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.

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.

Structural characteristics of gap junctions. I. Channel number in coupled and uncoupled conditions.

Gap junctions between crayfish lateral axons were studied by combining anatomical and electrophysiological measurements to determine structural changes associated during uncoupling by axoplasmic acidification. In basal conditions, the junctional resistance, Rj, was approximately 60-80 k omega and the synapses appeared as two adhering membranes; 18-20-nm overall thickness, containing transverse densities (channels) spanning both membranes and the narrow extracellular gap (4-6 nm). In freeze-fracture replicas, the synapses contained greater than 3 X 10(3) gap junction plaques having a total of approximately 3.5 X 10(5) intramembrane particles. "Single" gap junction particles represented approximately 10% of the total number of gap junction particles present in the synapse. Therefore, in basal conditions, most of the gap junction particles were organized in plaques. Moreover, correlations of the total number of gap junction particles with Rj suggested that most of the junctional particles in plaques corresponded to conducting channels. Upon acidification of the axoplasm to pH 6.7-6.8, the junctional resistance increased to approximately 300 k omega and action potentials failed to propagate across the septum. Morphological measurements showed that the total number of gap junction particles in plaques decreased approximately 11-fold to 3.1 X 10(4) whereas the number of single particles dispersed in the axolemmae increased significantly. Thin sections of these synapses showed that the width of the extracellular gap increased from 4-6 nm in basal conditions to 10-20 nm under conditions where axoplasmic pH was 6.7-6.8. These observations suggest that single gap junction particles dispersed in the synapse most likely represent hemi-channels produced by the dissasembly of channels previously arranged in plaques.

The structural organization and protein composition of lens fiber junctions.

The structural organization and protein composition of lens fiber junctions isolated from adult bovine and calf lenses were studied using combined electron microscopy, immunolocalization with monoclonal and polyclonal anti-MIP and anti-MP70 (two putative gap junction-forming proteins), and freeze-fracture and label-fracture methods. The major intrinsic protein of lens plasma membranes (MIP) was localized in single membranes and in an extensive network of junctions having flat and undulating surface topologies. In wavy junctions, polyclonal and monoclonal anti-MIPs labeled only the cytoplasmic surface of the convex membrane of the junction. Label-fracture experiments demonstrated that the convex membrane contained MIP arranged in tetragonal arrays 6-7 nm in unit cell dimension. The apposing concave membrane of the junction displayed fracture faces without intramembrane particles or pits. Therefore, wavy junctions are asymmetric structures composed of MIP crystals abutted against particle-free membranes. In thin junctions, anti-MIP labeled the cytoplasmic surfaces of both apposing membranes with varying degrees of asymmetry. In thin junctions, MIP was found organized in both small clusters and single membranes. These small clusters also abut against particle-free apposing membranes, probably in a staggered or checkerboard pattern. Thus, the structure of thin and wavy junctions differed only in the extent of crystallization of MIP, a property that can explain why this protein can produce two different antibody-labeling patterns. A conclusion of this study is that wavy and thin junctions do not contain coaxially aligned channels, and, in these junctions, MIP is unlikely to form gap junction-like channels. We suggest MIP may behave as an intercellular adhesion protein which can also act as a volume-regulating channel to collapse the lens extracellular space. Junctions constructed of MP70 have a wider overall thickness (18-20 nm) and are abundant in the cortical regions of the lens. A monoclonal antibody raised against this protein labeled these thicker junctions on the cytoplasmic surfaces of both apposing membranes. Thick junctions also contained isolated clusters of MIP inside the plaques of MP70. The role of thick junctions in lens physiology remains to be determined.

On the structural organization of isolated bovine lens fiber junctions.

Junctions between fiber cells of bovine lenses have been isolated in milligram quantities, without using detergents or proteases. The structure of the isolated junctions has been studied by thin-section, negative-stain, and freeze-fracture electron microscopy and by x-ray diffraction. The junctions are large and most often have an undulating surface topology as determined by thin sectioning and freeze-fracture. These undulations resemble the tongue-and-groove interdigitations between lens fiber cells previously seen by others (D. H. Dickson and G. W. Crock, 1972, Invest. Ophthalmol. 11:809-815). In sections, the isolated junctions display a pentalamellar structure approximately 13-14 nm in overall thickness, which is significantly thinner than liver gap junctions. Each junctional membrane contains in the plane of the lipid bilayers distinct units arranged in a square lattice with a center-to-center spacing of 6.6 nm. Freeze-fracture replicas of the junctions fractured transversely show that the repeating units extend across the entire thickness of each membrane. Each unit is probably constructed from four identical subunits, with each subunit containing a protein of an apparent molecular weight of 27,000. We conclude that the lens junctions are structurally and chemically, different from gap junctions and could represent a new kind of intercellular contact, not simply another crystalline state of the gap junction protein.

Influence of cholesterol on water penetration into bilayers.

X-ray diffraction and capacitance measurements have been used to calculate the depth to which water penetrates in fully hydrated bacterial phosphatidylethanolamine bilayers in the presence and absence of cholesterol. The data indicate that cholesterol decreases in depth of water penetration by about 2.5 angstroms.

The dielectric constant of phospholipid bilayers and the permeability of membranes to ions.

The Born charging equation predicts that the permeability of a phospholipid bilayer membrane to ions should depend markedly on the dielectric constant of the membrane. Increasing the dielectric constant of an artificial bilayer increases its permeability to perchlorate or thiocyanate by a factor of 1000, to a value comparable to that of mitochondrial membranes.

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.

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.

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.

Taste-specific neuronal ensembles in the gustatory cortex of awake rats.

In gustatory cortex, single-neuron activity reflects the multimodal processing of taste stimuli. Little is known, however, about the interactions between gustatory cortical (GC) neurons during tastant processing. Here, these interactions were characterized. It was found that 36% (85 of 237) of neuron pairs, including many (61%) in which one or both single units were not taste specific, produced significant cross-correlations (CCs) to a subset of tastants across a hundreds of milliseconds timescale. Significant CCs arose from the coupling between the firing rates of neurons as those rates changed through time. Such coupling significantly increased the amount of tastant-specific information contained in ensembles. These data suggest that taste-specific GC assemblies may transiently form and coevolve on a behaviorally appropriate timescale, contributing to rats' ability to discriminate tastants.

Dynamic and multimodal responses of gustatory cortical neurons in awake rats.

To investigate the dynamic aspects of gustatory activity, we recorded the responses of small ensembles of cortical neurons to tastants administered to awake rats. Multiple trials of each tastant were delivered during recordings made in oral somatosensory (SI) and gustatory cortex (GC). When integrated tastant responses (firing rates averaged across 2.5 sec) were compared with water responses, 14.4% (13/90) of the GC neurons responded in a taste-specific manner. When time was considered as a source of information, however, the incidence of taste-specific firing increased: as many as 41% (37/90) of the recorded GC neurons exhibited taste-specific patterns of response. For 17% of the neurons identified as responding with taste-specific patterns, the stimulus that caused the most significant response was a function of the time since stimulus delivery. That is, a single neuron might respond most strongly to one tastant in the first 500 msec of a response and then respond most strongly to another tastant later in the response. Further analysis of the time courses of GC and SI cortical neural responses revealed that modulations of GC firing rate arose from three separable processes: early somatosensory input (less than approximately 0.2 sec post-stimulus), later chemosensory input ( approximately 0.2-1 sec), and delayed somatosensory input related to orofacial responses (more than approximately 1.0 sec). These data demonstrate that sensory information is available in the time course of GC responses and suggest the viability of views of gustatory processing that treat the temporal structure of cortical responses as an integral part of the neural code.

Interdigitated hydrocarbon chain packing causes the biphasic transition behavior in lipid/alcohol suspensions.

It has been shown recently by Rowe ((1983) Biochemistry 22, 3299-3305) that ethanol has a 'biphasic' effect on the transition temperature (Tm) of phosphatidylcholine bilayers, reducing Tm at low concentrations but increasing Tm at high concentrations. Our X-ray diffraction data show that this reversal of Tm is a consequence of the induction of an unusual gel phase, where the lipid hydrocarbon chains from apposing monolayers fully interpenetrate or interdigitate. The properties of this interdigitated phase also explain the lipid chain length dependence of the reversal in the Tm versus ethanol concentration curves and the narrow width of the transition at high ethanol concentrations, as well as spectroscopic and calorimetric data from lipid suspensions containing other drugs such as methanol, benzyl alcohol, phenyl ethanol, and chlorpromazine.

Surface ripples cause the large fluid spaces between gel phase bilayers containing small amounts of cholesterol.

Previous studies have found that small concentrations of cholesterol, or several other molecules such as benzene and asialoganglioside, dramatically increase the fluid separation between gel phase phosphatidylcholine bilayers. These observations can not be explained in terms of changes in the repulsive and attractive pressures known to exist between flat gel phase bilayer surfaces. We show here that the increase in fluid space occurs as a consequence of cholesterol inducing large periodic ripples in the plane of the bilayer. The analysis of Mortensen et al. (Biochim. Biophys. Acta 945, 221-245) indicates that the sides of the ripples primarily contain gel phase phosphatidylcholine, whereas the apices are enriched in cholesterol and are liquid-crystalline. We argue that the large fluid spaces can be explained by steric repulsion between adjacent bilayers caused both by thermally induced accordion-like motions of these ripples and defects in the ripple organization. In addition, ripples potentially can decrease van der Waals attraction and change hydration repulsion between bilayers.

Solubility of carbon dioxide in lipid bilayer membranes and organic solvents.

Partition coefficients of carbon dioxide into lipid bilayers (liposomes) and organic solvents were measured as a function of temperature. The molar partition coefficient of CO2 into liposomes of egg lecithin at 25 degrees C was 0.95 (ml CO2/ml lipid)/(ml CO2/ml saline). The addition of an equimolar amount of cholesterol to the egg lecithin decreased the partition coefficient by about 25%. The partition coefficients for CO2 into liposomes at 25 degrees C were lower than the partition coefficients into octanol (1.3), hexadecane (1.5) and olive oil (1.7). The results are discussed in terms of the solubility-diffusion model of non-electrolyte transport through lipid bilayer membranes.

A simple model for calcium induced exocytosis.

We have developed a simple model showing how the presence or obsence of Ca2+ can determine whether an uncurved or curved membrane surface is favored energetically. The model shows why fusion of vesicles with the presynaptic membrane is favored in the presence of calcium and why the budding off of vesicles is favored in the absence of calcium inside of the presynaptic membrane. The model accurately predicts the radius of a synaptic vesicle using known properties of lipids and suggests consequences of temperature change, varied stimulation rate and addition of calcium by artificial means on rates of transmitter release.

The organization of n-alkanes in lipid bilayers.

The interaction of n-alkanes (C6--C16) with phosphatidylcholine has been studied by the combined use of differential scanning calorimetry, X-ray diffraction and monolayer techniques. It has been found that the thermal properties and ultrastructure of lipid-alkane vesicles are strongly dependent on the length of the n-alkanes. Long alkanes, such as tetradecane and hexadecane, increase the transition temperature of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, while the X-ray data indicate that these long alkanes align parallel to the lipid acyl chains. In contrast, shorter alkanes, such as hexane and octane, decrease and broaden the thermal transition and electron density profiles show that these alkanes increase bilayer width by partitioning between the apposing monolayers of the bilayer. For lipids in the gel and liquid crystalline states, the short alkanes form an alkane region in the geometric center of the bilayer.

Can regular solution theory be applied to lipid bilayer membranes?

Direct measurement of the partition coefficient of n-hexane into phosphatidylcholine and phosphatidylcholine-cholesterol bilayers showed that (a) isotropic liquids are not good models for lipid bilayers and (b), Regular Solution Theory cannot, in general, be applied to lipid bilayer membranes at temperatures above their phase transition. Theoretical and experimental evidence given.

One-dimensional crystals of (Na+ + K+)-ATPase dimers.

Preparations of purified (Na+ + K+)-ATPase contain both fragments of membranes and long and undulating cylindrical structures. These structures have been described as edgeways of membrane fragments. We have analyzed these structures using negative staining, thin sectioning and freeze-fracture-etch electron microscopy and describe their structure for the first time. Each cylinder is 12-19 nm in width and is comprised of an unstained core from which rows of distinct particles spaced 5-6 nm apart project on both sides. Each cylindrical structure was interpreted as a linear polymer of (alpha beta)2 dimers of (Na+ + K+)-ATPase molecules. Therefore, the particles that project from both sides are the cytoplasmic domains of the molecules of the enzyme, whereas the membrane-spanning domains form the unstained core of the cylinder. From considerations of the packing of the dimers in the cylinder we conclude that the cross-sectional area of the cytoplasmic domain should be larger than that of the membrane-spanning domain. Our results are consistent with the hypothesis that the (alpha beta) protomer is the native state of the enzyme. The (alpha beta)2 dimers observed in the fractions are the result of a secondary aggregation process occurring during the purification procedure.