Gustatory and reward brain circuits in the control of food intake.

Gustation is a multisensory process allowing for the selection of nutrients and the rejection of irritating and/or toxic compounds. Since obesity is a highly prevalent condition that is critically dependent on food intake and energy expenditure, a deeper understanding of gustatory processing is an important objective in biomedical research. Recent findings have provided evidence that central gustatory processes are distributed across several cortical and subcortical brain areas. Furthermore, these gustatory sensory circuits are closely related to the circuits that process reward. Here, we present an overview of the activation and connectivity between central gustatory and reward areas. Moreover, and given the limitations in number and effectiveness of treatments currently available for overweight patients, we discuss the possibility of modulating neuronal activity in these circuits as an alternative in the treatment of obesity.

Compounds from Sichuan and Melegueta peppers activate, covalently and non-covalently, TRPA1 and TRPV1 channels.

BACKGROUND AND PURPOSE: Oily extracts of Sichuan and Melegueta peppers evoke pungent sensations mediated by different alkylamides [mainly hydroxy-alpha-sanshool (alpha-SOH)] and hydroxyarylalkanones (6-shogaol and 6-paradol). We assessed how transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (TRPV1), two chemosensory ion channels, participate in these pungent sensations. EXPERIMENTAL APPROACH: The structure-activity relationships of these molecules on TRPA1 and TRPV1 was measured by testing natural and synthetic analogues using calcium and voltage imaging on dissociated dorsal root ganglia neurons and human embryonic kidney 293 cells expressing the wild-type channels or specific cysteine mutants using glutathione trapping as a model to probe TRPA1 activation. In addition, using Trpv1 knockout mice, the compounds' aversive responses were measured in a taste brief-access test. KEY RESULTS: For TRPA1 activation, the cis C6 double bond in the polyenic chain of alpha-SOH was critical, whereas no structural specificity was required for activation of TRPV1. Both 6-shogaol and 6-paradol were found to activate TRPV1 and TRPA1 channels, whereas linalool, an abundant terpene in Sichuan pepper, activated TRPA1 but not TRPV1 channels. Alkylamides and 6-shogaol act on TRPA1 by covalent bonding whereas none of these compounds activated TRPV1 through such interactions. Finally, TRPV1 mutant mice retained sensitivity to 6-shogaol but were not responsive to alpha-SOH. CONCLUSIONS AND IMPLICATIONS: The pungent nature of components of Sichuan and Melegueta peppers was mediated via interactions with TRPA1 and TRPV1 channels and may explain the aversive properties of these compounds.

The gustatory cortex and multisensory integration.

The central gustatory pathways are part of the brain circuits upon which rest the decision to ingest or reject a food. The quality of food stimuli, however, relies not only on their taste but also on properties such as odor, texture and temperature. We will review anatomical and functional evidence showing that the central gustatory system, in particular its cortical aspect, functions as an integrative circuit in which taste-responsive neurons also show sensitivity to somatosensory and olfactory stimulation. In addition, gustatory pathways are modulated by the internal state of the body, with neuronal responses to tastes changing according to variations in physiological parameters such as gastrointestinal hormones or blood glucose levels. Therefore, rather than working as the receptive field of peripheral taste receptor cells, the central gustatory pathways seem to operate as a multisensory system dedicated to evaluating the biological significance of intra-oral stimuli.

Evaluation of human fibroblast growth factor 23 (FGF-23) C-terminal and intact enzyme-linked immunosorbent-assays in end-stage renal disease patients.

Hyperphosphataemia, calcitriol deficency and secondary hyperparathyroidism (sHPT) are common complications in end-stage chronic kidney diseases (CKD). Fibroblast Growth Factor 23 (FGF-23) is a phosphaturic peptide, secreted by the osteoblast precursors, that also inhibits renal 1-alpha-hydroxylase activitiy and tubular phosphate reabsorption by the inhibition of sodium-dependant renal phosphate transport (Na-Pi-IIa). Consequences are a decreaese of serum 1,25 dihydroxyvitamin D3 and phosphaturia. Therefore, FGF-23 plays a role in hyperphosphataemia in association with CKD and may be involved in the pathogenesis of sHPT. Increased FGF-23 may contribute to maintaining a normal serum phoshpate level in face of a processing CKD, but if the creatinine clearance is reduced to lower than 30 ml/min the capacity of this regulative mechanism ends and hyperphosphataemia results. In our investigation of end-stage renal diseases markedly increased serum FGF-23, associated with hyperphosphataemia, phosphaturia and decreased serum calcitriol and sHPT, were found. Furthermore preanalytical testing for the stability of FGF-23 was performed by comparing samples which were stored at -20 degrees C with samples that have been stored for 6 days at +4 degrees C. The simultaneous investigation of serum and EDTA plasma FGF-23 certifies the advantage of EDTA plasma in subjects with an intact renal function.

Elasticity, strength, and water permeability of bilayers that contain raft microdomain-forming lipids.

Bilayers composed of phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol (CHOL) are commonly used as systems to model the raft-lipid domain structure believed to compartmentalize particular cell membrane proteins. In this work, micropipette aspiration of giant unilamellar vesicles was used to test the elasticities, water permeabilities, and rupture tensions of single-component PC, binary 1:1 PC/CHOL, and 1:1 SM/CHOL, and ternary 1:1:1 PC/SM/CHOL bilayers, one set of measurements with dioleoyl PC (DOPC; C18:1/C18:1 PC) and the other with stearoyloleoyl PC (SOPC; C18:0/C18:1 PC). Defining the elastic moduli (K(A)), the initial slopes of the increase in tension (sigma) versus stretch in lipid surface area (alpha(e)) were determined for all systems at low (15 degrees C) and high (32-33 degrees C) temperatures. The moduli for the single-component PC and binary phospholipid/CHOL bilayers followed a descending hierarchy of stretch resistance with SM/CHOL > SOPC/CHOL > DOPC/CHOL > PC. Although much more resistant to stretch than the single-component PC bilayers, the elastic response of vesicle bilayers made from the ternary phospholipid/CHOL mixtures showed an abrupt softening (discontinuity in slope), when immediately subjected to a steady ramp of tension at the low temperature (15 degrees C). However, the discontinuities in elastic stretch resistance at low temperature vanished when the bilayers were held at approximately 1 mN/m prestress for long times before a tension ramp and when tested at the higher temperature 32-33 degrees C. The elastic moduli of single-component PC and DOPC/CHOL bilayers changed very little with temperature, whereas the moduli of the binary SOPC/CHOL and SM/CHOL bilayers diminished markedly with increase in temperature, as did the ternary SOPC/SM/CHOL system. For all systems, increasing temperature increased the water permeability but decreased rupture tension. Concomitantly, the measurements of permeability exhibited a prominent correlation with the rupture tension across all the systems. Together, these micromechanical tests of binary and ternary phospholipid/CHOL bilayers demonstrate that PC hydrocarbon chain unsaturation and temperature are major determinants of the mechanical and permeation properties of membranes composed of raft microdomain-forming lipids.

Multisensory Processing of Gustatory Stimuli.

Gustatory perception is inherently multimodal, since approximately the same time that intra-oral stimuli activate taste receptors, somatosensory information is concurrently sent to the CNS. We review evidence that gustatory perception is intrinsically linked to concurrent somatosensory processing. We will show that processing of multisensory information can occur at the level of the taste cells through to the gustatory cortex. We will also focus on the fact that the same chemical and physical stimuli that activate the taste system also activate the somatosensory system (SS), but they may provide different types of information to guide behavior.

Changes in osmolality sensitize the response to capsaicin in trigeminal sensory neurons.

Changes in tonicity in the peripheral nervous system can activate nociceptors and produce pain. Under local inflammatory conditions the peripheral terminals of nociceptors are subject to deviations from isotonicity. Previously it was shown that several members of the TRP(V) family of ion channels are responsive to changes in tonicity. Here we explore how changes in tonicity affect TRPV1 receptor-mediated responses to capsaicin in dissociated rat trigeminal ganglion (TG) neurons. Using whole cell patch-clamp and calcium imaging, we found that mild anisotonicity (260 and 348 mOsm/kg for hypotonicity and hypertonicity, respectively) strikingly sensitized the capsaicin-evoked current, I(caps). Confocal immunolocalization studies also revealed a modest anisotonicity-mediated redistribution of TRPV1 toward the plasma membrane of TG neurons. With respect to downstream signaling pathways, tonicity-induced sensitization of I(caps) was dependent on whether hypo- or hypertonic stimuli were applied. Specifically, antagonism of PKA- and PI3K-activated pathways appreciably reduced the hypertonicity-induced sensitization of I(caps), whereas inhibition of PKC-mediated pathways selectively reduced the sensitization produced by hypotonic solutions. In summary, whereas the overall effects of hypo- and hypertonicity resulted in a similar pattern of potentiation of I(caps), intracellular signaling pathways were selective for hypo- versus hypertonicity-induced tuning of capsaicin-activated currents.

Dopamine levels modulate the updating of tastant values.

To survive, animals must constantly update the internal value of stimuli they encounter; a process referred to as incentive learning. Although there have been many studies investigating whether dopamine is necessary for reward, or for the association between stimuli and actions with rewards, less is known about the role of dopamine in the updating of the internal value of stimuli per se. We used a single-bottle forced-choice task to investigate the role of dopamine in learning the value of tastants. We show that dopamine transporter knock-out mice (DAT-KO), which have constitutively elevated dopamine levels, develop a more positive bias towards a hedonically positive tastant (sucrose 400 mM) than their wild-type littermates. Furthermore, when compared to wild-type littermates, DAT-KO mice develop a less negative bias towards a hedonically negative tastant (quinine HCl 10 mM). Importantly, these effects develop with training, because at the onset of training DAT-KO and wild-type mice display similar biases towards sucrose and quinine. These data suggest that dopamine levels can modulate the updating of tastant values, a finding with implications for understanding sensory-specific motivation and reward seeking.

Role of membrane curvature in mechanoelectrical transduction: ion carriers nonactin and valinomycin sense changes in integral bending energy.

We describe the phenomenon of mechanoelectrical transduction in macroscopic lipid bilayer membranes modified by two cation-selective ionophores, valinomycin and nonactin. We found that bulging these membranes, while maintaining the membrane tension constant, produced a marked supralinear increase in specific carrier-mediated conductance. Analyses of the mechanisms involved in mechanoelectrical transduction induced by the imposition of a hydrostatic pressure gradient or by an amphipathic compound chlorpromazine reveal similar changes in the charge carrier motility and carrier reaction rates at the interface(s). Furthermore, the relative change in membrane conductance was independent of membrane diameter, but was directly proportional to the square of membrane curvature, thus relating the observed phenomena to the bilayer bending energy. Extrapolated to biological membranes, these findings indicate that ion transport in cells can be influenced simply by changing shape of the membrane, without a change in membrane tension.

Conical electron tomography of a chemical synapse: vesicles docked to the active zone are hemi-fused.

We have used thin sectioning and conical electron tomography to determine the three-dimensional structure of synaptic vesicles that were associated (docked) at release sites of the presynaptic membrane, called active-zones. Vesicles docked at the active zone occupied a strategic location: they formed regions of contact with the plasma membrane on one side and with that of one or more vesicles located deeper within the presynaptic terminal on the other side. The region of contact with the active zone measured approximately 15 nm in diameter ( approximately 2% of the vesicle's surface) and contained a smaller approximately 6 nm region where the proximal leaflets merged (hemi-fused). Hemi-fusion was only observed on the side of vesicles in contact with the active zone; at the side of contact between neighboring vesicles, the membranes were not hemi-fused. Approximately three-fourths of the docked vesicles contained hemi-fused regions. Vesicles fully fused to the active zone (exhibiting pores that appeared as interruptions of a single membrane) were less frequently observed ( approximately 1 of 10 hemi-fused vesicles). In conclusion, our observations in cortical synapses strengthen the hypothesis that hemi-fusion is a stable intermediary that precedes full fusion and release.

Orbitofrontal ensemble activity monitors licking and distinguishes among natural rewards.

The classification of rhythmic licking into clusters has proved to be useful for characterizing brain mechanisms that modulate the ingestion of natural rewards (sucrose and water). One cortical area that is responsive to rewarding stimuli is the orbitofrontal cortex (OFC). However, it is not presently known how OFC neurons respond while rodents freely lick for natural rewards and whether these responses are related to the structure of licking clusters. We addressed these issues by showing that temporary inactivation of the OFC decreases the duration and increases the number of clusters and that the activity of OFC neurons changed at precise times before, during, and after the cluster terminates. Furthermore, analysis of the activity of OFC neuronal ensembles showed that they could discriminate cluster onset from termination, predict when a behaving animal will begin a cluster, and distinguish and anticipate between natural rewards. These results provide a new role for the OFC in influencing licking clusters and anticipating specific rewards.

Chronic IL-1beta signaling potentiates voltage-dependent sodium currents in trigeminal nociceptive neurons.

The proinflammatory cytokine interleukin-1beta (IL-1beta) mediates inflammation and hyperalgesia, although the underlying mechanisms remain elusive. To better understand such molecular and cellular mechanisms, we investigated how IL-1beta modulates the total voltage-dependent sodium currents (INa) and its tetrodotoxin-resistant (TTX-R) component in capsaicin-sensitive trigeminal nociceptive neurons, both after a brief (5-min) and after a chronic exposure (24-h) of 20 ng/ml IL-1beta. A brief exposure led to a 28% specific (receptor-mediated) reduction of INa in these neurons, which were found to contain type I IL-1 receptors (IL-1RI+) on both their soma and nerve endings. In marked contrast, after a 24-h exposure, the total sodium current was specifically increased by 67%, without significantly affecting the TTX-R component. This potentiation of INa was suppressed in the presence of selective inhibitors of protein kinase C and G-protein-coupled signaling pathways, thereby suggesting that INa can be modulated through multiple pathways. In summary, the potentiation of INa through chronic IL-1beta signaling in nociceptive sensory neurons may be a critical component of inflammatory-associated hyperalgesia.

Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores.

The membrane-lytic peptide melittin has previously been shown to form pores in lipid bilayers that have been described in terms of two different structural models. In the "barrel stave" model the bilayer remains more or less flat, with the peptides penetrating across the bilayer hydrocarbon region and aggregating to form a pore, whereas in the "toroidal pore" melittin induces defects in the bilayer such that the bilayer bends sharply inward to form a pore lined by both peptides and lipid headgroups. Here we test these models by measuring both the free energy of melittin transfer (DeltaG degrees ) and melittin-induced leakage as a function of bilayer elastic (material) properties that determine the energetics of bilayer bending, including the area compressibility modulus (K(a)), bilayer bending modulus (k(c)), and monolayer spontaneous curvature (R(o)). The addition of cholesterol to phosphatidylcholine (PC) bilayers, which increases K(a) and k(c), decreases both DeltaG degrees and the melittin-induced vesicle leakage. In contrast, the addition to PC bilayers of molecules with either positive R(o), such as lysoPC, or negative R(o), such as dioleoylglycerol, has little effect on DeltaG degrees , but produces large changes in melittin-induced leakage, from 86% for 8:2 PC/lysoPC to 18% for 8:2 PC/dioleoylglycerol. We observe linear relationships between melittin-induced leakage and both K(a) and 1/R(o)(2). However, in contrast to what would be expected for a barrel stave model, there is no correlation between observed leakage and bilayer hydrocarbon thickness. All of these results demonstrate the importance of bilayer material properties on melittin-induced leakage and indicate that the melittin-induced pores are defects in the bilayer lined in part by lipid molecules.

The protein tyrosine kinase inhibitor, genistein, decreases excitability of nociceptive neurons.

One mechanism by which neurons regulate their excitability is through ion channel phosphorylation. Compounds that increase nociceptive neuron excitability can cause hyperalgesia or allodynia whereas compounds that decrease nociceptive neuron excitability can be used as analgesics to relieve pain arising from inflammation or trauma. To identify targets that may cause a decrease in nociceptive neuron excitability, we have investigated the effects of genistein, a specific inhibitor of protein tyrosine kinases (PTKs), on capsaicin-sensitive neurons from cultured rat trigeminal ganglion neurons. It was found that genistein decreased the number of evoked action potentials, and hence their excitability. To determine whether genistein's effects occur through the inhibition of PTKs, we also tested the effects of two of its inactive analogues, daidzein and genistin. Whereas daidzein decreased excitability, albeit to a lower extent than genistein, excitability was unaffected by genistin. To determine which currents are involved in genistein's reduction in nociceptive neuron excitability, whole-cell voltage-clamp measurements were performed on voltage-gated sodium and potassium currents. One hundred micromolar genistein, daidzein and genistin inhibited tetrodotoxin-resistant voltage-gated sodium currents 74, 42, and 3%, respectively. Genistein markedly inhibited delayed rectifier (IK) and IA potassium currents, whereas daidzein and genistin were comparatively ineffective. In summary, we found that genistein's ability to inhibit nociceptive neuron excitability arises primarily from its non-specific inhibition of voltage-dependent sodium channels.

Voltage-gated ion channels in nociceptors: modulation by cGMP.

In tissue or nerve injury, proinflammatory mediators are released that can modulate a variety of ion channels found in nociceptors. The changes in channel activity, which primarily occurs through changes in intracellular pathways, may lead to the pathological states of hyperalgesia and allodynia. To understand further the regulatory mechanisms underlying the changes in channel activity, we used whole cell patch-clamp recordings from capsaicin-sensitive nociceptive neurons in rat trigeminal ganglion neurons to examine how the cGMP-dependent pathways may regulate ion channel function. Addition of the 8-(4-chlorophenylthio)-3',5' (CPT)-cGMP, a membrane permeant modulator of ion channels, decreased the number of evoked action potentials by 36% and inhibited the tetrodotoxin-resistant (TTX-R) sodium currents and IA potassium currents by 37 and 32%, respectively. Delayed rectifier potassium (IK) currents were unaffected, suggesting that the effects of CPT-cGMP are unlikely to arise from a nonspecific effect on channel activity as a consequence of the adsorption of amphipathic CPT-cGMP molecules to the membrane's bilayer component. This conclusion was reinforced by the lack of changes in gramicidin A channel function in the presence of CTP-cGMP. In summary, the activation of the cGMP-dependent pathways reduces nociceptor excitability, in part, by decreasing the activity of voltage-gated TTX-R sodium channels. This pathway may be a target for efforts to produce selective analgesics.

Nicotine inhibits voltage-dependent sodium channels and sensitizes vanilloid receptors.

Nicotine is an alkaloid that is used by large numbers of people. When taken into the body, it produces a myriad of physiological actions that occur primarily through the activation of neuronal nicotinic acetylcholine receptors (nAChRs). We have explored its ability to modulate TRPV1 receptors and voltage-gated sodium channels. The reason for investigating nicotine's effect on sodium channels is to obtain a better understanding of its anti-nociceptive properties. The reasons for investigating its effects on capsaicin-activated TRPV1 channels are to understand how it may modulate this channel that is involved in pain, inflammation, and gustatory physiology. Whole cell patch-clamp recordings from rat trigeminal ganglion (TG) nociceptors revealed that nicotine exhibited anesthetic properties by decreasing the number of evoked action potentials and by inhibiting tetrodotoxin-resistant sodium currents. This anesthetic property can be produced without the necessity of activating nAChRs. Nicotine also modulates TRPV1 receptors inducing a several-fold increase in capsaicin-activated currents in both TG neurons and in cells with heterologously expressed TRPV1 receptors. This sensitizing effect does not require the activation of nAChRs. Nicotine did not alter the threshold temperature (approximately 41 degrees C) of heat-activated currents in TG neurons that were attributed to arise from the activation of TRPV1 receptors. In this regard, its effect on TRPV1 receptors differs from those of ethanol that has been shown to increase the capsaicin-activated current but decrease the threshold temperature. These studies document several new effects of nicotine on channels involved in nociception and indicate how they may impact physiological processes involving pain and gustation.

TRPV1 receptors mediate particulate matter-induced apoptosis.

Exposure to airborne particulate matter (PM) is a world-wide health problem mainly because it produces adverse cardiovascular and respiratory effects that frequently result in morbidity. Despite many years of epidemiological and basic research, the mechanisms underlying PM toxicity remain largely unknown. To understand some of these mechanisms, we measured PM-induced apoptosis and necrosis in normal human airway epithelial cells and sensory neurons from both wild-type mice and mice lacking TRPV1 receptors using Alexa Fluor 488-conjugated annexin V and propidium iodide labeling, respectively. Exposure of environmental PMs containing residual oil fly ash and ash from Mount St. Helens was found to induce apoptosis, but not necrosis, as a consequence of sustained calcium influx through TRPV1 receptors. Apoptosis was completely prevented by inhibiting TRPV1 receptors with capsazepine or by removing extracellular calcium or in sensory neurons from TRPV1(-/-) mice. Binding of either one of the PMs to the cell membrane induced a capsazepine-sensitive increase in cAMP. PM-induced apoptosis was augmented upon the inhibition of PKA. PKA inhibition on its own also induced apoptosis, thereby suggesting that this pathway may be endogenously protective against apoptosis. In summary, it was found that inhibiting TRPV1 receptors prevents PM-induced apoptosis, thereby providing a potential mechanism to reduce their toxicity.

Vanilloid receptor activation by 2- and 10-microm particles induces responses leading to apoptosis in human airway epithelial cells.

Exposure to airborne particulate matter (PM) is associated with increased mortality and morbidity. It has been previously shown that PMs and synthetic particles (PC10 and PC2) that have similar characteristics to PMs induced depolarizing currents and increases in intracellular calcium ([Ca2+]i) in capsaicin- and acid-sensitive sensory neurons and in TRPV1-expressing HEK 293 cells. To determine whether such mechanisms also underlie PM-induced toxicity in epithelial cells lining the human airways, we tested the responses of PCs on BEAS-2B (immortalized human bronchial epithelial cells), NHBE (normal human bronchial/tracheal epithelial cells), and SAEC (normal human small airway epithelial cells from the distal airways). RT-PCR revealed that all these cell types expressed TRPV1 (VR1), ASIC1a, and ASIC3 subunits of proton-gated ion channels. Calcium imaging studies revealed that in all three cell types approximately 30% were activated by both capsaicin and acid. In these cells, PCs induced an increase in [Ca2+]i that was inhibited by capsazepine, a TRPV1 antagonist, and/or by amiloride, an ASIC antagonist. The capsazepine-sensitive contribution to PC-induced increases in [Ca2+]i was approximately 70%. Measurements of apoptosis revealed that exposure to PCs induced a time-dependent increase in the number of apoptotic cells. After incubation for 24 (PC10) or 48 h (PC2) approximately 60% of these cells were apoptotic. Pretreatment with capsazepine as well as removal of external calcium completely (approximately 100%) prevented PC-induced apoptosis. These data suggest that pharmacological inhibition of calcium-permeable vanilloid receptors could be used to prevent some of the pathological actions of PMs.