Chem. Senses 38: 685–704, 2013
doi:10.1093/chemse/bjt038 Advance Access publication July 31, 2013
The Fine Temporal Structure of the Rat Licking Pattern: What Causes the Variabiliy in the Interlick Intervals and How is it Affected by the Drinking Solution? Xiong Bin Lin1, Dwight R. Pierce2, Kim Edward Light2 and Abdallah Hayar1 1
Department of Neurobiology and Developmental Sciences, Center for Translational Neuroscience, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little 2 Rock, AR 72205, USA and Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA
Accepted June 27, 2013
Abstract Licking is a repetitive behavior controlled by a central pattern generator. Even though interlick intervals (ILIs) within bursts of licks are considered fairly regular, the conditions that affect their variability are unknown. We analyzed the licking pattern in rats that licked water, 10% sucrose solution, or 10% ethanol solution, in 90-min recording sessions after 4 h of water deprivation. The histograms of ILIs indicate that licking typically occurred at a preferred ILI of about 130−140 ms with evidence of bimodal or multimodal distributions due to occasional licking failures. We found that the longer the pause between bursts of licks, the shorter was the first ILI of the burst. When bursts of licks were preceded by a pause >4 s, the ILI was the shortest (~110 ms) at the beginning of the burst, and then it increased rapidly in the first few licks and slowly in subsequent licks. Interestingly, the first ILI of a burst of licks was not significantly different when licking any of the 3 solutions, but subsequent licks exhibited a temporal pattern characteristic of each solution. The rapid deceleration in intraburst licking rate was due to an increase from ~27 ms to ~56 ms in the tongue-spout contact duration while the intercontact interval was only slightly changed (80−90 ms). Therefore, the contact duration seems to be the major factor that increases the variability in the ILIs and could be another means for the rat to adjust the amount of fluid ingested in each individual lick. Key words: autocorrelograms, bursting, licking, rhythmicity
Introduction Although rodent fluid licking has no resemblance to human drinking, rat licking has been extensively studied in order to understand motor coordination of rhythmic movements and to reveal the mechanisms that control or modulate ingestive behavior (Smith 2001; reviewed by Davis 2004). In order to drink, rats dip their tongue repeatedly into the water and use it to scoop water into their mouth. Fluid licking is characterized by repetitive tongue and jaw movements that are controlled by a network of brainstem neurons forming a central pattern generator (reviewed by Travers et al. 1997) located in lateral medullary reticular formation (Chen et al. 2001). This central pattern generator is itself modulated by olivocerebellar neuronal firing that is time locked to licking events (Welsh et al. 1995; Bryant et al. 2010; Cao et al. 2012). Monitoring licking activity could also be a very useful
method for rating the physiological function of hypoglossal motoneurons that innervate the muscles of the tongue via the 12th cranial nerve. It may also serve to test the behavioral effects of acute or chronic drug treatments (e.g., Peachey et al. 1976; Hsiao and Spencer 1983; Genn et al. 2003). Moreover, differences in lick rate among different strains of mice have been exploited for mapping quantitative trait loci that are involved in controlling licking behavior and for identifying the genes that modulate the rhythm generated by the central pattern generators located in the brainstem (Boughter et al. 2007, 2012). Rats ingest fluids in a cluster of licks or bouts (Davis 1989). Different patterns in the rate of licking can generate similar intakes (Davis and Smith 1988). Therefore, to better understand ingestive behavior, it is important to consider the
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Correspondence to be sent to: Abdallah Hayar, Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, 4301 W. Markham Street Slot# 847, Little Rock, AR 72205, USA. e-mail:
[email protected]
686 X.B. Lin et al.
per burst, as well as the occurrence of subsequent bursts. We also found that these newly discovered licking features are distinctly altered when the oral somatosensory system is stimulated by a pleasant sucrose solution versus an unpleasant ethanol solution which is normally perceived by rats as aversive (Bice et al. 1992).
Materials and methods Animals and solutions
This study was approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. Female Sprague–Dawley rats of the same age (250−300 g) were used for experiments aimed at analyzing the effect of taste on the microstructure of licking. We used rats of the same sex and age because there is some evidence in previous studies indicating that females lick significantly more rapidly than males (Cone 1974), and an age-related difference in licking rate has been reported in gerbils (Schaeffer and David 1973). Rats were allowed to drink either tap water, 10% w/v sucrose solution (~0.3 M), or 10% v/v of ethanol solution (prepared using ethyl alcohol 190 proof, ACS/USP grade, purchased from Pharmaco-Aaper Company). Tap water was used to prepare sucrose and ethanol solutions. One milliliter of the 10% ethanol solution contained 0.075 g of ethanol. We used 20 naïve and untrained rats. On the 1st day, the first 10 rats were tested with ethanol solution, and then tested with water 2 days later. On the 2nd day, the remaining 10 rats were tested with sucrose solution and then tested with water 2 days later. For the purpose of analyzing licking data, rats were divided in 3 groups: group I, rats that licked tap water (n = 19 of 20; data from one rat were excluded because the ground wire was accidently disconnected during the experiment and this prevented the recording of all licks); group II, rats that licked 10% sucrose solution (n = 10); and group III: rats that licked 10% ethanol solution (n = 10). Rats that licked the ethanol solutions and sucrose solutions did not show any apparent difference in the licking pattern when tested with water 2 days later. Thus, the water licking data from all rats were pooled together. Rats were tested in 90-min drinking sessions (which constitute their only opportunity for fluid consumption) after 4 h of water deprivation. Animals were transferred from the animal facilities to the lab around 9:00 AM, and the water drinking bottles were removed from their cages around 9:30 AM. All rats were tested in the afternoon around 1:30 PM. Recording setup
For recording licking activity, we used methods similar to those published previously (Hayar et al. 2006; Bryant et al. 2010). During recording, each rat was placed in a cage
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microstructure of licking by analyzing several parameters such as the number of bouts and the number of licks/bouts in addition to the total number of licks (Galistu and D’Aquila 2012). Based on early work on the microstructure of licking behavior (Davis 1989), algorithms were designed to classify bouts into 2 categories, which were called bursts (sustained licking until a pause of 250 ms) and clusters (sustained licking until a pause of 500 ms). However, at that time, licking events were not acquired at high temporal resolution, and computer programs were not sufficiently developed to look for the subtle temporal features that characterize the pattern of licking. The concept that rats lick at a highly stable rate (6−7 Hz or not at all), independent of the level of water deprivation, has been prevalent for more than 5 decades (Stellar and Hill 1952; Keehn and Arnold 1960; Corbit and Luschei 1969). This notion persists in the present literature and computer programs for analyzing licking activity were designed based on this assumption (Houpt and Frankmann 1996). Welzl (1976) concluded that the amount of water consumed by rats is simply controlled by the length of time spent licking and not by changing the licking rate. For example, rats and mice can compensate for the narrowing of the drinking spout orifice by increasing the time spent drinking in order to maintain the same total intake volume (Freed and Mendelson 1977; Dotson and Spector 2005). However, recent studies have indicated that many environmental factors can change the interlick interval (ILI). For instance, rats can slow down their licking rate if the spout accessibility is restricted (Halpern 1977; Hernandez-Mesa et al. 1985), or if the distance between the rat and water source was increased (reviewed by Cone 1974; Weijnen 1998). Rats increase their lick rate under stress (Vajnerová et al. 2003) and change their licking pattern after identification of an aversive tastant, which could occur after a relatively few licks depending on tastant concentration, learning, and motivation to drink (Weiss and Lorenzo 2012). Therefore, a stable licking frequency is not the reflection of a rigid output of a central rhythm generator that is either active or not. In this study, we hypothesize that licking occurs with a defined temporal pattern characterized by a decremental intraburst rate that can be modulated by the taste of the ingested fluid. To test this hypothesis, we have analyzed the fine temporal structure of the licking pattern by investigating how the occurrence of each lick is affected by the occurrence of previous licks. For this purpose, we varied the classic definition of bursts and analyzed the autocorrelograms of all events as well as of bursts of events. We found that an in-depth investigation of the pattern of licking can reveal important features that reflect the degree of rhythmicity that is imposed by the central pattern generator. Using a high temporal resolution analysis, we showed that licking exhibits an influence of short-term memory that is revealed as a decrease in licking rate during a sustained burst of licks. This phenomenon, which has not been previously reported, seems to affect the determination of the number of licks
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Analysis of ILIs
The detection of licking events was performed off-line using Mini Analysis program (Synaptosoft Inc.). Consecutive
epochs of data (e.g., 100 ms) were searched for a peak voltage, and the average baseline voltage was calculated for an interval (e.g., 45−50 ms) before the peak. The event amplitude was calculated by subtracting the average baseline voltage from the peak voltage, and the event was rejected if the amplitude did not exceed a given threshold (e.g., 50 mV). The parameters characterizing the events were then imported into OriginLab 7.0 (Microcal Software Inc., Northampton, MA) for further analysis. The ILIs were calculated as the time difference between the onset times of occurrence of consecutive events. The Mini Analysis software does not automatically generate the values of the event onset times. Therefore, the onset time of each event was calculated as the time of peak minus the rise time. Typically, rats lick in bursts of licks (or bouts) with the number of licks per burst and the pauses between bursts being highly variable. When constructing histogram of ILIs, we were interested in analyzing relatively short ILIs, so we ignored all ILIs longer than 1000 ms. We also ignored ILIs shorter than 60 ms (which were due to artifacts when the rat touched the sipper tube with the paw or face without licking, and these intervals constituted less than 1% of the total number of intervals). We defined (N) as the total number of ILIs (i.e., total number of licks minus 1) collected during each 90-min recording session. Using Origin software, we calculated the statistical parameters for the ILIs for each rat tested. We wrote an algorithm using the “Labtalk” scripting language in OriginLab software to automate the analysis and calculate all of the different statistical parameters at once. These parameters included the mean, median, standard deviation (SD) and coefficient of variation (CV = SD/mean). We constructed the histograms of the ILIs using 90 min of recordings. The histograms were binned at 1 ms and smoothed by adjacent averaging of 10 points (i.e., each point was replaced by the average of itself, 10 points before and 10 points after) in order to easily detect the peak of the distribution, which enabled us to find the “mode,” defined as the ILI that occurs with the highest probability. In order to compare the ILI distributions in different rats and in different conditions, the histograms were normalized to the number of ILIs collected during each recording session. As a consequence of this normalization, the integral of each histogram (i.e., the area under the curve) was always equal to 1. Because of this normalization, a larger peak at the mode indicates a narrower distribution. Autocorrelograms were also binned at 1 ms and the zero lag time (i.e., the time interval between an event and itself) was ignored. All autocorrelograms, except those that showed sharp peaks (width 10−20 ms), were smoothed by adjacent averaging of 10 points. Autocorrelograms were normalized to the counts obtained with the largest (primary) peak. We used the skewness coefficient to determine how much the histograms deviated from a normal Gaussian distribution and to quantify the degree of their asymmetry around
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similar to that used for housing rats in the animal facility. A quarter-inch wire mesh fabric (Yardgard 2 ft × 5 ft × 0.25 inch 23-gauge galvanized hardware cloth, model # 308231B, Home Depot) was cut to fit the bottom of each cage. The central pin (core) of a (Bayonet Neill–Concelman) BNC cable input connector of an analog/digital converter (Digidata 1322A, Molecular Devices) was connected to a silver wire (0.5 mm diameter) that was introduced into the sipper tube from within the water bottle, and the wire exited the bottle via the stopper that firmly closed the bottle. The grounded housing (shield) of the BNC cable was connected to the galvanized screen which served to ground the rat while moving freely inside the cage. Each lick closed the electrical circuit for the duration of the tongue–sipper tube contact, and the junction potential between the sipper tube or the water and the rat’s saliva could be recorded. It has been shown previously that the mean licking frequency was lower if the rat’s access to the water source was restricted or if the distance between the rat and the water source was increased (reviewed by Weijnen 1998). For these reasons, we chose an unrestricted configuration, with the rat’s access to water similar as in its normal cage when housed in the animal facility, and all values reported here indicate that the rats were licking in the faster part of the range reported by other authors (Weijnen 1998; Vajnerová et al. 2003). In our setup, the stainless steel sipper tube was covered with a transparent hard plastic tube to prevent the rat from making contact unless it was an actual fluid consumption. Licks were recorded in the form of junction potentials created when the rat touched the waterspout with its tongue (Hayar et al. 2006), and no current or voltage was applied to the animal when the circuit was closed. The baseline noise was typically mode and ≤1000 ms). OriginLab (version 9) software was used for statistical analysis. Data were compared between groups using one-way ANOVA with post hoc Tukey pairwise comparisons unless otherwise stated. The main aim of this study was to compare the pattern of licking either an ethanol or a sucrose solution to that of water, which was considered the control fluid.
licking (0.18±0.03 licks/sec) was lower than that of water, but it did not reach statistical significance (P = 0.10). During the remaining 15-min bins of the session, the mean frequency of sucrose licking continued to be numerically higher than that of water, and the mean frequency of ethanol licking was either the same or numerically lower; however, no statistical significance was identified. We next studied the variation in the ILI by analyzing the ILI distribution histogram (Figure 2), a conventional method that has been used in almost all previous studies (e.g., Houpt and Frankmann 1996; Bryant et al. 2010; Boughter et al. 2007, 2012). However, unlike previous studies, we binned our histograms at 1 ms instead of 10 ms, and then we used adjacent averaging (21 ms) to filter out fast variations. This method allowed us to determine the mode of the distribution with relatively high temporal precision (1 ms resolution) and to detect differences between groups in the order of few milliseconds. The histograms were generated for ILIs >60 ms and ≤1000 ms, thus constituting 940 bins of 1 ms duration. They were then normalized to the total number of ILIs within the same time window to generate what is equivalent to a probability distribution function. This normalization procedure allowed us to compare the shapes of the distributions regardless of the differences in the total number of licks. When the histograms were averaged from all rats within each group, we identified bimodality for the water and ethanol groups (one peak can be identified in addition to the primary peak), and multimodality (multiple seemingly equidistant peaks) for the sucrose group. Because the ILI distributions were skewed, we used the nonparametric Kolmogorov–Smirnov (K–S) test to compare the cumulative probability distribution of ILIs from each group (Figure 2B). The distributions of ILIs from both the ethanol and sucrose groups were different from that of the water group (P 200 ms, Figure 2C) in rats of all groups indicates occasional failures of the rat tongue to establish contact with the water spout at the expected time. In order to determine how the ILI distributions of each group differed from each other, we calculated several statistical parameters that characterize the distribution of ILIs when the rats were drinking water, ethanol solution, or sucrose solution (Figure 3). When the mean licking frequency was calculated over the 90-min recording sessions (Figure 3A), it was found that rats licked the ethanol and sucrose solutions at lower (P = 0.13) and significantly higher (P 60 ms and ≤1000 ms, which constitute between 89% and 97% of the total number of ILIs obtained in each recording session (Figure 3C). The mean ILI in the sucrose group (155 ± 2.1 ms) was significantly
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smaller (P
