Early life experience shapes the functional organization of stress-responsive visceral circuits.

Emotions are closely tied to changes in autonomic (i.e., visceral motor) function, and interoceptive sensory feedback from body to brain exerts powerful modulatory control over motivation, affect, and stress responsiveness. This manuscript reviews evidence that early life experience can shape the structure and function of central visceral circuits that underlie behavioral and physiological responses to emotive and stressful events. The review begins with a general discussion of descending autonomic and ascending visceral sensory pathways within the brain, and then summarizes what is known about the postnatal development of these central visceral circuits in rats. Evidence is then presented to support the view that early life experience, particularly maternal care, can modify the developmental assembly and structure of these circuits in a way that impacts later stress responsiveness and emotional behavior. The review concludes by presenting a working hypothesis that endogenous cholecystokinin signaling and subsequent recruitment of gastric vagal sensory inputs to the caudal brainstem may be an important mechanism by which maternal care influences visceral circuit development in rat pups. Early life experience may contribute to meaningful individual differences in emotionality and stress responsiveness by shaping the postnatal developmental trajectory of central visceral circuits.

Early experience alters limbic forebrain Fos responses to a stressful interoceptive stimulus in young adult rats.

The present study examined whether manipulation of the early life experience of rat pups might alter the later ability of an interoceptive challenge to recruit central neural circuits that receive visceral sensory signals and generate stress responses. For this purpose, litters were exposed to daily maternal separation for either 15min (MS-15) or 180min (MS-180) from postnatal days (P)1 to P10. Pups in control litters were raised under standard conditions (i.e., no separations). Similar to previous reports in adult rats, adolescent rats (P35-45) with a developmental history of MS-15 displayed less anxiety-like behavior on the elevated plus maze compared to control and MS-180 rats. As young adults (P50-60), rats were anesthetized and perfused with fixative 90min after viscerosensory stimulation via lithium chloride (LiCl, 0.15M, 1% BW, i.p.) or saline control. In all three rearing groups, Fos activation within brainstem and forebrain regions of interest was significantly enhanced after LiCl vs. saline. MS-15 rats tended to display fewer LiCl-activated neurons in most brain regions compared with rats in the other two rearing groups. This trend reached significance within the dorsal bed nucleus of the stria terminalis. The ability of MS-15 to alter limbic forebrain activation in rats after an interoceptive challenge may contribute to the effect of early life experience to modulate physiological and behavioral stress responses more generally.

Pseudoreplication is a pseudoproblem.

Pseudoreplication is one of the most influential methodological issues in ecological and animal behavior research today. At its inception, the idea of pseudoreplication highlighted important concerns about the design and analysis of experiments in ecology. The doctrine purported to provide a unified view of experimental design and analysis, wherein precise criteria could be used to assess manuscripts and research proposals for acceptance or rejection. Few methodological doctrines have had as much impact as pseudoreplication, yet there has been very little critical analysis of it. In this paper, the authors extend the growing criticism of the concept of pseudoreplication. The authors argue that the core ideas behind pseudoreplication are based on a misunderstanding of statistical independence, the nature of control groups in science, and contexts of statistical inference. The authors also highlight how other areas of research have found and responded to similar issues in the design and analysis of experiments through the use of more advanced statistical methods. Ultimately, there are no universal criteria for accepting or rejecting experimental research; all research must be judged on its own merits.

An ancient black art.

The doctrine of pseudoreplication (DP) offers specific advice on how to ensure statistical independence and compute F-ratios properly when testing a null hypothesis. Our target article showed that this advice can lead to problems in experimental design and analysis. Though a few commenters attempt to defend DP, none offered substantive evidence that our modeling results were incorrect. In our response, we further highlight the complications surrounding definitions of experimental units. In particular, we show that the definition of independence assumed in DP is inconsistent with independence as defined in probability theory. We show that interconnectedness across levels of analysis is pervasive, and that no simple set of rules or procedures can help experimenters avoid this problem. We argue that the relevance or interference of a particular level of analysis can be determined only after an experiment is done. In our view, analytical methods must be designed to match experiments, the opposite of the advice offered in DP. Finally, we emphasize the weakness of null testing and the inability of p values to predict whether a result will generalize or be replicated.

Muscle anatomy is a primary determinant of muscle relaxation dynamics in the lobster (Panulirus interruptus) stomatogastric system.

We stained sarcomere thin filaments with fluorescently labeled phalloidin, measured sarcomere and muscle length, and calculated sarcomere number in pyloric and gastric mill muscles. A wide range of sarcomere lengths (3.25-12.29 microm), muscle lengths (5.9-21.1 mm), and sarcomere numbers (648-3,036) were observed. Sarcomere number differences occurred both because of changes in sarcomere length and muscle length, and sarcomere and muscle length varied independently. This independence, the wide range of sarcomere numbers present, and the muscles being all 'slow', graded muscles allowed us to use these data to test Huxley and Neidergerke's (1954) hypothesis that muscle dynamics depend on sarcomere number. The time constants of exponential fits to contraction relaxations were used to measure muscle dynamics, and comparison of theoretical predictions and experimental results quantitatively confirm the predicted dependence. The differing dynamics of the various pyloric muscles are likely functionally important, and the dependence of muscle dynamics on sarcomere number implies that sarcomere number is likely closely regulated in these muscles. The stomatogastric system may thus be an excellent model system for studying the mechanisms regulating muscle sarcomere number.

Progressive postnatal increases in Fos immunoreactivity in the forebrain and brain stem of rats after viscerosensory stimulation with lithium chloride.

Interoceptive signals have a powerful impact on the motivation and emotional learning of animals during stressful experiences. However, current insights into the organization of interoceptive pathways stem mainly from observation and manipulation of adults, and little is known regarding the functional development of viscerosensory signaling pathways. To address this, we have examined central neural activation patterns in rat pups after treatment with lithium chloride (LiCl), a malaise-inducing agent. Rat pups were injected intraperitoneally with 0.15 M LiCl or 0.15 M NaCl (2% body wt) on postnatal day (P)0, 7, 14, 21, or 28, perfused 60 to 90 min postinjection, and their brains assayed for Fos protein immunolabeling. Compared with saline treatment, LiCl increased Fos only slightly in the area postrema, nucleus of the solitary tract, and lateral parabrachial nucleus on P0. LiCl did not increase Fos above control levels in the central nucleus of the amygdala, bed nucleus of the stria terminalis (BNST), or paraventricular nucleus of the hypothalamus on P0 but did on P7 and later. Maximal Fos responses to LiCl were observed on P14 in all areas except the BNST, in which LiCl-induced Fos activation continued to increase through P28. These results indicate that central LiCl-sensitive interoceptive circuits in rats are not fully functional at birth, and show age-dependent increases in neural Fos responses to viscerosensory stimulation with LiCl.

Uncharged tRNA and sensing of amino acid deficiency in mammalian piriform cortex.

Recognizing a deficiency of indispensable amino acids (IAAs) for protein synthesis is vital for dietary selection in metazoans, including humans. Cells in the brain's anterior piriform cortex (APC) are sensitive to IAA deficiency, signaling diet rejection and foraging for complementary IAA sources, but the mechanism is unknown. Here we report that the mechanism for recognizing IAA-deficient foods follows the conserved general control (GC) system, wherein uncharged transfer RNA induces phosphorylation of eukaryotic initiation factor 2 (eIF2) via the GC nonderepressing 2 (GCN2) kinase. Thus, a basic mechanism of nutritional stress management functions in mammalian brain to guide food selection for survival.

Diets deficient in indispensable amino acids rapidly decrease the concentration of the limiting amino acid in the anterior piriform cortex of rats.

Diets deficient in an indispensable amino acid have long been known to suppress food intake in rats. Detection of dietary deficiency takes place in the anterior piriform cortex (APC). Recent studies showed that the response to amino acid deficiency takes as little as 15 min to develop, but few data exist to correlate the concentration of amino acids in the APC with this rapid response. The purpose of this study was to measure the concentration of amino acids in the APC in a behaviorally relevant time frame. Rats were preconditioned by consumption of a basal diet for 7-10 d, and then given a test diet with either a control or deficient amino acid profile. Both the threonine- and leucine-deficient diets reliably depleted threonine and leucine concentration in the APC within 30 min, respectively. The control diets and a diet lacking the dispensable amino acid glycine did not lead to amino acid depletion. In combination with previous studies, the present results show that the decrease in the concentration of indispensable amino acids in the APC may be the initial sensory signal for recognition of dietary amino acid deficiency.

Threonine-imbalanced diet alters first-meal microstructure in rats.

Diets limiting in an essential amino acid have long been known to suppress food intake. The purpose of this study was to examine the microstructure of feeding behavior of rats within the very first meal of an imbalanced diet. Rats were preconditioned for 12 days on a Baseline diet and were then given a test diet with either a corrected amino acid profile or a diet imbalanced with respect to the essential amino acid threonine. Overall, first-meal intake and first-meal duration were robustly and significantly reduced by the Imbalanced diet but not altered by the Corrected diet. The Corrected diet caused an increase in the number of feeding bouts during the first meal. The Imbalanced diet increased the duration of pauses during the first meal. Most rats in the Imbalanced group stopped eating after just 15 min of exposure to the diet, but those still eating after this time tended to have a lower rate of eating compared to those eating the Corrected diet. On the basis of these results, we conclude that changes in microstructure and meal duration contribute to the reduction in food intake upon exposure to amino-acid-deficient diets.

Threonine deprivation rapidly activates the system A amino acid transporter in primary cultures of rat neurons from the essential amino acid sensor in the anterior piriform cortex.

Omnivores show recognition of essential (indispensable) amino acid deficiency by changing their feeding behavior within 20 min, yet the cellular mechanisms of amino acid sensation in eukaryotes are poorly understood. The anterior piriform cortex (APC) of the brain in rats or its analog in birds likely houses the in vivo amino acid chemosensor. Because amino acid transporters adapt rapidly to essential amino acid deficiency in several cell models, we hypothesized that activation of electrogenic amino acid transport in APC neurons might contribute to the function of the amino acid sensor. We evaluated transport systems in primary cultures of neurons from the APC, hippocampus and cerebellum, or glia, incubated in complete or threonine-devoid (deficient) medium. After 10 min in deficient medium, uptake of threonine or a system A-selective substrate, methyl amino-isobutyric acid, was increased 60% in APC neurons only (P < 0.05). These results demonstrated upregulation of system A, an electrogenic amino acid-sodium symporter. This depletion-induced activation required sodium, intact intracellular trafficking, and phosphorylation of signal transduction-related kinases. Efflux studies showed that other transporter types were functional in the APC; they appeared to be altered dynamically in threonine-deficient cells in response to rapid increases in system A activity. The present data provided support for the chemical sensitivity of the APC and its role as the brain area housing the indispensable amino acid chemosensor. They also showed a region-specific, phosphorylation-dependent activation of the system A transporter in the brain in response to threonine deficiency.

Rats rapidly reject diets deficient in essential amino acids.

Omnivores must obtain diets balanced with respect to amino acids to support growth and protein synthesis. The standard paradigm used to study behavioral responses to amino acid deficiency combines deficient diets with dietary novelty. The objective of this study was to examine the effects of amino acid deficiency on the first meal of rats without the confounding effects of novelty. We report on a series of five studies of feeding behavior in rats. Rats were fed low protein diets for 5-7 d and then exposed to diets with and without essential amino acids. Rats consistently demonstrated recognition of essential amino acid deficiency within the first meal by a significant reduction in first meal duration, rejecting the deficient diets after just 12-16 min exposure. This is the first report of a rapid effect of amino acid-deficient diets without the confounding effects of dietary novelty.

The rapid anorectic response to a threonine imbalanced diet is decreased by injection of threonine into the anterior piriform cortex of rats.

Rats quickly recognize and reject diets deficient in an essential amino acid. The purpose of this study was to determine whether the anterior piriform cortex (APC), the site traditionally recognized as the amino acid chemosensor, plays a role in this early behavior. Rats had cannulae implanted bilaterally into the APC, and were injected with either saline vehicle or 2 nmoles of threonine (n = 6 per group). All rats were then fed a diet imbalanced with respect to threonine. The threonine-injected group had first meals of longer duration and consumed more food. These data conformed to expectations derived from earlier studies of responses to the first meal of an amino acid imbalanced diet. We conclude that the concentration of the dietary limiting amino acid in the APC regulates acceptance and rejection of amino acid deficient diets.