- Email: [email protected]
Taste responses in the nucleus tractus solitarius of the chronic decerebrate rat
Brain Research. 443 (1988) 137-148 Elsevier
137
BRE 13343
Taste responses in the nucleus tractus solitarius of the chronic decerebrate rat Gregory P. Mark i, Thomas R. Scott 2, Fat-Chun Tony Chang 3 and Harvey J. Grill 4 tDepartmem of Psychology, Princeton University, Princeton, NJ 085 44 ( U.S.A. ), 2Department of Psychology and Institute.for Neuroscience, University of Delaware, Newark, DE 19716 (U.S.A.), "~USAMRICD,APG, Aberdeen, MD 21010 (U.S.A.)and 4Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104 ( U.S. A. ) (Accepted 25 August 1987)
Key words: Decerebrate: Chronic; Rat: Nucleus tractus solitarii; Taste: Electrophysiology
The ingestive behavior of decerebrate rats has been studied for some time, yet little is known of its neural substrates. While taste fibers in rats proceed from hindbrain to thalamus and ventral forebrain, these regions return centrifugal fibers to the hindbrain by which lower-order taste activity may be influenced. We examined the functional characteristics of taste neurons in the nucleus tractus solitarii (NTS) of chronic decerebrate rats in which this reciprocal communication was disrupted and compared them with those of intact controls. Nine Wistar rats were decerebrated at the supracollicular level. After a minimum of one week recovery, they were immobilized with Flaxedil, anesthetized locally and prepared for recording. The responses of 50 taste cells were isolated bilaterally from the NTS of these animals, while the activity of 50 additional neurons was recorded from 12 intact rats under the same conditions. Taste stimuli included 7 Na-Li salts, 3 sugars, HCI and citric acids, quinine HCI and NaSaccharin. Mean spontaneous activity in decerebrates was 6.5 spikes/s, 36.0% lower than the level in intact animals. Mean evoked activity war, reduced by 32.6%. Analyses of the effects of stimulus quality, intensity and time course of the responses all indicated that the decrease in activity was attributable to the inability of taste cells in decerebrate rats to respond to demands for high discharge rates. This deficit could be responsible for the failure of these animals to develop conditioned taste aversions. Neurons from decerebrate preparations did, however, retain the broad sensitivity across stimuli that characterized taste cells in intact preparations. It was also typical that most neuron response profiles from decerebrates could be grouped into 3 loose clusters with peak sensitivities to acid-salt, salt or sugar. An analysis of similarities among stimulus activity profiles indicated that Na-Li salts, sugars and an acid-quinine complex represented 3 groups of stimulus quality: in intact animals, the primary distinction was between sweet and non-sweet stimuli. Moreover, the response to sodium saccharin lost its bitter component in decerebrates. These findings were in general agreement with those derived from ac;'e decerebm~e rats.
INTRODUCTION T h e h i n d b r a i n of the rat is i n v o l v e d in rather sophisticated ingestive functions, i n c l u d i n g those associated with taste a c c e p t a n c e or r e j e c t i o n , h o m e o s t a t -
a c o n d i t i o n e d taste aversion 3 or by the i m p o s i t i o n of satiety factors 7-~. Certain b e h a v i o r a l capacities, h o w e v e r , are disrupted by d e c e r e b r a t i o n . C o m p e n s a t o r y salt cons u m p t i o n , which a c c o m p a n i e s s o d i u m d e p r i v a t i o n in
ic processes and c o n d i t i o n i n g . D e c e r e b r a t e rats show
intact animals, is iost14: n o r d o d e c e r e b r a t e rats have
n o r m a l orofacial reactions to a variety of taste qualities 12. C o m p e n s a t o r y f e e d i n g r e a c t i o n s to glucoprivation 5 and c h o l e c y s t o k i n i n a d m i n i s t r a t i o n I° r e m a i n
the capacity to d e v e l o p or retain c o n d i t i o n e d taste aversions 13. It is a s s u m e d that these deficits are a
u n a l t e r e d with only h i n d b r a i n i n v o l v e m e n t . Moreover, electrophysiological studies in the nucleus tractus solitarii (NTS) of intact rats indicate that tastee v o k e d activity can be i n f l u e n c e d by d e v e l o p m e n t of
c o n s e q u e n c e of i n t e r r u p t i n g the reciprocal c o m m u n i cation b e t w e e n g u s t a t o r y - v i s c e r a l areas o f the hindbrain and forebrain regions associated with rei n f o r c e m e n t , notably the thalamus, h y p o t h a l a m u s and amygdala 24. T h u s d e c e r e b r a t i o n is likely to re-
Correspondence: T.R. Scott, Department of Psychology and Institute for Neuroscience, University of Delaware, Newark, DE 19716, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
138 duce some of the influences on hindbrain taste cells and so possibly modify their responses to gustatory stimuli. With a considerable literature on taste-related behavior in the decerebrate rat now available, it becomes important to determine the neural characteristics of the taste system in this preparation. MATERIALS AND METHODS
Subjects Twenty-one albino Wistar rats were used in this study. They were of both sexes and weighed 300-450 g. Of these, 12 were control animals and 9 were subjected to decerebration.
Surgery The surgical procedure for decerebration was modelled on that developed by Grill and Norgren 11 It proceeded under general anesthesia in two steps: a hemitransection followed in a week by completion of the cut. The entire parietal and approximately half of the temporal surface of the skull was exposed bilaterally. A thin transverse groove was made across the skull at a level corresponding to 40% of the distance from bregma to lambda (3.6-3.9 mm anterior to the interaural line). It was deepened over one hemisphere until only a thin layer of periosteum remained. This and the underlying dura were section~zd with a scalpel. An L-shaped spatula, ground to 0.5 mm thickness and left blunted, was inserted into the brain at the lateral margin of the groove and depressed to the cranial floor. Care was taken to compress rather than rupture the vasculature. The spatula was moved 2 mm medially with each successive stroke until the midline was reached. The steps were then retraced laterally to ensure a complete transection. Before exiting the brain, the spatula was angled laterally such that its heel sectioned all connections in the lateral convexity of the temporal lobe. The skin was sutured and the animal was permitted a minimum of one week to recover. The procedure was then repeated in the opposite hemisphere, following which the spatula was angled across the midline to complete the decerebration. This transection was designed to pass along the rostral border of the colliculi.
Care and maintenance Decerebrated rats were fully aphagic and adipsic.
Consequently each animal was fed by gavage 3-4 times per day. Each feeding consisted of 7-8 ml of equal parts sweetened condensed milk and water for a total daily volume of 25-30 ml. Body weight was monitored daily, with appropriate adjustments in volume as needed. Animals were housed in a climate-controlled chamber to minimize temperature fluctuations. Rectal temperature was recorded every 6 h Hyperthermia was treated by placing the rat on a cool surface, or, in more extreme cases, by spraying a mist of water on its fur. Hypothermia, usually a less serious concern, was controlled with a heat lamp or heating pad. Long-acting antibiotics were administered for precautionary and combative purposes following surgery, but their use was discontinued at least two days before the recording session.
Recording Decerebrate rats were permitted a minimum of one week to recover from the acute effects of surgery. Each of the 21 animals, decerebrate and control, was anesthetized to a surgical level using sodium pentobarbital. A tracheotomy was performed and the initial section of the esophagus was ligated to prevent ingestion of taste solutions. A 2 x 5 mm section of the occipital bone was removed to offer bilateral access to the NTS. Incisions were closed with wound clips and all wounds and pressure points were liberally treated with Lidocaine. The data presented here are eventually to be compared with those from decerabrates in which a conditioned taste aversion paradigm will be conducted. Since conditioning will involve conscious rats, considerations of state-dependent learning require that recordings take place under the same conditions. Consequently, the decerebrate rats in the present study (whose responses will serve as controls for those from conditioned decerebrates) and, in turn, the intact controls could not be administered a general anesthetic during recording. Thus, when surgery was complete, each animal was immobilized with an i.p. injection of Flaxedil (gallamine triethiodide, 60 mg/kg), respired using a small animal respirator and secured in a stereotaxic instrument with blunt, Lidocaine-treated earbars. The necessity of using an immobilized preparation required that extensive precautions be taken to ensure the relative comfort and physiological integrity of the rat. To this end, vital
139 signs were monitored and distress signals (pupillary dilation, blood pressure or heart rate changes) were attended to continuously throughout a recording session. Temperature was taken rectally and maintained at 36-38 °C. Heart rate, which provided the most sensitive index of physiological condition, was recorded through s.c. electrocardiographic electrodes and maintained at an interbeat interval of ca. 180 ms by adjusting respiration, level of paralysis and local anesthesia. Expired air was continuously monitored for CO2 content (Instrumentation Laboratory, IL 200 CO2 Monitor) which was maintained at 3.0-3.8%. In control animals the electrocorticogram was continuously monitored. The preponderance of the record showed desynchrony, but slow waves occasionally appeared and could be blocked by tail pinch. While decerebration precluded tne value of monitoring the ECoG in the experimental group, it also decreased the likelihood that these animals would be distressed during recording. Decerebration is, in fact, routinely performed in place of general anesthesia in some laboratories. With the stereotaxic bite bar positioned 5.5 mm below the interaural line, the NTS was typically located 11.3 mm posterior to bregma, 1.7 mm lateral to the midline and 7.5 mm below the surface of the cerebellum. One hundred single neurons, 50 in each group, were isolated with micropipettes (Z = 5-10 Mf~). Signals were amplified, filtered, displayed and stored using conventional techniques and analyzed off-line.
Stimuli and stimulus delivery The responses of each neuron to 12 chemicals were recorded. These included prototypes of the putative 4 basic tastes (sucrose, NaC!, HC!, quinine) plus 6 other saccharides, salts and acids and two intensities of sodium saccharin. Concentrations were those that elicited a half-maximum response from neural samples in the CT or NTS. In decerebrate animals 3 additional concentrations of NaC! were also applied. Stimuli and concentrations are listed in Table I. Stimulus solutions were delivered through a length of slender tubing, closed at its end and extensively perforated along its final 2 cm. The perforated end was slipped into the mouth so that the stimulus spray regularly cor~tacted nearly the entire taste-receptive surface. Stimulus onset was marked by the moment
TABLE I
Stimuli, abbreviations and molar concentrations 1 CA 2F 3G 4H 5L 6 NB 7 NC 8 NS 9Q 10 S 11 SA1 12 SA2 13 NCI* 14 NC2* 15 NC3*
0.01 M 0.5 M 0.5 M 0.01 M 0.1 M 0.1 M 0.1 M 0.1 M 0.005 M 0.5 M 0.0025 M 0.25 M 0.01 M 0.03 M 0.3 M
citric acid fructose glucose HCI LiC! NaBr NaCl Na2SO 4 quinine HC! sucrose NaSaccharin NaSaccharin NaCl NaCl NaCI
* Used only in decerebrates.
of contact with oral tissue 4. Five ml of solution were sprayed throughout the oral cavity in 3 s. The stimulus was then cleared from the tubing by air pressure and within 10 s a 25-ml distilled water rinse was initiated. A minimum of 90 s was allowed to elapse between stimulus applications to avoid the effects of adaptation.
A n alyses Primary analyses were performed by a PDP 11/03 computer. These included the summation of action potentials for 3 s prior to (spontaneous) and 5 s following (evoked) stimulus onset in 100-ms time bins and the generation of post-stimulus time histograms. Derived analyses were conducted on 3 computers: a DEC-10, a Burroughs 7700 and an IBM 3081D. These included the generation of interstimulus and interneuronal correlation matrices, multidimensional spaces, hierarchical cluster analyses and intensity-response functions.
Histology In 7 rats (4 intact, 3 decerebrate) electrolytic lesions were made at the final recording sites (20 I~A for 20 s, electrode negative). Animals were given a lethal injection of barbiturate and perfused with 10% formalin. Brains were removed, blocked and frozen for sectioning. Fifty-Ftm transverse sections, stained with Cresyi violet, were made from the mid-pontine level caudally until lesion sites were located. In addition, sagittal sections were made rostral to the mid-
140
.,...-:-. •
..-
: .... . ...,
~.::....,.~:..
;,a.
" ".~g
:~3:,
.
.
.
.
;"
"
Fig. 1. A representative sagittal section t h r o u g h the brain o f a d e c e r e b r a t e d rat.
pontine level in all decerebrated rats to confirm the full transection (Fig. 1). RESULTS
Basic responses Spontaneous activity and response criterion The mean spontaneous activity of taste cells in the NTS of control rats was 10.2 + 6.4 spikes/s. The mean rate in decerebrates was 6.5 + 5.6 spikes/s, a non-significant reduction of 36%. We adopted a response criterion of a change in neural activity of 1.28 S.D. from the mean spontaneous activity for that cell, sustained for 3 s.
Evoked activity Net evoked responses were calculated by counting the total spikes in 5 s, dividing by 5 to arrive at a gross rate per s, then subtracting the mean spontaneous rate for that neuron.
Breadth of responsiveness.
The characteristic broad sensitivity of taste cells was apparent in recordings from intact animals and was preserved in decerebrates. Of the 600 stimulus applications (50 cells, 12 stimuli), 504 (84.0%) fulfilled our criterion for excitation, 10 (1.7%) caused inhibition and 86 (14.3%) elicited no reaction from taste cells in control ani-
reals. The corresponding figures in decerebrates were 538 (89.7%) excitation, 5 (0.8%) inhibition and 57 (9.5%) no response. The breadth-of-tuning metric 2~was applied to both sets of data. In intact rats the mean coefficient was 0.86 while in decerebrates it was 0.75 (t = 1.60; non-significant). Both values indicate a wide range of responsiveness across the 4 prototypicai taste stimuli used in this analysis. Evoked response rates. For all stimulus-neuron interactions the mean response rate was 32.6% lower in decerebrates than in intact rats. But while reduced responsiveness was a rather consistent effect of decerebration, it did not occur equally across stimuli. There was a clear tendency for the cells of decerebrate rats to lag farther behind those of controls as higher discharge rates were demanded. In Fig. 2, stimuli are listed in ascending order of effectiveness for decerebrates and the mean response in each group is plotted. Where evoked responses are weak (sweet and bitter chemicals, n = 5), all differences are less than 20% and the overall reduction in response in decerebrates is non-significant (t = 0.69). Where they are robust (salty and sour stimuli, n = 7), the shortfalls are significant (t = 3.75; P < 0.01) and range from 34% to 41%. A single neuron analysis reinforces the notion that cells in decerebrate rats have difficulty meeting high response demands. In Fig. 3, neurons from decerebrate and from intact rats are ordered according to
141 70
w..
60 ~-
•
r~ ',,zk
t"
50
,v ffl
LU
i
40
i
i
O. Z
i
3O p
p
d/'~ .
LU
:E
2o
10
STIMULUS
Fig. 2. Mean net (evoked minus spontaneous) responses to each stimulus in intact (dashed line) and decerebrate (solid line) rats. Abbreviations are listed in Table I.
their responsiveness to each of the 4 prototypical taste stimuli. Individual differences that significantly exceed the mean discrepancy between each pair of curves (P < 0.05) are indicated with a filled circle above the abscissa. Almost without exception, the differences appear between cells in the most responsive one-third (17/50) of the profile. O f the 68 comparisons (4 x 17) between responses of these active neurons, 54 (79.4%) are significantly different. Of the remaining 132 comparisons (4 x 33) only two differences (1.5%) reach statistical significance. Thus there is not simply a suppression of activity that results in a linear reduction in evoked responses; rather, there is a gradual loss of capacity as the response requirements increase. In general, the failure to keep pace becomes significant as the demand exceeds 20 spikes/s to quinine, 30 spikes/s to sucrose, or 60 spikes/s to NaCi or HCI. A temporal analysis indicates that the attenuation of the phasic burst contributes disproportionately to the lower activity in decerebrates. During the 0.2-0.6 s a of evoked activity (defined, arbitrarily, as the phasic portion of the response), responses from decerebrate rats to all 4 stimuli are 42.2% lower than those of controls. During the succeeding 4.4 s (tonic)
the difference is only 31.1% (Fig. 4). Extending this analysis to include all stimuli, the mean difference in the phasic response is 42.8% compared to a tonic difference of 35.4%. This is, of course, much more striking in terms of absolute loss of spikes. Comparing responses across 50 cells to the four prototypicai stimuli over a 5-s period (a total of 1000 s of analysis), evoked activity in decerebrate rats falls 10,670 spikes short of that in controls. Fully 25% of that shortfall occurs during the 0.5 s (10%) of the response designated as phasic. Moreover, the phasic differences were quite reliable across the protypical stimuli: for sucrose, NaCI, HC! and quinine the phasic responses of decerebrates were lower by 42%, 39%, 46% and 41%, respectively. By contrast, comparisons of activity during the tonic period were less predictable, ranging from a 40% loss (to NaCI) to a 24% gain (to quinine) in decerebrate rats. Thus, even the similar total responses elicited from the two neural samples by quinine or by sucrose (see Fig. 2) result from a compensation during the tonic response for the consistent deficit in phasic activity. The 3 preceding analyses - - mean activity, responses of individual neurons, time course m all relate to the effect of decerebration on coding different stimulus qualities, with concentration held constant. To analyze the influence of decerebration on intensity coding, 4 concentrations of NaCI were used in decerebrate rats. The responses evoked by this intensity series were compared with those reported by Ganchrow and Erickson 6 from NTS of intact rats. At a concentration of 10 raM, neurons from both preparations responded at a mean rate of 33 spikes/s (Fig. 5b). As intensity increased, however, activity from decerebrates fell progressively behind, rising with a power function exponent of only 0.15 vs 0.33 in intact rats. Within one log unit of concentration (from 10 mM to 100 raM) the responses of decerebrates fell from parity to a deficit of 29% c relative to those of control animals. Thus four comparisons were made between the re-
a For sucrose, which typically has a slower onset, the (I.6-1.0 s of the evoked response was treated as the phasic portion. b The spike rates reported in this section are gross discharges (with no subtraction of spontaneous rate) during the period 0.4-1.3 s following stimulas onset, in conformity with the method of Ganchrow and Erickson~. Thus they are son'ex~'hat higher than would be expected for net spikes per s over 3 s as is used elsewhere in this manuscript. ¢ This is precisely the same percent difference to the standard 0.1 M NaCI as existed between dc,.ci'c,~rates and the intact rats whose responses were recorded in the present study.
60
m
0
0
10
10
!
Neurona
20
Neurons
Sucrose
20
**
30
3~
40
4,0
eoeoooeeeoeo
....
****,,.
50
eo
I I I
50
I ~ ~/11!// I I I
es e0
""
m ~D
=e
g,)
r.n e~
,Ad
~rJ
q)
40
60
80
100
--20
0
20
40
60
80
100
120
140
160
180
200
0
0
10
10 Neurons
20 Neurons
QHC1
~0
HCI
j
30
30
Fig. 3. Net responses to the 4 prototypical stimuli from each of 50 neurons in intact (dashed line) and decerebrate (solid line) rats. Filled circles above the abscissa indicate differences that exceeded the mean discrepancy between the curves by a significant amount (P < 0.05).
20
4O
oo
100
120
14.0
160
180
20
40
6O
80
lO0'
120
,~.
o~,.~
oe)
~m
m
140
160
180
NaC1
40
50
/
50
/
Il/l 1 /"///I oeoooooooeeo
40
eeooooeoooooooooo
/
-/
/ I I I I I
/ I
iX)
6'
0
6'
;
:
:
I!i
;
/
I
:
/
:
/
|
10
I
I
:
:
:
:
:
:
\
\
\
\
\/\\
Post--stimulus
:
I
:
Time
(xlO0
30
Sucrose
20
msee)
40
: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :
X\i/\\/... "~.
50
50
W
~d .M
Q)
0 0 M
q~
.,
4
0
8
0
20
Post--stimulus
10
Time
(xlO0
30
msee)
40
: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :
0
:
\I
Ix
0
:
\
10
u~ 2
;
\
12
2
0
,
/~
I% \
NaC1
0
o
i
II
!Ix
I~
I I I I \.
"~,'
i\
I'k \
~
i ~./
..,-...,. x\ "
20
Post--stimulus
10
IO 20 Post--stimulus
\/\,,2
.
Time
Time
/'----',
msec)
40
40 mseo)
\"'-•.
(xlO0
30
30 (xlO0
"\_-,"\."\
QHCI
/--',
HC1
Fig. 4. Post-stimulus time histograms showing the mean net responses to each of the prototypical stimuli in 100-ms time bins for intact (dashed lines) and decerebrate (solid lines) rats. The reduction in responsiveness in decerebrates occurred disproportionately during the phasic period of the response.
O O
CD
cJ 4 ¸
0p,,~
0 0
o
10,
12
50
5o
4~
144 11.18.5
120 "
tl.Z69 ,a
100
t). t53
11.4:1/
O LU r~
it-
80
CO LU n CO
t1.%21
,¢
SO
tl.
tl .(qSd
I
f
iI. ¢~/2
Z m
IAJ~i
~g t ~. fl,1 t I
20
iI"
-3".o
-~'.o LOG
MOLAR
-~'.o
o%
CONCENTRATION
B
....
Fig. 5. Mean responses across a NaCI co~ic~ntration series in intact (dashed lines) and decerebrate (solid lines) rats. Data from intact rats from Ganchrow and Erickson~. ,4. .4
,
r
sponses elicited from decerebrate rats and from two intact groups: (1) the mean effectiveness of various qualities (Fig. 2) and (2) different intensities of a single quality (Fig. 5), (3) the effects of 4 individual stimuli on single neurons (Fig. 3) and (4) the time courses of the mean responses to the same 4 chemicals (Fig. 4). Each comparison promotes a common conclusion: taste neurons of decerebrate rats are unable to respond normally to the peak demands placed upon them.
Derived analyses Categorization of neuronal profiles Each neuron generated a unique response profile across the array of 12 stimulus qualities used in this study. These profiles may be compared by calculating the Pearson product-moment correlation coefficient between each pair. The total of all such comparisons within a 50-neuron sample may be contained in a correlation matrix of 1225 entries (50 x 49)/2. This matrix serves as the basis for a hierarchical cluster analysis25 whereby related groups of prt;files may be identified. The number and size of clusters, and their degree of coherence have all been used to address the question of whether the taste system is composed of a discrete number of groups, or 'neuron types' within each of which the constituent neurons are functionally similar. The results of a cluster analysis may be expressed as a dendrogram, as shown in Fig. 6. Neurons, num-
t
* .4
~..
r-.
7~ ' I
'~
F~,~ ' -
,~'-:. ", '
T'~'I
,I'
,z.-,
Fig. 6. Dendrogn:ms ru,ating neurons to one another according to the correlations between their response profiles. A: analysis based on the responses of intact rats. B: decerebrate rats. In both groups, the basic clusters, and the numbers of cells assigned to them, are similar.
bered in the order in which they were isolated, are arranged horizontally. The correlation between the activity profiles of any two cells is expressed by the height of the horizontal line that connects them. Where groups are connected, the line is at a height which corresponds to the mean correlation between the constituents of the groups being connected. Thus the lower the horizontal line connecting any cell or group of cells, the more similar are their activity profiles across this stimulus array. The longer the vertical line leading to them, the more isolated the cells are from their neighbors. In Fig. 6 are shown dendrograms resulting from cluster analyses based on the responses of intact rats (top) and decerebrates (bottom). Although the absolute cueftlcients are somewhat lower in the analysis based on decerebrates (probably a consequence of reduced response rates, which normally lead to lower correlations), the forms of the two dendrograms are remarkably similar. In both, the primary distinction is between a large clus-
145
ter of neurons to the left and a smaller one to the right° This represents a dichotomy between cells that do not show a sweet-sensitive profile (left) vs those that do (r~g,ht). In control rats these groups number 37 and 13 respectively; in decerebrates, 39 and 11. Within the non-sweet sensitive cluster are two major groups. In both dendrograms ~:hese consist of neurons with combined salt and acid sensitivity to the left and more specific salt sensitivity to the right. In control tats these groups number 17 and 20, respecthely; in decerebrates, 23 and 16. Further subdivisions in each dendrogram continue to reveal the striking similarity between them. It is clear that the process of decerebration does not much alter the relative response profiles of taste neurons in the NTS.
1
20--
-2 0
-10
I
I
O
,
10
I
,
20
I
,
I
A
-20
• .25 NaSac Na2SO4
10
• Quinine
10
• .007~ NaSac ~Sucrose
NaOree,1 NaCI 2 o
LICI
• Fructose
o2
IGlucose Citric Acid •e HC -10
1o
2O
-20
I
I
i
-20
10
0
I
I 20
10
1
Relative taste quality Just as the profile of activity that each neuron generates across the stimulus array may be used to characterize its function, so the profile that each stimulus elicits across the neural sample may represent its taste quality. The matrix of correlaticn~ between each pair of stimulus profiles (n = (12 x 11)/2 = 66) permits an assessment of relative taste similarities within each group of rats. Multidimensional scaling routines can be used to generate spatial representations of these relative similarities. The results of these algorithms are multidimensional stimulus spaces of the type shown in Fig. 7 for intact rats (A) and decerebrates (B). In these figures, stimuli which elicit similar profiles are placed together while those with poorly correlated profiles are more distant. Within stimulus groups, coherence was maintained. Profiles among the 4 Na-Li salts had a mean intercorrelation of .-I-0.92 in decerebrate rats (vs +0.94 in controls) while the mean coefficient among sugars was +0.87 (vs +0.9i). 5imiiariy, the close relationship between the two acids and quinine was maintained (r = +0.75 vs +0.76 in intact animals). Sugars maintained their distinction from acids (mean r = +0.06 vs +0.211 and quinine (mean r = +0.03 vs +0.(191 with typically low correlations. One difference between the taste responses of intact rats and those of decerebrates, however, was in profiles generated by the Na-Li salts. These showed a marked increase in similarity to sugar-evoked profiles (mean r = +0.43 vs +0.19 in intact rats) and a sharp reduction in relation to profiles representing
1 20
I
tO
,
I
,
I
2O
1o
0
)
I
I
I
B
--
20
10
"o
)Sucrose • Fructose •Glucose
Quinine
2 o
02
Citric Acid • HCI
10
,0025 NoSac
10
.25 NaSac
.3 NaCI
Na2SO4 e 20
LleCI e.1 NaCI • .03 NaCi
NoBr•
-
e.01 NaCI
I
I
I
I
I
20
.I0
0
tO
20
20
1
Fig. 7. Two-dimensional representations of relative taste qualities of the stimuli used in this study. A: representation based on responses of neurons in intact rats. The two dimensions account for 97% of the data variance. B: representation based on responses from decerebrate rats; 92% of the data variance is accounted for. Sucrose is fixed at the same coordinates in each space to provide a degree of comparability. The axes of the spaces are not defined.
acids (mean r = +0.37 vs +0.85) and quinine (mean r = +0.23 vs +0.84). Thus what appears as a clear sweet vs non-sweet dichotomy in intact controls is modified in decerebrates to yield 3 distinct groups: sugars. Na-Li salts and acid-quinine. The same 3 stimulus clusters have been identified by others in intact hamsters, however-'-', so these results should
146 not necessarily be considered a peculiarity of decerebration. What may indeed prove to be anomalous in the decerebrate preparation is the coding of the complex taste of saccharin. In intact rats, weak (0.0025 M) NaSaccharin evoked a response profile which closely resembled those of the sugars (mean r = +0.79) with only poor cor_,elations to profiles associated with NaLi salts (mean r = +0.25), quinine (r = +0.20) and acids (mean r = +0.16). This is in accord with the clear preference rats show for this solution in behavioral experiments 23 and its description by humans as predominantly sweet ~. Strong (0.25 M) NaSaccharin generated a profile that was about equally related to those of sugars (mean r = +0.51), Na-Li salts (mean r = +0.74), quinine (r = +0.65) and the acids (mean r = +0.52). This also corresponds to the complex sweet-salty-bitter sensations reported to high concentrations of NaSaccharin in psychophysical experiments ~. In decerebrate rats, while correlations with sugars remained constant, the coefficients with Na-Li salts became closer at both concentrations while the association with quinine and acids 4~sappeared. At the lower intensity, the mean NaSaccharin vs salt coefficient, which was +0.25 in controls, was +0.62. The correlations with quinine and the acids changed from +0.20 to 0.00 and from +0.16 to +0,09, respectively. More dramatically, at the high concentration, NaSaccharin's mean correlation with Na-Li salts increased slightly from +0.74 in intact rats to +0.82 in decerebrates while correlations with quinine and the acids declined precipitously from +0.65 to -0.04 and from +0.52 to +0.08, respectively. The result of these differences is that both saccharin solutions assume positions adjacent to sweet and salty stimuli and widely separated from acids and quinine in the space derived from responses in decerebrate rats (Fig. 7B). Behavioral data are now being collected to determine whether this decisive shift away from profiles associated with quinine and acid is reflected in increased acceptance of high concentrations of NaSaccharin by decerebrates. DISCUSSION
Taste responses in the NTS of decerebrate rats may be compared with those of intact controls as follows. (1) The characteristic broad range of respon-
siveness is retained. (2) Evoked activity is lower in proportion to the discharge rate demanded by the quality and intensity of the stimulus and also during the phasic portion of the response. (3) The degree to which neuronal response profiles may be categorized is unchanged. (4) Stimulus profiles maintain their strong coherence within basic taste qualities. There is some suggestion of stimulus realignment in decerebrate rats, with Na-Li salt profiles perhaps showing less similarity to those of the acids and quinine and more to those of the sugars. Profiles elicited by two concentrations of NaSaccharin maintained their similarity to sugar-generated patterns while shifting toward those of the Na-Li salts and away from those representinf:, acids and quinine.
Decreased activity The most pervasive effect of decerebration is a reduction in taste cell activity. This is apparently not the result of a general metabolic deficiency since the group of neurons that fell significantly behind controls as the demand exceeded 20 spikes/s in response to quinine often proved themselves capable of generating twice that rate in response to NaCI. Also, mean spontaneous rate was lower in decerebrates by 36%, almost exactly the same as the mean deficit in evoked activity (33%) despite relatively low metabolic demands. It is more plausible that the shortfall results from a loss of centrifugal influences that have been shown to impinge on NTS taste cells. Taste-responsive neurons in the rat NTS send projections a few millimeters rostraily to the medial parabrachial nucleus 2°. From here, third-order taste cells project to the thalamic taste relay and thence to gustatory cortex. However, they also send axons to several ventral forebrain areas, mo.st prominently the hypothalamus, central nucleus of the amygdala, and bed nucleus of the stria terminalis 1~. These targets send reciprocal projections to both the parabrachial nuclei and gustatory NTS 19. Thus NTS receives not only afferent fibers from peripheral gustatory and autonomic sources, but also centrifugal input from both cortical and limbic sites. Knowledge of the physiology lags behind that of the anatomy. But while the functional value of these connections has not been adequately investigated, it is established that the activity of lateral hypothalamic neurons may influence NTS taste cells 17. In particular, there is evi-
147 dence to suggest that the reinforcing property of appetitive tastes is a product of hypothalamic activation 2, and that the very cells that mediate that reinforcement project back to NTS, facilitating the responses of taste neurons 16. Preliminary electrophysiological evidence, then, makes it plausible that the loss of communication between forebrain sites and the NTS could deprive taste neurons of a facilitating influence. Perhaps the most apparent taste-related deficit known in decerebrate rats is their inability to develop or retain a conditioned taste aversion (CTA) 13. Chang and Scott 3 described the major gustatory neural effect of a CTA in the NTS of intact rats as a sharp burst of activity to the conditioned stimulus (CS). Moreover, this burst was largely confined to cells that were most responsive to the solution used as CS. The present results indicate that this discharge occurs under just the conditions that would lead to its maximum attenuation in the decerebrate preparation: as a large, phasic response occurring in neurons from which the greatest activity is being demanded. If this signal does in fact represent a conditioned response to the CS, its reduction or loss through decerebration could render the subject incapable of identifying the salience of that taste and so of developing a CTA.
Comparison with other studies Hayama et ai.15 recently reported on the responses of NTS taste and somatosensory neurons in acute decerebrate rats. Recordings were made under Flaxedii, and the 4 basic taste stimuli were applied at about the same concentrations, though at twice the flow rate, as in the present study. The major methodological distinctions involved the means of decerebration
REFERENCES 1 Bartoshuk, L.M., Bitter taste of saccharin related to the genetic ability to taste the bitter substance 6-n-propylthiouracil, Science, 205 (1979) 934-935. 2 Blass, E.M., Opioids, sweets and a mechanism for positive affect: broad motivational implications. In J. Dobbing (Ed.), Sweemess, Springer, Berlin, 1986, pp. 115-124. 3 Chang, F.-C,T. and Scott, T.R., Conditioned taste aversions modify neural responses in the rat nucleus tractus ,,,olitarius, J. Neurosci.. 4 ( 19841 18511-1862. 4 Chang, F.-C.T. and Scott, T.R., A technique for gustatory stiJmulus delivery in the rodent, Chem. Senses. 9 119841 91i-96. 5 Flynn, F.W. trod Grill, H.J., Insulin elicits ingestion in de-
and the period of recovery. Hayama et al. aspirated all tissue rostral to the collicular level and also removed the cerebellum. Perhaps more critically, recording was begun in as little as 1 h following surgery and heart rate was used as the sole criterion for recovery from the acute effects of the operation. While the mean spontaneous rate of control animals was sharply lower in the Hayama et al. study than in the present one (2.3 vs 10.6 spikes/s), the effect of decerebration was familiar: a 65% loss of discharges (vs our 36% loss). Similarly, responses evoked by the 4 basic stimuli in control rats were lower in the Hayama et ai. report than in the present one (mean of 4.8 spikes/s vs 32.8 spikes/s), but decerebration had the same effect in both studies: a mean 27% loss of responsivity in the report of Hayama et al. and a mean loss of 33% in ours. There were clear differences in measures of response breadth between acute and chronic decerebrate groups, with cells in the present study being more broadly tuned (the lack of a stated response criterion in the Hayama et al. report prevents a quantitative comparison). This difference appears to reflect the dramatically higher discharge rates obtained in the present experiment. Thus, despite large and unexplained differences in absolute response rates, the common effect of decerebration was to reduce the spontaneous and evoked activity of NTS taste cells. ACKNOWLEDGEMENTS
This study was supported by Research Grant AM30964 and by a Biomedical Research Grant from the National Institutes of Health. We thank Ms. Patricia Uyeda for preparation of the manuscript.
cerebrate rats, Science, 221 (1983) 188-189. 6 Ganchrow. J.R. and Efickson, R.P., Neural correl~tes of gustatory intensity and quality, J. Neurophysiol.o 33 (1970) 768-784. 7 Giza, B.K. and Scott, T.R., Blood glucose selectively affects taste-evoked activity in the rat nucleus tractus solitarius, Physiol. Behav., 31 (1983)643-650. 8 Giza, B.K. and Scott, T.R., Intravenous insulin infusions in rats decrease gustatory-evoked responses to sugars, Am. J. Physiol.. 252 ( 19871 994-11|112. 9 Glenn, J.F. and Erickson, R.P., Gastric modulation of afferent activity, Physiol. Behav.. 16 ( 19761 561-568. ii} Grill, H.J., Ganster, D. and Smith, G.P., CCK-8 decreases sucrose intake in chronic deccrebrate rats, Soc. Neurosci. Abstr., 9 ( 19831 9113.
148 11 Grill, H.J. and Norgren, R., The taste reactivity test. I. Mimetic responses to gustatory stimuli in neurologically normal rats, Brain Research, 143 (1978) 263-279. 12 Grill, H.J. and Norgren, R., The taste reactivity test. II. Mimetic responses to gustatory stimuli in chronic thalamic and chronic decerebrate rats, Brain Research, 143 (1978) 281-297. 13 Grill, H.J. and Norgren, R., Chronic decerebrate rats demonstrated satiation but not baitshyness, Science, 201 (1978) 267-269. 14 Grill, H.J., Schulkin, J. and Fiynn, F.W., Sodium homeostasis in chronic decerebrate rats, Beh,v. Neurosci., 100 (1986) 536-543. 15 Hayama, T.. Ito, S. and Ogawa, H., Responses of solitary tract nucleus neurons to taste and mechanical stimulations of the oral cavity in decrebrate rats, Exp. Brain Res., 60 (1985) 235-242. 16 Murzi, E., Hernandez, L. and Baptista, T., Lateral hypothalamic sites eliciting eating affect medullary taste neurons in rats, Physiol. Behav., 36 (1986) 829-834. 17 Matsuo, R., Shimizu. N. and Kusano, K., Lateral hypothalamic modulation of oral sensory afferent activity in nucleus tractus solitarius neurons of rats, J. Neurosci., 4 (1984) 1201-1207.
18 Norgren, R., The central organization of the gustatory and visceral afferent systems in the nucleus of the solitary tract. In Y. Katsuki, R. Norgren and M. Sato (Eds.), Brain Mechanisms of Sensation, Wiley, New York, 1981, pp. 143-160. 19 Norgren, R., Taste and the autonomic nervous system, Chem. Senses, 10 (1985) 143-161. 20 Norgren, R. and Leonard, C.M., Taste pathways in rat brainstem, Science, 173 (1971) 1136-1139. 21 Smith, D.V. and Travers, J.B., A metric for the breadth of tuning of gustatory neurons, Chem. Senses, 4 (1979) 215-229. 22 Smith, D.V., Van Buskirk, R.L., Travers, J.B. and Bieber, S.L., Coding of taste stimuli by hamster brain stem neurons, J. Neurophysiol., 50 (1983) 541-558. 23 Strouthes, A., Thirst and saccharin preference in rats, Physiol. Behav., 6 (1971) 287-292. 24 Van der Kooy, D., Koda, L.Y., McGinty, J.F., Gerfen, C.R. and Bloom, F.E., The organization of projections from the cortex, amygdala, and hypothalamus to the nucleus of the solitary tract in rat, J. Comp. Neurol., 224 (1984) 1-24. 25 Wishart, D., Clustan, Program Library Unit, Edinburgh University, 1978.
137
BRE 13343
Taste responses in the nucleus tractus solitarius of the chronic decerebrate rat Gregory P. Mark i, Thomas R. Scott 2, Fat-Chun Tony Chang 3 and Harvey J. Grill 4 tDepartmem of Psychology, Princeton University, Princeton, NJ 085 44 ( U.S.A. ), 2Department of Psychology and Institute.for Neuroscience, University of Delaware, Newark, DE 19716 (U.S.A.), "~USAMRICD,APG, Aberdeen, MD 21010 (U.S.A.)and 4Department of Psychology, University of Pennsylvania, Philadelphia, PA 19104 ( U.S. A. ) (Accepted 25 August 1987)
Key words: Decerebrate: Chronic; Rat: Nucleus tractus solitarii; Taste: Electrophysiology
The ingestive behavior of decerebrate rats has been studied for some time, yet little is known of its neural substrates. While taste fibers in rats proceed from hindbrain to thalamus and ventral forebrain, these regions return centrifugal fibers to the hindbrain by which lower-order taste activity may be influenced. We examined the functional characteristics of taste neurons in the nucleus tractus solitarii (NTS) of chronic decerebrate rats in which this reciprocal communication was disrupted and compared them with those of intact controls. Nine Wistar rats were decerebrated at the supracollicular level. After a minimum of one week recovery, they were immobilized with Flaxedil, anesthetized locally and prepared for recording. The responses of 50 taste cells were isolated bilaterally from the NTS of these animals, while the activity of 50 additional neurons was recorded from 12 intact rats under the same conditions. Taste stimuli included 7 Na-Li salts, 3 sugars, HCI and citric acids, quinine HCI and NaSaccharin. Mean spontaneous activity in decerebrates was 6.5 spikes/s, 36.0% lower than the level in intact animals. Mean evoked activity war, reduced by 32.6%. Analyses of the effects of stimulus quality, intensity and time course of the responses all indicated that the decrease in activity was attributable to the inability of taste cells in decerebrate rats to respond to demands for high discharge rates. This deficit could be responsible for the failure of these animals to develop conditioned taste aversions. Neurons from decerebrate preparations did, however, retain the broad sensitivity across stimuli that characterized taste cells in intact preparations. It was also typical that most neuron response profiles from decerebrates could be grouped into 3 loose clusters with peak sensitivities to acid-salt, salt or sugar. An analysis of similarities among stimulus activity profiles indicated that Na-Li salts, sugars and an acid-quinine complex represented 3 groups of stimulus quality: in intact animals, the primary distinction was between sweet and non-sweet stimuli. Moreover, the response to sodium saccharin lost its bitter component in decerebrates. These findings were in general agreement with those derived from ac;'e decerebm~e rats.
INTRODUCTION T h e h i n d b r a i n of the rat is i n v o l v e d in rather sophisticated ingestive functions, i n c l u d i n g those associated with taste a c c e p t a n c e or r e j e c t i o n , h o m e o s t a t -
a c o n d i t i o n e d taste aversion 3 or by the i m p o s i t i o n of satiety factors 7-~. Certain b e h a v i o r a l capacities, h o w e v e r , are disrupted by d e c e r e b r a t i o n . C o m p e n s a t o r y salt cons u m p t i o n , which a c c o m p a n i e s s o d i u m d e p r i v a t i o n in
ic processes and c o n d i t i o n i n g . D e c e r e b r a t e rats show
intact animals, is iost14: n o r d o d e c e r e b r a t e rats have
n o r m a l orofacial reactions to a variety of taste qualities 12. C o m p e n s a t o r y f e e d i n g r e a c t i o n s to glucoprivation 5 and c h o l e c y s t o k i n i n a d m i n i s t r a t i o n I° r e m a i n
the capacity to d e v e l o p or retain c o n d i t i o n e d taste aversions 13. It is a s s u m e d that these deficits are a
u n a l t e r e d with only h i n d b r a i n i n v o l v e m e n t . Moreover, electrophysiological studies in the nucleus tractus solitarii (NTS) of intact rats indicate that tastee v o k e d activity can be i n f l u e n c e d by d e v e l o p m e n t of
c o n s e q u e n c e of i n t e r r u p t i n g the reciprocal c o m m u n i cation b e t w e e n g u s t a t o r y - v i s c e r a l areas o f the hindbrain and forebrain regions associated with rei n f o r c e m e n t , notably the thalamus, h y p o t h a l a m u s and amygdala 24. T h u s d e c e r e b r a t i o n is likely to re-
Correspondence: T.R. Scott, Department of Psychology and Institute for Neuroscience, University of Delaware, Newark, DE 19716, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
138 duce some of the influences on hindbrain taste cells and so possibly modify their responses to gustatory stimuli. With a considerable literature on taste-related behavior in the decerebrate rat now available, it becomes important to determine the neural characteristics of the taste system in this preparation. MATERIALS AND METHODS
Subjects Twenty-one albino Wistar rats were used in this study. They were of both sexes and weighed 300-450 g. Of these, 12 were control animals and 9 were subjected to decerebration.
Surgery The surgical procedure for decerebration was modelled on that developed by Grill and Norgren 11 It proceeded under general anesthesia in two steps: a hemitransection followed in a week by completion of the cut. The entire parietal and approximately half of the temporal surface of the skull was exposed bilaterally. A thin transverse groove was made across the skull at a level corresponding to 40% of the distance from bregma to lambda (3.6-3.9 mm anterior to the interaural line). It was deepened over one hemisphere until only a thin layer of periosteum remained. This and the underlying dura were section~zd with a scalpel. An L-shaped spatula, ground to 0.5 mm thickness and left blunted, was inserted into the brain at the lateral margin of the groove and depressed to the cranial floor. Care was taken to compress rather than rupture the vasculature. The spatula was moved 2 mm medially with each successive stroke until the midline was reached. The steps were then retraced laterally to ensure a complete transection. Before exiting the brain, the spatula was angled laterally such that its heel sectioned all connections in the lateral convexity of the temporal lobe. The skin was sutured and the animal was permitted a minimum of one week to recover. The procedure was then repeated in the opposite hemisphere, following which the spatula was angled across the midline to complete the decerebration. This transection was designed to pass along the rostral border of the colliculi.
Care and maintenance Decerebrated rats were fully aphagic and adipsic.
Consequently each animal was fed by gavage 3-4 times per day. Each feeding consisted of 7-8 ml of equal parts sweetened condensed milk and water for a total daily volume of 25-30 ml. Body weight was monitored daily, with appropriate adjustments in volume as needed. Animals were housed in a climate-controlled chamber to minimize temperature fluctuations. Rectal temperature was recorded every 6 h Hyperthermia was treated by placing the rat on a cool surface, or, in more extreme cases, by spraying a mist of water on its fur. Hypothermia, usually a less serious concern, was controlled with a heat lamp or heating pad. Long-acting antibiotics were administered for precautionary and combative purposes following surgery, but their use was discontinued at least two days before the recording session.
Recording Decerebrate rats were permitted a minimum of one week to recover from the acute effects of surgery. Each of the 21 animals, decerebrate and control, was anesthetized to a surgical level using sodium pentobarbital. A tracheotomy was performed and the initial section of the esophagus was ligated to prevent ingestion of taste solutions. A 2 x 5 mm section of the occipital bone was removed to offer bilateral access to the NTS. Incisions were closed with wound clips and all wounds and pressure points were liberally treated with Lidocaine. The data presented here are eventually to be compared with those from decerabrates in which a conditioned taste aversion paradigm will be conducted. Since conditioning will involve conscious rats, considerations of state-dependent learning require that recordings take place under the same conditions. Consequently, the decerebrate rats in the present study (whose responses will serve as controls for those from conditioned decerebrates) and, in turn, the intact controls could not be administered a general anesthetic during recording. Thus, when surgery was complete, each animal was immobilized with an i.p. injection of Flaxedil (gallamine triethiodide, 60 mg/kg), respired using a small animal respirator and secured in a stereotaxic instrument with blunt, Lidocaine-treated earbars. The necessity of using an immobilized preparation required that extensive precautions be taken to ensure the relative comfort and physiological integrity of the rat. To this end, vital
139 signs were monitored and distress signals (pupillary dilation, blood pressure or heart rate changes) were attended to continuously throughout a recording session. Temperature was taken rectally and maintained at 36-38 °C. Heart rate, which provided the most sensitive index of physiological condition, was recorded through s.c. electrocardiographic electrodes and maintained at an interbeat interval of ca. 180 ms by adjusting respiration, level of paralysis and local anesthesia. Expired air was continuously monitored for CO2 content (Instrumentation Laboratory, IL 200 CO2 Monitor) which was maintained at 3.0-3.8%. In control animals the electrocorticogram was continuously monitored. The preponderance of the record showed desynchrony, but slow waves occasionally appeared and could be blocked by tail pinch. While decerebration precluded tne value of monitoring the ECoG in the experimental group, it also decreased the likelihood that these animals would be distressed during recording. Decerebration is, in fact, routinely performed in place of general anesthesia in some laboratories. With the stereotaxic bite bar positioned 5.5 mm below the interaural line, the NTS was typically located 11.3 mm posterior to bregma, 1.7 mm lateral to the midline and 7.5 mm below the surface of the cerebellum. One hundred single neurons, 50 in each group, were isolated with micropipettes (Z = 5-10 Mf~). Signals were amplified, filtered, displayed and stored using conventional techniques and analyzed off-line.
Stimuli and stimulus delivery The responses of each neuron to 12 chemicals were recorded. These included prototypes of the putative 4 basic tastes (sucrose, NaC!, HC!, quinine) plus 6 other saccharides, salts and acids and two intensities of sodium saccharin. Concentrations were those that elicited a half-maximum response from neural samples in the CT or NTS. In decerebrate animals 3 additional concentrations of NaC! were also applied. Stimuli and concentrations are listed in Table I. Stimulus solutions were delivered through a length of slender tubing, closed at its end and extensively perforated along its final 2 cm. The perforated end was slipped into the mouth so that the stimulus spray regularly cor~tacted nearly the entire taste-receptive surface. Stimulus onset was marked by the moment
TABLE I
Stimuli, abbreviations and molar concentrations 1 CA 2F 3G 4H 5L 6 NB 7 NC 8 NS 9Q 10 S 11 SA1 12 SA2 13 NCI* 14 NC2* 15 NC3*
0.01 M 0.5 M 0.5 M 0.01 M 0.1 M 0.1 M 0.1 M 0.1 M 0.005 M 0.5 M 0.0025 M 0.25 M 0.01 M 0.03 M 0.3 M
citric acid fructose glucose HCI LiC! NaBr NaCl Na2SO 4 quinine HC! sucrose NaSaccharin NaSaccharin NaCl NaCl NaCI
* Used only in decerebrates.
of contact with oral tissue 4. Five ml of solution were sprayed throughout the oral cavity in 3 s. The stimulus was then cleared from the tubing by air pressure and within 10 s a 25-ml distilled water rinse was initiated. A minimum of 90 s was allowed to elapse between stimulus applications to avoid the effects of adaptation.
A n alyses Primary analyses were performed by a PDP 11/03 computer. These included the summation of action potentials for 3 s prior to (spontaneous) and 5 s following (evoked) stimulus onset in 100-ms time bins and the generation of post-stimulus time histograms. Derived analyses were conducted on 3 computers: a DEC-10, a Burroughs 7700 and an IBM 3081D. These included the generation of interstimulus and interneuronal correlation matrices, multidimensional spaces, hierarchical cluster analyses and intensity-response functions.
Histology In 7 rats (4 intact, 3 decerebrate) electrolytic lesions were made at the final recording sites (20 I~A for 20 s, electrode negative). Animals were given a lethal injection of barbiturate and perfused with 10% formalin. Brains were removed, blocked and frozen for sectioning. Fifty-Ftm transverse sections, stained with Cresyi violet, were made from the mid-pontine level caudally until lesion sites were located. In addition, sagittal sections were made rostral to the mid-
140
.,...-:-. •
..-
: .... . ...,
~.::....,.~:..
;,a.
" ".~g
:~3:,
.
.
.
.
;"
"
Fig. 1. A representative sagittal section t h r o u g h the brain o f a d e c e r e b r a t e d rat.
pontine level in all decerebrated rats to confirm the full transection (Fig. 1). RESULTS
Basic responses Spontaneous activity and response criterion The mean spontaneous activity of taste cells in the NTS of control rats was 10.2 + 6.4 spikes/s. The mean rate in decerebrates was 6.5 + 5.6 spikes/s, a non-significant reduction of 36%. We adopted a response criterion of a change in neural activity of 1.28 S.D. from the mean spontaneous activity for that cell, sustained for 3 s.
Evoked activity Net evoked responses were calculated by counting the total spikes in 5 s, dividing by 5 to arrive at a gross rate per s, then subtracting the mean spontaneous rate for that neuron.
Breadth of responsiveness.
The characteristic broad sensitivity of taste cells was apparent in recordings from intact animals and was preserved in decerebrates. Of the 600 stimulus applications (50 cells, 12 stimuli), 504 (84.0%) fulfilled our criterion for excitation, 10 (1.7%) caused inhibition and 86 (14.3%) elicited no reaction from taste cells in control ani-
reals. The corresponding figures in decerebrates were 538 (89.7%) excitation, 5 (0.8%) inhibition and 57 (9.5%) no response. The breadth-of-tuning metric 2~was applied to both sets of data. In intact rats the mean coefficient was 0.86 while in decerebrates it was 0.75 (t = 1.60; non-significant). Both values indicate a wide range of responsiveness across the 4 prototypicai taste stimuli used in this analysis. Evoked response rates. For all stimulus-neuron interactions the mean response rate was 32.6% lower in decerebrates than in intact rats. But while reduced responsiveness was a rather consistent effect of decerebration, it did not occur equally across stimuli. There was a clear tendency for the cells of decerebrate rats to lag farther behind those of controls as higher discharge rates were demanded. In Fig. 2, stimuli are listed in ascending order of effectiveness for decerebrates and the mean response in each group is plotted. Where evoked responses are weak (sweet and bitter chemicals, n = 5), all differences are less than 20% and the overall reduction in response in decerebrates is non-significant (t = 0.69). Where they are robust (salty and sour stimuli, n = 7), the shortfalls are significant (t = 3.75; P < 0.01) and range from 34% to 41%. A single neuron analysis reinforces the notion that cells in decerebrate rats have difficulty meeting high response demands. In Fig. 3, neurons from decerebrate and from intact rats are ordered according to
141 70
w..
60 ~-
•
r~ ',,zk
t"
50
,v ffl
LU
i
40
i
i
O. Z
i
3O p
p
d/'~ .
LU
:E
2o
10
STIMULUS
Fig. 2. Mean net (evoked minus spontaneous) responses to each stimulus in intact (dashed line) and decerebrate (solid line) rats. Abbreviations are listed in Table I.
their responsiveness to each of the 4 prototypical taste stimuli. Individual differences that significantly exceed the mean discrepancy between each pair of curves (P < 0.05) are indicated with a filled circle above the abscissa. Almost without exception, the differences appear between cells in the most responsive one-third (17/50) of the profile. O f the 68 comparisons (4 x 17) between responses of these active neurons, 54 (79.4%) are significantly different. Of the remaining 132 comparisons (4 x 33) only two differences (1.5%) reach statistical significance. Thus there is not simply a suppression of activity that results in a linear reduction in evoked responses; rather, there is a gradual loss of capacity as the response requirements increase. In general, the failure to keep pace becomes significant as the demand exceeds 20 spikes/s to quinine, 30 spikes/s to sucrose, or 60 spikes/s to NaCi or HCI. A temporal analysis indicates that the attenuation of the phasic burst contributes disproportionately to the lower activity in decerebrates. During the 0.2-0.6 s a of evoked activity (defined, arbitrarily, as the phasic portion of the response), responses from decerebrate rats to all 4 stimuli are 42.2% lower than those of controls. During the succeeding 4.4 s (tonic)
the difference is only 31.1% (Fig. 4). Extending this analysis to include all stimuli, the mean difference in the phasic response is 42.8% compared to a tonic difference of 35.4%. This is, of course, much more striking in terms of absolute loss of spikes. Comparing responses across 50 cells to the four prototypicai stimuli over a 5-s period (a total of 1000 s of analysis), evoked activity in decerebrate rats falls 10,670 spikes short of that in controls. Fully 25% of that shortfall occurs during the 0.5 s (10%) of the response designated as phasic. Moreover, the phasic differences were quite reliable across the protypical stimuli: for sucrose, NaCI, HC! and quinine the phasic responses of decerebrates were lower by 42%, 39%, 46% and 41%, respectively. By contrast, comparisons of activity during the tonic period were less predictable, ranging from a 40% loss (to NaCI) to a 24% gain (to quinine) in decerebrate rats. Thus, even the similar total responses elicited from the two neural samples by quinine or by sucrose (see Fig. 2) result from a compensation during the tonic response for the consistent deficit in phasic activity. The 3 preceding analyses - - mean activity, responses of individual neurons, time course m all relate to the effect of decerebration on coding different stimulus qualities, with concentration held constant. To analyze the influence of decerebration on intensity coding, 4 concentrations of NaCI were used in decerebrate rats. The responses evoked by this intensity series were compared with those reported by Ganchrow and Erickson 6 from NTS of intact rats. At a concentration of 10 raM, neurons from both preparations responded at a mean rate of 33 spikes/s (Fig. 5b). As intensity increased, however, activity from decerebrates fell progressively behind, rising with a power function exponent of only 0.15 vs 0.33 in intact rats. Within one log unit of concentration (from 10 mM to 100 raM) the responses of decerebrates fell from parity to a deficit of 29% c relative to those of control animals. Thus four comparisons were made between the re-
a For sucrose, which typically has a slower onset, the (I.6-1.0 s of the evoked response was treated as the phasic portion. b The spike rates reported in this section are gross discharges (with no subtraction of spontaneous rate) during the period 0.4-1.3 s following stimulas onset, in conformity with the method of Ganchrow and Erickson~. Thus they are son'ex~'hat higher than would be expected for net spikes per s over 3 s as is used elsewhere in this manuscript. ¢ This is precisely the same percent difference to the standard 0.1 M NaCI as existed between dc,.ci'c,~rates and the intact rats whose responses were recorded in the present study.
60
m
0
0
10
10
!
Neurona
20
Neurons
Sucrose
20
**
30
3~
40
4,0
eoeoooeeeoeo
....
****,,.
50
eo
I I I
50
I ~ ~/11!// I I I
es e0
""
m ~D
=e
g,)
r.n e~
,Ad
~rJ
q)
40
60
80
100
--20
0
20
40
60
80
100
120
140
160
180
200
0
0
10
10 Neurons
20 Neurons
QHC1
~0
HCI
j
30
30
Fig. 3. Net responses to the 4 prototypical stimuli from each of 50 neurons in intact (dashed line) and decerebrate (solid line) rats. Filled circles above the abscissa indicate differences that exceeded the mean discrepancy between the curves by a significant amount (P < 0.05).
20
4O
oo
100
120
14.0
160
180
20
40
6O
80
lO0'
120
,~.
o~,.~
oe)
~m
m
140
160
180
NaC1
40
50
/
50
/
Il/l 1 /"///I oeoooooooeeo
40
eeooooeoooooooooo
/
-/
/ I I I I I
/ I
iX)
6'
0
6'
;
:
:
I!i
;
/
I
:
/
:
/
|
10
I
I
:
:
:
:
:
:
\
\
\
\
\/\\
Post--stimulus
:
I
:
Time
(xlO0
30
Sucrose
20
msee)
40
: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :
X\i/\\/... "~.
50
50
W
~d .M
Q)
0 0 M
q~
.,
4
0
8
0
20
Post--stimulus
10
Time
(xlO0
30
msee)
40
: : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : :
0
:
\I
Ix
0
:
\
10
u~ 2
;
\
12
2
0
,
/~
I% \
NaC1
0
o
i
II
!Ix
I~
I I I I \.
"~,'
i\
I'k \
~
i ~./
..,-...,. x\ "
20
Post--stimulus
10
IO 20 Post--stimulus
\/\,,2
.
Time
Time
/'----',
msec)
40
40 mseo)
\"'-•.
(xlO0
30
30 (xlO0
"\_-,"\."\
QHCI
/--',
HC1
Fig. 4. Post-stimulus time histograms showing the mean net responses to each of the prototypical stimuli in 100-ms time bins for intact (dashed lines) and decerebrate (solid lines) rats. The reduction in responsiveness in decerebrates occurred disproportionately during the phasic period of the response.
O O
CD
cJ 4 ¸
0p,,~
0 0
o
10,
12
50
5o
4~
144 11.18.5
120 "
tl.Z69 ,a
100
t). t53
11.4:1/
O LU r~
it-
80
CO LU n CO
t1.%21
,¢
SO
tl.
tl .(qSd
I
f
iI. ¢~/2
Z m
IAJ~i
~g t ~. fl,1 t I
20
iI"
-3".o
-~'.o LOG
MOLAR
-~'.o
o%
CONCENTRATION
B
....
Fig. 5. Mean responses across a NaCI co~ic~ntration series in intact (dashed lines) and decerebrate (solid lines) rats. Data from intact rats from Ganchrow and Erickson~. ,4. .4
,
r
sponses elicited from decerebrate rats and from two intact groups: (1) the mean effectiveness of various qualities (Fig. 2) and (2) different intensities of a single quality (Fig. 5), (3) the effects of 4 individual stimuli on single neurons (Fig. 3) and (4) the time courses of the mean responses to the same 4 chemicals (Fig. 4). Each comparison promotes a common conclusion: taste neurons of decerebrate rats are unable to respond normally to the peak demands placed upon them.
Derived analyses Categorization of neuronal profiles Each neuron generated a unique response profile across the array of 12 stimulus qualities used in this study. These profiles may be compared by calculating the Pearson product-moment correlation coefficient between each pair. The total of all such comparisons within a 50-neuron sample may be contained in a correlation matrix of 1225 entries (50 x 49)/2. This matrix serves as the basis for a hierarchical cluster analysis25 whereby related groups of prt;files may be identified. The number and size of clusters, and their degree of coherence have all been used to address the question of whether the taste system is composed of a discrete number of groups, or 'neuron types' within each of which the constituent neurons are functionally similar. The results of a cluster analysis may be expressed as a dendrogram, as shown in Fig. 6. Neurons, num-
t
* .4
~..
r-.
7~ ' I
'~
F~,~ ' -
,~'-:. ", '
T'~'I
,I'
,z.-,
Fig. 6. Dendrogn:ms ru,ating neurons to one another according to the correlations between their response profiles. A: analysis based on the responses of intact rats. B: decerebrate rats. In both groups, the basic clusters, and the numbers of cells assigned to them, are similar.
bered in the order in which they were isolated, are arranged horizontally. The correlation between the activity profiles of any two cells is expressed by the height of the horizontal line that connects them. Where groups are connected, the line is at a height which corresponds to the mean correlation between the constituents of the groups being connected. Thus the lower the horizontal line connecting any cell or group of cells, the more similar are their activity profiles across this stimulus array. The longer the vertical line leading to them, the more isolated the cells are from their neighbors. In Fig. 6 are shown dendrograms resulting from cluster analyses based on the responses of intact rats (top) and decerebrates (bottom). Although the absolute cueftlcients are somewhat lower in the analysis based on decerebrates (probably a consequence of reduced response rates, which normally lead to lower correlations), the forms of the two dendrograms are remarkably similar. In both, the primary distinction is between a large clus-
145
ter of neurons to the left and a smaller one to the right° This represents a dichotomy between cells that do not show a sweet-sensitive profile (left) vs those that do (r~g,ht). In control rats these groups number 37 and 13 respectively; in decerebrates, 39 and 11. Within the non-sweet sensitive cluster are two major groups. In both dendrograms ~:hese consist of neurons with combined salt and acid sensitivity to the left and more specific salt sensitivity to the right. In control tats these groups number 17 and 20, respecthely; in decerebrates, 23 and 16. Further subdivisions in each dendrogram continue to reveal the striking similarity between them. It is clear that the process of decerebration does not much alter the relative response profiles of taste neurons in the NTS.
1
20--
-2 0
-10
I
I
O
,
10
I
,
20
I
,
I
A
-20
• .25 NaSac Na2SO4
10
• Quinine
10
• .007~ NaSac ~Sucrose
NaOree,1 NaCI 2 o
LICI
• Fructose
o2
IGlucose Citric Acid •e HC -10
1o
2O
-20
I
I
i
-20
10
0
I
I 20
10
1
Relative taste quality Just as the profile of activity that each neuron generates across the stimulus array may be used to characterize its function, so the profile that each stimulus elicits across the neural sample may represent its taste quality. The matrix of correlaticn~ between each pair of stimulus profiles (n = (12 x 11)/2 = 66) permits an assessment of relative taste similarities within each group of rats. Multidimensional scaling routines can be used to generate spatial representations of these relative similarities. The results of these algorithms are multidimensional stimulus spaces of the type shown in Fig. 7 for intact rats (A) and decerebrates (B). In these figures, stimuli which elicit similar profiles are placed together while those with poorly correlated profiles are more distant. Within stimulus groups, coherence was maintained. Profiles among the 4 Na-Li salts had a mean intercorrelation of .-I-0.92 in decerebrate rats (vs +0.94 in controls) while the mean coefficient among sugars was +0.87 (vs +0.9i). 5imiiariy, the close relationship between the two acids and quinine was maintained (r = +0.75 vs +0.76 in intact animals). Sugars maintained their distinction from acids (mean r = +0.06 vs +0.211 and quinine (mean r = +0.03 vs +0.(191 with typically low correlations. One difference between the taste responses of intact rats and those of decerebrates, however, was in profiles generated by the Na-Li salts. These showed a marked increase in similarity to sugar-evoked profiles (mean r = +0.43 vs +0.19 in intact rats) and a sharp reduction in relation to profiles representing
1 20
I
tO
,
I
,
I
2O
1o
0
)
I
I
I
B
--
20
10
"o
)Sucrose • Fructose •Glucose
Quinine
2 o
02
Citric Acid • HCI
10
,0025 NoSac
10
.25 NaSac
.3 NaCI
Na2SO4 e 20
LleCI e.1 NaCI • .03 NaCi
NoBr•
-
e.01 NaCI
I
I
I
I
I
20
.I0
0
tO
20
20
1
Fig. 7. Two-dimensional representations of relative taste qualities of the stimuli used in this study. A: representation based on responses of neurons in intact rats. The two dimensions account for 97% of the data variance. B: representation based on responses from decerebrate rats; 92% of the data variance is accounted for. Sucrose is fixed at the same coordinates in each space to provide a degree of comparability. The axes of the spaces are not defined.
acids (mean r = +0.37 vs +0.85) and quinine (mean r = +0.23 vs +0.84). Thus what appears as a clear sweet vs non-sweet dichotomy in intact controls is modified in decerebrates to yield 3 distinct groups: sugars. Na-Li salts and acid-quinine. The same 3 stimulus clusters have been identified by others in intact hamsters, however-'-', so these results should
146 not necessarily be considered a peculiarity of decerebration. What may indeed prove to be anomalous in the decerebrate preparation is the coding of the complex taste of saccharin. In intact rats, weak (0.0025 M) NaSaccharin evoked a response profile which closely resembled those of the sugars (mean r = +0.79) with only poor cor_,elations to profiles associated with NaLi salts (mean r = +0.25), quinine (r = +0.20) and acids (mean r = +0.16). This is in accord with the clear preference rats show for this solution in behavioral experiments 23 and its description by humans as predominantly sweet ~. Strong (0.25 M) NaSaccharin generated a profile that was about equally related to those of sugars (mean r = +0.51), Na-Li salts (mean r = +0.74), quinine (r = +0.65) and the acids (mean r = +0.52). This also corresponds to the complex sweet-salty-bitter sensations reported to high concentrations of NaSaccharin in psychophysical experiments ~. In decerebrate rats, while correlations with sugars remained constant, the coefficients with Na-Li salts became closer at both concentrations while the association with quinine and acids 4~sappeared. At the lower intensity, the mean NaSaccharin vs salt coefficient, which was +0.25 in controls, was +0.62. The correlations with quinine and the acids changed from +0.20 to 0.00 and from +0.16 to +0,09, respectively. More dramatically, at the high concentration, NaSaccharin's mean correlation with Na-Li salts increased slightly from +0.74 in intact rats to +0.82 in decerebrates while correlations with quinine and the acids declined precipitously from +0.65 to -0.04 and from +0.52 to +0.08, respectively. The result of these differences is that both saccharin solutions assume positions adjacent to sweet and salty stimuli and widely separated from acids and quinine in the space derived from responses in decerebrate rats (Fig. 7B). Behavioral data are now being collected to determine whether this decisive shift away from profiles associated with quinine and acid is reflected in increased acceptance of high concentrations of NaSaccharin by decerebrates. DISCUSSION
Taste responses in the NTS of decerebrate rats may be compared with those of intact controls as follows. (1) The characteristic broad range of respon-
siveness is retained. (2) Evoked activity is lower in proportion to the discharge rate demanded by the quality and intensity of the stimulus and also during the phasic portion of the response. (3) The degree to which neuronal response profiles may be categorized is unchanged. (4) Stimulus profiles maintain their strong coherence within basic taste qualities. There is some suggestion of stimulus realignment in decerebrate rats, with Na-Li salt profiles perhaps showing less similarity to those of the acids and quinine and more to those of the sugars. Profiles elicited by two concentrations of NaSaccharin maintained their similarity to sugar-generated patterns while shifting toward those of the Na-Li salts and away from those representinf:, acids and quinine.
Decreased activity The most pervasive effect of decerebration is a reduction in taste cell activity. This is apparently not the result of a general metabolic deficiency since the group of neurons that fell significantly behind controls as the demand exceeded 20 spikes/s in response to quinine often proved themselves capable of generating twice that rate in response to NaCI. Also, mean spontaneous rate was lower in decerebrates by 36%, almost exactly the same as the mean deficit in evoked activity (33%) despite relatively low metabolic demands. It is more plausible that the shortfall results from a loss of centrifugal influences that have been shown to impinge on NTS taste cells. Taste-responsive neurons in the rat NTS send projections a few millimeters rostraily to the medial parabrachial nucleus 2°. From here, third-order taste cells project to the thalamic taste relay and thence to gustatory cortex. However, they also send axons to several ventral forebrain areas, mo.st prominently the hypothalamus, central nucleus of the amygdala, and bed nucleus of the stria terminalis 1~. These targets send reciprocal projections to both the parabrachial nuclei and gustatory NTS 19. Thus NTS receives not only afferent fibers from peripheral gustatory and autonomic sources, but also centrifugal input from both cortical and limbic sites. Knowledge of the physiology lags behind that of the anatomy. But while the functional value of these connections has not been adequately investigated, it is established that the activity of lateral hypothalamic neurons may influence NTS taste cells 17. In particular, there is evi-
147 dence to suggest that the reinforcing property of appetitive tastes is a product of hypothalamic activation 2, and that the very cells that mediate that reinforcement project back to NTS, facilitating the responses of taste neurons 16. Preliminary electrophysiological evidence, then, makes it plausible that the loss of communication between forebrain sites and the NTS could deprive taste neurons of a facilitating influence. Perhaps the most apparent taste-related deficit known in decerebrate rats is their inability to develop or retain a conditioned taste aversion (CTA) 13. Chang and Scott 3 described the major gustatory neural effect of a CTA in the NTS of intact rats as a sharp burst of activity to the conditioned stimulus (CS). Moreover, this burst was largely confined to cells that were most responsive to the solution used as CS. The present results indicate that this discharge occurs under just the conditions that would lead to its maximum attenuation in the decerebrate preparation: as a large, phasic response occurring in neurons from which the greatest activity is being demanded. If this signal does in fact represent a conditioned response to the CS, its reduction or loss through decerebration could render the subject incapable of identifying the salience of that taste and so of developing a CTA.
Comparison with other studies Hayama et ai.15 recently reported on the responses of NTS taste and somatosensory neurons in acute decerebrate rats. Recordings were made under Flaxedii, and the 4 basic taste stimuli were applied at about the same concentrations, though at twice the flow rate, as in the present study. The major methodological distinctions involved the means of decerebration
REFERENCES 1 Bartoshuk, L.M., Bitter taste of saccharin related to the genetic ability to taste the bitter substance 6-n-propylthiouracil, Science, 205 (1979) 934-935. 2 Blass, E.M., Opioids, sweets and a mechanism for positive affect: broad motivational implications. In J. Dobbing (Ed.), Sweemess, Springer, Berlin, 1986, pp. 115-124. 3 Chang, F.-C,T. and Scott, T.R., Conditioned taste aversions modify neural responses in the rat nucleus tractus ,,,olitarius, J. Neurosci.. 4 ( 19841 18511-1862. 4 Chang, F.-C.T. and Scott, T.R., A technique for gustatory stiJmulus delivery in the rodent, Chem. Senses. 9 119841 91i-96. 5 Flynn, F.W. trod Grill, H.J., Insulin elicits ingestion in de-
and the period of recovery. Hayama et al. aspirated all tissue rostral to the collicular level and also removed the cerebellum. Perhaps more critically, recording was begun in as little as 1 h following surgery and heart rate was used as the sole criterion for recovery from the acute effects of the operation. While the mean spontaneous rate of control animals was sharply lower in the Hayama et al. study than in the present one (2.3 vs 10.6 spikes/s), the effect of decerebration was familiar: a 65% loss of discharges (vs our 36% loss). Similarly, responses evoked by the 4 basic stimuli in control rats were lower in the Hayama et ai. report than in the present one (mean of 4.8 spikes/s vs 32.8 spikes/s), but decerebration had the same effect in both studies: a mean 27% loss of responsivity in the report of Hayama et al. and a mean loss of 33% in ours. There were clear differences in measures of response breadth between acute and chronic decerebrate groups, with cells in the present study being more broadly tuned (the lack of a stated response criterion in the Hayama et al. report prevents a quantitative comparison). This difference appears to reflect the dramatically higher discharge rates obtained in the present experiment. Thus, despite large and unexplained differences in absolute response rates, the common effect of decerebration was to reduce the spontaneous and evoked activity of NTS taste cells. ACKNOWLEDGEMENTS
This study was supported by Research Grant AM30964 and by a Biomedical Research Grant from the National Institutes of Health. We thank Ms. Patricia Uyeda for preparation of the manuscript.
cerebrate rats, Science, 221 (1983) 188-189. 6 Ganchrow. J.R. and Efickson, R.P., Neural correl~tes of gustatory intensity and quality, J. Neurophysiol.o 33 (1970) 768-784. 7 Giza, B.K. and Scott, T.R., Blood glucose selectively affects taste-evoked activity in the rat nucleus tractus solitarius, Physiol. Behav., 31 (1983)643-650. 8 Giza, B.K. and Scott, T.R., Intravenous insulin infusions in rats decrease gustatory-evoked responses to sugars, Am. J. Physiol.. 252 ( 19871 994-11|112. 9 Glenn, J.F. and Erickson, R.P., Gastric modulation of afferent activity, Physiol. Behav.. 16 ( 19761 561-568. ii} Grill, H.J., Ganster, D. and Smith, G.P., CCK-8 decreases sucrose intake in chronic deccrebrate rats, Soc. Neurosci. Abstr., 9 ( 19831 9113.
148 11 Grill, H.J. and Norgren, R., The taste reactivity test. I. Mimetic responses to gustatory stimuli in neurologically normal rats, Brain Research, 143 (1978) 263-279. 12 Grill, H.J. and Norgren, R., The taste reactivity test. II. Mimetic responses to gustatory stimuli in chronic thalamic and chronic decerebrate rats, Brain Research, 143 (1978) 281-297. 13 Grill, H.J. and Norgren, R., Chronic decerebrate rats demonstrated satiation but not baitshyness, Science, 201 (1978) 267-269. 14 Grill, H.J., Schulkin, J. and Fiynn, F.W., Sodium homeostasis in chronic decerebrate rats, Beh,v. Neurosci., 100 (1986) 536-543. 15 Hayama, T.. Ito, S. and Ogawa, H., Responses of solitary tract nucleus neurons to taste and mechanical stimulations of the oral cavity in decrebrate rats, Exp. Brain Res., 60 (1985) 235-242. 16 Murzi, E., Hernandez, L. and Baptista, T., Lateral hypothalamic sites eliciting eating affect medullary taste neurons in rats, Physiol. Behav., 36 (1986) 829-834. 17 Matsuo, R., Shimizu. N. and Kusano, K., Lateral hypothalamic modulation of oral sensory afferent activity in nucleus tractus solitarius neurons of rats, J. Neurosci., 4 (1984) 1201-1207.
18 Norgren, R., The central organization of the gustatory and visceral afferent systems in the nucleus of the solitary tract. In Y. Katsuki, R. Norgren and M. Sato (Eds.), Brain Mechanisms of Sensation, Wiley, New York, 1981, pp. 143-160. 19 Norgren, R., Taste and the autonomic nervous system, Chem. Senses, 10 (1985) 143-161. 20 Norgren, R. and Leonard, C.M., Taste pathways in rat brainstem, Science, 173 (1971) 1136-1139. 21 Smith, D.V. and Travers, J.B., A metric for the breadth of tuning of gustatory neurons, Chem. Senses, 4 (1979) 215-229. 22 Smith, D.V., Van Buskirk, R.L., Travers, J.B. and Bieber, S.L., Coding of taste stimuli by hamster brain stem neurons, J. Neurophysiol., 50 (1983) 541-558. 23 Strouthes, A., Thirst and saccharin preference in rats, Physiol. Behav., 6 (1971) 287-292. 24 Van der Kooy, D., Koda, L.Y., McGinty, J.F., Gerfen, C.R. and Bloom, F.E., The organization of projections from the cortex, amygdala, and hypothalamus to the nucleus of the solitary tract in rat, J. Comp. Neurol., 224 (1984) 1-24. 25 Wishart, D., Clustan, Program Library Unit, Edinburgh University, 1978.