Imaging and Genomic Classification of Brain Alteration Induced By Gustatory Bitter Stimulus, A Pilot Study

Alteration Gustatory Abstract Objectives: Despite the exhaustive information concerning the role of physiological structures involved in gustatory stimuli, there is still a need to clarify as to whether the gustatory sense is processed in the human brain and how different kind of gustatory stimuli affect brain areas involved in taste processing. To characterize the networks that can be involved in shaping the properties of the gustatory pathways. Materials and Methods: In this fMRI study we investigated two populations characterized by their opposite response (sensitive/non-sensitive) to bitter gustatory stimulus induced by propylthiouracil molecule (PROP). In the MRI experiment PROP was delivered to the subjects by


Introduction
Bitter taste sensitivity varies greatly among individuals, and since many bitter substances are toxic, the genetic ability to taste the bitter quality confers an important survival advantage by allowing super-taster individuals, compared to non-tasters, to recognize potentially harmful substances. The best-known example of the bitter taste variability is the genetic ability to taste the bitter thiourea compounds, such as 6-n-Propylthiouracil (PROP) [1].
PROP sensitive and non-sensitive individuals are defined as tasters and non-tasters. The term "super-taster" is used to distinguish individuals who perceive PROP as extremely bitter from those who perceive it as moderately bitter. The allelic diversity of the gene codifying for TAS2R38 receptor can explain most phenotypic differences in PROP tasting [2,3]. Several investigations have attempted to elucidate the molecular basis and the genetic and psychophysical features of this human trait. Some studies have consistently reported that PROP super-tasters have a higher density of fungiform papillae on the anterior tongue surface, suggesting that they are also anatomically distinct from the other PROP taster groups in their peripheral taste system.
Although human brain pathways involved in the complex gustatory circuitry are well known, there is still the need to elucidate how taste signals are processed in the brain and whether stimuli may influence, in the neural processing, different brain areas depending on the meaning that they have for individuals.
We present data of a pilot study of Functional Magnetic Resonance Imaging (fMRI) aimed at determining if individual differences of taste perception of PROP can be represented in the brain by different activation patterns. The four major Regions of Interest (ROIs) constituting the Default Mode Network (DMN), e.g. the Posterior Cingulate Cortex (PCC), the Medial Prefrontal Cortex (MPFC), and left and Right Lateral Parietal Cortices (LLP and RLP), were examined as sources for the connectivity analysis [4]. Targets were the complete set of Broadman area.

Material and Methods
Subjects 13 (2 males, 11 females, mean age 31.6 ± 10.9 y) caucasian non-smoker subjects were recruited according to standard procedures. They were genotyped for TAS2R38 gene according to Cossu Movement Disorder [5], Melis Laryngoscope [6], and classified for their PROP taster status according to [7] and validated in several studies [8,9]. Five subjects were classified as PROP super-tasters and had a homozygous genotype for the taster receptor variant (PAV/PAV), while the others were classified as non-tasters and had homozygous genotype for the non-taster receptor variant (AVI/ AVI).

Image Acquisition
Images acquisition was performed on a GE Medical Signa

Experimental Protocol
Subjects underwent one fMRI run in the scanner. During the fMRI run they were requested to stay firm with relaxed muscles and their eyes closed. For each subjects the fMRI run included a 15 min recording of the brain activity at rest followed by another 15 min recording of the activity induced by PROP bitter taste stimulation.
PROP stimulation was performed by placing a filter paper disk impregnated with 50 mmol/L of the compound on the tip of the tongue. Taste stimulation induces brain activity revealed by fMRI.

Data Analysis
Data were processed with the SPM12 software for the general linear model ( ) GLM Y X β ε = + [10]. Following spatial-temporal adjustment of the volumes, the GLM technique allowed for statistical evaluation of activation. All contrasts were examined with a voxel wise significance level of p<0.05 (t-test) corrected for multiple comparisons across the brain volume. Minimum cluster size was settled at 20. The t-test was used to compare simultaneously differences between super-tasters and non-tasters, and rest and stimulation condition. As previously reported, BOLD signals from voxels of interest can be processed in CONN software using several indices of connectivity [11]. Connectivity measure is performed at the voxel-to-voxel level and, in order to discuss connectivity properties in connection with spatially segregated brain functions, a seed to voxel, and ROI to ROI analysis can be performed. In this experiment, brain areas of interest were spatially labelled as in Broadmann Areas (BA) in order characterize the source regions for the extraction of the time series of interest. The same areas were subsequently used as labels for targets in ROI-to-ROI, and seed to voxel analysis. Data presented in this paper focuses on the zero-lagged bivariate-correlation linear measure of functional connectivity between two sources defined as a bivariate correlation: This is the Voxel-Level Functional Connectivity MRI measures derived from the Voxel-to-Voxel Connectivity Matrix r(x,y); in this project the strength of the global connectivity pattern between each voxel and the rest of the brain with the Intrinsic Connectivity Contrast (ICC) was characterized. ICC is defined as: ICC represents the average r 2 connectivity of a given voxel i and all the other voxels, and the power map calculated provides a metric that requires no a priori information either in terms of arbitrary thresholds or ROI definitions [12]. We used the ICC metric to produce intrinsic connectivity contrast maps because this metric does not need a priori knowledge for defining any ROIs and ICC can be used as an exploratory tool for investigating the functional organization voxel by voxel; as reported in the formula, ICC is a L2 Norm. It is important to highlight that functional connectivity is modulated by the brain state. Our initial analyses compare voxel wise connectivity estimates within the Default Mode Network in a rest state condition. We supposed that regions within DMN should exhibit increased connectivity with other brain areas the network at rest compared with the state during the bitter solicitation, consistent with prior reports of elevated activation of DMN during rest [13].

Results
Our tests were performed to prove the validity of the hypothesis of greater activation in some cortical areas generated by the bit-    Given that the precuneus is a Functional Core of the Default-Mode Network our subjects are requested to do no tasks during fcMRI sessions, [14]. During the first part of the experiment they simply stay in idle, by standing still and trying to leave the mind wandering without a specific topic. In the second session of fcMRI they were requested to experiment the bitter taste of the  Figure 4.    Figure 5. Finally, we report the MPFC connections revealed in our experiments Figure 6.

Discussion
We used the fcMRI and the genotyping procedures to study the putative brain pathways of taste processing. In doing this investigation we hypothesized that, since many bitter natural substances are toxic, there would be a genetic ability to taste the bitter compounds. Moreover, it is likely that such biological ability confers an important survival advantage by allowing supertaster individuals to recognize potentially harmful substances, when compared to non-tasters, [15]. The individuals recruited for this study are 5 super-tester and 8 non-testers to the bitter stimulus, as defined by Zhao and coworkers [7] and validated in several studies [8,9].  [16]. Each system has its own organization, with sub-regions, that have varying degrees of specialization for cognitive and behavioral processes.
Studies on brain networking showed a network consisting of a set of functionally interconnected brain regions whose activity decreases during goal-oriented external tasks and increases during quiet waking state [17]. The network is active in the resting state of the brain with a high degree of functional connectivity between regions; this state has been termed as the default-mode of brain activity to denote a state during which an individual is awake and alert, but not actively involved in an attentional demanding or goal-directed task [18]. The brain's frontoparietal network region is shown to play a central role in routing the process of cognitive tasks among a range of specialized cognitive-processing network hubs which include the visual, auditory, and motor network hubs. Together, our study shows that the areas activated during the taste processing, those involved in processing the risk and possible behavioral conflicts, those In other words, the ability to taste the bitter in the Prop could relate to implications for food and drinks preference and dietary habits. Though speculative, it seems likely that our paleoanthropic ancestors would have been equipped with a fine biological sensor in order to discriminate the bitter taste, as an early detection of those often poisonous bitter, substances would have represented a real evolutionary advantage over subjects showing less sensitivity who were more exposed to the toxic action of bitter and poisonous substances. In the economy of this study it seems worth to mention from a neurological point of view is the connection of the MPFC with the BA34 since it is related to the peculiar processing of smell which is in the brain is strictly related to the taste [19]. In addition, the data acquired in our fcMRI experiment suggest an important connection between frontal and parietal networks. Various complex cognitive processes, such as memory acquisition and selection, rely on simultaneous activation of these macro and meso scale brain and "interpreting" gustatory information can reveal, at least in part, some creative aspect [20]. As a technical note, our study highlights the importance of using a whole-brain voxel-based measure of functional connectivity, a method that can turn out as a handy exploratory tool for investigating functional changes induced by particular brain states of solicitation as shown by one of the bitter stimuli in particular genotypes. Our results suggest that the voxelbased ICC measures seem to be the optimal approach [21] in order to delineate regions of interest for functional or network analyses. By moving to a voxel level, we are implicitly using a parcellation of the cortex as fine as that allowed in accordance with the acquisition strategy selected.

Conclusion
The present investigation on the gustatory effects of better taste in a population of subjects represented by super taster and nontaster, lead with the fc-MRI analysis offers a standard framework to perform a large set of connectivity-based experiments on brain functions under a variety of physiological and pathological conditions. Moreover, the calculation of several connectivity parameters and measures derived by the graph theory allow for the investigation of brain reactions/responses to diverse pathological and non-pathological conditions. Although the present study illustrated only preliminary data, the results obtained show the reliability of fcMRI investigation, in suggesting that the collaboration between studies on brain networking and "omic" sciences (postgenetic, genomic et al.) is a new exciting approach in exploiting such hard questions as the structural relationships between brain and mind [22].