Acta Physiologica Sinica, June 25, 2003, 55(3): 317－323
Received 2002-09-12 Accepted 2003-01-21
Corresponding author. Tel and Fax: +86-29-5275199; E-mail: yanjianqun@hotmail.com
Research Paper
Effects of the
central amygdaloid nucleus lesions on the gustatory responses in the
parabrachial nucleus in rats
KANG Yi, YAN Jian-Qun*, HUANG Tao
Department
of Physiology,
Abstract: To access the role of the central nucleus of the amygdala (CeA) in the gustatory activity in the pontine parabrachial nucleus (PBN), the responses to four prototypical taste stimuli (NaCl, HCl, QH2SO4 and sucrose) in the PBN were observed before and after bilateral electrolytic lesion of the CeA in the urethane-anesthetized rat. Of 29 neurons, 14 were classified as NaCl-best, 9 as HCl-best, 3 as QH2SO4-best and 3 sucrose-best. After CeA lesions, the response rates to HCl and QH2SO4 were statistically higher across all PBN neurons (P<0.01). According to the best-stimulus category, the effects on the responses to HCl and QH2SO4 were similarly subjected to these modulations in NaCl-best, HCl-best and QH2SO4-best neurons. Correlation analysis indicated that the CeA lesion depressed the effect on the chemical selection between NaCl and QH2SO4. These findings suggest that the CeA plays an important role in the taste coding at the pontine level and it may be involved in mediating the feeding behavior via modulating the gustatory responses.
Key words: parabrachial nucleus; gustatory neurons; central amygdaloid nucleus; electrolytic lesion; rat
电损毁大鼠杏仁中央核对脑桥臂旁核味觉神经元的影响
康怡, 闫剑群*, 黄涛
西安交通大学医学院生理教研室, 西安 710061
摘要: 应用细胞外记录的电生理学方法, 在乌拉坦麻醉的大鼠观察了电损毁双侧杏仁中央核前后脑桥臂旁核味觉神经元对四种基本味觉刺激(即氯化钠、 盐酸、 奎宁和蔗糖)反应的变化。根据对味觉刺激的优势反应, 29个记录的味觉神经元中, 有14个NaCl优势、9个HCl优势、3个QH2SO4优势和3个蔗糖优势反应神经元。损毁杏仁中央核明显增强臂旁核味觉神经元对盐酸和硫酸奎宁的反应(P<0.01)。氯化钠优势、盐酸优势和奎宁优势反应神经元对盐酸和硫酸奎宁的反应在电损毁杏仁中央核后也明显增强。 在破坏杏仁中央核后, 臂旁核味觉神经元对氯化钠和硫酸奎宁苦味的分辨能力降低。以上结果提示, 杏仁中央核在大鼠脑桥水平的味觉编码中发挥重要作用, 它可能是通过参与对味觉的影响来调节机体的摄食行为。
关键词: 臂旁核; 味觉神经元; 杏仁中央核; 电损毁; 大鼠
中图分类号: Q434
The amygdala is associated with control of emotion, motivation and hedonic tone[1,3]. It is characterized as a region where representations of the internal and external worlds overlap, permitting the animal to assess its needs in relation to the external resources available to fulfill them[8]. Behavioral study has shown that hypophagia and weight loss often accompany lesions of the amygdala in the rat[2]. Since feeding behavior is largely guided by the sense of taste, involvement of the amygdala in the control of feeding implies its sensitivity to gustatory input. In spite of the structural and functional heterogeneity of the amygdala, one subregion, the central nucleus of the amygdala (CeA), plays an important role in the central evaluation of gustatory inputs in the rat. Behavioral studies have shown that lesion of the CeA changes the taste reactivity and sodium appetite of the rat[6,20]. One interpretation of this modulation is that CeA normally regulates gustatory processing in the brain stem. Anatomical evidence has shown that the CeA receives gustatory information from the pontine parabrachial nucleus (PBN)[11] and, in turn, sends fibers to the PBN and the nucleus of the tractus solitarius (NTS)[19,24], both of which constitute major relays of the taste pathways in rodents. By using electrophysiological method, Lundy et al.[9] have shown that stimulation of the CeA depresses the pontine taste activity in rat. However, no reports have so far been made on the effect of the CeA lesion on gustatory response in the PBN. The aim of the present study was to assess the effect of CeA on gustatory processing at the pontine level and to make a better understanding how the CeA is involved in the normal response to exteroceptive food stimuli. We recorded responses of single PBN neurons to gustatory stimuli applied before and after the bilateral electrolytic lesion of the CeA.
1 MATERIALS AND METHODS
1.1 Subjects. Twenty-nine Sprague-Dawley rats weighing 220－290 g were housed individually in stainless steel cages with free access to food; water was available ad lib except on the experiment day. Room lights were on 12 h per day and temperature was maintained at (25±1)℃.
1.2 Surgery． The rats were anesthetized with ethyl carbamate (urethane) (1.4－1.5 g/kg, i.p.), and were secured in a stereotaxic instrument by using nonpuncture ear bars and a bite bar. For the bilateral CeA lesions, two 2 mm trephine holes were centered 4.0 mm lateral to the midline, 2.4 mm posterior to bregma. Two stainless steel wires (insulated except at the last 0.5 mm of the tip) were penetrated so that the ventral tips were located in the CeA (7.1 mm below the brain surface). The parameters for the CeA lesion were 0.4 mA constant current for 60 s. For access to the taste responsive area of the PBN, the transverse sinus overlying the PBN was ligated and retracted. The electrocardiogram was continuously monitored and the rectal temperature was maintained at about 37℃ by using a heating pad throughout the experiment.
1.3 Recording. Extracellular activity from PBN
neurons was isolated using a glass microelectrode filled with 2% pontamine sky
blue in 0.5 mol/L sodium acetate (15－20 MΩ impedance at 1 kHz). During the
search procedure, the gustatory neuron was identified by applying a mixture of
four standard tastants to the tongue. Action potentials were recorded through
conventional physiologic equipment consisting of a preamplifier (MEZ-8201,
1.4 Gustatory stimuli. Gustatory stimuli consisted of NaCl (0.3 mol/L), HCl (0.01 mol/L), Quinine-sulfate (QH2SO4) (0.0015 mol/L), and sucrose (0.5 mol/L). Fluid stimuli were delivered through a length of slender tubing, closed at the end of the tubing which was extensively perforated along its final 2 cm. The perforated end was slipped into the mouth, held just slightly agape, so that the tastants could be delivered by gravity flow from a system of overhead funnel attached at one end to a reservoir containing a particular tastant or water. Each stimulus trial consisted of a 10 s flow of distilled water, a 10 s taste stimulus, a 10 s wait, and a 20 s rinse with distilled water. The flow rate was 2 ml/s for all stimuli including the rinse. Ninety seconds were allowed to elapse between stimuli to avoid adaptation. If the gustatory responses were tested more than once, means were used. Markers of the taste stimulus onset were stored concurrently in another channel to facilitate off-line analysis.
1.5 Data analysis. Neural responses to a taste stimulus were calculated by subtracting the 5 s discharge rate to each stimulus, beginning with the onset of a stimulus infusion, from its 5 s discharge rate to water. An increase in firing rate in the first 5 s of the stimulus presentation that exceeded the spontaneous spike by at least 50% was defined as a response. An inhibitory response was defined as a fall in the spontaneous firing rate of at least 50%. The neurons in the present study were classified as inhibited ones or augmented ones if these response measures differed from their corresponding control rates by <20% or >20%, respectively. After the recording sessions, the recording site was marked. Rats were perfused transcardially, first with saline followed by 10% formalin. The location of each recording and lesion site was histologically examined.
To assess the across-unit differences among stimuli, the Pearson produce-moment correlations across stimuli were determined for PBN units before and after amygdaloid lesion[14]. As an indication of the breadth of tuning, an uncertainty measure was calculated with the following formula H=－1.661∑pI log pI, where pI is the proportion of the response to each of the stimuli against the total response to all the stimuli[17]. Based on its response to a standard concentration of each of the four sapid chemicals, each neuron was categorized according to its best stimulus. Since the main response in best-stimulus category plays a more important role in taste coding [16], the effects of the CeA lesions on the response in best-stimulus categories were examined in the present study. One-way ANOVA was applied where it was appropriate for comparisons of neural responses. Data analyses were made by using SPSS software and P<0.05 was considered significant.
2 RESULTS
Taste-responsive neurons were located in the caudomedial quadrant of the PBN extending from near the dorsal surface of the pons through the brachium conjunctivum (BC) and into the compact layer of cells between the BC and the mesencephalic trigeminal nucleus. Figure 1 shows line drawings that correspond to three different levels of the PBN. The area matched the classically defined taste responsive zone of the PBN[14]. Neurophysiological data in the present study were obtained from animals in which the bilateral electrolytic lesions histologically conformed to terminate in the CeA. If other subnuclei surrounding the CeA were damaged, the results of these rats were excluded from the analysis.
Fig.1.The areas in which taste-responsive neurons were isolated. The approximate area in which taste-responsive neurons were isolated at different PBN levels is illustrated by a filled oval in the 3 line drawings. LPB, lateral parabrachial nucleus; Me5, mesencephalic trigeminal nucleus; MPB, medial parabrachial nucleus.
2.1 Effects of the CeA lesion on general response characteristics in the PBN
Gustatory responses from 29 PBN neurons were observed before and after bilateral electrolytic lesion of the CeA. Of the 29 PBN neurons, the CeA lesion augmented firing rates in 21, inhibited the rates in 2, and were ineffective in 6. In general, PBN units tended to respond to more stimuli after the CeA was destroyed. However, Uncertainty measures, an indication of the breadth of tuning, were not significantly different (P=0.12, n=29) (mean Uncertainty before CeA lesion, 0.70 ± 0.05 SEM; after CeA lesion, 0.72 ± 0.05).
Mean spontaneous firing rate (7.67 ± 1.06 Hz) of the PBN units after the CeA lesions was significantly higher than that (4.21 ± 0.79 Hz) before lesions (F1, 57 = 6.86, P<0.05, n=29). As shown in Fig.2, the mean evoked-firing rates to HCl (33.1±4.66 Hz) and to QH2SO4 (17.7±3.46 Hz) after CeA lesions were significantly higher than those (13.2±2.07 Hz and 7.36±1.61 Hz) before lesions (P<0.01, n=29), but there were no significant differences for responses to NaCl and sucrose (P>0.05).
2.2 Effects of the CeA lesion on the different types of gustatory neurons of the PBN
On the basis of their largest responses to the four sapid stimuli in the 29 PBN taste neurons, 14 were NaCl-best, 9 HCl-best, 3 QHCl-best and 3 sucrose-best in the intact rats. However, after the CeA lesions, 7 neurons responding to the NaCl as their best stimulus before the CeA lesion changed into HCl-best neurons, as shown in Fig. 3. The gustatory responses of NaCl-, HCl-, QH2SO4-, and sucrose-best neurons in the intact and CeA lesioned rats were calculated respectively (Fig.4). Separate one-way ANOVA tests revealed that the main responses in HCl-best (F1,17=7.16, P<0.05) and QH2SO4-best units (F1,5=115.9, P<0.01) were significantly enhanced after the bilateral lesions of the CeA. Furthermore, the response to HCl (F1,27=7.39, P<0.05) in NaCl-best and the response to QH2SO4 (F1,17=5.89, P<0.05) in HCl-best also significantly increased. However, the responses in sucrose-best units in the CeA damaged rats were not significantly different from those in the intact rats. The spontaneous firing rates in each best-stimulus category of PBN neurons after the CeA lesions did not significantly change.
Fig.2.Mean responses of all PBN taste neurons before and after CeA lesions. Bar graphs showing the mean response rates (spikes/s±S.E.M) for all PBN units before and after bilateral electrolytic lesion of CeA. Abbreviations: H, HCl; N, NaCl; Q, QH2SO4; S, sucrose. **P<0.01 indicates statistical significances compared with that before CeA lesions.
2.3 Effects of the CeA lesion on across-unit patterns of responses in the PBN
The analyses of Pearson produce-moment interstimulus correlations before and after the CeA lesion are presented in Table 1.Except for the correlation between NaCl and QH2SO4, which decreased in some degree, the interstimulus correlations among the other basic tastants were comparable before and after the bilateral electrolytic lesion of CeA.
Fig.3.Responses of a NaCl-best neuron before and after CeA lesions. Oscilloscope recordings show the responses of a NaCl-best neuron before and after the bilateral CeA lesions. Abbreviations: Qu, QH2SO4; Su, sucrose. Arrow indicates the onset of the taste stimuli. Scale bar， 1 s.
Fig.4.Mean gustatory responses of four types of PBN neurons. Bar graphs showing the mean response rates of four types of neurons in the PBN responding to 4 basic tastants before and after the lesions of the CeA. *P<0.05 and **P<0.01 indicate statistical significance compared with that before CeA lesion. Abbreviations: H, HCl; N, NaCl; Q, QH2SO4; S, sucrose.
Table １.Pearson produce-moment interstimulus correlations
|
|
NaCl |
HCl |
QH2SO4 |
|
Before CeA stimulation (n=29) |
|
|
|
|
HCl |
0.783 |
|
|
|
QH2SO4 |
0.485 |
0.512 |
|
|
Sucrose |
0.347 |
0.306 |
0.353 |
|
After CeA stimulation (n=29) |
|
|
|
|
HCl |
0.792 |
|
|
|
QH2SO4 |
0.694 |
0.559 |
|
|
Sucrose |
0.361 |
0.320 |
0.377 |
3 DISCUSSION
In the present study, we provided clear evidence for a significant role of the CeA in the control of activities of the PBN taste neurons. Although the PBN taste neurons were more responsive after the bilateral CeA lesion, only the magnitudes of the response to HCl and QH2SO4 significantly increased. Based on the best-stimulus category, the main responses of HCl- and QH2SO4-best neurons were also significantly facilitated after the CeA damage. Analysis of Pearson produce-moment interstimulus correlations showed that the correlation between NaCl and QH2SO4 was larger after the CeA damaged, which indicates the CeA lesion makes the discrimination between NaCl and QH2SO4 more difficult. The spontaneous activity of the PBN taste neurons was significantly enhanced after the CeA lesion. It suggests that the CeA may exert a nonspecific effect on the PBN taste neurons.
The apparent influence of the CeA on the gustatory responses in the PBN reveals that the CeA plays a significant role in the central evaluation of taste. Also in our experiments, the results of 14 rats were not included owing to the damaged areas outside the CeA. Among the 14 rats, taste responses in PBN were inhibited only in 5. The proportion (5/14) is greatly lower than that reported above, which may indicate that the effects of CeA on PBN are different from that of adjacent nuclei surrounding the CeA. In previous study, Sun et al.[18] showed that the neurons providing the major CeA outputs to the brainstem received an extensive GABAergic innervations and these neurons are under strong tonic inhibition by intrinsic GABAergic neurons. Furthermore, the input from the GC to the CeA preferentially innervates these intrinsic GABAergic neurons. These anatomic evidence may interpret the reason why the CeA lesion enhanced most gustatory responses in the PBN.
However, although there is only a light projection from the CeA to the PBN[19,24], most PBN units record-ed in the present study were affected by the bilateral electrolytic lesion of the CeA. This may indicate that the activation of the CeA influences the PBN neurons through either a direct contact or an indirect projection via some other forebrain regions. The amygdala connects not only with the PBN but also with other taste responsive regions, such as the GC[22], LH[12] and NTS[13,23]. Previous studies have suggested that the neurons in the CeA may serve as an interface between the principal inputs and outputs of the GC[18]. Furthermore, electrolytic lesion cannot differentiate between activation of intrinsic neurons and fibers of passage. As already reported, the GC[4,5] or the LH[10] can modulate the gustatory responses in the PBN or the NTS. The elimination of the CeA output might affect these taste-related areas that in turn project to the PBN.
In the present study, it is clear that CeA can selectively modify the responses to aversive taste such as sourness and bitterness without affecting the responses to other tastants. This result essentially accords with the findings of Touzani et al.[20], which suggest that the aversive value of taste stimuli is enhanced following lesion of the CeA. Combined with the fact that the Pearson produce-moment correlation between HCl and QH2SO4 with the CeA lesion is not different from that in the control rats, it reveals that the CeA may be more concerned with general distinctions between palatable and unpalatable stimuli rather than the fine-grained distinctions among the various taste qualities. It has been postulated that ingestion is a complex central evaluation that is influenced by the internal state of the animal[15]. The anatomical connections of the CeA are well suited to modulate gustatory information processing in the PBN based on physiological demand. As one of primary taste relays, the PBN plays an important role in feeding behavior, and the gustatory responses of which can be greatly changed in different internal states[7]. The CeA is a taste relay between the primary gustatory cortex, where neurons mainly receive taste inputs, and the LH where are implicated in the central evaluation of palatability and may adjust the level of palatability to the internal state[21]. Taken together, it suggests that the CeA, probably acting in concert with the LH, regulates the feeding behavior of animals via altering the gustatory responses of pontine neurons. Despite a lack of knowledge about how rostral brain structures might interpret this altered gustatory information, the present results provide clues to a neurophysiological relationship between the CeA and the PBN in the control of feeding.
Interstimulus correlation analyses indicate that CeA lesion reduces the discrimination between NaCl and QH2SO4. Bitterness was commonly evoked by toxin, so the sensitivity to the bitterness is directly proportional to the susceptibility to toxicosis. As we know, the CeA participates in the maintenance of body fluid balance. For instance, bilateral lesions of the CeA disrupt the sodium appetite that develops following furosemide[6]. The role of the gustatory system is to carry the neural information that signals the presence of the Na+ ion. It suggests that the CeA may maintain the body fluid balance via modulating the gustatory responses of neurons in the PBN.
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