In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents

ASH sensory neurons are required in Caenorhabditis elegans for a wide range of avoidance behaviors in response to chemical repellents, high osmotic solutions and nose touch. The ASH neurons are therefore hypothesized to be polymodal nociceptive neurons. To understand the nature of polymodal sensory response and adaptation at the cellular level, we expressed the calcium indicator protein cameleon in ASH and analyzed intracellular Ca(2+) responses following stimulation with chemical repellents, osmotic shock and nose touch. We found that a variety of noxious stimuli evoked strong responses in ASH including quinine, denatonium, detergents, heavy metals, both hyper- and hypo-osmotic shock and nose touch. We observed that repeated chemical stimulation led to a reversible reduction in the magnitude of the sensory response, indicating that adaptation occurs within the ASH sensory neuron. A key component of ASH adaptation is GPC-1, a G-protein gamma-subunit expressed specifically in chemosensory neurons. We hypothesize that G-protein gamma-subunit heterogeneity provides a mechanism for repellent-specific adaptation, which could facilitate discrimination of a variety of repellents by these polymodal sensory neurons.     

The C. elegans D2-Like Dopamine Receptor DOP-3 Decreases Behavioral Sensitivity to the Olfactory Stimulus 1-Octanol

We previously found that dopamine signaling modulates the sensitivity of wild-type C. elegans to the aversive odorant 1-octanol. C. elegans lacking the CAT-2 tyrosine hydroxylase enzyme, which is required for dopamine biosynthesis, are hypersensitive in their behavioral avoidance of dilute concentrations of octanol. Dopamine can also modulate the context-dependent response of C. elegans lacking RGS-3 function, a negative regulator of Gα signaling. rgs-3 mutant animals are defective in their avoidance of 100% octanol when they are assayed in the absence of food (E. coli bacterial lawn), but their response is restored when they are assayed in the presence of food or exogenous dopamine. However, it is not known which receptor might be mediating dopamine''s effects on octanol avoidance. Herein we describe a role for the C. elegans D2-like receptor DOP-3 in the regulation of olfactory sensitivity. We show that DOP-3 is required for the ability of food and exogenous dopamine to rescue the octanol avoidance defect of rgs-3 mutant animals. In addition, otherwise wild-type animals lacking DOP-3 function are hypersensitive to dilute octanol, reminiscent of cat-2 mutants. Furthermore, we demonstrate that DOP-3 function in the ASH sensory neurons is sufficient to rescue the hypersensitivity of dop-3 mutant animals, while dop-3 RNAi knockdown in ASH results in octanol hypersensitivity. Taken together, our data suggest that dopaminergic signaling through DOP-3 normally acts to dampen ASH signaling and behavioral sensitivity to octanol.     

Dopamine mediates context-dependent modulation of sensory plasticity in C. elegans

Dopamine has been implicated in the modulation of diverse forms of behavioral plasticity, including appetitive learning and addiction. An important challenge is to understand how dopamine's effects at the cellular level alter the properties of neural circuits to modify behavior. In the nematode C. elegans, dopamine modulates habituation of an escape reflex triggered by body touch. In the absence of food, animals habituate more rapidly than in the presence of food; this contextual information about food availability is provided by dopaminergic mechanosensory neurons that sense the presence of bacteria. We find that dopamine alters habituation kinetics by selectively modulating the touch responses of the anterior-body mechanoreceptors; this modulation involves a D1-like dopamine receptor, a Gq/PLC-beta signaling pathway, and calcium release within the touch neurons. Interestingly, the body touch mechanoreceptors can themselves excite the dopamine neurons, forming a positive feedback loop capable of integrating context and experience to modulate mechanosensory attention.     

Inositol 1,4,5-Trisphosphate Signalling Regulates the Avoidance Response to Nose Touch in Caenorhabditis elegans

When Caenorhabditis elegans encounters an unfavourable stimulus at its anterior, it responds by initiating an avoidance response, namely reversal of locomotion. The amphid neurons, ASHL and ASHR, are polymodal in function, with roles in the avoidance responses to high osmolarity, nose touch, and both volatile and non-volatile repellents. The mechanisms that underlie the ability of the ASH neurons to respond to such a wide range of stimuli are still unclear. We demonstrate that the inositol 1,4,5-trisphosphate receptor (IP₃R), encoded by itr-1, functions in the reversal responses to nose touch and benzaldehyde, but not in other known ASH-mediated responses. We show that phospholipase Cβ (EGL-8) and phospholipase Cγ (PLC-3), which catalyse the production of IP₃, both function upstream of ITR-1 in the response to nose touch. We use neuron-specific gene rescue and neuron-specific disruption of protein function to show that the site of ITR-1 function is the ASH neurons. By rescuing plc-3 and egl-8 in a neuron-specific manner, we show that both are acting in ASH. Imaging of nose touch–induced Ca²⁺ transients in ASH confirms these conclusions. In contrast, the response to benzaldehyde is independent of PLC function. Thus, we have identified distinct roles for the IP₃R in two specific responses mediated by ASH.     

Dopamine counteracts octopamine signalling in a neural circuit mediating food response in C. elegans

Animals assess food availability in their environment by sensory perception and respond to the absence of food by changing hormone and neurotransmitter signals. However, it is largely unknown how the absence of food is perceived at the level of functional neurocircuitry. In Caenorhabditis elegans, octopamine is released from the RIC neurons in the absence of food and activates the cyclic AMP response element binding protein in the cholinergic SIA neurons. In contrast, dopamine is released from dopaminergic neurons only in the presence of food. Here, we show that dopamine suppresses octopamine signalling through two D2‐like dopamine receptors and the G protein Gi/o. The D2‐like receptors work in both the octopaminergic neurons and the octopamine‐responding SIA neurons, suggesting that dopamine suppresses octopamine release as well as octopamine‐mediated downstream signalling. Our results show that C. elegans detects the absence of food by using a small neural circuit composed of three neuron types in which octopaminergic signalling is activated by the cessation of dopamine signalling.     

Bidirectional Modulation of Substantia Nigra Activity by Motivational State

A major output nucleus of the basal ganglia is the substantia nigra pars reticulata, which sends GABAergic projections to brainstem and thalamic nuclei. The GABAergic (GABA) neurons are reciprocally connected with nearby dopaminergic neurons, which project mainly to the basal ganglia, a set of subcortical nuclei critical for goal-directed behaviors. Here we examined the impact of motivational states on the activity of GABA neurons in the substantia nigra pars reticulata and the neighboring dopaminergic (DA) neurons in the pars compacta. Both types of neurons show short-latency bursts to a cue predicting a food reward. As mice became sated by repeated consumption of food pellets, one class of neurons reduced cue-elicited firing, whereas another class of neurons progressively increased firing. Extinction or pre-feeding just before the test session dramatically reduced the phasic responses and their motivational modulation. These results suggest that signals related to the current motivational state bidirectionally modulate behavior and the magnitude of phasic response of both DA and GABA neurons in the substantia nigra.     

Dissecting the serotonergic food signal stimulating sensory-mediated aversive behavior in C. elegans

Nutritional state often modulates olfaction and in Caenorhabditis elegans food stimulates aversive responses mediated by the nociceptive ASH sensory neurons. In the present study, we have characterized the role of key serotonergic neurons that differentially modulate aversive behavior in response to changing nutritional status. The serotonergic NSM and ADF neurons play antagonistic roles in food stimulation. NSM 5-HT activates SER-5 on the ASHs and SER-1 on the RIA interneurons and stimulates aversive responses, suggesting that food-dependent serotonergic stimulation involves local changes in 5-HT levels mediated by extrasynaptic 5-HT receptors. In contrast, ADF 5-HT activates SER-1 on the octopaminergic RIC interneurons to inhibit food-stimulation, suggesting neuron-specific stimulatory and inhibitory roles for SER-1 signaling. Both the NSMs and ADFs express INS-1, an insulin-like peptide, that appears to cell autonomously inhibit serotonergic signaling. Food also modulates directional decisions after reversal is complete, through the same serotonergic neurons and receptors involved in the initiation of reversal, and the decision to continue forward or change direction after reversal is dictated entirely by nutritional state. These results highlight the complexity of the "food signal" and serotonergic signaling in the modulation of sensory-mediated aversive behaviors.     

Redundant neural circuits regulate olfactory integration

Olfactory integration is important for survival in a natural habitat. However, how the nervous system processes signals of two odorants present simultaneously to generate a coherent behavioral response is poorly understood. Here, we characterize circuit basis for a form of olfactory integration in Caenorhabditis elegans. We find that the presence of a repulsive odorant, 2-nonanone, that signals threat strongly blocks the attraction of other odorants, such as isoamyl alcohol (IAA) or benzaldehyde, that signal food. Using a forward genetic screen, we found that genes known to regulate the structure and function of sensory neurons, osm-5 and osm-1, played a critical role in the integration process. Loss of these genes mildly reduces the response to the repellent 2-nonanone and disrupts the integration effect. Restoring the function of OSM-5 in either AWB or ASH, two sensory neurons known to mediate 2-nonanone-evoked avoidance, is sufficient to rescue. Sensory neurons AWB and downstream interneurons AVA, AIB, RIM that play critical roles in olfactory sensorimotor response are able to process signals generated by 2-nonanone or IAA or the mixture of the two odorants and contribute to the integration. Thus, our results identify redundant neural circuits that regulate the robust effect of a repulsive odorant to block responses to attractive odorants and uncover the neuronal and cellular basis for this complex olfactory task.     

Antagonistic sensory cues generate gustatory plasticity in Caenorhabditis elegans

Caenorhabditis elegans shows chemoattraction to 0.1-200 mM NaCl, avoidance of higher NaCl concentrations, and avoidance of otherwise attractive NaCl concentrations after prolonged exposure to NaCl (gustatory plasticity). Previous studies have shown that the ASE and ASH sensory neurons primarily mediate attraction and avoidance of NaCl, respectively. Here we show that balances between at least four sensory cell types, ASE, ASI, ASH, ADF and perhaps ADL, modulate the response to NaCl. Our results suggest that two NaCl-attraction signalling pathways exist, one of which uses Ca(2+)/cGMP signalling. In addition, we provide evidence that attraction to NaCl is antagonised by G-protein signalling in the ASH neurons, which is desensitised by the G-protein-coupled receptor kinase GRK-2. Finally, the response to NaCl is modulated by G-protein signalling in the ASI and ADF neurons, a second G-protein pathway in ASH and cGMP signalling in neurons exposed to the body fluid.     

Membrane voltage and neurotransmitter regulation of neuronal growth cone motility

The neurotransmitters serotonin and dopamine inhibit growth cone motility and neurite elongation of specific identified neurons of the pond snail Helisoma. Similarly, experimentally evoked action potentials inhibit motility of these growth cones. Here we explore the possibility that the motility- and elongation-inhibiting actions of serotonin and dopamine derive from the electrophysiological responses of the respective neurons. Evidence of three types in support of this hypothesis is presented: (1) Only those identified neurons for which motility is inhibited by serotonin or dopamine respond to the transmitter with sustained electrical excitation. (2) The magnitude of the electrical excitation response correlates with the degree of inhibition of growth cone motility. (3) The injection of hyperpolarizing current enables motility to continue as in the absence of transmitters. We conclude that membrane voltage is an important level of control of growth cone motility, at which neurotransmitters exert a regulatory influence.     

Convergent processing of both positive and negative motivational signals by the VTA dopamine neuronal populations

Dopamine neurons in the ventral tegmental area (VTA) have been traditionally studied for their roles in reward-related motivation or drug addiction. Here we study how the VTA dopamine neuron population may process fearful and negative experiences as well as reward information in freely behaving mice. Using multi-tetrode recording, we find that up to 89% of the putative dopamine neurons in the VTA exhibit significant activation in response to the conditioned tone that predict food reward, while the same dopamine neuron population also respond to the fearful experiences such as free fall and shake events. The majority of these VTA putative dopamine neurons exhibit suppression and offset-rebound excitation, whereas ～25% of the recorded putative dopamine neurons show excitation by the fearful events. Importantly, VTA putative dopamine neurons exhibit parametric encoding properties: their firing change durations are proportional to the fearful event durations. In addition, we demonstrate that the contextual information is crucial for these neurons to respectively elicit positive or negative motivational responses by the same conditioned tone. Taken together, our findings suggest that VTA dopamine neurons may employ the convergent encoding strategy for processing both positive and negative experiences, intimately integrating with cues and environmental context.     

Inhibitory and activatory effects of dopamine on neurons in slices of the compact zone of theSubstantia nigra of rats

Effects of dopamine on the background spike activity of functionally (according to their electrophysiological characteristics) identified dopaminergic (DA) or non-dopaminergic (non-DA) neurons of the compact zone of thesubstantia nigra were studied on slices of the midbrain of adult rats. In the majority of DA neurons, dopamine suppressed the background activity in a dose-dependent manner. Sensitivity of these cells to dopamine varied within a wide range: IC₅₀, the concentration providing the 50% maximum effect, equalled from 3 to 3,000 µM in different units. A part of DA neurons responded to dopamine with an increase in their activity. Mixed responses, in which an initial suppression of impulsation was replaced by a slow-developing activation, was observed in some neurons. Non-DA neurons usually responded to dopamine by an enhancement of impulsation; yet, the cells with inhibitory or mixed responses similar to those of DA neurons could be found in this population as well. Sensitivity of non-DA neurons to dopamine was about the same as that of DA-cells, without the dependence on the direction of responses. S(–)-suipiride, a blocker of D₂ receptors, decreased the inhibitory component of all tested responses to dopamine both in DA and non-DA neurons and evoked no changes in the excitatory component. At the same time, R(+)-SCH 23390 HC1, a blocker of D₁ receptors, suppressed the activatory component of responses with no effect on the inhibitory component. We conclude that both types of DA receptors, D₂ and D₁ receptors, can be present on the DA and non-DA neurons. Dopamine, influencing these receptors, suppresses or facilitates, respectively, the spike activity of these cells. The relative amount of such receptors is the main factor determining the pattern and dynamics of the integral response to dopamine application. The possible functional role of the presence of both D₁ and D₂ receptors on the membrane of a single neuron is discussed.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 268–277, July–August, 1995.     

The G Protein regulators EGL-10 and EAT-16, the G<Subscript>i</Subscript>α GOA-1 and the G<Subscript>q</Subscript>α EGL-30 modulate the response of the <Emphasis Type="Italic">C. elegans</Emphasis>ASH polymodal nociceptive sensory neurons to repellents

Background  

Polymodal, nociceptive sensory neurons are key cellular elements of the way animals sense aversive and painful stimuli. In Caenorhabditis elegans, the polymodal nociceptive ASH sensory neurons detect aversive stimuli and release glutamate to generate avoidance responses. They are thus useful models for the nociceptive neurons of mammals. While several molecules affecting signal generation and transduction in ASH have been identified, less is known about transmission of the signal from ASH to downstream neurons and about the molecules involved in its modulation.     

Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans

The nematode C. elegans is an excellent model organism for studying behavior at the neuronal level. Because of the organism's small size, it is challenging to deliver stimuli to C. elegans and monitor neuronal activity in a controlled environment. To address this problem, we developed two microfluidic chips, the 'behavior' chip and the 'olfactory' chip for imaging of neuronal and behavioral responses in C. elegans. We used the behavior chip to correlate the activity of AVA command interneurons with the worm locomotion pattern. We used the olfactory chip to record responses from ASH sensory neurons exposed to high-osmotic-strength stimulus. Observation of neuronal responses in these devices revealed previously unknown properties of AVA and ASH neurons. The use of these chips can be extended to correlate the activity of sensory neurons, interneurons and motor neurons with the worm's behavior.     

Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans

An animal's ability to detect and avoid toxic compounds in the environment is crucial for survival. We show that the nematode Caenorhabditis elegans avoids many water-soluble substances that are toxic and that taste bitter to humans. We have used laser ablation and a genetic cell rescue strategy to identify sensory neurons involved in the avoidance of the bitter substance quinine, and found that ASH, a polymodal nociceptive neuron that senses many aversive stimuli, is the principal player in this response. Two G protein alpha subunits GPA-3 and ODR-3, expressed in ASH and in different, nonoverlapping sets of sensory neurons, are necessary for the response to quinine, although the effect of odr-3 can only be appreciated in the absence of gpa-3. We identified and cloned a new gene, qui-1, necessary for quinine and SDS avoidance. qui-1 codes for a novel protein with WD-40 domains and which is expressed in the avoidance sensory neurons ASH and ADL.     

Amphidial neurons ADL and ASH initiate sodium dodecyl sulphate avoidance responses in the infective larva of the dog hookworm Anclyostoma caninum

Ablations of specific amphidial neuron pairs with a laser microbeam were conducted to understand better the neurological basis of the behaviours of larval parasitic nematodes. To date, the functions of the amphidial neurons of Caenorhabditis elegans and their counterparts in parasitic nematodes have been found to be remarkably conserved allowing the possibility to predict the relationships between neurons and their functions. Therefore, we anticipated that ablation of neuron pairs ASH and ASK would abrogate avoidance of sodium dodecyl sulphate (SDS) by infective larvae (L3i) of Anclyostoma caninum. Instead, we have found that laser microbeam ablation of these neuron pairs did not eliminate SDS avoidance in A. caninum, but that neuron pairs ASH and ADL are the amphidial neurons responsible for SDS repulsion. When a droplet of the repellent is placed in the direct path of a normal A. caninum L3i, a strong backward avoidance response is triggered. However, when the ASH and ADL neurons are ablated, the nematodes demonstrate the opposite reaction, increasing their movement in a forward direction.     

Behavioral and electrophysiological studies of peptide-induced emesis in dogs

The electrophysiological responses of neurons in the canine area postrema (AP) to ionophoretic application of neuropeptides and transmitters were studied and correlated with the presence or absence of an emetic response on systemic administration. Of 17 common neuropeptides 11 were emetic when applied systemically at doses of 0.03-0.35 mg/kg. The emesis was dose dependent and was no longer observed in animals with chronic ablation of the AP. The responses of 122 AP single units were recorded. Neurons were silent at rest, and most were excited by glutamate, apomorphine, and dopamine. Excitatory responses to each of eight emetic peptides were recorded in 22-65% of cells studied; no responses were found to two peptides that were not emetic. The response to glutamate was always a brief, high-frequency discharge; the responses to all 13 other excitatory substances were of long latency, low frequency, and long duration. With high ionophoretic current or multiple applications, units would frequently become spontaneously active for many minutes or longer. The similarity of response of so many substances on small neurons suggests a common ionic or metabolic mechanism underlying the response. The direct correlation between the occurrence of emesis on systemic administration and the presence of excitatory receptors on AP neurons provides strong support for the proposed role of the AP as the chemoreceptor trigger zone for emesis.     

A role of dopamine-dependent activity reorganizations in the cortico-basal ganglia-thalamocortical loops in visual attention (hypothetical mechanism)

A mechanism of attention is proposed according to which its influence on visual processing is switched on by release of dopamine into the striatum. A dopamine release during involuntary attention is promoted by visual activation of striatonigral cells via the thalamus and subsequent disinhibition through the basal ganglia of the superior colliculus. A dopamine release during voluntary attention is promoted by activation of prefrontal cortex. The strengthening of responses of neocortical neurons to attended stimulus, and suppression of responses to other stimuli is the result of opposite modulatory action of dopamine on the efficacy of strong and weak corticostriatal inputs. This leads to changes in the output basal ganglia signals ("attentional filter") that exert disinhibitory and inhibitory influence (via the thalamus) on neocortical cells that initially were strongly and weakly activated by a stimulus, respectively. From proposed mechanism follows, that attention modulates only those components of responses of cortical neurons which latency exceeds the latency of reactions of dopaminergic cells (80-100 ms).     

Optogenetic mimicry of the transient activation of dopamine neurons by natural reward is sufficient for operant reinforcement

Activation of dopamine receptors in forebrain regions, for minutes or longer, is known to be sufficient for positive reinforcement of stimuli and actions. However, the firing rate of dopamine neurons is increased for only about 200 milliseconds following natural reward events that are better than expected, a response which has been described as a "reward prediction error" (RPE). Although RPE drives reinforcement learning (RL) in computational models, it has not been possible to directly test whether the transient dopamine signal actually drives RL. Here we have performed optical stimulation of genetically targeted ventral tegmental area (VTA) dopamine neurons expressing Channelrhodopsin-2 (ChR2) in mice. We mimicked the transient activation of dopamine neurons that occurs in response to natural reward by applying a light pulse of 200 ms in VTA. When a single light pulse followed each self-initiated nose poke, it was sufficient in itself to cause operant reinforcement. Furthermore, when optical stimulation was delivered in separate sessions according to a predetermined pattern, it increased locomotion and contralateral rotations, behaviors that are known to result from activation of dopamine neurons. All three of the optically induced operant and locomotor behaviors were tightly correlated with the number of VTA dopamine neurons that expressed ChR2, providing additional evidence that the behavioral responses were caused by activation of dopamine neurons. These results provide strong evidence that the transient activation of dopamine neurons provides a functional reward signal that drives learning, in support of RL theories of dopamine function.     

Mean field model of acetylcholine mediated dynamics in the cerebral cortex

A recent continuum model of the large scale electrical activity of the cerebral cortex is generalized to include cholinergic modulation. In this model, dynamic modulation of synaptic strength acts over the time scales of nicotinic and muscarinic receptor action. The cortical model is analyzed to determine the effect of acetylcholine (ACh) on its steady states, linear stability, spectrum, and temporal responses to changes in subcortical input. ACh increases the firing rate in steady states of the system. Changing ACh concentration does not introduce oscillatory behavior into the system, but increases the overall spectral power. Model responses to pulses in subcortical input are affected by the tonic level of ACh concentration, with higher levels of ACh increasing the magnitude firing rate response of excitatory cortical neurons to pulses of subcortical input. Numerical simulations are used to explore the temporal dynamics of the model in response to changes in ACh concentration. Evidence is seen of a transition from a state in which intracortical inputs are emphasized to a state where thalamic afferents have enhanced influence. Perturbations in ACh concentration cause changes in the firing rate of cortical neurons, with rapid responses due to fast acting facilitatory effects of nicotinic receptors on subcortical afferents, and slower responses due to muscarinic suppression of intracortical connections. Together, these numerical simulations demonstrate that the actions of ACh could be a significant factor modulating early components of evoked response potentials.