustatory lesions and learned taste aversions: unconditioned stimuli.

The efficacy of two different unconditioned stimuli (US) in producing conditioned taste aversion (CTA) was tested in rats after bilateral ibotenic acid (IBO) lesions of the gustatory nucleus of thalamus (TTAx) and the medial and lateral parabrachial nuclei (mPBNx, lPBNx). An initial study determined an equivalent dose for the two USs, LiCl and cyclophosphamide (CY), using non-lesioned rats. Subsequently, using a separate set of lesioned animals and their sham controls (SHAM), injections of CY were paired 3 times with one of two taste stimuli (CSs), 0.1 M NaCl for half the rats in each group, 0.2 M sucrose for the other half. After these conditioning trials, the CS was presented twice more without the US, first in a 1-bottle test, then in a 2-bottle choice with water. The acquisition and test trials had 2 intervening water-only days to assure complete rehydration. Two weeks later, the same rats were tested again for acquisition of a CTA using LiCl as the US and the opposite CS as that used during the CY trials. The SHAM and TTAx groups learned to avoid consuming the taste associated with either CY or LiCl treatment. The two PBNx groups failed to learn an aversion regardless of the US.

A novel method for induction of salt appetite in rats.

A number of procedures exist for the experimental induction of sodium appetite. With the exception of a low sodium diet, nearly all of these methods are invasive, requiring injections, surgery, or both. In addition to stimulating intake of concentrated salt, some of them produce substantial side-effects like reduced food intake and weight gain. The present experiment was designed to evaluate the efficacy of a novel non-invasive method to induce a salt appetite. We investigated the effects of ingesting 100 microM amiloride, a diuretic and natriuretic compound, on urine quantity, electrolyte balance, 0.5 1M sodium chloride (NaCl) intake, water intake, and Na(+)-free chow intake, and weight gain in rats. A water ingestion only group served as control. Consumption of amiloride in mixture with water produced greater loss of urinary Na(+) and intake of 0.51 M NaCl compared with controls. This treatment was without effect on food intake and only modestly influenced weight gain. These results demonstrate a rapid and non-invasive method for the induction of salt appetite free of unwanted side-effects.

Furosemide-induced food avoidance: evidence for a conditioned response.

Furosemide (Furo) is a potent natriuretic drug that is often used experimentally to investigate the brain mechanisms underlying salt appetite. Within this experimental paradigm, however, Furo also has anorectic activity that has received only modest attention. In Experiment 1 we varied two things-administering a 10-mg dose of Furo in a single or a divided dose and preinjection exposure to a Na-free diet. In the 24 h after Furo, all four groups of rats reduced ingestion of Na-free diet. Both the division of the Furo dose and the preexposure to Na-free diet reduced the amount of food consumed even more than a single dose or continuous access to normal chow did. The fact that preexposure to Na-free diet increased the post-Furo anorexia implied an associative component to the phenomenon. Experiments 2 and 3 investigated the ability of Furo (2 and 10 mg) to serve as an unconditioned stimulus in taste aversion learning using 0.2 M sucrose as the conditioned stimulus. A saline (Sal) injection group served as control in both experiments. The results show that animals avoided sucrose when its ingestion was immediately followed by 10 mg Furo but not with 2 mg Furo or Sal. An aversion to sucrose did not develop when 10 mg Furo was administered the day prior to sucrose access. Thus, the suppressive effects of high-dose Furo on food intake might be due to a conditioned response.

Activity in the hypothalamus, amygdala, and cortex generates bilateral and convergent modulation of pontine gustatory neurons.

Evidence suggests that centrifugal modulation of brain stem gustatory cells might play a role in the elaboration of complex taste-guided behaviors like conditioned taste aversion and sodium appetite. We previously showed that activity in one forebrain area, the central nucleus of the amygdala (CeA), increased the chemical selectivity of taste cells in the parabrachial nucleus (PBN). The present study investigates how activity in 2 other similarly interconnected forebrain sites, the lateral hypothalamus (LH) and gustatory cortex (GC), might influence PBN gustatory processing in rats. The potential convergence of descending inputs from these sites, as well as the CeA, was also evaluated. After anesthesia (35 mg/kg Nembutal ip), 70 PBN gustatory neurons were tested before, during, and after electrical stimulation of these forebrain sites, while responding to 0.3 M sucrose, 0.1 M NaCl, 0.01 M citric acid, and 0.003 M QHCl. Although each forebrain site modulated taste-evoked responses, more PBN neurons were influenced by stimulation of the GC (67%) and CeA (73%) than of the LH (48%). Activation of cortex (71%) and amygdala (85%) most often produced inhibition, whereas inhibition and excitation occurred equally often during hypothalamic stimulation. Of the neurons tested for convergence (n = 60), 88% were influenced by > or =1 of the 3 sites. Twenty were modulated by stimulation at all 3 sites and another 17 by 2 of the 3 sites. The net effect of centrifugal modulation was to sharpen the across-stimulus response profiles of PBN cells, particular with regard to the NaCl- and citric acid-best cells.

Furosemide, sodium appetite, and ingestive behavior.

Sodium appetite is often produced experimentally by using the diuretic furosemide (Furo) to induce a rapid loss of urinary sodium. The present experiments were designed to investigate the dose-dependent relationship between renal and behavioral responses to Furo. We compared the effects of five different Furo doses (0.5, 1, 2, 6, and 10 mg) on 3% NaCl intake, water intake, Na(+)-free chow intake, urine quantity, electrolyte balance, and weight gain in rats. The Na(+) loss produced by Furo injection was dose dependent from 0.5 to 10 mg and did not change across repeated depletions. There was only a weak correspondence, however, between these dose-dependent changes in renal function and subsequent sodium appetite. This suggests that net Na(+) loss is not the only determinant of sodium intake. Moreover, at the two higher doses of Furo, both food intake and weight dropped significantly, but these did not change following the three lower ones. Given these substantial side effects, the preferred dose of Furo for inducing a salt appetite should not exceed 2.0 mg.