Electrical Stimulation of Gustatory Cortex Causes the Activation of Motor Output in Conscious Rats

Electrical Stimulation of Gustatory Cortex Causes the Activation of Motor Output in Conscious Rats Sara Greco Dr. Michael King Biology Department, Stetson University Abstract The role of the gustatory cortex (GC), a forebrain region that receives taste and other orosensory input, in the control of taste-related behaviors is unclear. The goal of the present study was to determine if electrical stimulation of the GC, that elicits taste reactivity (TR) behaviors, causes perception of a taste or activates a motor output. Conditioned taste aversion (CTA) was used as a way to measure possible taste perception. Under sodium pentobarbital anesthesia, an electrode was placed into the GC using a stereotaxic device and intraoral cannulas were implanted into seven male Wistar rats. Following a week of recovery, rats received an i.p. injection of either LiCl (experimental group) or NaCl (controls) followed by electrical stimulation of the GC and intraoral infusion of taste solutions. A two-bottle preference test comparing water and sucrose intake also was performed. Immunohistochemistry for the Fos protein was used to identify neurons activated within the GC. During electrical stimulation, the number of aversive taste reactivity (TR) behaviors expressed by experimental rats (n=4) increased over time as compared to control rats (n=3; p<0.05). TR behaviors in response to intraoral solutions were not different between treatment groups, however, HCl elicited the most aversive responses and sucrose elicited the most ingestive responses among all animals regardless of treatment group. The two-bottle preference test showed that both groups preferred sucrose over water. The center of the electrode tract typically was located within the GC 1.0-1.3 mm anterior to bregma with some variation among animals. Specifically, the Fos protein was expressed in neurons that mainly were spread throughout the granular and dysgranular insular GC. These data indicate that electrical stimulation of the GC did not result in the perception of taste and that the observed TR behaviors were most likely attributed to stimulation of a motor output. Introduction Taste perception plays a key role in the survival and health of animals. Taste is responsible for the evaluation of food in deciding whether something is nutritious or poisonous. When taste stimuli are delivered through a surgically implanted intraoral cannula, rats elicit affective behaviors known as taste reactivity (Grill and Norgren, 1978). Taste reactivity (TR) behaviors are the immediate, stereotypical, physiological responses to a taste in the oral cavity (Grill and Norgren, 1978). TR behaviors are reflex-like and can be used to indicate the palatability of an intraoral substance and measure the response to a specific taste (Grill and Norgren, 1978; Spector et al., 1988). The most basic tastes that can be perceived are sweet, salty, bitter, sour, and umami. Normally preferred stimuli such as sweet, salty, and umami tastes usually induce an ingestive taste response (Flynn et al., 1991; Grill and Norgren, 1978). Bitter and sour tastes tend to elicit aversive taste responses even at low concentrations (Flynn et al., 1991; Grill and Norgren, 1978). Information regarding a particular tastant is taken from the taste receptor cells on the tongue and transmitted through different neural stations to the gustatory cortex (Peng et al., 2015). Specifically, the information is sent to the insula via the facial, glossopharyngeal, and vagus nerves (Flynn et al., 1991). Qualitative identification of taste is associated with the gustatory thalamocortical pathway (King et al., 2015). The insular cortex is responsible for visceroautonomic functions such as gastrointestinal, respiratory, and cardiovascular functions (Yamamoto and Kitamura, 1990). The gustatory cortex (GC) acts as the central neural hub that integrates taste information from the external world with other areas of the brain for interpretation (Norgren, 1995). The GC is highly integrated and its roles in taste function include qualitative discrimination, affect, and physiological reflexes (Bales et al., 2015). The GC is a subregion in the insular cortex and contains the agranular insular cortex (AI), dysgranular insular cortex (DI), and the granular insular cortex (GI) (Accolla et al., 2007; Bales et al., 2015; Norgren, 1995). The GI lies ventral to the somatosensory areas of SI and SII and is characterized by having a fully developed layer IV; DI has scattered granule cells observable in laver V; the AI is situated between the DI and piriform cortex (Yamamoto and Kitamura, 1990). Electrophysiological, histological, and behavioral studies indicate the cortical taste area containing gustatory neurons is located in the GI, DI, and AI regions (Bales et al., 2015; Norgren, 1995; Yamamoto and Kitamura, 1990). These neurons are involved in processing palatability of specific taste stimuli (Accolla et al., 2007; Accolla and Carleton, 2008; Chen et al., 2011; King et al., 2015; Yamamoto et al., 1989). The functional role of the GC has been studied as it relates to taste memory (Bales et al., 2015). The GC plays a key role in the acquisition and retention of conditioned taste aversion in rats (Bales et al., 2015; Yamamoto and Kitamura, 1990). Conditioned taste aversion (CTA) occurs when there is ingestion of a substance (conditioned stimulus [CS]) followed by visceral malaise (unconditioned stimulus [US]) causing the animal to learn to avoid the CS (Accolla and Carleton, 2008; Bales et al., 2015; Spector et al., 1988). CTA has experimentally been induced via intraperitoneal injection of LiCl (US) after ingestion of a tastant such as sucrose (CS) (Accolla and Carleton, 2008; Bales et al., 2015; Spector et al., 1988). An i.p. injection of an equivalent amount of saline is given to control rats to attribute the CTA to gastric malaise produced by LiCl (Bales et al., 2015; Spector et al., 1988). Recent studies show that lesions of the GC do not alter CTA (Schier et al., 2015). Stimulating electrodes have been used to determine the behavioral importance of activated neurons located in the GC (King et al., 2014). Immunohistochemistry of the Fos protein paired with electrical stimulation can be used to identify neurons that have been activated by taste stimuli in the gustatory cortex (Harrison, 2001). Different taste stimuli elicit different patterns of Fos-immunoreactive neurons (Accolla et al. 2007; Chen et al., 2011). Intrinsic signal maps of the same rat have shown distinct patterning within the GC that corresponded to the four different taste stimuli (Accolla et al., 2007; Chen et al., 2011). Sweet tastes are typically perceived in the rostral GC, salty is perceived in between the rostral and caudal sections of the GC, sour is perceived in the caudal GC, and bitter is perceived in the most caudal part of the GC. However, there is topographic separation between sweet and bitter taste with no overlap of gustatory fields (Chen et al., 2011). Although TR behaviors can be elicited through stimulation of the GC, the GC may not be a necessary component for processing taste or it may be a minor component. A previous study indicated that when there was excessive damage to 94% of the GC through lesions there was no change in the number of TR behaviors observed (King et al., 2015). Such a finding indicates that the part of the GC that was destroyed may not be necessary for normal expression of TR behaviors (King et al., 2015). There is a possibility that taste perception may occur in another part of the insula and production of TR behaviors may be due to activation of a motor output by a stimulus. The present study was designed to determine whether activation of a specific part of the GC via electrical stimulation would lead to the perception of taste, not just sensorimotor output. This was done by pairing electrical stimulation with gastric malaise in an attempt to induce a CTA. Methods Animals Data from 8 male Wistar rats (275-325 g) from Charles River Laboratories were used in this report. Three rats were used as controls and the other five were designated experimental. The rats were housed individually in standard, plastic cages (40cm x 24cm x 20 cm) in a secluded room with a 12h light-12h dark cycle. The rats had constant access to block rodent food (Harlan Teklad) and water. The conditions and procedures for housing were approved by the Institutional Animal Care and Use Committee and conform to guidelines specified by the National Institutes of Health. Surgical procedures All rats were implemented with an electrode placed in the right GC and bilateral intra-oral cannulas. Electrodes consisted of two stainless steel Formvar-insulated wires that were twisted around each other and protruded 9 mm from a plastic pedestal that contained electrical mounts (Plastics One). The uninsulated tips of the wires were 150 m apart. Rats were anesthetized by intraperitoneal injection of 60mg/kg sodium pentobarbital and placed in a stereotaxic device with nontraumatic ear bars (Stoelting) to hold the skull horizontal. Surgical procedures were similar to those previously described (Grill and Norgren, 1978; Morganti et al., 2007) to implant the stainless steel electrodes into the GC (Figure 1). The scalp was shaved, cleaned with betadine, and a 2 cm incision was made in the scalp. A 1 mm burr hole was made in the skull above the right GC. The stimulating electrode tip was inserted into the right GC at 1.0 mm anterior to the bregma, 3.6 mm lateral to the midline, and 7.0 mm ventral to the skull (Paxinos and Watson, 2006). The electrode was secured using dental acrylic and screws embedded in the skull. A cap was placed over the electrical mount. Intraoral cannulas to deliver tastes were implanted bilaterally during the same surgery. The cannulas were formed from PE-100 tubing approximately 1.0 cm in length that had a Teflon washer threaded onto one end and heat flanged to secure the washer. One side of the washer was cut flat to sit comfortably beside the gum. The opposite end of the tubing was connected to a syringe needle that allowed for the tubing to be inserted through the temporal muscle anterolateral to the first maxillary molar and brought up the side of the skull, under the skin, and exited the incision in the scalp (Riley and King, 2013). The PE tubing was cut and connected to 1.0 cm of 19-gauge stainless steel tubing and secured with dental acrylic. A topical antibiotic was applied and the skin was sutured shut. The rats were given a three-day recovery period after the surgery in which the cannulae were flushed with dH2O (King et al., 2015; Yamamoto et al., 1989). Figure 1. Highlighted area of gustatory cortex for visual representation of size and position in relation to the rat brain (Accolla et al., 2007). Stimulation and behavioral testing The rats had an additional three-day adaptation period to acclimate to the behavioral arena. The behavioral arena was located in a separate, isolated room. The behavioral arena consisted of a cylinder (26 cm tall with a 26 cm diameter approximately) with a mirrored bottom that allowed for easy viewing of ingestive and aversive taste responses (TR) exhibited by the rat. Rats were allowed to acclimate individually in the behavioral arena for 30 minutes each day. On the day of testing, control rats received an injection of NaCl i.p. (Bales et al., 2015) and experimental rats received an i.p. injection of LiCl at concentrations of 3 mEq/kg each (Yamamoto and Kitamura, 1990). Both control and experimental rats received microstimulation at a current of 10-20 A, 50 Hz to the right anterior GC with a pulse duration of 0.4 ms. Stimulation occurred for one minute each time at time = 0, 5, 15, and 30 minutes following the i.p. injection. During stimulation rats were in the behavioral arena and TR behaviors were videotaped. The following day, rats acclimated in the behavioral arena for approximately 10-15 minutes before room temperature taste solutions (Yamamoto et al., 1989) were delivered into the mouth via intraoral cannulae. Intraoral 0.10 M sucrose, 0.10 M NaCl, 0.03 M HCl, 0.16 M MSG, were administered for one minute each at a rate of 1mL/min. DH2O was used in between solutions to rinse the cannula (Yamamoto and Kitamura, 1990). To test if a taste aversion had been acquired, a two-bottle preference test (Grill and Norgren, 1978) was performed the night after the taste solution experiment. The two-bottle preference test was also repeated a day later to test for retention of a potential CTA. Control and experimental rats had access to a bottle that contained 200 mL of dH2O in addition to a bottle that contained a 200 mL 0.10 M sucrose solution. In the first trial, if the experimental rat drank a similar amount of sucrose as the control rat the taste solution was altered to a 200 mL 0.10 M solution of NaCl to test for aversion. The volume of liquid in each bottle was measured before and after the two-bottle preference test to establish the volume consumed by each animal. The bottles also were switched around the second night to eliminate placement preference by the rats. Histology and Fos immunohistochemistry The rats were placed back in the behavioral arena two days after the initial two-bottle preference test. The GC in both control and experimental groups was stimulated for five minutes using the same parameters. After waiting 45 minutes for the Fos protein to be expressed, the rats were sacrificed with an overdose of sodium pentobarbital (80mg/kg) until unresponsive to toe pinch and then perfused transcardially with heparinized 0.15 NaCl followed by 4% paraformaldehyde (King et al., 2015). The brains were removed and fixed overnight in the same fixative at 4oC (King et al., 2015; Riley and King, 2013). A vibratome was used to slice 75m thick coronal sections that were placed into their own individual wells in a 12-well plate. Every other section was treated according to Fos immunohistochemistry as previously mentioned (Morganti et al., 2007; Riley and King, 2013). These sections were soaked in 1% sodium borohydride in KPBS for 20 minutes. The sections were rinsed in KPBS before incubation in a Fos primary antibody raised in rabbit (Santa Cruz Biotech) diluted 1:10,000 in KPBS with 0.4% Triton X-100 for 72 hours at 4oC. The sections were then rinsed with KPBS and incubated in goat antirabbit IgG (Vector Labs) diluted to 1:600 in KPBS with Triton X-100 at room temperature for 4 hours. KPBS was used to rinse the sections before they were incubated in reagents of a Vectastain ABC kit (Vector Labs) at 4oC overnight. The sections were rinsed and then reacted in 0.1 M sodium phosphate buffer that contained 0.03% diaminobenzidine, 0.008% nickel ammonium sulfate, 0.008% cobalt chloride, and 0.0075% hydrogen peroxide at room temperature for 9 minutes. A final rinse in KPBS was performed before affixing the sections to gelatin and chrome alum-coated glass slides. The slides were set to dry at room temperature overnight before coverslips were applied using Permount (Fisher Scientific). The other sections that were not stained with Fos were put on slides and Nissl-stained with 0.1% thionin. Cover slips were applied. Data analysis Recorded TR behaviors were reviewed frame by frame according to the procedures described (Grill and Norgren, 1978; Spector et al., 1988). The mimetic responses of interest were rhythmic mouth movements, tongue protrusions, lateral tongue movements, and gapes (Figure 2), in addition to headshaking and forelimb flailing (Grill and Norgren, 1978). Figure 2. Mimetic responses of taste reactivity (Grill and Norgren, 1978). Data were pooled with data from a colleague to increase the overall sample size to three control rats and five experimental for a total of eight animals. The data gathered from this experiment were analyzed using ANOVAs and a Two-Sample t-Test. Specifically, behavioral data collected during the microstimulation portion of the experiment were analyzed using two Two-Factor ANOVAs. This was to evaluate the difference between mean number of aversive behaviors present between control and experimental groups as well as the difference in mean number of ingestive behaviors exhibited between control and experimental animals at the various time points. Two Univariate ANOVAs were performed to analyze the mean difference in aversive behaviors present between control and experimental groups when exposed to the different taste solutions. The same procedure was done to examine the difference in ingestive behaviors between control and experimental groups in relation to the taste solutions. A post hoc Tukey test was performed between taste solutions and number of behaviors to investigate the source of significance found from both ANOVAs. A Two-Sample t-Test was used to compare the data gathered from the two-bottle preference tests to determine if there was a statistical significance between the mean volumes of either dH2O or sucrose consumed by the control rats when compared to the experimental rats. The location of the electrode tract was identified in each rat brain and notated in relation to its distance from the bregma. Attention was also paid to whether or not the electrode had stimulated the GC by identifying Fos-IR neurons and their location in relation to the GI, DI, and AI. Results TR behaviors elicited from GC stimulation Overall, there was no difference between aversive behaviors elicited by control (n=3) or experimental (n=4) animals at each time point (Figure 3A). However, at time T=30 there was a visual difference between the average aversive behaviors of control as compared to experimental (Figure 3A). The difference is not significant (p=0.087). There is a trend of increasing aversive behaviors seen in the experimental group as time increases, whereas the control group stays fairly consistent. Ingestive behaviors elicited by control and experimental groups also showed no statistical significance over time (Figure 3B). Figure 3. Graphs of the behavioral effects of microstimulation at time points after intraperitoneal injection of either NaCl for control (n=3) or LiCl for experimental (n=4) animals. (A) Graph of the total number (+SEM, standard errors of mean) of aversive TR behaviors exhibited between control and experimental animals for one minute each at time points 0, 5, 15, and 30 minutes. (B) Graph of the total number of ingestive behaviors observed between control and experimental animals at each time point. TR behaviors elicited from intraoral taste solutions without GC stimulation Without stimulation of the GC, TR behaviors were dependent upon the intraoral taste solutions that were delivered. HCl elicited the most aversive responses from both control and experimental groups (Figure 4A). The number of aversive behaviors expressed by control and experimental groups to each solution was not different. Sucrose elicited the most ingestive responses from both control and experimental groups (Figure 4B). 0 10 20 30 40 50 60 70 80 0 5 15 30 Number of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors Observed Time (minutes)Time (minutes) Time (minutes)Time (minutes)Time (minutes) Time (minutes) Time (minutes)Time (minutes) Time (minutes) Aversive ControlAversive Control Aversive Control Aversive ControlAversive Control Aversive Control Aversive ControlAversive ControlAversive Control Aversive Control Aversive ExperimentalAversive Experimental Aversive Experimental Aversive ExperimentalAversive Experimental Aversive Experimental Aversive ExperimentalAversive ExperimentalAversive ExperimentalAversive ExperimentalAversive ExperimentalAversive Experimental Aversive ExperimentalAversive ExperimentalAversive ExperimentalAversive Experimental 0 10 20 30 40 50 60 70 80 0 5 15 30 Number of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors Observed Time (minutes)Time (minutes) Time (minutes)Time (minutes)Time (minutes) Time (minutes) Time (minutes)Time (minutes) Time (minutes) Ingestive Control Ingestive ControlIngestive ControlIngestive ControlIngestive Control Ingestive Control Ingestive Control Ingestive ControlIngestive Control Ingestive ControlIngestive Control Ingestive Experimental Ingestive ExperimentalIngestive ExperimentalIngestive ExperimentalIngestive Experimental Ingestive Experimental Ingestive Experimental Ingestive ExperimentalIngestive ExperimentalIngestive ExperimentalIngestive Experimental Ingestive Experimental Ingestive ExperimentalIngestive Experimental A. B. Figure 4. Graph of behavioral responses to various intra-oral taste solutions. (A) Graph of total number (+ SEM, standard error of means) of aversive behaviors to administered taste solutions between control (n=3) and experimental (n=4) groups. (B) Graph of total number (+ SEM) of ingestive behaviors between control and experimental groups to intra-oral taste solutions. An increase in the ingestive behaviors for sucrose is mainly due to an increased number of mouth movements and tongue protrusions (Figure 4B, first doublet). There was no difference between control and experimental groups for ingestive behaviors to each taste solution. Two-bottle preference test When given the option to choose between water and sucrose, the volume of sucrose consumed was greater than the volume of water consumed (Figure 5). There was no difference between control or experimental consumption of water (p=0.10). There was also no difference between control or experimental consumption of sucrose (p=0.25). It was noted that control 0 5 10 15 20 25 30 35 SucroseSucroseSucroseSucrose SucroseSucrose NaClNaCl HClHCl MSG MSG Number of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors Observed Tastant SolutionTastant Solution Tastant Solution Tastant Solution Tastant Solution Tastant Solution Control Aversive Control AversiveControl Aversive Control AversiveControl AversiveControl Aversive Control Aversive Control Aversive Control AversiveControl Aversive Experimental AversiveExperimental AversiveExperimental AversiveExperimental AversiveExperimental Aversive Experimental Aversive Experimental AversiveExperimental Aversive Experimental Aversive Experimental AversiveExperimental AversiveExperimental Aversive Experimental AversiveExperimental Aversive 0 50 100 150 200 250 SucroseSucroseSucroseSucrose SucroseSucrose NaClNaCl HClHCl MSG MSG Number of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors ObservedNumber of Behaviors Observed Tastant SolutionTastant Solution Tastant Solution Tastant SolutionTastant Solution Tastant Solution Tastant Solution Control Ingestive Control IngestiveControl Ingestive Control IngestiveControl IngestiveControl Ingestive Control IngestiveControl IngestiveControl IngestiveControl Ingestive Control IngestiveControl Ingestive Experimental IngestiveExperimental IngestiveExperimental IngestiveExperimental IngestiveExperimental Ingestive Experimental Ingestive Experimental IngestiveExperimental Ingestive Experimental Ingestive Experimental IngestiveExperimental IngestiveExperimental IngestiveExperimental IngestiveExperimental IngestiveExperimental Ingestive B. A. animals typically preferred the sucrose solution and consumed more than experimental animals. Experimental animals consumed more water than control animals, however, experimental animals still preferred the sucrose solution. Although sucrose was typically preferred by all rats, there was a single experimental rat out of the sample that displayed aversion to sucrose. Following the electrical stimulation and gastric malaise produced from the LiCl, the experimental rat consumed 46 mL of water and 0 mL of sucrose, compared to the control rat that consumed 0 mL of water and 68 mL of sucrose. Figure 5. Graph comparing the average volumes (+SEM, standard error of means) of water and sucrose consumed by control (n=3) and experimental (n=4) animals during a two-bottle preference test. On the second day, the experimental rat’s aversion had decreased as it consumed 22 mL of water and 50 mL of sucrose. The control rat’s response was unchanged and still preferred the sucrose (174 mL) over water (0 mL). 0 20 40 60 80 100 120 140 dH2OdH2OdH2O SucroseSucroseSucroseSucrose SucroseSucrose Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL)Volume Consumed (mL) Solution Solution Solution Control ControlControl ControlControl ExperimentalExperimentalExperimentalExperimentalExperimental Experimental ExperimentalExperimentalFigure 6. Images of coronal sections ranging from 0.5-2.2 mm anterior to Bregma taken at 2.5x magnification. (A) The central electrode tract (arrow) in all 8 animals labeled A-H. A is 1.7-1.9 mm anterior to Bregma. B-D, G are located 1.0-1.3 mm anterior to Bregma. F is 2.0-2.2 mm anterior to Bregma. H is 0.7-0.8 mm anterior to Bregma. Left column contains experimental rats, right column contains corresponding control rats. This is for all except image D, which is an experimental rat. (B) The difference in concentration of Fos-IR neurons around the electrode stimulation site. Location of electrode stimulation site The central location of the electrode tract was variable among animals (Figure 6A). In image C there are few Fos labeled cells, some of which are located in the medial part of the GI. Image D and E show some Fos labeled neurons in the GI and DI. Image H depicted Fos cells in the medial GI, with most in the DI, and some in the AID. Image G corresponds to rat SG3 and A. B. mostly had cells in the GI, however, some were present in the AID. The same phenomena can be observed at higher magnification for the rat SG3 (Figure 6B Image B). There was a continued presence of Fos-IR neurons in this animal until about 0.7 mm anterior to Bregma. There is a lack of Fos-IR neurons present for experimental rat JL3 (Image C of Figure 6A, Image A of Figure 6B). Further investigation of this animal concluded that the electrode was not located in the GC, resulting in very few TR behaviors from electrical stimulation and therefore this rat was removed from the data set. A high concentration of Fos-IR neurons was found surrounding the electrode stimulation site and extending throughout the GC of experimental rat SG3 (Image G of Figure 6A, Image B of Figure 6B). SG3 was the same rat that had a CTA to sucrose during the first round of the two-bottle preference test. Discussion The goal of the present study was to determine if electrical stimulation of the GC would elicit a perception of taste in the animals tested. Although there was a single event that demonstrated a CTA evoked from electrical stimulation of the GC, the hypothesis that GC stimulation would lead to the perception of taste was not supported. Electrical stimulation of the GC without exposure to intraoral taste solutions elicited both ingestive and aversive TR behaviors among control and experimental animals. As time progressed, experimental animals showed more aversive TR behaviors than control animals which can be attributed to the induced gastric malaise from the intraperitoneal injection of LiCl. The data were not significant, however, a trend that there were more aversive TR behaviors produced by experimental rats than control rats could suggest that an increase in the sample size has the potential to yield a significant p-value. Exposure to intraoral taste solutions elicited ingestive and aversive TR behaviors from control and experimental groups. The number of TR behaviors elicited in response to a taste solution was statistically different between tastes and determined the treatment played no effect. Sucrose was found to be the most palatable taste, followed by NaCl, HCl, and MSG as least palatable. An investigation into the location of the electrode tract and spread of activated neurons throughout the GC was done using Fos immunohistochemistry to identify the range of activation in relation to the Bregma as a landmark. The obvious advantage of using Fos to highlight activated neurons is for easy visualization to determine how far the electrical stimulation spread through the GC. There was a high amount of variability in the electrode location between rats leading to the incomplete activation of the GC in some animals. Since the two-bottle preference test was looking specifically at aversion to sucrose, in order to evoke a CTA, the activation of neurons would need to be located in the rostral end of the GC (Accolla et al., 2007). The stimulation site present in the successful experimental rat was located 1.1-1.3mm anterior to the Bregma and had activated neurons throughout the GI, DI, and AID. Although some stimulation sites in the other rats were similar and ranged from 1.0-1.3 mm anterior to the bregma, only some areas of the GI and DI had activated Fos-IR neurons. A larger sample size and a method to increase accuracy for implanting electrodes into this specific section of the GC could increase the possibility of developing a CTA through electrical stimulation. The labeled activated neurons present in the coronal sections of the rats that are not associated with the perception of sucrose are due to the electrical stimulation which may cause perception of a different taste. This would be consistent with previous studies that provided evidence for taste differentiation even though there may not be a distinct pattern. Bitter tastes are usually represented by activation in the more caudal region of the GC, whereas sucrose tends to be located more rostral (Accolla et al., 2007). Previous research has found that there are broadly tuned neurons scattered through highly activated regions, contributing to the overlapping of taste modalities represented in topographic maps (Accolla et al., 2007). However, sweet and bitter tastes tend to have distinct separation and do not have overlapping regions (Chen et al., 2011). Topographic mapping of the GC can become difficult due to the overlap and spatial patterning associated with different taste solutions. Studies have sought to determine the specific areas within the GC that perceive and correspond to different tastes. Some have developed topographic maps, whereas others have come back with inconclusive findings (Accolla et al., 2007; Chen et al., 2011). Other research has proposed that the GC may not be entirely necessary for the perception of taste (King et al., 2015). The purpose of the present study was to expand upon previous research in determining if activation of the GC can result in a perception of taste that corresponds to what would be expected based on the area of activation according to these studies. Although the GC plays a role in taste processing, the results indicate that the area of electrical stimulation within the GC did not elicit a taste perception and the TR behaviors produced were sensorimotor outputs from the stimulation. References Accolla, R., Bathellier, B., Petersen, C. C., & Carleton, A. 2007. Differential spatial representation of taste modalities in the rat gustatory cortex. Journal of Neuroscience 27: 1396-1404. Accolla, R., Carleton, A. 2008. Internal body state influences topographical plasticity of sensory representations in the rat gustatory cortex. Proceedings of the National Academy of Sciences 105: 4010-4015. Bales, M. B., Schier, L. A., Blonde, G. D., & Spector, A. C. 2015. 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