William R. Schafer

Guidelines on nicotine dose selection for in vivo research

RationaleThis review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure.ObjectivesThis review capitalizes on the authors’ collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models.ResultsSeven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses.ConclusionsThe selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose–response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

Optical Imaging of Calcium Transients in Neurons and Pharyngeal Muscle of C. elegans

Electrophysiology and optical indicators have been used in vertebrate systems to investigate excitable cell firing and calcium transients, but both techniques have been difficult to apply in organisms with powerful reverse genetics. To overcome this limitation, we expressed cameleon proteins, genetically encoded calcium indicators, in the pharyngeal muscle of the nematode worm Caenorhabditis elegans. In intact transgenic animals expressing cameleons, fluorescence ratio changes accompanied muscular contraction, verifying detection of calcium transients. By comparing the magnitude and duration of calcium influx in wild-type and mutant animals, we were able to determine the effects of calcium channel proteins on pharyngeal calcium transients. We also successfully used cameleons to detect electrically evoked calcium transients in individual C. elegans neurons. This technique therefore should have broad applications in analyzing the regulation of excitable cell activity in genetically tractable organisms.

In Vivo Imaging of C. elegans Mechanosensory Neurons Demonstrates a Specific Role for the MEC-4 Channel in the Process of Gentle Touch Sensation

In the nematode C. elegans, genes encoding components of a putative mechanotransducing channel complex have been identified in screens for light-touch-insensitive mutants. A long-standing question, however, is whether identified MEC proteins act directly in touch transduction or contribute indirectly by maintaining basic mechanoreceptor neuron physiology. In this study, we used the genetically encoded calcium indicator cameleon to record cellular responses of mechanosensory neurons to touch stimuli in intact, behaving nematodes. We defined a gentle touch sensory modality that adapts with a time course of approximately 500 ms and primarily senses motion rather than pressure. The DEG/ENaC channel subunit MEC-4 and channel-associated stomatin MEC-2 are specifically required for neural responses to gentle mechanical stimulation, but do not affect the basic physiology of touch neurons or their in vivo responses to harsh mechanical stimulation. These results distinguish a specific role for the MEC channel proteins in the process of gentle touch mechanosensation.

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 Ca2+ 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 γ‐subunit expressed specifically in chemosensory neurons. We hypothesize that G‐protein γ‐subunit heterogeneity provides a mechanism for repellent‐specific adaptation, which could facilitate discrimination of a variety of repellents by these polymodal sensory neurons.

The insulin/PI 3-kinase pathway regulates salt chemotaxis learning in Caenorhabditis elegans.

The insulin-like signaling pathway is known to regulate fat metabolism, dauer formation, and longevity in Caenorhabditis elegans. Here, we report that this pathway is also involved in salt chemotaxis learning, in which animals previously exposed to a chemoattractive salt under starvation conditions start to show salt avoidance behavior. Mutants of ins-1, daf-2, age-1, pdk-1, and akt-1, which encode the homologs of insulin, insulin/IGF-I receptor, PI 3-kinase, phosphoinositide-dependent kinase, and Akt/PKB, respectively, show severe defects in salt chemotaxis learning. daf-2 and age-1 act in the ASER salt-sensing neuron, and the activity level of the DAF-2/AGE-1 pathway in this neuron determines the extent and orientation of salt chemotaxis. On the other hand, ins-1 acts in AIA interneurons, which receive direct synaptic inputs from sensory neurons and also send synaptic outputs to ASER. These results suggest that INS-1 secreted from AIA interneurons provides feedback to ASER to generate plasticity of chemotaxis.

Control of Alternative Behavioral States by Serotonin in Caenorhabditis elegans

Serotonin has been implicated in the regulation of a wide range of brain functions involving alternative behavioral states, including the control of mood, aggression, sex, and sleep. Here, we report that in the nematode Caenorhabditis elegans, serotonin controls a switch between two distinct, on/off states of egg-laying behavior. Through quantitative analysis of the temporal pattern of egg-laying events, we determined that egg laying can be modeled as a novel random process, in which animals fluctuate between discrete behavioral states: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs are retained. Single-cell ablation experiments indicate that two pairs of motor neurons, HSNL/HSNR and VC4/VC5, can induce the active phase by releasing serotonin. These neurons also release acetylcholine, which appears to trigger individual egg-laying events within the active phase. Genetic experiments suggest that determination of the behavioral states observed for C. elegans egg laying may be mediated through protein kinase C-dependent (PKC-dependent) modulation of voltage-gated calcium channels.

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine‐modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine‐mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1‐like dopamine receptor gene (dop‐1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop‐1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild‐type copy of the gene. The dop‐1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop‐1 gene in mechanosensory neurons using the mec‐7 promoter rescues the mechanosensory deficit in dop‐1 mutant animals. The tyrosine hydroxylase‐deficient C. elegans mutant (cat‐2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.

Functional asymmetry in Caenorhabditis elegans taste neurons and its computational role in chemotaxis.

Chemotaxis in Caenorhabditis elegans, like chemotaxis in bacteria, involves a random walk biased by the time derivative of attractant concentration, but how the derivative is computed is unknown. Laser ablations have shown that the strongest deficits in chemotaxis to salts are obtained when the ASE chemosensory neurons (ASEL and ASER) are ablated, indicating that this pair has a dominant role. Although these neurons are left–right homologues anatomically, they exhibit marked asymmetries in gene expression and ion preference. Here, using optical recordings of calcium concentration in ASE neurons in intact animals, we demonstrate an additional asymmetry: ASEL is an ON-cell, stimulated by increases in NaCl concentration, whereas ASER is an OFF-cell, stimulated by decreases in NaCl concentration. Both responses are reliable yet transient, indicating that ASE neurons report changes in concentration rather than absolute levels. Recordings from synaptic and sensory transduction mutants show that the ON–OFF asymmetry is the result of intrinsic differences between ASE neurons. Unilateral activation experiments indicate that the asymmetry extends to the level of behavioural output: ASEL lengthens bouts of forward locomotion (runs) whereas ASER promotes direction changes (turns). Notably, the input and output asymmetries of ASE neurons are precisely those of a simple yet novel neuronal motif for computing the time derivative of chemosensory information, which is the fundamental computation of C. elegans chemotaxis. Evidence for ON and OFF cells in other chemosensory networks suggests that this motif may be common in animals that navigate by taste and smell.

Caenorhabditis elegans TRPA-1 functions in mechanosensation

Members of the transient receptor potential (TRP) ion channel family mediate diverse sensory transduction processes in both vertebrates and invertebrates. In particular, members of the TRPA subfamily have distinct thermosensory roles in Drosophila, and mammalian TRPA1 is postulated to have a function in noxious cold sensation and mechanosensation. Here we show that mutations in trpa-1, the C. elegans ortholog of mouse Trpa1, confer specific defects in mechanosensory behaviors related to nose-touch responses and foraging. trpa-1 is expressed and functions in sensory neurons required for these mechanosensory behaviors, and contributes to neural responses of these cells to touch, particularly after repeated mechanical stimulation. Furthermore, mechanical pressure can activate C. elegans TRPA-1 heterologously expressed in mammalian cells. Collectively, these data demonstrate that trpa-1 encodes an ion channel that can be activated in response to mechanical pressure and is required for mechanosensory neuron function, suggesting a possible role in mechanosensory transduction or modulation.

C. elegans TRP Family Protein TRP-4 Is a Pore-Forming Subunit of a Native Mechanotransduction Channel

Mechanotransduction channels mediate several common sensory modalities such as hearing, touch, and proprioception; however, very little is known about the molecular identities of these channels. Many TRP family channels have been implicated in mechanosensation, but none have been demonstrated to form a mechanotransduction channel, raising the question of whether TRP proteins simply play indirect roles in mechanosensation. Using Caenorhabditis elegans as a model, here we have recorded a mechanosensitive conductance in a ciliated mechanosensory neuron in vivo. This conductance develops very rapidly upon mechanical stimulation with its latency and activation time constant reaching the range of microseconds, consistent with mechanical gating of the conductance. TRP-4, a TRPN (NOMPC) subfamily channel, is required for this conductance. Importantly, point mutations in the predicted pore region of TRP-4 alter the ion selectivity of the conductance. These results indicate that TRP-4 functions as an essential pore-forming subunit of a native mechanotransduction channel.