Jennifer M. Stratford

Chorda tympani nerve transection alters linoleic acid taste discrimination by male and female rats

Taste is intimately associated with food choice, yet little is known about the role of taste in preferences for dietary fat, a major component of many foods. We measured the taste threshold for linoleic acid (LA), an essential free fatty acid found in dietary fat, before and after bilateral transections of the chorda tympani nerve (CTX) in adult male and female rats. We conditioned a taste aversion to 88 microM LA and assessed the generalization of the aversion to lower LA concentrations to determine LA discrimination thresholds. We discovered that female rats had a lower LA discrimination threshold (approximately 2.75 microM LA) than did male rats (approximately 11 microM LA). In another set of animals, we performed CTX and found that CTX elevated LA threshold to the same level (approximately 22 microM LA) in male and female rats. Finally, we evaluated licking responses to 11, 22, 44 and 88 microM LA mixed in sucrose by male rats and ovariectomized (OVX) female rats treated with estradiol benzoate or oil vehicle. All rats increased licking to increasing LA concentrations, but OVX rats responded to a lower LA concentration (22 microM) than did males (44 microM) in 10-s trials. However, estradiol did not affect this outcome. Collectively, these experiments show that male and female rats use taste to discriminate LA and that the chorda tympani nerve, which innervates taste buds on the anterior tongue, plays a role in this discrimination. Furthermore, sex differences in fat preferences may depend on differences in fatty acid taste thresholds as well as on the taste stimuli with which fat is combined.

Central Representation of Postingestive Chemosensory Cues in Mice That Lack the Ability to Taste

The gustatory nerves of mice lacking P2X2 and P2X3 purinergic receptor subunits (P2X-dblKO) are unresponsive to taste stimulation (Finger et al., 2005). Surprisingly, P2X-dblKO mice show residual behavioral responses to concentrated tastants, presumably via postingestive detection. Therefore, the current study tested whether postingestive signaling is functional in P2X-dblKO mice and if so, whether it activates the primary viscerosensory nucleus of the medulla, the nucleus of the solitary tract (nTS). Like WT animals, P2X-dblKO mice learned to prefer a flavor paired with 150 mm monosodium glutamate (MSG) over a flavor paired with water. This preference shows that, even in the absence of taste sensory input, postingestive cues are detected and associated with a flavor in P2X-dblKO mice. MSG-evoked neuronal activation in the nTS was measured by expression of the immediate early gene c-Fos [c-Fos-like immunoreactivity (Fos-LI)]. In rostral, gustatory nTS, P2X-dblKO animals, unlike WT animals, showed no taste quality-specific labeling of neurons. Furthermore, MSG-evoked Fos-LI was significantly less in P2X-dblKO mice compared with WT animals. In contrast, in more posterior, viscerosensory nTS, MSG-induced Fos-LI was similar in WT and P2X-dblKO mice. Together, these results suggest that P2X-dblKO mice can form preferences based on postingestive cues and that postingestive detection of MSG does not rely on the same purinergic signaling that is crucial for taste.

Residual Chemoresponsiveness to Acids in the Superior Laryngeal Nerve in ''Taste-Blind'' (P2X2/P2X3 Double-KO) Mice

Mice lacking both the P2X2 and the P2X3 purinergic receptors (P2X-dblKO) exhibit loss of responses to all taste qualities in the taste nerves innervating the tongue. Similarly, these mice exhibit a near total loss of taste-related behaviors in brief access tests except for a near-normal avoidance of acidic stimuli. This persistent avoidance of acids despite the loss of gustatory neural responses to sour was postulated to be due to continued responsiveness of the superior laryngeal (SL) nerve. However, chemoresponses of the larynx are attributable both to taste buds and to free nerve endings. In order to test whether the SL nerve of P2X-dblKO mice remains responsive to acids but not to other tastants, we recorded responses from the SL nerve in wild-type (WT) and P2X-dblKO mice. WT mice showed substantial SL responses to monosodium glutamate, sucrose, urea, and denatonium-all of which were essentially absent in P2X-dblKO animals. In contrast, the SL nerve of P2X-dblKO mice exhibited near-normal responses to citric acid (50 mM) although responsiveness of both the chorda tympani and the glossopharyngeal nerves to this stimulus were absent or greatly reduced. These results are consistent with the hypothesis that the residual avoidance of acidic solutions by P2X-dblKO mice may be attributable to the direct chemosensitivity of nerve fibers innervating the laryngeal epithelium and not to taste.

Linoleic acid increases chorda tympani nerve responses to and behavioral preferences for monosodium glutamate by male and female rats

Previous studies suggest that the chorda tympani nerve (CT) is important in transmitting fat taste information to the central nervous system. However, the contribution of the CT in this process may depend upon the presence of other taste stimuli and/or differ in males and females. Accordingly, the present study investigated the role of the CT in free fatty acid taste processing by examining electrophysiological activity of the CT in response to the free fatty acid linoleic acid (LA), as well as by measuring behavioral responses to LA-taste mixtures. We recorded whole nerve responses from the CT in response to lingual application of LA with or without monosodium glutamate (MSG) in anesthetized male and female rats. In addition, we examined preferences for MSG + LA taste mixtures in behavioral tests. Although lingual application of LA alone did not produce CT whole nerve responses, coapplication of LA and MSG elicited greater CT responses than did MSG alone. These findings were paralleled by greater preferences for MSG + LA taste mixtures than for MSG alone. In both cases, the effect was particularly pronounced in male rats. Thus LA enhances CT activity and behavioral responses to LA + MSG taste mixtures, although there are sex differences in the effects. These results suggest that CT input is important in mediating behavioral responses to fat taste, but the effects depend upon other taste stimuli and differ in males and females.

A Relationship between Reduced Nucleus Accumbens Shell and Enhanced Lateral Hypothalamic Orexin Neuronal Activation in Long-Term Fructose Bingeing Behavior

Fructose accounts for 10% of daily calories in the American diet. Fructose, but not glucose, given intracerebroventricularly stimulates homeostatic feeding mechanisms within the hypothalamus; however, little is known about how fructose affects hedonic feeding centers. Repeated ingestion of sucrose, a disaccharide of fructose and glucose, increases neuronal activity in hedonic centers, the nucleus accumbens (NAc) shell and core, but not the hypothalamus. Rats given glucose in the intermittent access model (IAM) display signatures of hedonic feeding including bingeing and altered DA receptor (R) numbers within the NAc. Here we examined whether substituting fructose for glucose in this IAM produces bingeing behavior, alters DA Rs and activates hedonic and homeostatic feeding centers. Following long-term (21-day) exposure to the IAM, rats given 8–12% fructose solutions displayed fructose bingeing but unaltered DA D1R or D2R number. Fructose bingeing rats, as compared to chow bingeing controls, exhibited reduced NAc shell neuron activation, as determined by c-Fos-immunoreactivity (Fos-IR). This activation was negatively correlated with orexin (Orx) neuron activation in the lateral hypothalamus/perifornical area (LH/PeF), a brain region linking homeostatic to hedonic feeding centers. Following short-term (2-day) access to the IAM, rats exhibited bingeing but unchanged Fos-IR, suggesting only long-term fructose bingeing increases Orx release. In long-term fructose bingeing rats, pretreatment with the Ox1R antagonist SB-334867 (30 mg/kg; i.p.) equally reduced fructose bingeing and chow intake, resulting in a 50% reduction in calories. Similarly, in control rats, SB-334867 reduced chow/caloric intake by 60%. Thus, in the IAM, Ox1Rs appear to regulate feeding based on caloric content rather than palatability. Overall, our results, in combination with the literature, suggest individual monosaccharides activate distinct neuronal circuits to promote feeding behavior. Specifically, long-term fructose bingeing activates a hyperphagic circuit composed in part of NAc shell and LH/PeF Orx neurons.

Saliva and other taste stimuli are important for gustatory processing of linoleic acid

Paradoxically, bilateral transection of the chorda tympani nerve (CTX) raises the taste discrimination threshold for the free fatty acid, linoleic acid (LA), yet the chorda tympani nerve (CT) is unresponsive to lingual application of LA alone. LA may require a background of saliva to activate taste cells, since CTX decreases saliva production through denervation of the submaxillary and sublingual salivary glands. To assess the role of saliva, we measured LA taste discrimination thresholds for animals whose submaxillary and sublingual salivary glands were removed and also recorded CT responses to LA mixed in artificial saliva. Partial desalivation shifted LA discrimination thresholds from between 5.5 and 11 microM to between 11 and 22 microM. However, this effect was not as pronounced as previously seen with CTX animals. Surprisingly, the CT was unresponsive to LA mixed with artificial saliva, suggesting that artificial saliva may lack components necessary for LA taste. Additionally, fats may primarily enhance other tastes. We previously reported that LA increases CT responses to monosodium glutamate (MSG). Thus we also recorded CT whole nerve responses to taste mixtures of LA and sodium chloride (NaCl), sucrose (SUC), citric acid (CA), or quinine hydrochloride (QHCl) in anesthetized rats. We found that LA increased CT responses to NaCl but did not alter CT responses to SUC, CA, and QHCl. Thus CT recordings either lack the sensitivity to detect small changes to SUC, CA, and QHCl or LA may affect CT responses to MSG and NaCl only, perhaps by specifically modulating gustatory processing of Na(+).

Immunocytochemical organization and sour taste activation in the rostral nucleus of the solitary tract of mice.

Sensory inputs from the oropharynx terminate in both the trigeminal brainstem complex and the rostral part of the nucleus of the solitary tract (nTS). Taste information is conveyed via the facial and glossopharyngeal nerves, while general mucosal innervation is carried by the trigeminal and glossopharyngeal nerves. In contrast, the caudal nTS receives general visceral information largely from the vagus nerve. Although the caudal nTS shows clear morphological and molecularly delimited subdivisions, the rostral part does not. Thus, linking taste‐induced patterns of activity to morphological subdivisions in the nTS is challenging. To test whether molecularly defined features of the rostral nTS correlate with patterns of taste‐induced activity, we combined immunohistochemistry for markers of various visceral afferent and efferent systems with c‐Fos–based activity maps generated by stimulation with a sour tastant, 30 mM citric acid. We further dissociated taste‐related activity from activity arising from acid‐sensitive general mucosal innervation by comparing acid‐evoked c‐Fos in wild‐type and “taste blind” P2X2/P2X3 double knockout (P2X‐dbl KO) mice. In wild‐type mice, citric acid stimulation evoked significant c‐Fos activation in the central part of the rostral nTS—activity that was largely absent in the P2X‐dbl KO mice. P2X‐dbl KO mice, like wild‐type mice, did exhibit acid‐induced c‐Fos activity in the dorsomedial trigeminal brainstem nucleus situated laterally adjacent to the rostral nTS. This dorsomedial nucleus also showed substantial innervation by trigeminal nerve fibers immunoreactive for calcitonin gene‐related peptide (CGRP), a marker for polymodal nociceptors, suggesting that trigeminal general mucosal innervation carries information about acids in the oral cavity. J. Comp. Neurol. 525:271–290, 2017.

MSG-Evoked c-Fos Activity in the Nucleus of the Solitary Tract Is Dependent upon Fluid Delivery and Stimulation Parameters

The marker of neuronal activation, c-Fos, can be used to visualize spatial patterns of neural activity in response to taste stimulation. Because animals will not voluntarily consume aversive tastes, these stimuli are infused directly into the oral cavity via intraoral cannulae, whereas appetitive stimuli are given in drinking bottles. Differences in these 2 methods make comparison of taste-evoked brain activity between results that utilize these methods problematic. Surprisingly, the intraoral cannulae experimental conditions that produce a similar pattern of c-Fos activity in response to taste stimulation remain unexplored. Stimulation pattern (e.g., constant/intermittent) and hydration state (e.g., water-restricted/hydrated) are the 2 primary differences between delivering tastes via bottles versus intraoral cannulae. Thus, we quantified monosodium glutamate (MSG)-evoked brain activity, as measured by c-Fos, in the nucleus of the solitary tract (nTS; primary taste nucleus) across several conditions. The number and pattern of c-Fos neurons in the nTS of animals that were water-restricted and received a constant infusion of MSG via intraoral cannula most closely mimicked animals that consumed MSG from a bottle. Therefore, in order to compare c-Fos activity between cannulae-stimulated and bottle-stimulated animals, cannulated animals should be water restricted prior to stimulation, and receive taste stimuli at a constant flow.

Measurement of Behavioral Taste Responses in Mice: Two-Bottle Preference, Lickometer, and Conditioned Taste-Aversion Tests.

The natural like and dislike of foods based on taste is one of the most easily observed behaviors in animals. Animals eat palatable foods and reject aversive foods, which makes measurement of taste perception possible using various behavioral techniques. Three different methods to accurately measure taste behavior are described here. First, two‐bottle preference tests evaluate whether a taste compound (tastant) is preferred over water. Second, lickometer tests quantify the like and dislike for multiple concentrations of the same tastant or multiple tastants at the same time. Finally, conditioned taste aversion tests accurately determine the perceived taste threshold for palatable tastants. Together, these diverse methods enable researchers to observe and measure behavioral taste responses in mice to any tastant.

5-HT3A -driven GFP delineates Gustatory Fibers innervating Sour-responsive Taste Cells: A Labeled Line for Sour Taste?

Taste buds contain multiple cell types with each type expressing receptors and transduction components for a subset of taste qualities. The sour sensing cells, Type III cells, release serotonin (5‐HT) in response to the presence of sour (acidic) tastants and this released 5‐HT activates 5‐HT3 receptors on the gustatory nerves. We show here, using 5‐HT3AGFP mice, that 5‐HT3‐expressing nerve fibers preferentially contact and receive synaptic contact from Type III taste cells. Further, these 5‐HT3‐expressing nerve fibers terminate in a restricted central‐lateral portion of the nucleus of the solitary tract (nTS)—the same area that shows increased c‐Fos expression upon presentation of a sour tastant (30 mM citric acid). This acid stimulation also evokes c‐Fos in the laterally adjacent mediodorsal spinal trigeminal nucleus (DMSp5), but this trigeminal activation is not associated with the presence of 5‐HT3‐expressing nerve fibers as it is in the nTS. Rather, the neuronal activation in the trigeminal complex likely is attributable to direct depolarization of acid‐sensitive trigeminal nerve fibers, for example, polymodal nociceptors, rather than through taste buds. Taken together, these findings suggest that transmission of sour taste information involves communication between Type III taste cells and 5‐HT3‐expressing afferent nerve fibers that project to a restricted portion of the nTS consistent with a crude mapping of taste quality information in the primary gustatory nucleus.