The tactile receptive fields of freely moving Caenorhabditis elegans nematodes.

Sensory neurons embedded in skin are responsible for the sense of touch. In humans and other mammals, touch sensation depends on thousands of diverse somatosensory neurons. By contrast, Caenorhabditis elegans nematodes have six gentle touch receptor neurons linked to simple behaviors. The classical touch assay uses an eyebrow hair to stimulate freely moving C. elegans, evoking evasive behavioral responses. This assay has led to the discovery of genes required for touch sensation, but does not provide control over stimulus strength or position. Here, we present an integrated system for performing automated, quantitative touch assays that circumvents these limitations and incorporates automated measurements of behavioral responses. The Highly Automated Worm Kicker (HAWK) unites a microfabricated silicon force sensor holding a glass bead forming the contact surface and video analysis with real-time force and position control. Using this system, we stimulated animals along the anterior-posterior axis and compared responses in wild-type and spc-1(dn) transgenic animals, which have a touch defect due to expression of a dominant-negative α-spectrin protein fragment. As expected from prior studies, delivering large stimuli anterior and posterior to the mid-point of the body evoked a reversal and a speed-up, respectively. The probability of evoking a response of either kind depended on stimulus strength and location; once initiated, the magnitude and quality of both reversal and speed-up behavioral responses were uncorrelated with stimulus location, strength, or the absence or presence of the spc-1(dn) transgene. Wild-type animals failed to respond when the stimulus was applied near the mid-point. These results show that stimulus strength and location govern the activation of a characteristic motor program and that the C. elegans body surface consists of two receptive fields separated by a gap.

Artificial dirt: microfluidic substrates for nematode neurobiology and behavior.

With a nervous system of only 302 neurons, the free-living nematode Caenorhabditis elegans is a powerful experimental organism for neurobiology. However, the laboratory substrate commonly used in C. elegans research, a planar agarose surface, fails to reflect the complexity of this organism's natural environment, complicates stimulus delivery, and is incompatible with high-resolution optophysiology experiments. Here we present a new class of microfluidic devices for C. elegans neurobiology and behavior: agarose-free, micron-scale chambers and channels that allow the animals to crawl as they would on agarose. One such device mimics a moist soil matrix and facilitates rapid delivery of fluid-borne stimuli. A second device consists of sinusoidal channels that can be used to regulate the waveform and trajectory of crawling worms. Both devices are thin and transparent, rendering them compatible with high-resolution microscope objectives for neuronal imaging and optical recording. Together, the new devices are likely to accelerate studies of the neuronal basis of behavior in C. elegans.

Child abuse quilts: revealing and healing the pain of child abuse.

According to the recent government report, Child Maltreatment 1997: Reports From the States to the National Child Abuse and Neglect Data System, almost 3 million reports of child abuse and neglect were filed in the United States in 1997. The number of substantiated or indicated maltreatment cases decreased from more than 1 million child victims in 1996 to about 984,000 in 1997. More than half of the reports involved neglect, about one-fourth involved physical abuse, and 12% included sexual abuse, and an estimated 1,197 fatalities resulted from child maltreatment in 1997. In response to the issue of child maltreatment, the Child Abuse Quilts Project was conceived. At least 28 quilts were made for this project, visually addressing the subject of child abuse, child abuse prevention, and violence against children. Mary Beth Goodman, curator of the quilt exhibit says, "The quilters put these quilts before you, not to make you turn away from their quilts in distaste, but to make you think about child abuse and its impact on the child, the survivor, the family, the community. Change comes slowly, heart by heart. We hope that each person who sees these quilts with [be]... re-awakened to the tragedy of child abuse and resolved to prevent it" Ten of these quilts are displayed here; all 28 are displayed on the Internet at members.tripod.com/mbgoodman/ caqpics/caqindex.html.

Pressure polishing: a method for re-shaping patch pipettes during fire polishing.

The resolution of patch-clamp recordings is limited by the geometrical and electrical properties of patch pipettes. The ideal whole-cell patch pipette has a blunt, cone-shaped tip and a low resistance. The best glasses for making patch pipettes are low noise, low capacitance glasses such as borosilicate and aluminasilicate glasses. Regrettably, nearly all borosilicate glasses form pipettes with sharp, cone-shaped tips and relatively high resistance. It is possible, however, to reshape the tip during fire polishing by pressurizing the pipette lumen during fire polishing, a technique we call 'pressure polishing.' We find that this technique works with pipettes made from virtually any type of glass, including thick-walled aluminasilicate glass. We routinely use this technique to make pipettes suitable for whole-cell patch-clamp recording of tiny neurons (1-3 microm in diameter). Our pipettes are made from thick-walled, borosilicate glass and have submicron tip openings and resistances <10 MOmega. Similar pipettes could be used to record from subcellular neuronal structures such as axons, dendrites and dendritic spines. Pressure polishing should also be useful in patch-clamp applications that benefit from using pipettes with blunt tips, such as perforated-patch whole-cell recordings, low-noise single channel recordings and experiments that require internal perfusion of the pipette.

Active currents regulate sensitivity and dynamic range in C. elegans neurons.

Little is known about the physiology of neurons in Caenorhabditis elegans. Using new techniques for in situ patch-clamp recording in C. elegans, we analyzed the electrical properties of an identified sensory neuron (ASER) across four developmental stages and 42 unidentified neurons at one stage. We find that ASER is nearly isopotential and fails to generate classical Na+ action potentials. Rather, ASER displays a high sensitivity to input currents coupled to a depolarization-dependent reduction in sensitivity that may endow ASER with a wide dynamic range. Voltage clamp revealed depolarization-activated K+ and Ca2+ currents that contribute to high sensitivity near the zero-current potential. The depolarization-dependent reduction in sensitivity can be attributed to activation of K+ current at voltages where it dominates the net membrane current. The voltage dependence of membrane current was similar in all neurons examined, suggesting that C. elegans neurons share a common mechanism of sensitivity and dynamic range.

Variations in the ensemble of potassium currents underlying resonance in turtle hair cells.

1. Potassium currents were characterized in turtle cochlear hair cells by whole-cell voltage clamp during superfusion with the potassium channel antagonists, tetraethylammonium (TEA) and 4-aminopyridine (4-AP). The estimated resonant frequency, f0, was inferred from tau, the time constant of deactivation of outward current upon repolarization to -50 mV, according to the empirical relation, f0 = k1 tau-1/2 + k2. 2. Dose-response relations for TEA and 4-AP were obtained by exposing single cells to ten concentrations exponentially distributed over four orders of magnitude. Potassium current in cells tuned to low frequencies was carried by a single class of channels with an apparent affinity constant, K1, for TEA of 35.9 mM. Half-blocking concentrations of 4-AP were correlated with the time constant of deactivation and varied between 26.2 and 102 microM. In cells tuned to higher frequencies, K+ current was carried by a single class of channels with high affinity for TEA (K1 = 0.215 mM) and low affinity for 4-AP (K1 = 12.3 mM). This pharmacological profile suggests that K+ current in low frequency cells is purely voltage gated and in high frequency cells, it is gated by both Ca2+ and voltage. 3. For each current type, the voltage dependence of activation was determined from tail current amplitude at -50 mV. The purely voltage-gated current, IK(V), was found to increase e-fold in 4.0 +/- 0.3 mV (n = 3) in low frequency cells exposed to TEA (25 mM). The Ca(2+)- and voltage-gated current, IK(Ca), was more steeply voltage dependent, increasing e-fold in 1.9 mV (n = 2) in high frequency cells exposed to 4-AP (0.8 mM). 4. IK(V) was found to inactivate slowly during prolonged voltage steps (approximately 10 s). Steady-state inactivation increased with depolarization from -70 mV and was incomplete such that on average IK(v) did not fall below approximately 0.39 of its maximum value. 5. Superfusion of 4-AP (0.8 mM) reversibly depolarized a low frequency cell and eliminated steady voltage oscillations, while TEA (6 mM) had no effect. In a high frequency cell, voltage oscillations were abolished by TEA, but not by 4-AP. 6. The differential pharmacology of IK(V) and IK(Ca) was used to measure their contribution to K+ current in cells tuned to different frequencies. Both currents exhibited a frequency-dependent increase in maximum conductance. IK(V) accounted for nearly all K+ current in cells tuned to less than 60 Hz, while IK(Ca) was the dominant current in higher frequency cells. 7. Mapping resonant frequency onto epithelial position suggests an exponential relation between K+ current size and position. IK(V) appeared to be limited to the apical or low frequency portion of the basilar papilla and coincided with maximal expression of a K(+)-selective inward rectifier, IK(IR). This finding is consistent with the notion that low frequency resonance is produced by interaction of IK(V) and IK(IR) with the voltage-gated Ca2+ current, ICa, and the cell's capacitance. The ionic events underlying higher frequency resonance are dominated by the action of IK(Ca) and ICa and include a contribution from IK(IR).

Positive feedback by a potassium-selective inward rectifier enhances tuning in vertebrate hair cells.

Electrical resonance in vertebrate hair cells shapes receptor potentials and tunes each cell to a narrow band of frequencies. We have investigated the contribution of a potassium-selective inward rectifier (IR) to electrical resonance, isolating outward current carried by IR from other ionic currents active in the physiological voltage range (-75 to -30 mV) using a combination of potassium and calcium channel antagonists. IR expression is tightly regulated in the turtle's auditory epithelium, as revealed by the observation that its size declines systematically with resonant frequency. A critical feature of IR is the rapid inhibition produced by depolarization, which results in a negative slope in the steady-state current-voltage relation in the vicinity of the resting potential (-50 mV). The increasing block of outward current produced by depolarization is functionally equivalent to activating an inward current, suggesting that IR provides positive feedback and, in hair cells, serves an electrical function ordinarily reserved for voltage-dependent sodium and calcium currents. Additional support for this idea comes from the observation that superfusion with cesium selectively reduces IR and eliminates resonance in cells tuned to low frequencies and degrades resonant quality in cells tuned to more than 50 Hz.

Activation of the inositol trisphosphate second messenger system by cAMP in a mouse fibroblast cell line.

Intracellular Ca2+ mobilization events were assessed in mouse L cells, which contain native prostaglandin E1 receptors and transfected human beta 2 adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca2+]i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca2+]i in response to isoproterenol (0.1 nM-100 nM), which is inhibited by the beta-adrenergic antagonist, propranolol, and is a result of intracellular Ca2+ release. [Ca2+]i in these cells was also increased by prostaglandin E1, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP1, IP2, and IP3. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP3 production which leads to an elevation in [Ca2+]i. We propose that this cyclic AMP dependent activation of the IP3 generating system occurs at a post-receptor site.

Inositol trisphosphate mediates cloned muscarinic receptor-activated conductances in transfected mouse fibroblast A9 L cells.

1. The mechanism by which cloned m1 and m3 muscarinic receptor subtypes activate Ca2+-dependent channels was investigated with whole-cell and cell-attached patch-clamp recording techniques and with Fura-2 Ca2+ indicator dye measurements in cultured A9 L cells transfected with rat m1 and m3 cDNAs. 2. The Ca2+-dependent K+ and Cl- currents induced by muscarinic receptor stimulation were dependent on GTP. Responses were reduced when GTP was excluded from the intracellular recording solution or when GDP-beta-S was added. Intracellular GTP-gamma-S activated spontaneous fluctuations and permitted only one acetylcholine-(ACh) induced current response. These results implicate GTP-binding proteins (G protein) in the signal transduction pathway. This G protein is probably not pertussis toxin-sensitive as the ACh-induced electrical response was not abolished by pertussis toxin treatment. 3. Cell-attached single-channel recordings revealed activation of ion channels within the patch during application of ACh outside the patch, implying that second messengers might be involved in the ACh-induced response. Two types of K+ channel were activated, a discrete channel of 36 pS and channel activity calculated to be about 5 pS. 4. Application of 8-bromo cyclic AMP or 1-oleoyl-1,2-acetylglycerol (OAG) produced no electrical response and did not affect the ACh-induced responses. Phorbol myristic acetate (PMA) evoked no electrical response, but reduced the ACh-induced responses. 5. Inclusion of inositol 1,4,5-trisphosphate (IP3) in the intracellular pipette solution activated outward currents at -50 mV associated with an increase in conductance. The IP3-induced current response reversed polarity at -65 mV and showed a dependence on K+. Increasing the intracellular free Ca2+ concentration ([Ca2+]i) from 20 nM to 1 microM also induced an outward current response associated with an increase in conductance. Inclusion of inositol 1,3,4,5-tetrakisphosphate (IP4) in the intracellular solution had no effect on the A9 L cells. 6. Fura-2 measurements revealed ACh-induced increases in Cai2+. The Ca2+ responses were abolished by atropine showing that they were muscarinic in nature. Removal of extracellular Ca2+ did not affect the initial ACh-induced increase in Cai2+ but subsequent Cai2+ responses to ACh were depressed, suggesting depletion of Ca2+ intracellular stores. Residual though small responses continued to be elicited by ACh. Barium (5 mM) had little effect and cobalt slightly reduced the ACh-induced Ca2+ response. 7. The ACh-induced currents recorded at -50 mV were unaffected by removal of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium currents and fura-2 signals in fluorescence-activated cell sorted lactotrophs and somatotrophs of rat anterior pituitary.

Optical and electrical recording techniques were applied to single primary pituitary cells to characterize the types of voltage-dependent calcium currents (ICa) and levels of intracellular calcium ([Ca2+]i). GH-containing somatotrophs and PRL-containing lactotrophs were isolated from adult female rats using fluorescence-activated cell-sorting techniques and were maintained in culture for 1-4 days. Whole cell patch-clamp recordings were made to analyze the ICa, and [Ca2+]i was measured with fura-2. Cell type was verified after each recording by indirect immunocytochemistry. GH and PRL cells could be divided into two groups: silent and spontaneously active. Silent cells had stable membrane potentials and stable levels of [Ca2+]i. Spontaneously active cells exhibited spontaneous action potentials and large fluctuations in [Ca2+]i. Two types of ICa were found: a low threshold, transient current which was insensitive to the dihydropyridine -Bay 5417 (the negative isomer of Bay K 8644), and a high threshold, sustained current which was enhanced by -Bay 5417. Both types of ICa were present in PRL and GH cells, but each cell type differed quantitatively in the proportion of each current type. While the GH cells had a more prominent, low threshold, transient ICa, the PRL cells had a more prominent, high threshold, sustained ICa. The enhancement of ICa by -Bay 5417 was greater in the PRL cells, which have a larger dihydropyridine-sensitive ICa. Parallel fura-2 measurements showed an increase in [Ca2+]i in response to 50 mM KCl and -Bay 5417 for both lactotrophs and somatotrophs.