Multiple liquid stimulus dispenser

Physiology & Behamor, Vol 20, pp 9 5 - 9 8

Pergamon Press and Brain Research P u b l , 1978 Printed m the U S.A

BRIEF COMMUNICATION Multiple Liquid Stimulus Dispenser JACOB P P WEBBERS AND EDWARD E LITTLE

Department of Bzologtcal Scwnce, Sensory Physiology, Florida State Unwerstty, Tallahassee, FL 32306 (Recewed 27 November 1976) WEBBERS, J P P AND E E LITTLE Mulnplehqutd stimulus dispenser PHYSIOL BEHAV 20(1) 95-98, 1978 - An eight valve dispenser was developed for admmistenng low concentrations of hqmd sUmuh The ¢hspenser and its electrical ctrcmtry Is described The dispenser was used m behaxaoral studies of the chemical senses of channel catfish, Ictalurus punctatus The concentration threshold of catfish trained to suppress heart rate m response to the amino acid, L- sterne, was found to be 5 × 10-9 Molar Aqueous stimulus dispenser Relay ctrcuitry program Chemical senses Cardmc condlUonmg Amino acid Channel catfish, [ctaluruspunctatus

A MAJOR problem m studying the chemical senses is presenting adequate concentrations of chemical stunuh while rigorously controlling contamination The problem becomes partlcularly acute m behavioral studies where extended periods of training and testing are required Channel catfish (Ictalurus punctatus}, for example, can be trained to respond to extremely dilute solutions of amino acids Durmg the training period up to 1000 1 of solutions and rinses must be apphed Previously described chemostimulators have been used mainly in electrophyslologtcal studies [ 1,2] Application of these m behavtoral experiments were hmlted since they required lmmoblhzed fish These devices were also hmlted m the number of different solutions that could be dispensed without contamination during a testing session Generally, the test shmuh were rejected into the water supply of the test chamber through pipettes or solenoid valves. Manual operation of the apparatus was generally required Thus accurate control of the procedural sequence was difficult and lengthly experiments were laborious One apparatus, recently described for use in physiological experiments [3] can be automatically controlled, however, it could not handle large volumes of solutions reqmred for behavioral procedures The automatic stimulus delivery system described below was developed to facthtate our experiments on chemical sensitivity m unanesthetlzed aquatic ammals With slight modifications tins system could be used m studies on taste m terrestrial organisms as well as m electrophysiologlcal studies of the chemical senses The dispenser is capable of dehvenng eight separate solutions without detectable cross contamination and can be automatically operated by conventional programming apparatus Presently, the system is used for dispensing

Sensmvlty threshold

solutions of various amino acids m concentrations rangmg from lO"3 to 10-9 Molar APPARATUS With the exception of speclficaUy noted components, the dispenser was developed and constructed by the authors The dispenser is constructed of stainless steel As shown m Fig l, it has eight valves and a central valve hfter (4) Each valve 1s normally closed by a stamless steel spring The valve inlet (7) Is connected v~th a solution container winch provides a constant flow rate Well water flows continuously through the hollow valve stems (5) and the common valve outlet (9) This continuous nnsmg prevents cross contamination between channels of the dispenser During the release of a solution there is a ten-fold decrease m the stimulus concentration, as it becomes diluted by the continuous rinse A solution can be selected via position 1 of switch S II and one of the positions of Switch S I (Fig 2) An automatic programming apparatus can also be connected to the nghthand terminals of Fig 2 with the Switch S I I m position 3 Either pathway results m 24 V D C being applied to the motor (1) and to the coil of relay A Whale the motor is runnmg, the homing switch (3, Fig 1), central valve hfter (4, Fig 1) and central outlet rotor (8, Fig 1) turn to the chosen position At that point the motor stops and the coll of relay A is de-energized Relay A disconnects the valve clrcmt during the time the motor is running A delay in the release of the stimulus occurs when delay relay 1 and relay B are energized by normally open (N O) contacts of a - 1 after the homing switch has closed Relay C, activated via the normally closed (N C ) contacts 95

96

WEBBERS AND LITTLE

FIG 1 Photograph (A) and diagram (B) of the multiple chermcal sUmulus dispenser The motor (1) moves the homing switch (3), valve hfter (4), and central outlet rotor (8) When the homing switch reaches the selected dlspensor channel the solenoid (2) hfts the valve (6) to release the soluUon (7) The valve Is contmuously rmsed through the hollow valve stems (5), central outlet rotor, and common valve outlet (9) (GLOBE f h p motor, and GUARDIAN solenoid) of delay relay 2 stays energized after delay relay 1 operates wa the contacts of C 2 and delay relay 2 The central valve hfter opens the valve and the stimulus enters the dispenser when delay relay 2 is energized through the second N O contacts of delay relay 1 Thus as delay relay 2 Is energized the solenoid (2, Fig 1) is activated, rotating the spindle (10, Fig 1) and the central valve hfter (11, Fig 1) The termmatmn of the stimulus occurs when delay relay 2 operates The valve closes and the tnal cycle is completed as relay C and the solenoid (2) are de-energ/zed Both timing relays can be adjusted between 1 2 and 120 sec A switch (S 3) can be pressed to manually dispense solutions through the N C contacts of C 1 The contacts C 3 can be used for monitoring the opening and closing of the valves BEHAVIORAL STUDIES The dispenser was used m cardiac condltlomng studies to determine c o n c e n t r a t m n thresholds for L-cysteme Channel catfish fingerhngs (15 to 20 cm) were tethered m short plastic tubes Two silver wires chromcally inserted subcutaneously over the cardiac region were anchored to the tube The fish were anesthetized with tncame methanesulfonate (1 6000) dilution while being tethered The fish were held seml-mobde m a 400 ml chamber which received a constant flow of unchlormated water (100 ml/mn) Heart rate responses were amplified and filtered with conventional cathode follower and preamplifier and were observed and

recorded on an oscilloscope and chart recorder m an adjacent room The dispenser was programmed wath a relay apparatus to release a random number of plato water control stlmuh preceedmg the release of cysteme solutions The fish were shocked at the conclusmn of the cysteme presentation through sliver wtres held against the sides of the fish Figure 3a illustrates a typical heart rate response to 10-4 Molar cysteme (L-cysteme readily oxidizes to form another amino acid, L-cystme The observed behavioral response may have been made to the latter stimulus) The heart rate changed from a resting rate of 90 beats per mm to 30 beats per mm m response to cysteme The heart rate was shghtly suppressed during the onset of the control trial, probably m response to nonchem~cal stimulation such as tactile After the heart rate response to a particular concentratmn of cystelne became constant the solution was diluted tenfold for the next stimulus Heart rate suppression became less pronounced as the concentration of the cysteme solution was reduced (Fig 3b) Ascending and descending concentration ser~es were used to insure that the dechne m the cardiac response to lower stimulus concentrations was not due to sensory fatigue or adaptation At stimulus concentrations ranging from 18-8 to 10-9 M the heart rate response could not be dlstmgutshed from that made to plato water control solutmns This threshold is somewhat lower than that reported for olfactory receptors m electrophysmloglcal studies [4] Cardmc responses to cystelne and other amino acids occur within 5 to 15 tnals

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CONTROLCIRCUIT FIG 2 Diagram of valve and control box ctrcmts Switch 1 (S I) selects the dispenser channel Switch 2 (S ll) selects the automaUc or manual mode of operation Svntch 3 (S l i d allows solutions to be released manually After the motor has stopped, delay relay 1 provides an mterval before the sUmulus Is released Delay relay 2 controls the duraUon of sUmulus release Relay A prevents operaUon of the valve ctrcmt whtle the motor is runnmg Relay B momtors output from the homing svatch and starts the tmung relays whtle relay C acUvates the second ttmmg relay (POTTER AND BRUMFIELD ctol-38 Delay Relays and KR-5965 Relays )

of training and are e x t m g m s h e d within five trials if the a m i n o acxd stimulus is n o longer paired with shock Behavtoral sensitivity to a m i n o acids and sensory c o n t r o l of the cardzac response wall be discussed m a later paper (Little, s u b m i t t e d ) S p e c t r o p h o t o m e t n c m e a s u r e m e n t s of rinse water taken 5 sec after valve had passed 1 0-4 M adenosine m o n o p h o s p h o n c acid or 10- 3 M m e t h y l e n e blue indicated n o cont a m i n a t i o n The h m l t o f sensltlvlW of the s p e c t r o p h o t o m e t e r was 10- 7 M for these solutions The behavioral

threshold for cysteme, b e t w e e n 10 -s and 10 -9 M, indicate that the c o n t a m i n a t i n g c o n c e n t r a t i o n m the n n s e water was less than 10 -8 M after handling 10- s M

ACKNOWLEDGEMENTS We thank Dr L M Beldler for encouragmg the development of this dispenser and Dr Don Tucker for help vath the manuscnpt Tins work was supported by NIH Grant No NS-7010

98

WEBBERS AND LITTLE

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FIG 3 (a) Supenmposed on the EKG record are the electrical artifacts of the relay program and dispenser ctrcmtry wh]ch mark the temporal sequence of the dispenser operation The resting heart rate of an unanesthettzed fish was measured at the beginning of the trial Heart rate changes m response to plato water (Control) and 10-4 M cysteme were measured at the concluszon of the release of these solutions (Response) The dispenser motor had moved the hftmg mechamsm to the selected channel (Motor Operation), there was a period of silence (Delay Relay 1) preceding the release of a solution (b) Relationship between the magmtude of the cardiac response and a descending concentratzon series of L - c y s t e m e solutions Response = 11 39 Log concentration + 101 97, r = 95 H 2 0 indicates cardiac response to plato water control, and Cardiac reduction = Resting heart rate Heart rate response after stimulation - Restmg heart rate

REFERENCES 1 Capno, J High sensltwlty of catfish taste receptors to amino acids Comp Biochem Physzo! 52A" 2 4 7 - 2 5 1 , 1975 2 Dovmg, K B The influence of olfactory stamuh upon the actlwty of secondary neurons m the Burbot (Lota Iota L) Acta phystol scand 66: 2 9 0 - 2 9 9 , 1966

3

Hara, T J , Y M C Law and E Van Der Veen Stlmulatory apparatus for studying the olfactory acUvlty m fishes J Fish Res Bd Canada 30" 2 8 3 - 2 8 5 , 1973 4 Suzuki, N and D Tucker Amino acids as olfactory stimuli m fresh water cat fish, Ictalurus catus Comp Blochem Physzol 40A" 3 9 9 - 4 0 4 , 1971