- Email: [email protected]
Concentration-dependent licking of sucrose and sodium chloride in rats with parabrachial gustatory lesions
Physiology&Behavior.Vol. 53, pp. 277-283, 1993
0031-9384/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Printed in the USA.
Concentration-Dependent Licking of Sucrose and Sodium Chloride in Rats With Parabrachial Gustatory Lesions ALAN
C. S P E C T O R , * t I H A R V E Y
J. G R I L L t : I : A N D
RALPH
NORGREN§
*Department of Psychology, University of Florida, Gainesville, FL 32611, f'Smell and Taste Center and ~:Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104, and §Department of Behavioral Science, The Pennsylvania State University, Hershey, PA 1 7033
R e c e i v e d 19 M a r c h 1992 SPECTOR, A. C., H. J. GRILL AND R. NORGREN. Concentration-dependent lickingof sucrose and sodium chloridein rats with parabrachialgustatory lesions. PHYSIOL BEHAV 53(2) 277-283, 1993.--The medial zone of parabrachial nuclei (PBN) serves as an obligatory synapse in the central gustatory system in rodents. Lesions in the PBN impair taste aversion learning and depletion-induced sodium appetite in rats, and also alter the ingestion of sapid stimuli. Interpretation of these lesion-induced behavioral deficits requires an evaluation of whether taste function is compromised. The present study examined whether rats with PBN lesions could show normal concentration-dependent changes in licking behavior to very small volumes of NaCI and sucrose. Physiological state was also varied; taste responsivity was examined in water-deprived and nondeprived rats. In a specially designed gustometer, nine rats with electrophysiologically guided lesions in the PBN and five surgical controls were trained to lick a drinking spout to receive 10-s access to various concentrations of NaCI (0.03-1.0 M) and sucrose (0.01-1,0 M) during 30min sessions. Water-deprived control rats progressively decreased their responses compared with water as the concentration of NaCl was raised. In contrast, water-deprived PBNX rats did not decrease their licking responses to NaCl relative to water until the concentration reached 1.0 M. In the nondeprived state, control and PBNX rats decreased their responsiveness as a function of NaC1 concentration, and the two groups did not differ. The licking responses of water-deprived PBNX rats did not differ from control rats when sucrose was the stimulus. In the nondeprived condition, both groups monotonically increased their licking to sucrose as a function of concentration, but PBNX rats were significantly less responsive than controls. These findings demonstrate that rats with lesions centered in the parabrachial gustatory area are not ageusic, but under certain physiological conditions these lesions do appear to blunt responsivity to sapid stimuli. Sugar
Salt
Taste
Motivation
Central gustatory system
IN rodents, the pontine parabrachial nuclei (PBN) serve as an obligatory synapse in the central gustatory system [see (10)]. Several investigations have demonstrated that both permanent and temporary lesions of the PBN severely impair, if not abolish, a rat's ability to learn a taste aversion (1,2,9,15). Similar parabrachial lesions also eliminate the expression of sodium appetite following acute sodium depletion (2). One obvious explanation for these deficits is that PBN lesions render the animals ageusic. Several other studies, however, indicate that rats with similar PBN lesions do distinguish sapid chemicals from one another. Nowlis et al. (i2) reported that, when tested in a one-bottle test, rats with PBN lesions exhibited normal aversions to HC1 and quinine HC1, but exaggerated preference for sucrose and weak saline solutions. Similarly, using an one-bottle test, Flynn et al. (3) observed that, compared with controls, rats with PBN lesions overconsumed both normally preferred and normally avoided solutions, at least at some concentrations. When taste
Dorsal pons
stimuli were infused directly into the oral cavity and the immediate oral motor responses were measured, these same animals appeared to be less sensitive than controls to sucrose and quinine HCI, as indicated by rightward shifts in their concentration-response functions. In another study, the PBN lesions were made in 10-day-old rat pups that were then tested as adults using several 24-h intake paradigms (8). In a three-bottle test, the rats with lesions drank slightly more saccharin than water and virtually no quinine HCI. In the same situation, however, the control rats preferred water to saccharin, an u n c o m m o n observation at the stimulus concentration employed (0.15%). In the same experiment, the rats with PBN lesions drank less 2.5% NaCl in a twobottle test with water, but more quinine HC1 when it was the only fluid available. Given the differences in the experimental procedures (e.g., brief or long duration, intake or immediate responses), the results of these studies are difficult to compare. Nevertheless, they do
Requests for reprints should be addressed to Alan C. Spector.
277
278
SPECTOR. GRILl, AND NORGREN
imply that while bilateral parabrachial lesions do not render an animal ageusic, they do alter its responsiveness to sapid stimuli. As with neurologically normal animals, the extent of the behavioral changes produced by PBN lesions depends on the taste stimuli and the methodology used to assess them (6). The purpose of the present experiment was to determine whether rats with PBN lesions could demonstrate concentration-dependent changes in their appetitive licking responses to sucrose and NaC1. In addition, the rats were tested in both a water-deprived and a nondeprived state to examine the possibility that PBN lesions alter the relationship between physiological condition and taste responsivity. In order to increase our confidence that the behavior was guided by immediate orosensory factors, taste trials consisted of small volumes and the dependent variable was licking. METHOD
Subjects Fourteen naive male Sprague-Dawley albino rats (CD, Charles River, Wilmington, MA) served as subjects (246-298 g at start of the experiment). They were individually housed in stainless steel cages in a room where temperature, humidity, and lighting were controlled automatically. All rats had water and Purina rat chow (5001 ) freely available except where noted otherwise. Four days prior to the start of behavioral testing, all rats received, for 2 days, sodium-deficient diet (Teklad, #170950, sodium content: 0.005-0.015%) in addition to their normal Purina rat chow. The rats were kept on a 12/12 light/dark cycle and all manipulations were performed during the light phase.
Surgery Nine deeply anesthetized rats received electrophysiologically guided bilateral lesions in the PBN (PBNX) using procedures that have been described in detail elsewhere (15). Rats were pretreated with atropine (0.1 mg, IP) and then injected with Nembutal anesthesia (50.0 mg/kg, IP). Supplemental doses of Nembutal were delivered as necessary. The rat was secured in a stereotaxic (Kopf) headholder; nontraumatic ear bars were used. Two holes were drilled through the skull on either side of the midline and centered approximately 11.5 mm posterior to bregma. The recording electrode (tungsten-in-glass, Z = 0.51.0 Mohm at 1 kHz) was positioned (20 ° off vertical in the AP axis with tip anterior, - 11.5 mm AP from bregma, 1.8 mm ML) and lowered through the meninges and cerebellum to penetrate into the dorsal pons. The exact location of the gustatory neurons was identified by recording multiunit responses to 0.3 M NaCl applied to the anterior tongue. The sapid stimulus always was preceded and followed by a rinse of distilled water. Once the gustatory area had been identified on both sides, the recording electrode was replaced with a lesion electrode that consisted of a 130 #m diameter teflon-insulated stainless steel wire with only the crosssection at its tip bared. This electrode was then lowered into the PBN using the coordinates obtained with the recording electrode. In the vast majority of cases, the gustatory area also could be electrophysiologically identified via the lesion electrode. Two lesions were made on each side, one ventral (60 #A/20 s) and one 200-300 #m more dorsally (40 #A/20 s), in an attempt to destroy taste neurons both below and above the brachium conjunctivum (l 1). The holes in the skull were packed with sterile gelfoam and the incision closed with wound clips. Five rats served as full surgical controls (CON). These rats received several penetrations of the recording electrode on both sides. The electrode was lowered only into the cerebellum and did not penetrate the dorsal pons. All rats in this experiment
were given at least 24 days to recover from surger.~ bclbre behavioral manipulations. Two rats, one PBNX and one control developed mild respiratory, infections just prior to and during behavioral testing and were treated with oxytetracycline (0.05 cc, SC) twice a day.
Procedure In a specially designed gustometer described elsewhere (13), rats were trained to lick a drinking spout to receive 10-s access to various sapid stimuli. Food was not available during these sessions. On day 1, all rats were weighed and had their water bottles removed. There were no significant differences in body weight between the two groups (PBNX = 407 g; controls = 421 g). On day 2, the rats were placed in the gustometer and allowed to lick the drinking spout to receive water. During this 30-rain session, the control rats drank 9.56 ml and PBNX rats drank 10.0 ml on the average. The purpose of this session was to familiarize rats with the testing apparatus. On days 3-6, rats received 10-s trials during which they had access to either water or various concentrations of NaC1 (0.03, 0.1, 0.3. 1.0 M) and sucrose (0.01, 0.03, 0.06, 0.1, 0.3, 1.0 M). The 11 fluid stimuli were randomly presented in blocks of I 1 trials. In order to insure that the rat was attending to the spout, it was required to lick the drinking spout twice with an interlick interval of no greater than 500 ms to initiate a trial. The minimum intertrial interval was 6 s--the amount of time required to clean the drinking spout [see ( 13)]. The session length was 30 min and the rat could initiate as many trials as possible within this period. On days 2-5, the rats received their daily fluid allotment during the behavioral training and testing sessions. On day 6, the rats were 23.5 h water deprived at the start of the session, but water bottles were replaced on the home cages for the remainder of the experiment. On average, the rats lost 10% of their body weight over the first 2 days of the water deprivation schedule and then stabilized their body weight on days 3-6 at about 90% of their ad lib drinking value. On days 7 and 8, the rats were allowed to rehydrate. On days 9-11, the nondeprived rats were tested in the gustometer using the same paradigm and the same sapid stimuli.
Data Analysis The licks elicited during the 10-s trial were measured, but only the licks during the last 8 s of the trial were used in the analysis. This was done so that the analysis could concentrate on a rat's affective response to the stimulus and minimize the contribution of the mere motor acts involved in the initial fluid acquisition. The mean number of licks for water and for each taste stimulus during the water-deprived and nondeprived test sessions was computed for each rat. These means were then used to compute a lick difference score between a given taste stimulus and water for each rat: Lick Difference Score~Tastex~ = Licks(Taste x) - Licks~water) This score was then used in the various statistical analyses of variance (ANOVAs). The statistical rejection criterion (i.e., alpha) was set at 0.05. The data from one session in the water-deprived testing phase were discarded for four rats (day 5 for one rat and day 6 for the others) due to technical problems with the apparatus. The spout was not properly positioned on some of the trials during this session for two of these rats, and there was a pressure drop in the system during some of these trials for the other two rats. Consequently, the lick difference scores during the water-deprived testing phase for these four rats were computed based on
TASTE-RELATED LICKING IN RATS WITH PARABRACHIAL LESIONS
three sessions rather than four. One rat in the PBNX group failed to initiate at least one trial for each taste stimulus during the nondeprived testing phase. Consequently, a complete concentration-response profile could not be composed for this animal and its data were excluded from analyses involving data collected during the nondeprived testing phase (with the exception of number of trials).
Histology At the completion of the experiment, rats were deeply anesthetized (100 mg/kg Nembutal, IP) and perfused intracardially with saline and 10% buffered formalin. The brains were removed, stored in 30% sucrose-10% formalin for several days, then cut coronally on a freezing microtome (50 um sections). These sections were mounted on slides and alternate series stained with the cresyl Lecht violet and Weil procedures. The volume of the lesions was estimated by measuring the area of necrosis and gliosis on each section in the cresyl violet series (JAVA), multiplying those areas by 100 urn, and then totalling the results. Each lesion also was assessed qualitatively for its overlap with the parabrachial gustatory area defined by Norgren and Pfaffmann (11). Three categories of overlap were used--complete (>50% of the taste area), partial (<50% of the taste area), or n o n e - - a n d the preparation was judged on the basis of the least complete lesion. Both procedures were conducted without knowledge of the behavioral results. RESULTS In general, the lesions in the dorsal pons were large (average volume = 0.40 cu. mm. + 0.044). Eight out of nine rats had bilateral lesions that involved the gustatory zone of the PBN (Fig. 1). The one remaining rat had a complete lesion on one side, but on the other side the lesion damaged the lateral portion of the brachium conjunctivum, and did not encroach upon the gustatory area (Fig. 1G, H). This rat was discarded from the statistical analysis. In many cases, damage extended into the supratrigeminal area, the lateral subnuclei of the PBN, the brachium conjunctivum, and the mesencephalic trigeminal nucleus. The mean number of trials initiated by PBNX rats during the water-deprived testing phase was not significantly different from that initiated by controls, F(1, 11) = 1.70, p = 0.22 (Fig. 2). Although, strictly speaking, there was no significant difference between the number of trials initiated by PBNX and control rats during the nondeprived testing phase, F(1, 11) = 4.50, p = 0.057, the p value just missed the statistical rejection criterion. These data suggest that both PBNX and control rats were similarly motivated to drink when water deprived. It appears, however, that the overall incentive value of the taste stimuli during the nondeprived testing phase was lower for PBNX rats compared with controls. Water-deprived control rats progressively decreased their responses as the concentration of NaC1 was raised (Fig. 3, left panel). In contrast, water-deprived PBNX rats did not decrease their licking responses to NaC1 relative to water until the concentration reached 1.0 M. A two-way ANOVA indicated a significant effect of lesion, F(I, 11) = 9.06, p = 0.012, and concentration, F(3, 33) = 22.7, p < 0.0001, as well as a significant interaction, F(3, 33) = 5.12, p = 0.005. It appears that waterdeprived PBNX rats are compromised in their ability to appropriately modify their licking behavior based on the concentration of the salt stimulus. Note, however, that PBNX rats do significantly reduce their licking responses to 1.0 M NaC1. In the nondeprived state (Fig. 3, right panel), PBNX and control rats did not differ in their response to NaCI, F(1, 10) =
279
0.024, p = 0.879. Despite the relatively low frequency of licks to water, there was a significant decrease in responses as a function of concentration, F(3, 30) = 3.76, p = 0.021. This observation should be regarded with some caution, however, because a floor effect may have obscured differences between the two groups. Water-deprived PBNX and control rats did not differ in their response to sucrose, F(1, 11) = 0.548, p = 0.475 (Fig. 4, left panel). Interestingly, despite the relatively high frequency of licks to water, both groups significantly increased their licking as a function of concentration, F(5, 55) = 6.32, p = 0.0001. The lack of difference between the two groups may be attributable to a ceiling effect. In the nondeprived state, PBNX rats had significantly lower sucrose lick scores than controls, F(1, 10) = 5.33, p = 0.044 (Fig. 4, right panel). Both groups, however, increased their licking responses relative to water as the concentration of sucrose was increased, F(5, 50) = 19.9, p < 0.0001. This differential responding to sucrose based on concentration provides strong evidence against a lesion-induced ageusia hypothesis. With one exception, rats with PBN lesions appeared to have no gross motor impairments in licking (Fig. 5). One animal did lick at a lower rate during the first 2 days of the water-depfivaton phase, but thereafter appeared to be normal. Even with these data included in the analysis, there were no significant differences in the number of licks to water between the PBNX and control rats in either the water-deprived testing phase, F(l, 1l) = 0.80, p = 0.391, or the nondeprived testing phase, F(1, 10) = 0.36, p = 0.561. DISCUSSION These findings, particularly the sucrose licking responses of nondeprived PBNX rats, demonstrate that rats with lesions centered in the parabrachial gustatory area are clearly not ageusic. Rats with PBN lesions demonstrated concentration-dependent licking of sucrose and NaC1 in both the water-deprived and nondeprived states. On the other hand, under certain physiological conditions these lesions do appear to blunt responsivity to sapid stimuli. In the water-deprived state, rats with PBN lesions licked sucrose in a manner similar to that of controls, but the water deprivation might have produced a ceiling effect that obscured potential differences between water- and sucrose-elicited licking. When tested with sucrose in the nondeprived state, the PBNX rats had lower lick difference scores than the control animals. Also in contrast to controls, deprived PBNX rats failed to reduce their intake of NaC1, relative to water, until the concentration reached 1.0 M. Conversely, in the nondeprived state, the PBNX and control rats did not differ in their relative intake of NaC1 solutions; both groups showed similar concentration-dependent decreases in licking. In the nondeprived condition, however, both groups drank very little water or salt, so the apparent similarity could reflect a floor effect. Nevertheless, it is clear that rats with PBN lesions can taste. Depending on the deprivation conditions, rats with PBN lesions exhibited either no differences in their response to sapid stimuli or their responses were blunted. When response deficits occur, the underlying basis may involve impairment of motor, sensory (i.e., taste), visceral (i.e., fluid balance), or affective neural processes. With respect to these alternative explanations, lingual motor dysfunction seems an unlikely hypothesis. When in a highly motivated water-deprived state, PBNX rats did not differ from controls in either the number of taste trials initiated during sessions or the number of licks to water and sucrose. It is not possible with the present experimental design to distinguish be-
ig
r
FIG. 1. Photomicrographs of the lesions in the pontine parabrachial nuclei of tbur rats that illustrate the range of damage present in the nine experimental animals in this study (coronal sections; cresyl Lecht violet stain). The let~hand lesions are in the left column; the right lesions, on the right. The margins of the gliosis are marked with solid circles. The sections in Panel A and B are from rat 9077. This animal had the smallest lesion (0.347 and 0.306 cu. m m , left and right, respectively) that were judged to include more than half of the gustatory area bilaterally. The lesions in rat 9078 (C and D) were both well placed, but the one on the left was small (0.054 cu. m m ) and was judged to involve only a part of the gustatory area. The lesions in rat 9073 (E and F) were both large (0.648 and 0.552 cu. mm), but the one on the left was far enough lateral that it only damaged part of the gustatory area. In rat 9051 (G and H), the lesion placements were almost identical to those in rat 9073 but the volumes were smaller (0.105 and 0.205 cu. mm). For that reason, the lesion on the left barely impinged on the gustatory area, so the data from this animal were discarded. Abbreviations: BC, brachium conjunctivum; LC, locus coeruleus; MesV, mesencephalic trigeminal nucleus: MV, trigeminal motor nucleus; PV, principal trigeminal sensory nucleus. The scale in H equals 0.5 m m .
TASTE-RELATED LICKING IN RATS WITH PARABRACHIAL LESIONS
9O z
80
O
70 I.d
60
\\\'q
-...\'-.~
n~
o_ 50 rv
\ \ \ \ \ \ \ \ \\\X \ \ \ \ \\\',
~- 30
\\\', \ \ \ \ \ \
~ 20 p-
lo
\ \ \ \ \ \
\ \ \ \ \ \
I IPBNX ~CONTROL
\ \ \ \ \ \
WATER-DEPRIVED
NON-DEPRIVED
FIG. 2. Mean (_+SE) number of taste trials per session initiated by rats
with lesionsin parabrachial nucleus lesions(PBNX) and surgicalcontrols tested in both the water-deprivedand nondeprived conditions.
tween a sensory or affective basis for the deficits observed. This is because the animal's behavior is driven by the affective nature of the taste solutions. In other words, this procedure relies on the inherent hedonic characteristics of the taste solutions to generate the changes in licking behavior. Therefore, a depression in licking at a given concentration of, say sucrose, can be interpreted as merely a decrease in the perceived intensity (sensory), which, in turn, has less hedonic value (affective). On the other hand, the perceived intensity may not be influenced at all by the lesion, but the affective responsiveness to the solution may be disrupted. The possibility of distinguishing between a sensory and affective basis for such a lesion-induced impairment may be possible in future experiments. This could be accomplished by pro-
281
cedures that employ taste stimuli as signals for other reinforcing events. For example, we are currently training water-deprived rats to suppress licking to sodium chloride and to maintain licking to water. The avoidance of a brief shock to the feet motivates the rats to suppress their licking of NaC1. Once the animals learn to discriminate NaC1 from water we begin to lower the concentration of the NaC1 and, thus, can measure sensory thresholds. In this example, the water deprivation motivates the animal to lick water and the avoidance of the shock motivates the animal not to lick NaCI; the inherent hedonic characteristics of the taste solution are irrelevant. This procedure has been employed successfully to measure sucrose and NaCI detection thresholds in rats with and without peripheral nerve damage (16). If PBN lesions are interfering with strictly sensory mechanisms that are involved with the processing of taste signal intensity, then such rats should have higher sensory thresholds. Based on the available evidence, in rodents, the parabrachial nuclei serve as an obligatory synapse in the central gustatory system [see (10)]. The capacity of the caudal brain stem to support taste-guided behavior has been demonstrated using chronically decerebrate rats, but these preparations can make use of local connections from both the nucleus of the solitary tract (NST) and the PBN (5). After damage to the PBN, any remaining gustatory function must rely on a) pre-PBN circuitry, which consists of the nucleus of the solitary tract (NST) and its local connections, or b) the NST and any undamaged parabrachial gustatory neurons along with their connections. The possibility that some PBN gustatory neurons remained functionally intact after some or all of our lesions cannot be discounted. First, only one mapping study of gustatory responsiveness in the parabrachial region of the rat has been completed (11). Although more extensive than most, the stimulation procedures used in that study may not have reached all of the gustatory receptors in the oral cavity. Subsequent investigations emphasized the ditSculty of stimulating the foliate, circumvallate, and nasoincisor duct buds without techniques specifically de-
WATER-DEPRIVED; NQCI
NON-DEPRIVED; NaCI
05 ................................ 0o (O w
-10 -15
I
-20
oo 0m .J
v)
0
-25
CO
0
CONTROL (n=5)
•
PBNX (n=7)
-30 -55 -40 [
0.01
........
I
........
0.1
CONCENTRATION (U)
I
1
. . . . . . .
0.01
I
........
0.1
I
1
CONCENTRATION (M)
FIG. 3. Mean (+SE) lick difference scores (licks to water subtracted from licks to taste solution) based on the last 8 s of 10-s trials with various concentrations of sodium chloride for rats with parabrachial nucleus lesions(PBNX) and surgicalcontrols. Left panel:water-deprivedcondition. Rightpanel:nondeprivedcondition.
282
SPECTOR. GRILL ANI) N()RGREN WATER-DEPRIVED; SUCROSE r -
I
40 4 Ii
.
.
.
.
0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
NON-DEPRIVED; SUCROSE .
.
.
.
.
.
.
.
CONTROL (n=5)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
,
[) CONTROL (n=5)
f
35_4
~
-
,PBNX(o=8)
/ o .......
,PBNX(r,=7)
25
20 -5 ~_ 10
o
..........................
-5
....... I 0.01
................................................................................................. i ................
........
I
........
0.1
I
i
, , t . . . . . . ,~
1
0.01
CONCENTRATION (M)
........
I
........
O.1
I
, ,
1
CONCENTRATION (M)
FIG. 4. Mean (_+SE)lick difference scores (licks to water subtracted from licks to taste solution) based on the last 8 s of 10-s trials with various concentrations of sucrose for rats with parabrachial nucleus lesions (PBNX) and surgical controls. Left panel: water-deprived condition. Right panel: nondeprived condition.
signed for that purpose (4,17). Thus, the gustatory area in the PBN may have been underestimated. Second, recent anatomical studies have raised the possibility that some gustatory neurons from the NST project to the external medial subnucleus of the PBN, a small area at the ventrolateral tip of the brachium conjunctivum (7). The gustatory function of this subnucleus, however, has yet to be electrophysiologically confirmed in the rat. Nevertheless, the lesions in the current series of animals did not involve this area consistently. In an attempt to gauge the possibility of residual PBN gustatory function, we correlated the fit and size of the lesions with the magnitude of the behavioral deficits. Our normal histological analysis consists of measuring the volume of the lesions and estimating their degree of bilateral overlap with the gustatory area as defined by Norgren and Pfaffmann ( 11 ). These two measures are then combined by arranging the lesions by volume within categories of overlap, independent of the behavioral resuits, to provide a rough, qualitative ordering. When the lick difference scores within the PBNX group were ranked for 1.0 M NaCI and 1.0 M sucrose (the highest concentrations used), they correlated with the lesion ranks at r = 0.65 and r = 0.71, respectively. While neither correlation reached conventional levels for statistical significance, both were close (p = 0.066 and p = 0.058, respectively). At best, the relative size and placement of the lesions can account for 50% of the variance in the behavioral measures. Thus, while the issue remains ambiguous, it seems unlikely that residual function can account for all of the concentration-dependent licking behavior evident after electrolytic lesions electrophysiologically centered in the gustatory area of the parabrachial nuclei. It is important to recognize that lesions in the PBN not only compromise any processing that occurs in this structure, but also deprive forebrain of its direct source of gustatory input. Therefore, any behavioral impairments resulting from PBN lesions may involve taste-responsive areas rostral to the pons. It is also worth emphasizing that the functional characterization
of PBN lesions and their underlying basis requires the use of a variety of taste-related behavioral tasks. Each task possesses particular assumptions, limitations and attributes; each task focuses on various aspects of taste processing. The collective findings from such experiments illustrate a profile of function that ultimately helps reveal the basic organization of information processing across levels of the central gustatory system. At present, it appears that the pre-PBN gustatory circuitry is capable of influencing ingestive behavior as a function of taste. This influence, however, lacks an amplification that presumably reflects the missing contribution of more rostral gustatory circuits. Furthermore, based on results from experiments examining conditioned taste aversion (1,2,15) and depletion-induced sodium appetite (2), it has been suggested that the pre-PBN circuitry is
45 40 55 30
[~PBNX
25
[~CONTROL
20 15 10 5 0
WATER-DEPRIVED
NON-DEPRIVED
FIG. 5. Mean (+_SE)number of licks on the last 8 s of 10-s water trials by rats with lesions in parabrachial nucleus lesions (PBNX) and surgical controls tested in both the water-deprived and nondeprived conditions.
T A S T E - R E L A T E D L I C K I N G IN R A T S W I T H P A R A B R A C H I A L LESIONS
not capable of integrating taste with physiological state (15). The present experiment suggests that the relationship between water balance and taste-guided behavior is altered as a result of lesions in the PBN. The water-deprived P B N X rat's quest for hypotonic fluid clearly drives its licking behavior, but appears to overshadow the inhibitory influence that concentrated NaC1 solutions (hypertonic) normally exert on taste responsiveness. It is likely that as more behavioral experiments on the central gustatory system are conducted, this profile of function will become more well defined.
283 ACKNOWLEDGEMENTS
We would like to thank Ms. Christine Kornet, Ms. Kathleen J. Smith, and Dr. Takeshi Kasagi for their technical assistance, and Dr. Madhavi R. Prakash for help with surgery. Portions of this work were presented at the Thirteenth Annual Meeting of the Association for Chemoreception Sciences in Sarasota, FL, 1991 [see (14)]. This research was supported by PHS grants DC-00161 (A.C.S.), MH-43787 (H.J.G.), and DC-00240 (R.N.). Ralph Norgren is a recipient of a Research Scientist Award from the National Institute of Mental Health (MH 00563).
REFERENCES 1. DiLorenzo, P. M. Long-delay learning in rats with parabrachial pontine lesions. Chem. Senses 13:219-229; 1988. 2. Flynn, F. W.; Grill, H. J.; Schulkin, J.; Norgren, R. Central gustatory lesions. II. Effects on salt appetite, taste aversion learning and feeding behaviors. Behav. Neurosci. 105:944-954; 1991. 3. Flynn, F. W.; Grill, H. J.; Schwartz, G. J.; Norgren, R. Central gustatory lesions. I. Preference and taste reactivity tests. Behav. Neurosci. 105:933-943; 1991. 4. Frank, M. E. Taste-responsive neurons of the glossopharyngeal nerve of the rat. J. Neurophysiol. 65:1452-1463; 1991. 5. Grill, H. J.; Norgren, R. The taste reactivity test. II. Mimetic responses to gustatory stimuli in chronic thalamic and chronic decerebrate rats. Brain Res. 143:281-297; 1978. 6. Grill, H. J.; Spector, A. C.; Schwartz, G. S.; Kaplan, J. M.; Flynn, F. W. Evaluating taste effects on ingestive behavior. In: Toates, F.; Rowland, N., eds. Techniques in the behavioral and neural sciences, vol. 1: Feeding and drinking. Amsterdam: Elsevier; 1987:151-188. 7. Herbert, H.; Moga, M. M.; Saper, C. B. Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat. J. Comp. Neurol. 293:540-580; 1990. 8. Hill, D. L.; Almll, C. R. Parabrachial nuclei damage in infant rats produces residual deficits in gustatory preferences/aversions and sodium appetite. Dev. Psychobiol. 16:519-533; 1983. 9. Ivanova, S. F.; Bures, J. Conditioned taste aversion is disrupted by prolonged retrograde effects of intracerebral injection of tetrodotoxin in rats. Behav. Neurosci. 104:948-954; 1990.
10. Norgren, R. Taste: Central neural mechanisms. In: Darien-Smith, I., ed. Handbook of physiology: The nervous system Ill--Sensory processes. Washington, DC: American Physiological Society; 1984: 1087-1128. 11. Norgren, R.; Pfaffmann, C. The pontine taste area in the rat. Brain Res. 91:99-117; 1975. 12. Nowlis, G. H.; Braun, J. J.; Norgren, R. The central gustatory system: Ingestion and rejection functions after lesions. Paris, France: Sixth International Conference on the Physiology of Food and Fluid Intake; 1977 (Abstr.). 13. Spector, A. C.; Andrews-Labenski, J.; Letterio, F. C. A new gustometer for psychophysical taste testing in the rat. Physiol. Behav. 47:795-803; 1990. 14. Spector, A. C.; Norgren, R.; Grill, H. J. Concentration-dependent changes in appetitive responsivity to sucrose and NaCI in rats with parabrachial nucleus lesions. Chem. Senses 16:584; 1991 (Abstr.). 15. Spector, A. C.; Norgren, R.; Grill, H. J. Parabrachial gustatory lesions impair taste aversion learning in rats. Behav. Neurosci. 106:147161; 1992. 16. Spector, A. C.; Schwartz, G. J.; Grill, H. J. Chemospecific deficits in taste detection after selective gustatory deafferentation in rats. Am. J. Physiol. 258:R820-R826; 1990. 17. Travers, S. P.; Pfaffmann, C.; Norgren, R. Convergence of lingual and palatal gustatory neural activity in the nucleus of the solitary tract. Brain Res. 365:305-320; 1986.
0031-9384/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.
Printed in the USA.
Concentration-Dependent Licking of Sucrose and Sodium Chloride in Rats With Parabrachial Gustatory Lesions ALAN
C. S P E C T O R , * t I H A R V E Y
J. G R I L L t : I : A N D
RALPH
NORGREN§
*Department of Psychology, University of Florida, Gainesville, FL 32611, f'Smell and Taste Center and ~:Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104, and §Department of Behavioral Science, The Pennsylvania State University, Hershey, PA 1 7033
R e c e i v e d 19 M a r c h 1992 SPECTOR, A. C., H. J. GRILL AND R. NORGREN. Concentration-dependent lickingof sucrose and sodium chloridein rats with parabrachialgustatory lesions. PHYSIOL BEHAV 53(2) 277-283, 1993.--The medial zone of parabrachial nuclei (PBN) serves as an obligatory synapse in the central gustatory system in rodents. Lesions in the PBN impair taste aversion learning and depletion-induced sodium appetite in rats, and also alter the ingestion of sapid stimuli. Interpretation of these lesion-induced behavioral deficits requires an evaluation of whether taste function is compromised. The present study examined whether rats with PBN lesions could show normal concentration-dependent changes in licking behavior to very small volumes of NaCI and sucrose. Physiological state was also varied; taste responsivity was examined in water-deprived and nondeprived rats. In a specially designed gustometer, nine rats with electrophysiologically guided lesions in the PBN and five surgical controls were trained to lick a drinking spout to receive 10-s access to various concentrations of NaCI (0.03-1.0 M) and sucrose (0.01-1,0 M) during 30min sessions. Water-deprived control rats progressively decreased their responses compared with water as the concentration of NaCl was raised. In contrast, water-deprived PBNX rats did not decrease their licking responses to NaCl relative to water until the concentration reached 1.0 M. In the nondeprived state, control and PBNX rats decreased their responsiveness as a function of NaC1 concentration, and the two groups did not differ. The licking responses of water-deprived PBNX rats did not differ from control rats when sucrose was the stimulus. In the nondeprived condition, both groups monotonically increased their licking to sucrose as a function of concentration, but PBNX rats were significantly less responsive than controls. These findings demonstrate that rats with lesions centered in the parabrachial gustatory area are not ageusic, but under certain physiological conditions these lesions do appear to blunt responsivity to sapid stimuli. Sugar
Salt
Taste
Motivation
Central gustatory system
IN rodents, the pontine parabrachial nuclei (PBN) serve as an obligatory synapse in the central gustatory system [see (10)]. Several investigations have demonstrated that both permanent and temporary lesions of the PBN severely impair, if not abolish, a rat's ability to learn a taste aversion (1,2,9,15). Similar parabrachial lesions also eliminate the expression of sodium appetite following acute sodium depletion (2). One obvious explanation for these deficits is that PBN lesions render the animals ageusic. Several other studies, however, indicate that rats with similar PBN lesions do distinguish sapid chemicals from one another. Nowlis et al. (i2) reported that, when tested in a one-bottle test, rats with PBN lesions exhibited normal aversions to HC1 and quinine HC1, but exaggerated preference for sucrose and weak saline solutions. Similarly, using an one-bottle test, Flynn et al. (3) observed that, compared with controls, rats with PBN lesions overconsumed both normally preferred and normally avoided solutions, at least at some concentrations. When taste
Dorsal pons
stimuli were infused directly into the oral cavity and the immediate oral motor responses were measured, these same animals appeared to be less sensitive than controls to sucrose and quinine HCI, as indicated by rightward shifts in their concentration-response functions. In another study, the PBN lesions were made in 10-day-old rat pups that were then tested as adults using several 24-h intake paradigms (8). In a three-bottle test, the rats with lesions drank slightly more saccharin than water and virtually no quinine HCI. In the same situation, however, the control rats preferred water to saccharin, an u n c o m m o n observation at the stimulus concentration employed (0.15%). In the same experiment, the rats with PBN lesions drank less 2.5% NaCl in a twobottle test with water, but more quinine HC1 when it was the only fluid available. Given the differences in the experimental procedures (e.g., brief or long duration, intake or immediate responses), the results of these studies are difficult to compare. Nevertheless, they do
Requests for reprints should be addressed to Alan C. Spector.
277
278
SPECTOR. GRILl, AND NORGREN
imply that while bilateral parabrachial lesions do not render an animal ageusic, they do alter its responsiveness to sapid stimuli. As with neurologically normal animals, the extent of the behavioral changes produced by PBN lesions depends on the taste stimuli and the methodology used to assess them (6). The purpose of the present experiment was to determine whether rats with PBN lesions could demonstrate concentration-dependent changes in their appetitive licking responses to sucrose and NaC1. In addition, the rats were tested in both a water-deprived and a nondeprived state to examine the possibility that PBN lesions alter the relationship between physiological condition and taste responsivity. In order to increase our confidence that the behavior was guided by immediate orosensory factors, taste trials consisted of small volumes and the dependent variable was licking. METHOD
Subjects Fourteen naive male Sprague-Dawley albino rats (CD, Charles River, Wilmington, MA) served as subjects (246-298 g at start of the experiment). They were individually housed in stainless steel cages in a room where temperature, humidity, and lighting were controlled automatically. All rats had water and Purina rat chow (5001 ) freely available except where noted otherwise. Four days prior to the start of behavioral testing, all rats received, for 2 days, sodium-deficient diet (Teklad, #170950, sodium content: 0.005-0.015%) in addition to their normal Purina rat chow. The rats were kept on a 12/12 light/dark cycle and all manipulations were performed during the light phase.
Surgery Nine deeply anesthetized rats received electrophysiologically guided bilateral lesions in the PBN (PBNX) using procedures that have been described in detail elsewhere (15). Rats were pretreated with atropine (0.1 mg, IP) and then injected with Nembutal anesthesia (50.0 mg/kg, IP). Supplemental doses of Nembutal were delivered as necessary. The rat was secured in a stereotaxic (Kopf) headholder; nontraumatic ear bars were used. Two holes were drilled through the skull on either side of the midline and centered approximately 11.5 mm posterior to bregma. The recording electrode (tungsten-in-glass, Z = 0.51.0 Mohm at 1 kHz) was positioned (20 ° off vertical in the AP axis with tip anterior, - 11.5 mm AP from bregma, 1.8 mm ML) and lowered through the meninges and cerebellum to penetrate into the dorsal pons. The exact location of the gustatory neurons was identified by recording multiunit responses to 0.3 M NaCl applied to the anterior tongue. The sapid stimulus always was preceded and followed by a rinse of distilled water. Once the gustatory area had been identified on both sides, the recording electrode was replaced with a lesion electrode that consisted of a 130 #m diameter teflon-insulated stainless steel wire with only the crosssection at its tip bared. This electrode was then lowered into the PBN using the coordinates obtained with the recording electrode. In the vast majority of cases, the gustatory area also could be electrophysiologically identified via the lesion electrode. Two lesions were made on each side, one ventral (60 #A/20 s) and one 200-300 #m more dorsally (40 #A/20 s), in an attempt to destroy taste neurons both below and above the brachium conjunctivum (l 1). The holes in the skull were packed with sterile gelfoam and the incision closed with wound clips. Five rats served as full surgical controls (CON). These rats received several penetrations of the recording electrode on both sides. The electrode was lowered only into the cerebellum and did not penetrate the dorsal pons. All rats in this experiment
were given at least 24 days to recover from surger.~ bclbre behavioral manipulations. Two rats, one PBNX and one control developed mild respiratory, infections just prior to and during behavioral testing and were treated with oxytetracycline (0.05 cc, SC) twice a day.
Procedure In a specially designed gustometer described elsewhere (13), rats were trained to lick a drinking spout to receive 10-s access to various sapid stimuli. Food was not available during these sessions. On day 1, all rats were weighed and had their water bottles removed. There were no significant differences in body weight between the two groups (PBNX = 407 g; controls = 421 g). On day 2, the rats were placed in the gustometer and allowed to lick the drinking spout to receive water. During this 30-rain session, the control rats drank 9.56 ml and PBNX rats drank 10.0 ml on the average. The purpose of this session was to familiarize rats with the testing apparatus. On days 3-6, rats received 10-s trials during which they had access to either water or various concentrations of NaC1 (0.03, 0.1, 0.3. 1.0 M) and sucrose (0.01, 0.03, 0.06, 0.1, 0.3, 1.0 M). The 11 fluid stimuli were randomly presented in blocks of I 1 trials. In order to insure that the rat was attending to the spout, it was required to lick the drinking spout twice with an interlick interval of no greater than 500 ms to initiate a trial. The minimum intertrial interval was 6 s--the amount of time required to clean the drinking spout [see ( 13)]. The session length was 30 min and the rat could initiate as many trials as possible within this period. On days 2-5, the rats received their daily fluid allotment during the behavioral training and testing sessions. On day 6, the rats were 23.5 h water deprived at the start of the session, but water bottles were replaced on the home cages for the remainder of the experiment. On average, the rats lost 10% of their body weight over the first 2 days of the water deprivation schedule and then stabilized their body weight on days 3-6 at about 90% of their ad lib drinking value. On days 7 and 8, the rats were allowed to rehydrate. On days 9-11, the nondeprived rats were tested in the gustometer using the same paradigm and the same sapid stimuli.
Data Analysis The licks elicited during the 10-s trial were measured, but only the licks during the last 8 s of the trial were used in the analysis. This was done so that the analysis could concentrate on a rat's affective response to the stimulus and minimize the contribution of the mere motor acts involved in the initial fluid acquisition. The mean number of licks for water and for each taste stimulus during the water-deprived and nondeprived test sessions was computed for each rat. These means were then used to compute a lick difference score between a given taste stimulus and water for each rat: Lick Difference Score~Tastex~ = Licks(Taste x) - Licks~water) This score was then used in the various statistical analyses of variance (ANOVAs). The statistical rejection criterion (i.e., alpha) was set at 0.05. The data from one session in the water-deprived testing phase were discarded for four rats (day 5 for one rat and day 6 for the others) due to technical problems with the apparatus. The spout was not properly positioned on some of the trials during this session for two of these rats, and there was a pressure drop in the system during some of these trials for the other two rats. Consequently, the lick difference scores during the water-deprived testing phase for these four rats were computed based on
TASTE-RELATED LICKING IN RATS WITH PARABRACHIAL LESIONS
three sessions rather than four. One rat in the PBNX group failed to initiate at least one trial for each taste stimulus during the nondeprived testing phase. Consequently, a complete concentration-response profile could not be composed for this animal and its data were excluded from analyses involving data collected during the nondeprived testing phase (with the exception of number of trials).
Histology At the completion of the experiment, rats were deeply anesthetized (100 mg/kg Nembutal, IP) and perfused intracardially with saline and 10% buffered formalin. The brains were removed, stored in 30% sucrose-10% formalin for several days, then cut coronally on a freezing microtome (50 um sections). These sections were mounted on slides and alternate series stained with the cresyl Lecht violet and Weil procedures. The volume of the lesions was estimated by measuring the area of necrosis and gliosis on each section in the cresyl violet series (JAVA), multiplying those areas by 100 urn, and then totalling the results. Each lesion also was assessed qualitatively for its overlap with the parabrachial gustatory area defined by Norgren and Pfaffmann (11). Three categories of overlap were used--complete (>50% of the taste area), partial (<50% of the taste area), or n o n e - - a n d the preparation was judged on the basis of the least complete lesion. Both procedures were conducted without knowledge of the behavioral results. RESULTS In general, the lesions in the dorsal pons were large (average volume = 0.40 cu. mm. + 0.044). Eight out of nine rats had bilateral lesions that involved the gustatory zone of the PBN (Fig. 1). The one remaining rat had a complete lesion on one side, but on the other side the lesion damaged the lateral portion of the brachium conjunctivum, and did not encroach upon the gustatory area (Fig. 1G, H). This rat was discarded from the statistical analysis. In many cases, damage extended into the supratrigeminal area, the lateral subnuclei of the PBN, the brachium conjunctivum, and the mesencephalic trigeminal nucleus. The mean number of trials initiated by PBNX rats during the water-deprived testing phase was not significantly different from that initiated by controls, F(1, 11) = 1.70, p = 0.22 (Fig. 2). Although, strictly speaking, there was no significant difference between the number of trials initiated by PBNX and control rats during the nondeprived testing phase, F(1, 11) = 4.50, p = 0.057, the p value just missed the statistical rejection criterion. These data suggest that both PBNX and control rats were similarly motivated to drink when water deprived. It appears, however, that the overall incentive value of the taste stimuli during the nondeprived testing phase was lower for PBNX rats compared with controls. Water-deprived control rats progressively decreased their responses as the concentration of NaC1 was raised (Fig. 3, left panel). In contrast, water-deprived PBNX rats did not decrease their licking responses to NaC1 relative to water until the concentration reached 1.0 M. A two-way ANOVA indicated a significant effect of lesion, F(I, 11) = 9.06, p = 0.012, and concentration, F(3, 33) = 22.7, p < 0.0001, as well as a significant interaction, F(3, 33) = 5.12, p = 0.005. It appears that waterdeprived PBNX rats are compromised in their ability to appropriately modify their licking behavior based on the concentration of the salt stimulus. Note, however, that PBNX rats do significantly reduce their licking responses to 1.0 M NaC1. In the nondeprived state (Fig. 3, right panel), PBNX and control rats did not differ in their response to NaCI, F(1, 10) =
279
0.024, p = 0.879. Despite the relatively low frequency of licks to water, there was a significant decrease in responses as a function of concentration, F(3, 30) = 3.76, p = 0.021. This observation should be regarded with some caution, however, because a floor effect may have obscured differences between the two groups. Water-deprived PBNX and control rats did not differ in their response to sucrose, F(1, 11) = 0.548, p = 0.475 (Fig. 4, left panel). Interestingly, despite the relatively high frequency of licks to water, both groups significantly increased their licking as a function of concentration, F(5, 55) = 6.32, p = 0.0001. The lack of difference between the two groups may be attributable to a ceiling effect. In the nondeprived state, PBNX rats had significantly lower sucrose lick scores than controls, F(1, 10) = 5.33, p = 0.044 (Fig. 4, right panel). Both groups, however, increased their licking responses relative to water as the concentration of sucrose was increased, F(5, 50) = 19.9, p < 0.0001. This differential responding to sucrose based on concentration provides strong evidence against a lesion-induced ageusia hypothesis. With one exception, rats with PBN lesions appeared to have no gross motor impairments in licking (Fig. 5). One animal did lick at a lower rate during the first 2 days of the water-depfivaton phase, but thereafter appeared to be normal. Even with these data included in the analysis, there were no significant differences in the number of licks to water between the PBNX and control rats in either the water-deprived testing phase, F(l, 1l) = 0.80, p = 0.391, or the nondeprived testing phase, F(1, 10) = 0.36, p = 0.561. DISCUSSION These findings, particularly the sucrose licking responses of nondeprived PBNX rats, demonstrate that rats with lesions centered in the parabrachial gustatory area are clearly not ageusic. Rats with PBN lesions demonstrated concentration-dependent licking of sucrose and NaC1 in both the water-deprived and nondeprived states. On the other hand, under certain physiological conditions these lesions do appear to blunt responsivity to sapid stimuli. In the water-deprived state, rats with PBN lesions licked sucrose in a manner similar to that of controls, but the water deprivation might have produced a ceiling effect that obscured potential differences between water- and sucrose-elicited licking. When tested with sucrose in the nondeprived state, the PBNX rats had lower lick difference scores than the control animals. Also in contrast to controls, deprived PBNX rats failed to reduce their intake of NaC1, relative to water, until the concentration reached 1.0 M. Conversely, in the nondeprived state, the PBNX and control rats did not differ in their relative intake of NaC1 solutions; both groups showed similar concentration-dependent decreases in licking. In the nondeprived condition, however, both groups drank very little water or salt, so the apparent similarity could reflect a floor effect. Nevertheless, it is clear that rats with PBN lesions can taste. Depending on the deprivation conditions, rats with PBN lesions exhibited either no differences in their response to sapid stimuli or their responses were blunted. When response deficits occur, the underlying basis may involve impairment of motor, sensory (i.e., taste), visceral (i.e., fluid balance), or affective neural processes. With respect to these alternative explanations, lingual motor dysfunction seems an unlikely hypothesis. When in a highly motivated water-deprived state, PBNX rats did not differ from controls in either the number of taste trials initiated during sessions or the number of licks to water and sucrose. It is not possible with the present experimental design to distinguish be-
ig
r
FIG. 1. Photomicrographs of the lesions in the pontine parabrachial nuclei of tbur rats that illustrate the range of damage present in the nine experimental animals in this study (coronal sections; cresyl Lecht violet stain). The let~hand lesions are in the left column; the right lesions, on the right. The margins of the gliosis are marked with solid circles. The sections in Panel A and B are from rat 9077. This animal had the smallest lesion (0.347 and 0.306 cu. m m , left and right, respectively) that were judged to include more than half of the gustatory area bilaterally. The lesions in rat 9078 (C and D) were both well placed, but the one on the left was small (0.054 cu. m m ) and was judged to involve only a part of the gustatory area. The lesions in rat 9073 (E and F) were both large (0.648 and 0.552 cu. mm), but the one on the left was far enough lateral that it only damaged part of the gustatory area. In rat 9051 (G and H), the lesion placements were almost identical to those in rat 9073 but the volumes were smaller (0.105 and 0.205 cu. mm). For that reason, the lesion on the left barely impinged on the gustatory area, so the data from this animal were discarded. Abbreviations: BC, brachium conjunctivum; LC, locus coeruleus; MesV, mesencephalic trigeminal nucleus: MV, trigeminal motor nucleus; PV, principal trigeminal sensory nucleus. The scale in H equals 0.5 m m .
TASTE-RELATED LICKING IN RATS WITH PARABRACHIAL LESIONS
9O z
80
O
70 I.d
60
\\\'q
-...\'-.~
n~
o_ 50 rv
\ \ \ \ \ \ \ \ \\\X \ \ \ \ \\\',
~- 30
\\\', \ \ \ \ \ \
~ 20 p-
lo
\ \ \ \ \ \
\ \ \ \ \ \
I IPBNX ~CONTROL
\ \ \ \ \ \
WATER-DEPRIVED
NON-DEPRIVED
FIG. 2. Mean (_+SE) number of taste trials per session initiated by rats
with lesionsin parabrachial nucleus lesions(PBNX) and surgicalcontrols tested in both the water-deprivedand nondeprived conditions.
tween a sensory or affective basis for the deficits observed. This is because the animal's behavior is driven by the affective nature of the taste solutions. In other words, this procedure relies on the inherent hedonic characteristics of the taste solutions to generate the changes in licking behavior. Therefore, a depression in licking at a given concentration of, say sucrose, can be interpreted as merely a decrease in the perceived intensity (sensory), which, in turn, has less hedonic value (affective). On the other hand, the perceived intensity may not be influenced at all by the lesion, but the affective responsiveness to the solution may be disrupted. The possibility of distinguishing between a sensory and affective basis for such a lesion-induced impairment may be possible in future experiments. This could be accomplished by pro-
281
cedures that employ taste stimuli as signals for other reinforcing events. For example, we are currently training water-deprived rats to suppress licking to sodium chloride and to maintain licking to water. The avoidance of a brief shock to the feet motivates the rats to suppress their licking of NaC1. Once the animals learn to discriminate NaC1 from water we begin to lower the concentration of the NaC1 and, thus, can measure sensory thresholds. In this example, the water deprivation motivates the animal to lick water and the avoidance of the shock motivates the animal not to lick NaCI; the inherent hedonic characteristics of the taste solution are irrelevant. This procedure has been employed successfully to measure sucrose and NaCI detection thresholds in rats with and without peripheral nerve damage (16). If PBN lesions are interfering with strictly sensory mechanisms that are involved with the processing of taste signal intensity, then such rats should have higher sensory thresholds. Based on the available evidence, in rodents, the parabrachial nuclei serve as an obligatory synapse in the central gustatory system [see (10)]. The capacity of the caudal brain stem to support taste-guided behavior has been demonstrated using chronically decerebrate rats, but these preparations can make use of local connections from both the nucleus of the solitary tract (NST) and the PBN (5). After damage to the PBN, any remaining gustatory function must rely on a) pre-PBN circuitry, which consists of the nucleus of the solitary tract (NST) and its local connections, or b) the NST and any undamaged parabrachial gustatory neurons along with their connections. The possibility that some PBN gustatory neurons remained functionally intact after some or all of our lesions cannot be discounted. First, only one mapping study of gustatory responsiveness in the parabrachial region of the rat has been completed (11). Although more extensive than most, the stimulation procedures used in that study may not have reached all of the gustatory receptors in the oral cavity. Subsequent investigations emphasized the ditSculty of stimulating the foliate, circumvallate, and nasoincisor duct buds without techniques specifically de-
WATER-DEPRIVED; NQCI
NON-DEPRIVED; NaCI
05 ................................ 0o (O w
-10 -15
I
-20
oo 0m .J
v)
0
-25
CO
0
CONTROL (n=5)
•
PBNX (n=7)
-30 -55 -40 [
0.01
........
I
........
0.1
CONCENTRATION (U)
I
1
. . . . . . .
0.01
I
........
0.1
I
1
CONCENTRATION (M)
FIG. 3. Mean (+SE) lick difference scores (licks to water subtracted from licks to taste solution) based on the last 8 s of 10-s trials with various concentrations of sodium chloride for rats with parabrachial nucleus lesions(PBNX) and surgicalcontrols. Left panel:water-deprivedcondition. Rightpanel:nondeprivedcondition.
282
SPECTOR. GRILL ANI) N()RGREN WATER-DEPRIVED; SUCROSE r -
I
40 4 Ii
.
.
.
.
0
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
NON-DEPRIVED; SUCROSE .
.
.
.
.
.
.
.
CONTROL (n=5)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
,
[) CONTROL (n=5)
f
35_4
~
-
,PBNX(o=8)
/ o .......
,PBNX(r,=7)
25
20 -5 ~_ 10
o
..........................
-5
....... I 0.01
................................................................................................. i ................
........
I
........
0.1
I
i
, , t . . . . . . ,~
1
0.01
CONCENTRATION (M)
........
I
........
O.1
I
, ,
1
CONCENTRATION (M)
FIG. 4. Mean (_+SE)lick difference scores (licks to water subtracted from licks to taste solution) based on the last 8 s of 10-s trials with various concentrations of sucrose for rats with parabrachial nucleus lesions (PBNX) and surgical controls. Left panel: water-deprived condition. Right panel: nondeprived condition.
signed for that purpose (4,17). Thus, the gustatory area in the PBN may have been underestimated. Second, recent anatomical studies have raised the possibility that some gustatory neurons from the NST project to the external medial subnucleus of the PBN, a small area at the ventrolateral tip of the brachium conjunctivum (7). The gustatory function of this subnucleus, however, has yet to be electrophysiologically confirmed in the rat. Nevertheless, the lesions in the current series of animals did not involve this area consistently. In an attempt to gauge the possibility of residual PBN gustatory function, we correlated the fit and size of the lesions with the magnitude of the behavioral deficits. Our normal histological analysis consists of measuring the volume of the lesions and estimating their degree of bilateral overlap with the gustatory area as defined by Norgren and Pfaffmann ( 11 ). These two measures are then combined by arranging the lesions by volume within categories of overlap, independent of the behavioral resuits, to provide a rough, qualitative ordering. When the lick difference scores within the PBNX group were ranked for 1.0 M NaCI and 1.0 M sucrose (the highest concentrations used), they correlated with the lesion ranks at r = 0.65 and r = 0.71, respectively. While neither correlation reached conventional levels for statistical significance, both were close (p = 0.066 and p = 0.058, respectively). At best, the relative size and placement of the lesions can account for 50% of the variance in the behavioral measures. Thus, while the issue remains ambiguous, it seems unlikely that residual function can account for all of the concentration-dependent licking behavior evident after electrolytic lesions electrophysiologically centered in the gustatory area of the parabrachial nuclei. It is important to recognize that lesions in the PBN not only compromise any processing that occurs in this structure, but also deprive forebrain of its direct source of gustatory input. Therefore, any behavioral impairments resulting from PBN lesions may involve taste-responsive areas rostral to the pons. It is also worth emphasizing that the functional characterization
of PBN lesions and their underlying basis requires the use of a variety of taste-related behavioral tasks. Each task possesses particular assumptions, limitations and attributes; each task focuses on various aspects of taste processing. The collective findings from such experiments illustrate a profile of function that ultimately helps reveal the basic organization of information processing across levels of the central gustatory system. At present, it appears that the pre-PBN gustatory circuitry is capable of influencing ingestive behavior as a function of taste. This influence, however, lacks an amplification that presumably reflects the missing contribution of more rostral gustatory circuits. Furthermore, based on results from experiments examining conditioned taste aversion (1,2,15) and depletion-induced sodium appetite (2), it has been suggested that the pre-PBN circuitry is
45 40 55 30
[~PBNX
25
[~CONTROL
20 15 10 5 0
WATER-DEPRIVED
NON-DEPRIVED
FIG. 5. Mean (+_SE)number of licks on the last 8 s of 10-s water trials by rats with lesions in parabrachial nucleus lesions (PBNX) and surgical controls tested in both the water-deprived and nondeprived conditions.
T A S T E - R E L A T E D L I C K I N G IN R A T S W I T H P A R A B R A C H I A L LESIONS
not capable of integrating taste with physiological state (15). The present experiment suggests that the relationship between water balance and taste-guided behavior is altered as a result of lesions in the PBN. The water-deprived P B N X rat's quest for hypotonic fluid clearly drives its licking behavior, but appears to overshadow the inhibitory influence that concentrated NaC1 solutions (hypertonic) normally exert on taste responsiveness. It is likely that as more behavioral experiments on the central gustatory system are conducted, this profile of function will become more well defined.
283 ACKNOWLEDGEMENTS
We would like to thank Ms. Christine Kornet, Ms. Kathleen J. Smith, and Dr. Takeshi Kasagi for their technical assistance, and Dr. Madhavi R. Prakash for help with surgery. Portions of this work were presented at the Thirteenth Annual Meeting of the Association for Chemoreception Sciences in Sarasota, FL, 1991 [see (14)]. This research was supported by PHS grants DC-00161 (A.C.S.), MH-43787 (H.J.G.), and DC-00240 (R.N.). Ralph Norgren is a recipient of a Research Scientist Award from the National Institute of Mental Health (MH 00563).
REFERENCES 1. DiLorenzo, P. M. Long-delay learning in rats with parabrachial pontine lesions. Chem. Senses 13:219-229; 1988. 2. Flynn, F. W.; Grill, H. J.; Schulkin, J.; Norgren, R. Central gustatory lesions. II. Effects on salt appetite, taste aversion learning and feeding behaviors. Behav. Neurosci. 105:944-954; 1991. 3. Flynn, F. W.; Grill, H. J.; Schwartz, G. J.; Norgren, R. Central gustatory lesions. I. Preference and taste reactivity tests. Behav. Neurosci. 105:933-943; 1991. 4. Frank, M. E. Taste-responsive neurons of the glossopharyngeal nerve of the rat. J. Neurophysiol. 65:1452-1463; 1991. 5. Grill, H. J.; Norgren, R. The taste reactivity test. II. Mimetic responses to gustatory stimuli in chronic thalamic and chronic decerebrate rats. Brain Res. 143:281-297; 1978. 6. Grill, H. J.; Spector, A. C.; Schwartz, G. S.; Kaplan, J. M.; Flynn, F. W. Evaluating taste effects on ingestive behavior. In: Toates, F.; Rowland, N., eds. Techniques in the behavioral and neural sciences, vol. 1: Feeding and drinking. Amsterdam: Elsevier; 1987:151-188. 7. Herbert, H.; Moga, M. M.; Saper, C. B. Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat. J. Comp. Neurol. 293:540-580; 1990. 8. Hill, D. L.; Almll, C. R. Parabrachial nuclei damage in infant rats produces residual deficits in gustatory preferences/aversions and sodium appetite. Dev. Psychobiol. 16:519-533; 1983. 9. Ivanova, S. F.; Bures, J. Conditioned taste aversion is disrupted by prolonged retrograde effects of intracerebral injection of tetrodotoxin in rats. Behav. Neurosci. 104:948-954; 1990.
10. Norgren, R. Taste: Central neural mechanisms. In: Darien-Smith, I., ed. Handbook of physiology: The nervous system Ill--Sensory processes. Washington, DC: American Physiological Society; 1984: 1087-1128. 11. Norgren, R.; Pfaffmann, C. The pontine taste area in the rat. Brain Res. 91:99-117; 1975. 12. Nowlis, G. H.; Braun, J. J.; Norgren, R. The central gustatory system: Ingestion and rejection functions after lesions. Paris, France: Sixth International Conference on the Physiology of Food and Fluid Intake; 1977 (Abstr.). 13. Spector, A. C.; Andrews-Labenski, J.; Letterio, F. C. A new gustometer for psychophysical taste testing in the rat. Physiol. Behav. 47:795-803; 1990. 14. Spector, A. C.; Norgren, R.; Grill, H. J. Concentration-dependent changes in appetitive responsivity to sucrose and NaCI in rats with parabrachial nucleus lesions. Chem. Senses 16:584; 1991 (Abstr.). 15. Spector, A. C.; Norgren, R.; Grill, H. J. Parabrachial gustatory lesions impair taste aversion learning in rats. Behav. Neurosci. 106:147161; 1992. 16. Spector, A. C.; Schwartz, G. J.; Grill, H. J. Chemospecific deficits in taste detection after selective gustatory deafferentation in rats. Am. J. Physiol. 258:R820-R826; 1990. 17. Travers, S. P.; Pfaffmann, C.; Norgren, R. Convergence of lingual and palatal gustatory neural activity in the nucleus of the solitary tract. Brain Res. 365:305-320; 1986.