The coding of temperature in the Drosophila brain

Thermosensation is an indispensable sensory modality. Here, we study temperature coding in Drosophila, and show that temperature is represented by a spatial map of activity in the brain. First, we identify TRP channels that function in the fly antenna to mediate the detection of cold stimuli. Next, we identify the hot-sensing neurons and show that hot and cold antennal receptors project onto distinct, but adjacent glomeruli in the Proximal-Antennal-Protocerebrum (PAP) forming a thermotopic map in the brain. We use two-photon imaging to reveal the functional segregation of hot and cold responses in the PAP, and show that silencing the hot- or cold-sensing neurons produces animals with distinct and discrete deficits in their behavioral responses to thermal stimuli. Together, these results demonstrate that dedicated populations of cells orchestrate behavioral responses to different temperature stimuli, and reveal a labeled-line logic for the coding of temperature information in the brain.     

TRPM8 is required for cold sensation in mice

ThermoTRPs, a subset of the Transient Receptor Potential (TRP) family of cation channels, have been implicated in sensing temperature. TRPM8 and TRPA1 are both activated by cooling; however, it is unclear whether either ion channel is required for thermosensation in vivo. We show that mice lacking TRPM8 have severe behavioral deficits in response to cold stimuli. In thermotaxis assays of temperature gradient and two-temperature choice assays, TRPM8-deficient mice exhibit strikingly reduced avoidance of cold temperatures. TRPM8-deficient mice also lack behavioral response to cold-inducing icilin application and display an attenuated response to acetone, an unpleasant cold stimulus. However, TRPM8-deficient mice have normal nociceptive-like responses to subzero centigrade temperatures, suggesting the presence of at least one additional noxious cold receptor. Finally, we show that TRPM8 mediates the analgesic effect of moderate cooling after administration of formalin, a painful stimulus. Therefore, depending on context, TRPM8 contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia.     

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 °C to 5 °C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.     

Feeding preferences in 2 disjunct populations of tiger snakes, Notechis scutatus (Elapidae)

Variations at both the genetic and phenotypic levels play animportant role in responses to food and food-related stimuli.Knowledge of such variations is crucial to understanding howpopulations adapt to changing environments. We investigatedthe dietary preferences of 2 tiger snake populations and comparedthe responses of diet-naive animals (laboratory-born neonates),diet-controlled animals (laboratory-reared juveniles), and naturaldiet–experienced animals (wild-caught adults) to visualand chemical cues from 6 prey types (mouse, skink, silver gull,chicken, shearwater, and frog). The mainland population inhabitsa swamp, feeds mostly on frogs, and suffers heavy predation.The second population inhabits a small nearby offshore islandwith no standing water (no frogs); feeds mostly on skinks, mice,and, as adults, silver gull chicks; and suffers no known predation.Although different prey are eaten in the 2 populations, adultwild-caught snakes from both populations showed a significantpreference for 3 types of prey (frog, mouse, and chick), irrespectiveof their natural diet. Neonates responded to all prey cues morethan they did to control stimuli in both populations. However,the island neonates showed significantly higher interest insilver gull chick stimuli (the main prey of the island adultsnakes) than did their mainland conspecifics. Laboratory-bredjuveniles displayed behavioral plasticity by significantly increasingtheir response to mice after being fed baby mice for 7 months.We conclude that genetic-based differences in food-related cuesare important in tiger snakes but that they are also capableof behavioral plasticity. Island adult and neonate snakes exhibitedresponses to prey types no longer consumed naturally (frog),suggesting that behavioral characters may have been retainedfor long periods under relaxed selection. Island neonates showeda strong interest in a novel prey item (silver gull). This resultcomplements previous work describing how island snakes havedeveloped the ability to swallow larger prey than usual, aswell as seemingly developing a taste for them.     

Differences in spontaneously avoiding or approaching mice reflect differences in CB1-mediated signaling of dorsal striatal transmission

Approach or avoidance behaviors are accompanied by perceptual vigilance for, affective reactivity to and behavioral predisposition towards rewarding or punitive stimuli, respectively. We detected three subpopulations of C57BL/6J mice that responded with avoiding, balancing or approaching behaviors not induced by any experimental manipulation but spontaneously displayed in an approach/avoidance conflict task. Although the detailed neuronal mechanisms underlying the balancing between approach and avoidance are not fully clarified, there is growing evidence that endocannabinoid system (ECS) plays a critical role in the control of these balancing actions. The sensitivity of dorsal striatal synapses to the activation of cannabinoid CB1 receptors was investigated in the subpopulations of spontaneously avoiding, balancing or approaching mice. Avoiding animals displayed decreased control of CB1 receptors on GABAergic striatal transmission and in parallel increase of behavioral inhibition. Conversely, approaching animals exhibited increased control of CB1 receptors and in parallel increase of explorative behavior. Balancing animals reacted with balanced responses between approach and avoidance patterns. Treating avoiding animals with URB597 (fatty acid amide hydrolase inhibitor) or approaching animals with AM251 (CB1 receptor inverse agonist) reverted their respective behavioral and electrophysiological patterns. Therefore, enhanced or reduced CB1-mediated control on dorsal striatal transmission represents the synaptic hallmark of the approach or avoidance behavior, respectively. Thus, the opposite spontaneous responses to conflicting stimuli are modulated by a different involvement of endocannabinoid signaling of dorsal striatal neurons in the range of temperamental traits related to individual differences.     

A novel behavioral assay for measuring cold sensation in mice

Behavioral models of cold responses are important tools for exploring the molecular mechanisms of cold sensation. To complement the currently cold behavioral assays and allow further studies of these mechanisms, we have developed a new technique to measure the cold response threshold, the cold plantar assay. In this assay, animals are acclimated on a glass plate and a cold stimulus is applied to the hindpaw through the glass using a pellet of compressed dry ice. The latency to withdrawal from the cooled glass is used as a measure of the cold response threshold of the rodents, and the dry ice pellet provides a ramping cold stimulus on the glass that allows the correlation of withdrawal latency values to rough estimates of the cold response threshold temperature. The assay is highly sensitive to manipulations including morphine-induced analgesia, Complete Freund's Adjuvant-induced inflammatory allodynia, and Spinal Nerve Ligation-induced neuropathic allodynia.     

The Effects of Tail Biopsy for Genotyping on Behavioral Responses to Nociceptive Stimuli

Removal of a small segment of tail at weaning is a common method used to obtain tissue for the isolation of genomic DNA to identify genetically modified mice. When genetically manipulated mice are used for pain research, this practice could result in confounding changes to the animals'' responses to noxious stimuli. In this study, we sought to systematically investigate whether tail biopsy representative of that used in standard genotyping methods affects behavioral responses to a battery of tests of nociception. Wild-type littermate C57BL/6J and 129S6 female and male mice received either tail biopsies or control procedural handling at Day 21 after birth and were then tested at 6–9 weeks for mechanical and thermal sensitivity. C57BL/6J mice were also tested in the formalin model of inflammatory pain. In all tests performed (von Frey, Hargreaves, modified Randall Selitto, and formalin), C57BL/6J tail-biopsied animals'' behavioral responses were not significantly different from control animals. In 129S6 animals, tail biopsy did not have a significant effect on behavioral responses in either sex to the von Frey and the modified Randall-Selitto tests of mechanical sensitivity. Interestingly, however, both sexes exhibited small but significant differences between tail biopsied and control responses to a radiant heat stimulus. These results indicate that tail biopsy for genotyping purposes has no effect on nocifensive behavioral responses of C57BL/6J mice, and in 129S6 mice, causes only a minor alteration in response to a radiant heat stimulus while other nocifensive behavioral responses are unchanged. The small effect seen is modality- and strain-specific.     

TRPV1-lineage neurons are required for thermal sensation

The ion-channel TRPV1 is believed to be a major sensor of noxious heat, but surprisingly animals lacking TRPV1 still display marked responses to elevated temperature. In this study, we explored the role of TRPV1-expressing neurons in somatosensation by generating mice wherein this lineage of cells was selectively labelled or ablated. Our data show that TRPV1 is an embryonic marker of many nociceptors including all TRPV1- and TRPM8-neurons as well as many Mrg-expressing neurons. Mutant mice lacking these cells are completely insensitive to hot or cold but in marked contrast retain normal touch and mechanical pain sensation. These animals also exhibit defective body temperature control and lose both itch and pain reactions to potent chemical mediators. Together with previous cell ablation studies, our results define and delimit the roles of TRPV1- and TRPM8-neurons in thermosensation, thermoregulation and nociception, thus significantly extending the concept of labelled lines in somatosensory coding.     

Decreased sensory responses in osteocalcin null mutant mice imply neuropeptide function

Osteocalcin, the most abundant member of the family of extracellular mineral binding gamma-carboxyglutamic acid proteins is synthesized primarily by osteoblasts. Its affinity for calcium ions is believed to limit bone mineralization. Several of the numerous hormones that regulate synthesis of osteocalcin, including glucocorticoids and parathyroid hormone, are also affected by stressful stimuli that require energy for an appropriate response. Based on our observations of OC responding to stressful sensory stimuli, the expression of OC in mouse and rat sensory ganglia was confirmed. It was thus hypothesized that the behavioral responses of the OC knockout mouse to stressful sensory stimuli would be abnormal. To test this hypothesis, behaviors related to sensory aspects of the stress response were quantified in nine groups of mice, aged 4-14 months, comparing knockout with their wild-type counterparts in six distinctly different behavioral tests. Resulting data indicated the following statistically significant differences: open field grooming frequency following saline injection, wild-type > knockout; paw stimulation with Von Frey fibers, knockout < wild-type; balance beam, knockout mobility < WT; thermal sensitivity to heat (tail flick), knockout < wild-type; and cold, knockout < wild-type. Insignificant differences in hanging wire test indicate that these responses are unrelated to reduced muscle strength. Each of these disparate environmental stimuli provided data indicating alterations of responses in knockout mice that suggest participation of osteocalcin in transmission of information about those sensory stimuli.     

A variable-response model for parasitoid foraging behavior

An important factor inducing variability in foraging behavior in parasitic wasps is experience gained by the insect. Together with the insect's genetic constitution and physiological state, experience ultimately defines the behavioral repertoire under specified environmental circumstances. We present a conceptual variable-response model based on several major observations of a foraging parasitoid's responses to stimuli involved in the hostfinding process. These major observations are that (1) different stimuli evoke different responses or levels of response, (2) strong responses are less variable than weak ones, (3) learning can change response levels, (4) learning increases originally low responses more than originally high responses, and (5) hostderived stimuli serve as rewards in associative learning of other stimuli. The model specifies how the intrinsic variability of a response will depend on the magnitude of the response and predicts when and how learning will modify the insect's behavior. Additional hypotheses related to the model concern how experience with a stimulus modifies behavioral responses to other stimuli, how animals respond in multistimulus situations, which stimuli act to reinforce behavioral responses to other stimuli in the learning process, and finally, how generalist and specialist species differ in their behavioral plasticity. We postulate that insight into behavioral variability in the foraging behavior of natural enemies may be a help, if not a prerequisite, for the efficient application of parasitoids in pest management.     

Moderate prenatal alcohol exposure impairs cognitive control,but not attention,on a rodent touchscreen continuous performance task

A common feature associated with fetal alcohol spectrum disorders is the inability to concentrate on a specific task while ignoring distractions. Human continuous performance tasks (CPT), measure vigilance and cognitive control simultaneously while these processes are traditionally measured separately in rodents. We recently established a touchscreen 5-choice CPT (5C-CPT) that measures vigilance and cognitive control simultaneously by incorporating both target and nontargets and showed it was sensitive to amphetamine-induced improvement in humans and mice. Here, we examined the effects of moderate prenatal alcohol exposure (PAE) in male and female mice on performance of the 5-choice serial reaction time task (5-CSRTT), which contained only target trials, and the 5C-CPT which incorporated both target and nontarget trials. In addition, we assessed gait and fine motor coordination in behavioral naïve PAE and control animals. We found that on the 5-CSRTT mice were able to respond to target presentations with similar hit rates regardless of sex or treatment. However, on the 5C-CPT PAE mice made significantly more false alarm responses vs controls. Compared with control animals, PAE mice had a significantly lower sensitivity index, a measure of ability to discriminate appropriate responses to stimuli types. During 5C-CPT, female mice, regardless of treatment, also had increased mean latency to respond when correct and omitted more target trials. Gait assessment showed no significant differences in PAE and SAC mice on any measure. These findings suggest that moderate exposure to alcohol during development can have long lasting effects on cognitive control unaffected by gross motor alterations.     

Systems genetics of sensation seeking

Sensation seeking is a multifaceted, heritable trait which predicts the development of substance use and abuse in humans; similar phenomena have been observed in rodents. Genetic correlations among sensation seeking and substance use indicate shared biological mechanisms, but the genes and networks underlying these relationships remain elusive. Here, we used a systems genetics approach in the BXD recombinant inbred mouse panel to identify shared genetic mechanisms underlying substance use and preference for sensory stimuli, an intermediate phenotype of sensation seeking. Using the operant sensation seeking (OSS) paradigm, we quantified preference for sensory stimuli in 120 male and 127 female mice from 62 BXD strains and the C57BL/6J and DBA/2J founder strains. We used relative preference for the active and inactive levers to dissociate preference for sensory stimuli from locomotion and exploration phenotypes. We identified genomic regions on chromosome 4 (155.236‐155.742 Mb) and chromosome 13 (72.969‐89.423 Mb) associated with distinct behavioral components of OSS. Using publicly available behavioral data and mRNA expression data from brain regions involved in reward processing, we identified (a) genes within these behavioral QTL exhibiting genome‐wide significant cis‐eQTL and (b) genetic correlations among OSS phenotypes, ethanol phenotypes and mRNA expression. From these analyses, we nominated positional candidates for behavioral QTL associated with distinct OSS phenotypes including Gnb1 and Mef2c. Genetic covariation of Gnb1 expression, preference for sensory stimuli and multiple ethanol phenotypes suggest that heritable variation in Gnb1 expression in reward circuitry partially underlies the widely reported relationship between sensation seeking and substance use.     

Peripheral and central aspects of trigeminal nociceptive systems

Three aspects of trigeminal pain are considered: the peripheral mechanisms of pain from teeth and from the cornea, and the role of the trigeminal brainstem nuclei in pain. Pain is probably the only sensation that can be evoked by stimulation of dentin or dental pulp in man. Five nerve-endings enter dentinal tubules from the pulp but do not extend into the outer dentine, which is nevertheless sensitive. In teeth of limited growth in experimental animals, the dental pulp is supplied by A beta, A delta and C fibres and these are associated with two categories of receptor: one responds to cooling and to other stimuli that cause displacement of the contents of the dentinal tubules such as probing and drying the dentine, and the other group responds most vigorously to heating. Some cold sensitive units have A beta fibres and the evidence suggests that stimulation of these is capable of evoking both muscle reflexes and pain and, near threshold, 'pre-pain' sensations. Thermal stimulation of the cornea produces sensations of pain and, with less intense stimuli, irritation, Mechanical stimulation also produces pain but it is not clear whether, below the pain threshold, such stimuli produce touch sensation or some other sensation related to pain. Histologically, the nerve-endings in the corneal epithelium consist of fine, bare processes closely associated with the surface of the epithelial cells. Recordings in experimental animals have shown that many of the receptors respond to several different forms of stimulus and their properties correlate well with those predicted from psychophysical experiments in man. The results of trigeminal tractotomy in man and recordings from the trigeminal brainstem nuclei in anaesthetized animals, have generally indicated that nucleus caudalis is the main relay in the pain pathway from the face and associated structures. Recent observations have, however, shown that tractotomy does not produce complete analgesia of this region and responses to thermal stimulation of teeth and noxious stimulation of other oro-facial tissues have been recorded from the more rostral parts of the brainstem nuclear complex. The surgical procedures employed to set up an animal for stereotaxic recording may induce long-lasting depression in the excitability of neurons in these nuclei, which masks some of their properties. The mechanism of this depression has not been established.     

Pharmacological blockade of TRPM8 ion channels alters cold and cold pain responses in mice

TRPM8 (Transient Receptor Potential Melastatin-8) is a cold- and menthol-gated ion channel necessary for the detection of cold temperatures in the mammalian peripheral nervous system. Functioning TRPM8 channels are required for behavioral responses to innocuous cool, noxious cold, injury-evoked cold hypersensitivity, cooling-mediated analgesia, and thermoregulation. Because of these various roles, the ability to pharmacologically manipulate TRPM8 function to alter the excitability of cold-sensing neurons may have broad impact clinically. Here we examined a novel compound, PBMC (1-phenylethyl-4-(benzyloxy)-3-methoxybenzyl(2-aminoethyl)carbamate) which robustly and selectively inhibited TRPM8 channels in vitro with sub-nanomolar affinity, as determined by calcium microfluorimetry and electrophysiology. The actions of PBMC were selective for TRPM8, with no functional effects observed for the sensory ion channels TRPV1 and TRPA1. PBMC altered TRPM8 gating by shifting the voltage-dependence of menthol-evoked currents towards positive membrane potentials. When administered systemically to mice, PBMC treatment produced a dose-dependent hypothermia in wildtype animals while TRPM8-knockout mice remained unaffected. This hypothermic response was reduced at lower doses, whereas responses to evaporative cooling were still significantly attenuated. Lastly, systemic PBMC also diminished cold hypersensitivity in inflammatory and nerve-injury pain models, but was ineffective against oxaliplatin-induced neuropathic cold hypersensitivity, despite our findings that TRPM8 is required for the cold-related symptoms of this pathology. Thus PBMC is an attractive compound that serves as a template for the formulation of highly specific and potent TRPM8 antagonists that will have utility both in vitro and in vivo.     

Toxoplasma gondii: cold stress-induced modulation of antibody responses.

Physical or psychological stressors have been shown to have significant consequences in the immune function and the outcome of disease in human and animal models. Recent work has demonstrated that products released during stress, such as glucocorticoids and catecholamines, can profoundly influence the in vitro growth of pathogens by modulating immune responses. The present study examined the effects of a physical stressor (cold stress) on antigens of Toxoplasma gondii that elicits an antibody-mediated immune response during the acute and chronic phases of infection. Sera obtained from different groups of mice subjected to cold stress during the acute and chronic phases of T. gondii infection were used to measure the levels of antibodies and to localize by Western blot the dominant antigens eliciting IgG and IgM antibody responses. Serum antibodies collected from stressed and infected mice recognized antigens different from those recognized by infected mice without stress. During the acute phase, a stronger IgM antibody response against antigens of 30, 42, 54, and 60 kDa was detected in stressed animals at 3 weeks postinfection. In addition, a 5-kDa antigen was specifically detected in mice subjected to stress during the acute and chronic phases of infection. Levels of specific IgG were increased in infected and in infected and stressed animals that underwent stress in the chronic phase. IgM production did not increase following cold stress in the chronic phase. These results suggest that the strong antibody response in stressed animals is associated with longer parasite persistence in circulation. Stress modulated not only the host immune response but also the ability of parasite antigens to elicit specific antibody responses by the host.     

Attenuated cold sensitivity in TRPM8 null mice

Thermosensation is an essential sensory function that is subserved by a variety of transducer molecules, including those from the Transient Receptor Potential (TRP) ion channel superfamily. One of its members, TRPM8 (CMR1), a ligand-gated, nonselective cation channel, is activated by both cold and chemical stimuli in vitro. However, its roles in cold thermosensation and pain in vivo have not been fully elucidated. Here, we show that sensory neurons derived from TRPM8 null mice lack detectable levels of TRPM8 mRNA and protein and that the number of these neurons responding to cold (18 degrees C) and menthol (100 microM) is greatly decreased. Furthermore, compared with WT mice, TRPM8 null mice display deficiencies in certain behaviors, including icilin-induced jumping and cold sensation, as well as a significant reduction in injury-induced responsiveness to acetone cooling. These results suggest that TRPM8 may play an important role in certain types of cold-induced pain in humans.     

HEN-1, a secretory protein with an LDL receptor motif,regulates sensory integration and learning in Caenorhabditis elegans

Animals sense many environmental stimuli simultaneously and integrate various sensory signals within the nervous system both to generate proper behavioral responses and also to form relevant memories. HEN-1, a secretory protein with an LDL receptor motif, regulates such processes in Caenorhabditis elegans. The hen-1 mutants show defects in the integration of two sensory signals and in behavioral plasticity by paired stimuli, although their sensation capability seems to be identical to that of the wild-type. The HEN-1 protein is expressed in two pairs of neurons, but expression in other neurons is sufficient for wild-type behavior. In addition, expression of HEN-1 at the adult stage is sufficient. Thus, HEN-1 regulates sensory processing non-cell-autonomously in the mature neuronal circuit.     

Behaviorally measured audiograms and gap detection thresholds in CBA/CaJ mice

Tone detection and temporal gap detection thresholds were determined in CBA/CaJ mice using a Go/No-go procedure and the psychophysical method of constant stimuli. In the first experiment, audiograms were constructed for five CBA/CaJ mice. Thresholds were obtained for eight pure tones ranging in frequency from 1 to 42 kHz. Audiograms showed peak sensitivity between 8 and 24 kHz, with higher thresholds at lower and higher frequencies. In the second experiment, thresholds for gap detection in broadband and narrowband noise bursts were measured at several sensation levels. For broadband noise, gap thresholds were between 1 and 2 ms, except at very low sensation levels, where thresholds increased significantly. Gap thresholds also increased significantly for low pass-filtered noise bursts with a cutoff frequency below 18 kHz. Our experiments revised absolute auditory thresholds in the CBA/CaJ mouse strain and demonstrated excellent gap detection ability in the mouse. These results add to the baseline behavioral data from normal-hearing mice which have become increasingly important for assessing auditory abilities in genetically altered mice.     

Evolutionary Aspects of the Neuromodulation of Nociceptive Behaviors

There is accumulating evidence for a phylogentic continuityin the expression and regulation of fundamental behaviors ofessential survival value. The ability to detect and respondto aversive environmental stimuli is a basic feature of allanimals that is expressed in the term "nociception." Nociceptiveresponses provide an index of the sensitivity of individualsto actual or potential aversive physical stimuli. Measurementsof alterations in nociceptive responses (antinociception oranalgesia, hyperanalgesia) are commonly used to monitor thebehavioral and physiological status of animals following exposureto either noxious or potentially damaging stimuli. In this paperthe neuromodulation of the nociceptive and analgesic behaviorsof molluscs (the land snail, Cepaea nemoralis) and mammals (rodents)is considered. Behavioral and pharmacological evidence is presentedto suggest that opioid neuropeptides are similarly involvedin the modulation of the nociceptive responses of rodents andsnails. The FMRFamide-related family of neuropeptides are alsoshown to be involved in the modulation of nociceptive behaviors,though with apparently different roles in molluscs and mammals.It is proposed that comparative investigations of the mediationof basic phylogenetically conserved functions, such as nociception,are a useful means to determine and analyse, general featuresof behavioral neuromodulation by neuropeptides.