Physiological Psychology 1977. Vol. 5 (1).21-26

Effect of dypsogenic stimuli on licking response execution of the rat

P. BART VRTUNSKI, TOVI COMET, and LEE R. WOLIN Laboratory of Neuropsychology, Ohio MH & MR Research Center, Cleveland, Ohio 44109

In two experiments, effect of several dypsogenic stimuli upon licking response force in rats was investigated. Independent variables were preferred solutions vs water, stimulus intensity, length of water deprivation, and extinction conditions. Measurements of response forces were made with a water spout fitted on a pressure transducer and its output fed into a signal averager or a digital computer. Results indicated the response force to be primarily a function of the stimulus intensity. A tenfold reduction in stimulus intensity accounted for close to a three-and-one-half-fold increase in licking response force. Although less pronounced, water deprivation also affected the response force . The animals' preference for NaCI and glucose solutions was not reflected on licking response force . Finally, contrary to some suggestions from literature, extinction condition did not generate higher response forces than the control conditions. Results are interpreted in terms of role of feed­back information in generation of licking responses and its potential value in further exploration of other aspects of water regulating functions.

Numerous reports have indicated the licking response in the rat to be a simple, reflex-like tongue movement (Hulse, 1966), executed with high and constant rate (Stellar & Hill, 1952; Corbit & Luschei, 1969) and, presumably, controlled by a relatively simple, on-off type of neural circuit (Corbit & Luschei, 1969). Such description of the response well corresponds to Glickman and Schiff's (1967) under­standing of species-specific responses, postulated to be preprogrammed and stored in the brainstem. In their theoretical framework, initiation of a response, and presumably of the behavior in general as well, could be interpreted as an act of triggering of these preprogrammed patterns. In a context of motivational and reinforcing mechanisms, Valenstein (1970) also postulated preprogrammed ("fixed action") response patterns and indicated the structures caudal to the hypothalamus to be the most likely locus of their storage. Since these models of response emission are rather general in their out­lines, we considered it necessary to characterize the licking response in greater detail than the traditional approach with drinkometer or lever-dipper arrange­ment yields. Most of these traditional approaches do not contain any qualitative information about the response itself but, rather, treat it as an event on the time continuum. Its proximity to a stimulus, or to a just preceding response, is virtually the only attribute of such a behavioral measure. The need

Preliminary report presented at the Society for Neuroscience Annual Meeting, San Diego, November 1973. P.B. Y_ is presently at Neurobehavior Research, Y A Hospital, 10701 East Boulevard, Cleveland, Ohio 44106. T. C., a participant in the Case Western Reserve University Undergraduate Research Program, is presently at the Albert Einstein School of Medicine, New York, New York.

21

for increasing the power of resolution in our analysis of response execution parameters was rightly em­phasized by Allison (1971). Relying only on duration­related measures, he established several important microbehavioral adjustments present in licking re­sponse execution. Investigation of other responses, such as the food-reinforced barpressing response in rats (Notterman & Mintz, 1%5), the biting response (Daunton, 1973), and the intracranially reinforced barpressing response (Vrtunski & Gluck, 1974; Vrtunski, Murray, & Wolin, 1973), amply illustrates significant information toward the understanding of antecedent factors in the given motor per­formance.

This study was performed in order to examine the forces rats use in licking the water spout. The ex­tensive literature on thirst and drinking behavior does not contain any systematic investigation of this parameter. In addition to tests with such fre­quently investigated independent variables as pre­ferred vs control drinking solution and length of water deprivation interval, a' novel technique of controlling the stimulus intensity (amount of avail­able water for each licking response) was designed. Specifically, the question we tried to answer with this study was whether the licking response was an invariable, reflex-like behavioral event or a con­trolled one, i.e., in part affected by dypsogenic stimuli. If the former is true, dypsogenic stimuli should have no effect on the licking response force.

The report contains two experiments. In the first experiment, for estimate of licking response force, a signal averager was used. In the second, an on-line computer performed all the measurements. Both techniques are discussed elsewhere (Vrtunski & Wolin, 1974).

22 VRTUNSKI, COMET, AND WOLIN

GENERAL METHOD

Subjects The subjects were experimentally naive male Sprague-Dawley

rats, 100 days old at the beginning of the experiment. The animals were maintained in individual cages with continuous access to Purina Laboratory Chow.

Apparatus The test chamber consisted of an aluminum box (H45 x

W16 x D28 cm, Zero Mfg. Co.) fitted with a grid floor and Plexiglas door. On the narrow vertical wall, a pressure transducer was installed with the water spout mounted on its sensitive shaft. The spout was 9.5 cm above the floor and connected to the variable speed infusion pump (Harvard, Type 600-900) with a plastic tubing. The transducer signal was amplified and fed into control circuitry (K series modules, Digital Equipment Corp.) and a signal averager (Nuclear Data, Type ND-1024). Any pressure with the tongue upon the spout exceeding criterion set at I g was interpreted as a licking response. The control cir­cuitry served both to synchronize the sweep of the signal averager and to issue a 200-msec pulse to the unfusion pump. In this manner, the test solution was presented to the animal contingent upon response emission. A distinct advantage of such an arrangement is that, by option of varying speed of the in­fusion pump, the amount of test liquid to be presented for each lick of the spout, i.e., stimulus intensity, could be adequately controlled.

The signal averager sweep was set a 125 or 250 msec. With the 125-msec sweep, the average covered one response and the time immediately following. With the 250-msec sweep, two responses were included. Each average contained 800 or 400 sweeps, depending on the time base used. Hard copy of the average was drawn with an X-Y recorder (Houston Instrument, Type 2000) on a 25 x 38 cm chart and measured with a plani­meter (Alvin). So-obtained square centimeter values, following calibration procedure, were converted to gram-second values of time integral of force emitted by the tongue (throughout the report the time integral of force is abbreviated and referred to as force).

Procedure Except for water-deprivation experiments, each licking response

measurement was taken following 18 h of water deprivation. In order to habituate various interfering behaviors, before an animal was included in testing, it was allowed for 15 min in the test chamber on three separate occasions. Unless otherwise noted, each test session was 3 min long. Where the experiment called for repeated tests, the animal was allowed free access to water for 30 h between test sessions. In all experiments, in order to avoid the possible confounding effect of temperature changes, two groups of animals were tested with identical test conditions at different times.

EXPERIMENT 1

Effect of Stimulus Intensity on Licking Response Execution

With rare exceptions (Hulse, 1967), studies involv­ing the licking response do not control the amount of water which comes in contact with the rat's tongue in a single licking response (the lever-dipper arrange­ment usually provides a greater amount of water, and it is consumed in several licks). In our preliminary tests, we· observed rapid and sizable changes in the force of the tongue's pressure upon the spout follow­ing change in the infusion pump rate of water supply.

Figure 1. Two samples of voltage analog record of licking response in the same rat. The upper tracing was obtained with the stimulus intensity set at 1.91 mllmin, the lower tracing with 3.82 ml/min. Calibration mark 5 g and 1 sec.

It would take not more than two to four responses before a new level of force emission was reached. Within a given stimulus presentation rate, licking response force seemed to be maintained on a stable level (Figure 1). This observation led us to present more systematic exploration of the effect of stimulus intensity on licking response force.

Method Stimulus intensity was defined as the amount of water presented

at the spout for each licking response. As already indicated, it was achieved by a variable speed infusion pump. Seven rates of water presentation were used: 0.247, 0.382, 0.764, 1.230, 1.910,2.470, and 3.820 mllmin. Due to some variability in licking rate, the above presentation rates cannot be translated into exact amount of water the animal obtains with each lick. Never­theless, assuming the licking rate is constant and identical for all animals, i.e. 4OO/min, the calculated values of stimulus in­tensities with the above water presentation covered the range from approximately 0.6 to 9.5 iii of water per lick.

Results The obtained results are summarized in Figure 2.

The observed changes in licking response clearly demonstrated an inverse relationship between stimulus intensity and force used in response emission. The repeated measures design of ANOV A for two groups yielded F ratio values of 15.02 and 38.53, well exceeding critical limits of 3.83 and 3.51 respectively, for the probability level of 1070.

TWO ATTEMPTS WITH NEGATIVE OUTCOME

(1) Effect of Preferred Solution on Licking Response Execution

It is well established that rats prefer isotonic solu­tions of NaCI (Stellar, Hyman, & Samet, 1954) and glucose (McCleary, 1953) to water. The preference, usually expressed in terms of volume of solution consumed over that of water, approaches the ratio of 2: 1 with both solutions.

Two groups of 10 animals each were trained to lick the spout with the water presentation rate set at 1.23 mllmin. For the first group, the test consisted of one session with tap water and one session with 0.9070 NaCI solution used as a stimulus. Order of tests was reversed for the second group of animals. After a 7 -day rest, the same two groups of animals were used for the glucose test. The sequence, rate of solution presentation, and number of runs were also identical to the saline test. Tests were performed with aqueous solution of glucose, in a concentration of5.3%.

Measurements of the licking response force indicated no difference between force levels reached with water, on the one hand, and NaCI and glucose, on the other. In tests with saline, a t test yielded a value of 0.152.

Similarly, the licking response force was not differ­entially affected by the glucose solution. The value of t obtained was 0.663.

(2) Effect of Water Deprivation on Licking Response Execution

Since it was established that the longer the depriva­tion period, the larger the amount of water con­sumed, it is customary to define drive in terms of hours the animal was deprived from access to water. Here, we tested the possibility that increase in "drive" might be reflected on licking response force.

Stimulus intensity was set at 1.23 mllmin, and licking response measurements were performed after 18,42, and 66 h of water deprivation. Obtained data indicated an increase in force of the licking response following 42 h of water deprivation when compared with the 18-h deprivation level, and subsequent de­crease in force when tested at 66 h of deprivation. Analysis of variance, however, yielded an F ratio of 2.04, well below the 5% critical limit of 3.32.

DISCUSSION

The experiments with saline and glucose solution, water deprivation, and variable stimulus intensities were designed in order to test the effect of differ­ent reinforcers, different drive levels, and variation in peripheral sensory level, respectively, on force emission in the licking response. Tests with various

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LICKING RESPONSE EXECUTION 23

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HOH, ml/min Figure 2. Means and respective standard errors for time integral

of force as a function of stimulus intensity. Upper numbers on abscissa indicate calculated values of ,dllick of water, available at the spout.

reinforcements were clearly negative, i.e., various reinforcement solutions did not differentiate the response force. Comparison of force levels emitted with water vs glucose and saline for individual animals shows the changes with the latter to be virtually random, both in size and direction.

Concerning the drive-level manipulation, the results, although statistically negative, do justify discussion of a certain trend in changes in the lick­ing response force. An immediate question that arises is whether such small differences between depriva­tion levels would be observed with a higher stimulus intensity, namely a higher rate of water presentation. One may speculate that, with the 1.23-mllmin rate presentation we used, response force was already considerably elevated, thus precluding response force differentiation due to length of time the animal was kept without water.

Another problem involved in the experiment is with stimulus intensity. Our measurements covered the range of water presentation from 0.247 to 3.820 mllmin (Figure 2). When individual forces were examined, however, some animals reached the peak at 0.764 mllmin intensity, and both lower intensities, i.e., 0.247 and 0.382 mllmin, yielded lower force emissions. It led us to assume that further reduction in stimulus intensity, i.e., extinction condi­tion, would further reduce the licking response force. This hypothesis was tested in Experiment 2(1).

In their extensive studies of the food-reinforced barpressing response, Notterman and his collabor­ators (DiLollo, Ensminger, & Notterman, 1965; Notterman & Mintz, 1965) reported the extinction

24 VRTUNSKI, COMET, AND WOLIN

condition to generate barpressing responses of the highest amplitude. Increase in the amount of re­inforcement resulted in a proportional drop in emitted response force. The above authors suggested that the amount of reinforcement determines the ability of the animal to discriminate the force threshold required to produce the reinforcement. In this context, the extinction was considered as a simple zero point on the scale for the amount of reinforcement.

We do not intend to challenge this interpretation here. Differences between the food-reinforced bar­pressing response and a lick of a watering spout are sufficiently large to preclude simple comparisons.

The level of force emission in the licking response, however, is rapidly changed when the rate of water presentation is changed. Whether it is an increase or decrease, the inversely proportional change in licking force emission, except at the very low in­tensities, is so noticeable and immediate that it could be called a "dial-a-response" controlled behavioral output. In other words, for the licking response, as opposed to the barpressing response, an inter­pretation on a more elementary level of sensory control of the motor output seems more appropriate. We will return to these aspects of response execution following Experiment 2.

EXPERIMENT 2

This experiment was designed and performed in order to examine the possibility that the statistically negative results in the water-deprivation test in the preceding experiment were due to a relatively low stimulus intensity, and to compare the licking re­sponse execution under extinction and control stimulus conditions.

General Method To a large extent, the method and procedure in this experiment

were identical to those used in Experiment I. The only significant change made was in the technique of data gathering and response execution measurement. Instead of a signal averager, the voltage analog of the tongue's pressure upon the spout was fed into a PDP-12 (Digital Equipment Corp.) computer. The analog signal sampling rate was set at 1 kHz. Following each test session, the typed output contained several parameters of the licking response sequence (Vrtunski & Wolin, 1974). As in Experiment 1, only the time integral of force will be discussed here.

Experiment 2(1): Licking Response Execution During Extinction

In already cited studies of Notterman and his associates with the food-reinforced barpressing response, extinction generated responses of higher force than did any other stimulus condition. Here, in Experiment 1, it was demonstrated that the licking response increased as an accelerated and inversely proportional function of stimulus intensity to a certain point (0.382 mllmin of water). Further reduc-

tion in the stimulus reduced the response force. This observation raised the possibility that the extinc­tion condition would generate responses with even lower forces.

Method. Two different procedures were used. In one (N = 8), the animals were trained to drink in the test chamber for 3 days with· 6 h of free access to water afterwards. On the test day, following 200-250 emitted responses under control condition, the water supply was stopped and the animal was left in the test chamber for an additional 15 min. In the second procedure (N = 10), the 15-min test session was administered with no initially reinforced responses.

Results. Inspection of the strip-chart recording of the transducer output indicated that as soon as the extinction condition is introduced, the licking re­sponse force indeed tends to increase within the on­going burst of responses. This increase in force is not maintained for more than a few seconds, how­ever. The principal changes within the licking se­quence are an intermittent appearance of licks of very low force level and a relatively large number of short pauses interspersed with a few emitted responses, rapidly followed by complete cessation of response. While under the reinforcement condi­tion, the animal licks the spout several hundred times per minute; in a 15-min extinction test, the mean number of licks recorded was 98 (both groups). Considering the licking response force, both proce­dures failed to differentiate the reinforcing and non­reinforcing stimulus conditions. In both groups, t tests yielded values below the critical limits of 5Ofo.

Experiment 2(2): Effect of Water Deprivation on Licking Response Execution-A Replication

The replication of this experiment was performed in order to find whether or not the response force established with higher stimulus intensity could differentiate better various levels of water depriva­tion than the lower intensity did in Experiment 1.

Method. The subjects were two groups of five experimentally naive animals each. Procedure and sequence of testing were identical to those used in the previous attempt (part of Experi­ment I). Stimulus intensity was set at 2.47 mllmin.

Results. Obtained mean values of the licking re­sponse force were 0.2906, 0.3249, and 0.3341 g-sec, for 18, 42, and 66 h of deprivation, respectively. Although the differences between means are not greater than those observed in Experiment 1, it should be noted that there was a steady increase in force levels generated and, due to smaller variability, a repeated measures ANOV A design yielded an F ratio of 4.81, thus exceeding the 5 Ofo critical limit of 3.55.

DISCUSSION

The above experiments demonstrated a sizable range of changes in the force animals exert in the course of licking behavior. The largest effect was observed when the amount of water, i.e., stimulus

intensity, was varied. A tenfold reduction in stimulus intensity accounted for an almost three-and-one­half-fold increase in licking response force (Experi­ment 1). In two attempts in varying "drive," although a discernible effect in response force increase was observed, the size of that increase is rather small. Among 10 animals in Experiment 2(2), the smallest increase in response force was from 0.0782 to 0.0887 g-sec, and the largest increase was from 0.2415 to 0.3683 g-sec from 18 to 66 h of depriva­tion, respectively. These changes were therefore well within a rather narrow band of the total range of forces these animals are capable of emitting. Finally, dipsogenic stimuli considered to be more reinforcing than water did not affect the licking response execu­tion at all. Parenthetically, we should add that, as part of Experiment 2, an attempt was made to per­form a test with 30/0 NaCI solution preloading (300 mg/kg). Eventually, the experiment was terminated because of an aversive effect of the intra­peritoneal administration of the solution. Neverthe­less, on the basis of data collected on 12 animals (6 with NaCI and 6 controls), preloading had virtually no effect on licking response force. At the same time, NaCl administration did increase the number of emitted responses for over 500/0 (vs 12% for controls).

In an attempt to interpret the above results, we may start from a functional description of a single licking response. The rapid extrusion and retraction of the tongue serves as a kind of vacuum pump. Friction of the tongue against the orifice of the watering tube creates a vacuum which, in turn, transfers a certain amount of water from the tube to the tongue surface. In this manner, the only role of increasing licking response force would be to in­crease the efficiency of that pump, i.e., water transfer function. In a natural setting, where the animal would lick the water from a presumably flat surface, a cup-like container, or a stream, increase in licking response force would represent a physically greater extrusion of the tongue and, again, a pro­portional increase in water volume, i.e., water transfer function. Thus, going back to Experiment 1, where the amount of water was under control of the experimenter, accompanying changes in licking response could be interpreted as the animal's control (or attempt to control) of this water transfer func­tion under changing conditions.

Instead of considering the licking response to be a "preprogrammed" event released by some oscillator, we suggest the response to be a muscular contraction akin to the stretch reflex (Granit, 1970), a response guided by simultaneous sensory input. As such, the licking response would conform to rules of what is commonly called a closed-loop feedback system, and in most particulars is similar to what Powers (1973) described as the first-order control system. Its prin-

LICKING RESPONSE EXECUTION 25

cipal function is defined in terms of sensory input intensity control.

Obviously, we derive support for this interpreta­tion from Experiment 1. The inversely proportional relationship between the stimulus intensity and the motor output of the tongue warrants the conclusion. In this context, the reduction of water intensity re­sults in increased error signal and the subsequent increase in licking forces has a role to compensate for that error, i.e., to reduce the difference between the sensory input signal and the set reference level.

There are several problems with the use of the feedback model in interpreting our data. In the first place, if the inverse relationship between the stimulus intensity and motor output indeed represents a behavioral compensation for sensory input changes, why would that relationship change its course on very low stimulus intensities (as observed in Experi­ment 1) or under extinction conditions [Experi­ment 2(1)]? Powers' (Note 1) explanation is that behavioral output has a certain maximum value in attempting to compensate for the error signal in­crease. Further increase in that error tends to produce an overload of the feedback loop and eventual reduction at the behavioral output, i.e., a "giving-up" type of effect. This interpretation is certainly congruent with the very definition of extinction. If the same interpretation is applied to data from the already mentioned studies of DiLollo, Ensminger, and Notterman (1965), and Notterman and Mintz (1965), the most likely reason they ob­served maximal response forces under the extinc­tion condition is probably in the relative complexity of the barpressing response, i.e., participation of other than purely sensory processes in control of response forces. In both studies, 35 barpressing responses represented the sample under investiga­tion. It may well be that barpressing force would drop only after several test sessions. This certainly raises an important problem of time scale against which various responses could be compared.

The second major problem in interpreting our data according to control theory principles is in the nature of information from higher nervous centers and its role in maintaining the licking response. If the licking response is indeed similar to a spinal type of reflex, additional neuronal elements such as primary and secondary fibers and gamma efferents should be assumed to play a role in the response execution (Granit, 1970). The link between the peripheral motor elements, on one hand, and diencephalic integrators such as the septal area and lateral hypothalamus (Mogenson, 1973), on the other, still remains to be established. Our experi­ments with preferred dypsogenic stimuli and vari­ation of drive levels were specifically designed to shed a light on this link. With the benefit of hind­sight, it may be surmised that in order to do so more

26 VRTUNSKI, COMET, AND WOLIN

fully one must follow other than traditional experi­mental approaches. For instance, in most experiments involving drinking and licking response recording, animals are deprived of water prior to testing. It is taken for granted to be the most appropriate way to initiate and maintain the response. The reason behind it, however, is purely practical. A more careful scrutiny of the experimental situation would show how large a load upon the water regulating mechanisms this deprivation must represent.

We believe the results of the study support the following conclusions. First, investigation of the forces involved in the licking response execution provides important information both about the character of the response and about internal and external conditions which influence the response. Secondly, by accepting the control theory's feedback model, investigation of response execution does shed light upon an orderly chain of events, or parts of that sequence, which define a behavioral response, i.e., maintenance of the water balance function.

REFERENCE NOTE

1. Powers. W. T. Personal communication, July 21, 1975.

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(Received for publication April 19, 1976; revision accepted October 28, 1976.)