2009. The Journal ofArachnology 37:379-382 SHORT COMMUNICATION A new approach to examining scorpion peg sensilla: the mineral oil flood technique Elizabeth D. Knowlton and Douglas D. Gaffin: Department ofZoology, University of Oklahoma, Norman, OK 73019-0235, USA. E-mail; [email protected] Abstract. All scorpionspossessjointed, ventralappendagescalledpectines. Theseorgans havechemosensory, peg-shaped sensillathatdetectsubstrate-bornechemicals. Previousphysiological studiesshow thatneuronswithin pegsensilla respond to an assortment ofvolatile organic chemical stimulants blown across the sensillar opening. We developed an improved method ofchemical stimulant delivery called the mineral oil flood technique to further investigate the neural circuitry of scorpion pectines. The new mineral oil flood technique allows us to deliver chemical stimulants directly to individual sensilla by introducing a polar, liquid substance under non-polar mineral oil. Unlike previous methods of stimulant delivery, the mineral oil flood technique allows for precise control over the duration ofdirect contact between a liquid stimulant ofknown concentration and a sensillum. Keywords: Pectines, chemosensory, electrophysiology, stimulant, Scorpiones Pectinesaremovablesensoryappendagesthatextendfromthemid- 55-60%). We fed each scorpion one early instar cricket biweekly and ventralsurfaceofallscorpions(CIoudsley-Thompson 1955).Theyare watered the sand of individual containers with 5 ml of deionized similar in function to the antennae ofmandibulate arthropods that water twice a week. At the conclusion of our study, we deposited a detect airborne chemicals (Kaissling 1987; Itagaki & Hildebrand voucher specimen in the Sam Noble Oklahoma Museum ofNatural 1990), except that pectines are ground-directed and respond to History (OMNH-16279). substrate-borne chemicals. When scorpions move in their environ- For preparing a scorpion for electrophysiological study, we briefly ment, thepectines sweep intermittentlyagainstthesubstrate todetect anesthetized a live animal by cooling it for two minutes inside a food (Krapf 1986; Skutelsky 1995) and pheromones (Gaffin & freezerat —5° C. Thenweimmobilized theanimalwithmodelingclay Brownell 1992, 2001). on a microscope slide (7.62 X 2.54 cm), ventral side up to expose the The primary sensory elements on pectines are hundreds ofminute pectines. We positioned a modified cover glass (5 X 18 mm) with structures called peg sensilla, which adorn the ground-facing surface walls ofwax approximately 1 mm high caudal to where the pectines ofeach pectinal tooth (Carthy 1966, 1968; Ivanov & Balashov 1979; join the body (Fig. la). A notch in the wax wall allowed access ofa Foelix & Miiller-Vorholt 1983). Each peg sensillum has a single slit- pecten to the chamber (Fig. lb). We secured the pecten spine and like pore that allows chemical stimulants access to receptor neurons teeth to the cover glass with double-sided adhesive tape and fine inside the peg shaft. Approximately 10 sensory neurons innervate application of less than 5 pi of quick-drying adhesive glue (Instant eachpeg(Foelix & Miiller-Vorholt 1983), and synapticcontacts exist Krazy Glue^''^). Next, we applied additional wax to the area where between neurons within a sensillum (Gaffin & Brownell 1997a). the pecten spine crossed the edge ofthe cover glass to complete the Peg sensilla respond with different neural activity patterns to wall ofthe chamber. We then placed one drop ofmineral oil (~5 pi) alcohols, aldehydes, ketones, and esters (Gaffin & Brownell 1997b). over the pecten with a 0.25 ml syringe (Fig. Ic). We secured the Thesensillawerestimulated indirectlythrough puffs ofstimuli blown animalontoanadjustableplatformandlocatedpegfieldswithahigh- acrossentire pegfields (Gaffin & Brownell 1997b) or by staticclouds powered compound microscope equipped with epi-illumination ofvolatile organic compounds brought near the peg tips (Gaffin & (Olympus BX50-WI). Lastly, we used an electrolytically-sharpened Walvoord 2004). Although these methods ofstimulus delivery elicit tungstenelectrodeto makeextracellular recordingsofchemosensitive neural responses, they have limitations. Most importantly, it is neurons within individual sensilla (Gaffin & Brownell 1997b). impossible to introduce a stimulant to a single peg sensillum without Torecordpegneurons,wedigitizedelectricalactivitywithananalog stimulating its neighbors. There is also no way, as such, to tell if to digital converter (1401-plus digitizing hardware, CED, Cambridge, stimulating a neighboring sensillum influences the response of the England) and analyzed the record using Spike 2 laboratory software recorded sensillum. In addition, the concentration of stimulant (CED, Cambridge, England). Impulses (“spikes”) from each sponta- reaching the sensillar pore is unknown, and removal ofthe stimulant neouslyactivechemosensitiveneuronwereidentifiedandseparatedinto from the peg field is uncontrollable. three classes (Al, A2, and B), based on the characteristic spike To overcome these limitations, we developed an improved method waveforms of the impulses from each neuron (Gaffin & Brownell ofchemical stimulus delivery called the mineral oil flood technique, 1997b). We used auto-correlation analysis to determine the purity of which uses non-polar mineral oil as a medium for delivering polar, each spike class and cross-correlation analysis to detect synaptic liquid stimulants to an individualsensillum. Inthisstudy, wedescribe interactions among spike classes (for details on auto- and cross- our new method and compare spontaneous and chemically-induced correlation analyses, see Gaffin & Brownell 1997a; Eggermont 1990). neural activity ofpeg sensilla in air and under oil. To stimulatepegsensilla chemically, we filled a glasscapillary tube Mature female Paruroctoniis utahensis (Williams, 1968) (Scor- (stimulant pipette) with an approximately 10 pm diameter tip with piones: Vaejovidae) collected from Crane County (31°28'59"N, 95% ethanol (Gaffin & Walvoord 2004). We used a nonmetallic 102°40'38"W), Texas, in March of 2008 were the animals used for syringe needle (World Precision Instruments, Inc. MicroFiF*^ this study. We individually housed each scorpion in 3.8 glass jars MF34G) to transfer the ethanol to the stimulant pipette. We then 1 containing approximately 250 ml of sand from the scorpion’s placed the stimulant pipette into a glass electrode holder positioned collection site. The animals were on a 15:9 h L:D cycle and kept in on a mechanical micromanipulator (Sutter Instrument Corp. 1140). a room with a steady temperature and relative humidity (22° C, RH For fluid stimulant introduction, we maneuvered the pipette tip with 379 380 THE JOURNAL OF ARACHNOLOGY — Figure 1. Scorpion pectenconfiguration, a. The right pecten (outlined) as positioned forelectrophysiological examination, b. Close-upview ofthe right pecten in a stimulation chamber. A piece ofthe wax barrier (dashed outline) is removed for pecten placement, c. Left, an expanded fieldofviewofpatchesofpegsensilla(ps). Right, amineraloil overlayofanexpandedfieldofviewofapegfield. Amicroelectrode(e)isinserted at the base ofa single peg, under oil, to record baseline neural activity in the presence or absence ofa chemical stimulant (cs). the micromanipulator so that it touched the pore of the recorded within the barrier and provided sufficient overlay for stimulant sensillum. Fig. Ic shows the general configuration of the peg field, introduction. microelectrode, and chemical stimulus delivery device during chem- The presence ofmineral oil on peg sensilla did not affect baseline ical stimulation. neural activity. Fig. 2 compares the spontaneous firing pattern ofa In general, we were able to record spontaneous neural activity peg sensillum in air with that of another peg sensillum under oil. under oil for extended periods, some longer than six hours. Because Cross-correlation analyses reveal the same synaptic interactions in thestability ofarecordingoftendepended on theanimal’s inabilityto each record: when spike B fired, it inhibited spikes A1 and A2 for move its pecten, we improved the method ofadhering the pecten to approximately 0.1 s. the cover glass. The most effective method was careful application of Using the mineral oil flood technique, we confined chemical quick-drying adhesive to the pecten spine and the distal-lateral stimulation to a single peg sensillum and controlled the onset and surface of each pectinal tooth. The least effective adhesives were removal of the stimulant. For example, the right panel of Figure 3 paraffin wax and silicone gel. shows the introduction and removal of liquid ethanol to a peg Because the spread ofmineral oil beyond the pecten and onto the sensillum under oil for durations of one, two, and three seconds. animal’s body induced pecten movement, we assembled a barrier Stimulations were consecutive and spaced approximately 20 s apart, betweenan isolatedpectenandtherestoftheanimal. Themost useful which produced receptor adaptation in the third response. In barriers were about one millimeter high, which still allowed easy contrast, the left panel of Fig. 3 shows a prolonged neural recovery access of an electrode and stimulant pipette to the sensillum, while after introduction ofa drop ofethanol to a peg sensillum in air. The preventing the spread ofmineral oil beyond the pecten. The amount time-expanded view of stimulant introduction shows the extent of of mineral oil applied to the pecten also affected the quality of a recorddisturbance; recordedelectricalactivitywasinconsistentacross preparation. Volumes ofoil greater than or equal to 50 pi were not all stimulations. contained within the barrier. Excessive oil also blurred the field of Our study represents the first account of selective chemical view. In contrast, 5 pi ofoil actually improved the resolution ofthe stimulation of individual scorpion peg sensilla with a known peg field; we could discern individual peg sensillar shafts, which is concentration ofaqueous stimulant. Because the presence ofmineral difficult when viewingsensilla in air. In addition, 5 pi ofoil remained oil overa sensillumdid notaffect thebaselineneural activity, weused KNOWLTON & GAFFIN—NEW METHOD FOR SCORPION PECTEN RESEARCH 381 — Figure 2. Processed recordings of spontaneous neural activity. Shown are recordings ofchemosensitive neurons (B, Al, and A2) from a sensillum in air(leftpanel) and adifferent sensillum underoil (rightpanel). Each line represents a neural impulse within the parsed 50 s record. Theenlarged figures at the left ofeach recording indicate the B, Al, and A2 impulse waveforms. Cross-correlograms reveal that the firing ofB (white arrow) inhibits the activity ofAl and A2 (black arrow) both in air and under oil. oil as a medium through which to directly stimulate individual pegs activityofthe recorded sensillum as we stimulate its neighbor. Such a with 95% ethanol for controlled durations. situationwould provideevidenceoflateral inhibition, whichisaform Onelimitationofthemineraloilfloodtechniqueisthatwecanonly ofperipheral processingseen mostcommonly in thevertebrate retina. use polarliquidsas stimulants. Non-polarstimulants would mix with ACKNOWLEDGMENTS non-polar mineral oil. Therefore, future studies on peg sensillar function will use varying concentrations of polar solutions, such as We thank Drs. Marielle Hoefnagels, Don Wilson, and Ari saltsandorganiccompoundsthatcontainfewerthanthreecarbonsas Berkowitz for their helpful suggestions and use of equipment. We stimulants. also thank Nataliya Popokina for assistance in collecting and In forthcoming experiments, we will use the mineral oil technique maintaining our animals. Finally, we thank the Life Fund of the to further our understanding of peg sensillar function. This new University of Oklahoma Foundation and the OU Zoology Adams method should generate the quantifiable data necessary for compar- Scholarship Fund for providing support for this work. ingsensillarneuralresponses. Forexample, weaim tostimulatemany LITERATURE CITED peg sensilla individually to compare response intensities to varying concentrations of stimulants. This will help us determine if all peg Carthy, J.D. 1966. 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