Takashi Yamamoto

Conditioned taste aversion in rats with excitotoxic brain lesions

Conditioned taste aversion (CTA) is well known to be a robust and long-lasting learning after a single conditioned stimulus (CS) (taste)--unconditioned stimulus (US) (malaise) pairing. The neural mechanisms of this taste aversion learning still remain to be resolved. To elucidate the basic brain mechanisms of the taste aversion learning, we examined the effects of lesions of various sites of the rat brain on the acquisition and retention of CTAs. Confined brain lesions were made by injections of a small amount of excitotoxic drug, ibotenic acid. CTAs were established to saccharin (CS) by pairing its ingestion with an i.p. injection of LiCl (US). Rats lacking the parabrachial nucleus (PBN) almost completely failed to acquire CTAs. The second most effective lesion was in the medial thalamus including the parvocellular part of the ventral posteromedial nucleus of the thalamus (VPMpc) and the midline part, followed by the damage of the lateral nuclear group of the amygdala including the basolateral amygdaloid nucleus. Lesions of the gustatory cortex (GC) and hippocampus induced moderate effects, but lesions in the other subnuclei of the amygdala, such as the medial and central amygdaloid nuclei, entorhinal cortex, lateral hypothalamic area, and ventromedial hypothalamic nucleus induced slight or no effects. On the other hand, paired lesions among the amygdala, medial thalamus and GC caused severe impairment of CTAs; in particular, lesions of amygdala and VPMpc completely disrupted acquisition of CTAs. These results suggest that the PBN, medial thalamus and the lateral nuclear group of the amygdala play an essential role in the formation of taste aversion learning.

Conditioned taste aversion and c-fos expression in the rat brainstem after administration of various USs

CONDITIONED taste aversion (CTA) is a form of association learning established when an animal associates a conditioned stimulus (taste) with a subsequent unconditioned stimulus (US, illness). We have studied the relationship between the efficacy of inducing CTA to 0.1% saccharin and c-fos expression in the lower brain stem following administration of 13 different USs in rats. The effective USs were grouped into abdominal irritants, rewarding drugs and emetic agents. Regardless of the properties of USs, good correlation was detected between the strength of CTA and c-fos expression within the area postrema, caudal and intermediate subdivisions of nucleus tractus solitarius and the external lateral subnucleus of the parabrachial nucleus. Only hypertonic saline was exceptional because of the experimental procedure: it induced strong c-fos expression, but was not an effective US for CTA formation. Different dosages of the emetic LiCl induced CTA and c-fos expression in a dose-dependent manner.

Different disruptive effects on the acquisition and expression of conditioned taste aversion by blockades of amygdalar ionotropic and metabotropic glutamatergic receptor subtypes in rats.

Conditioned taste aversion (CTA) is based on the gustatory long-term memory established after association of the taste of food (conditioned stimulus, CS) with visceral signals of poisoning (unconditioned stimulus, US). After the acquisition of CTA, hedonics of the taste CS changes from positive to negative as indicated by reduced ingestive and increased aversive taste reactivities in response to re-exposures to the CS. We examined the effects of reversible and selective blockades of the amygdalar glutamate receptor subtypes, AMPA, NMDA and metabotropic glutamate receptors, on the formation of CTA. Blockades of each of the three receptor subtypes between ingestion of saccharin (CS) and malaise-inducing LiCl (US) disrupted the acquisition of CTA. After the acquisition of CTA, however, blockades of only AMPA receptors, but not NMDA or metabotropic receptors, impaired the expression of CTA. This effect was seen only during the period when the antagonistic action to AMPA receptors lasted. These results indicate that both ionotropic and metabotropic glutamate receptor subtypes in the amygdala are indispensable for the acquisition of CTA, but that the expression of acquired CTA is mediated only by AMPA receptors. The present results also suggest that the amygdalar glutamatergic neural transmission is involved in the formation and storage of long-term gustatory memory associated with the altered hedonics from positive to negative.

Comparison of C-fos-like immunoreactivity in the brainstem following intraoral and intragastric infusions of chemical solutions in rats

To examine whether the activation of brainstem neurons during ingestion is due to orosensory afferents or post-ingestive factors, neuronal activation in response to intraoral and intragastric infusions of taste stimuli was compared in the area postrema (AP), nucleus tractus solitarius (NTS) and parabrachial nucleus (PBN) by the c-fos immunohistochemical method. An aliquot (7.5 ml) of 0.5 M sucrose, 5 mM sodium saccharin, 1 mM quinine hydrochloride and distilled water was delivered into the oral cavity or the stomach in each rat, which had been deprived of water and food overnight. Water induced little c-Fos-like immunoreactivity (c-FLI), but both intraoral and intragastric infusions of sucrose, but not non-caloric saccharin, induced strong c-FLI in the AP, caudal NTS and the external lateral subnucleus of the rostral PBN, suggesting that these areas receive general visceral inputs. Other areas in the NTS and PBN may receive gustatory inputs since more dominant c-FLI was detected by intraoral rather than intragastric infusions of the stimuli. Functional segregation of neurons reflecting qualitative and hedonic aspects of sweeteners (sucrose and saccharin) and bitter-tasting substance (quinine) was suggested in the PBN, but less evident in the NTS. These results indicate that c-fos induction in brainstem neurons during ingestion reflects gustatory inputs and postingestional factors depending on the kind of food ingested.

Effects of taste stimulation on β-endorphin levels in rat cerebrospinal fluid and plasma.

Opioids are suggested to be involved in generation of palatability and facilitation of consumption of food and fluid. We measured the level of an endogenous opioid, beta-endorphin, in the cerebrospinal fluid (CSF) and plasma after free drinking of water and taste solutions in Wistar rats. When the water-deprived animals were allowed to drink 10 mL of water, the level of beta-endorphin increased significantly 60 and 90 min after the start of drinking in both samples. beta-Endorphin in the CSF increased most after ingestion of 0.5 M sucrose and 0.005 M saccharin followed by 0.1 M NaCl, 0.1 mM quinine and water. An intragastric infusion of 7 mL of water did not change the beta-endorphin level. Essentially the same results were obtained for plasma samples except that NaCl and quinine solutions did not increase beta-endorphin levels. Sucrose became ineffective in releasing beta-endorphin in both samples after the establishment of conditioned taste aversions to this taste stimulus. These results suggest that the release of beta-endorphin is positively correlated with the palatability of taste stimuli, and that CSF beta-endorphin also reflects the reinforcement of fluid intake in thirsty animals.

Gustatory and visceral inputs to the amygdala of the rat: conditioned taste aversion and induction of c-fos-like immunoreactivity

Expression of proto-oncogene c-fos was immunohistochemically examined in the central and basolateral amygdaloid nuclei in rats after ingestion of taste solutions (0.5 M sucrose or 0.005 M saccharin), intragastric infusion of these solutions, or an intraperitoneal injection of malaise-inducing lithium chloride (LiCl). C-Fos-like immunoreactive neurons were distributed most densely in the central nucleus in response to the LiCl injection, followed by the ingestion and intragastric infusion of sucrose. The intraoral infusion of sucrose, but not of saccharin, elicited intense c-fos expression in the central nucleus after establishment of conditioned taste aversion to these taste stimuli. The results are discussed in terms of post-ingestional factors and the conditioned illness reaction after taste aversion learning.

Possible routes of visceral information in the rat brain in formation of conditioned taste aversion.

When ingestion of a taste stimulus is paired with internal malaise, the animal remembers the taste and rejects its ingestion thereafter. This learning is referred to as conditioned taste aversion (CTA). To establish CTA in adult male Wistar rats, 0.1% saccharin and an i.p. injection of 0.15 M LiCl were used as the conditioned and unconditioned stimuli, respectively. Neuroanatomical study using the tracer method was performed to examine the ascending routes from the lateral part of the parabrachial nucleus (PBlat) which receives general visceral information and suggested the three possible routes to the amygdala: (1) direct route to the central nucleus of the amygdala (CeA); (2) diencephalic route to the basolateral nucleus of the amygdala (BLA) involving the zona incerta (ZI) and the midline and intralaminar thalamic complex (MITC); and (3) cortical route to the BLA involving insular cortex (IC). Rats with excitotoxic lesions of each of the CeA, ZI, MITC or IC had only a small or negligible effect on the acquisition of CTA. However, single lesions of the BLA and combined lesions of the ZI and IC, but not CeA and IC, almost completely abolished the acquisition of CTA. These results together with previous findings suggest that visceral (or unconditioned stimulus) information in the PBlat is sent to the BLA which is essential for the acquisition of CTA via the functionally important two parallel routes, the diencephalic and cortical routes, with either being able to create the aversion.

Single unit responses of the amygdala after conditioned taste aversion in conscious rats

Amygdalar neuronal responses to sodium saccharin used as the conditioned stimulus (CS) and to other taste stimuli including sucrose, NaCl, HCl and quinine hydrochloride were recorded before and after the acquisition of conditioned taste aversion (CTA) in freely behaving rats. Of 73 units recorded from the basolateral nucleus of the amygdala (BLA), 17 (23%) and 1 (1%) exhibited facilitatory and inhibitory responses, respectively, to both the CS and sucrose after aversive conditioning to the CS. On the other hand, 3 (5%) and 11 (17%) of 64 units recorded from the central nucleus of the amygdala (Ce) exhibited facilitatory and inhibitory responses, respectively. The responsiveness of these BLA and Ce units to other taste stimuli did not change significantly. These findings that the facilitatory effect was dominant in the BLA, while the inhibitory effect was more frequent in the Ce suggest that the BLA and Ce are differentially involved in CTA.

Rat gustatory memory requires protein kinase C activity in the amygdala and cortical gustatory area

WE have studied the physiological involvement of protein kinase C (PKC) in the formation of conditioned taste aversion (CTA) by means of microinjections of PKC inhibitors into the gustatory cortex (GC), amygdala (AMY) and thalamic gustatory area at various timewindows of the CTA paradigm. Rats injected between the CS–US interval with PKC inhibitors into the GC and AMY, but not into the thalamic gustatory area, failed to acquire CTA. Injections of PKC inhibitors 4 h after the US presentation or just before the retention test elicited no disruptive effect. Injections of PKC inhibitor into the AMY, but not into the GC, 30 min after the CS–US pairing impaired CTA formation. These results show that PKC activity in the GC and AMY has a key role in the acquisition phase of CTA, but not in the retrieval phase. The findings also suggest that the GC is concerned with information processing of the CS, and that the AMY is involved in the CS–US association.

Role of the medial and lateral parabrachial nucleus in acquisition and retention of conditioned taste aversion in rats.

When ingestion of a taste stimulus is paired with internal malaise, the animal remembers the taste and rejects its ingestion thereafter. This learning is referred to as conditioned taste aversion (CTA). To establish CTA in adult male Wistar rats, 0.1% saccharin and an i.p. injection of 0.15 M LiCl were used as the conditioned and unconditioned stimuli, respectively. To elucidate the functional role of the medial part of the parabrachial nucleus (PBmed) which receives taste information and the lateral part (PBlat) which receives general visceral information, confined electrolytic lesions were made to either of these regions. Rats with bilateral lesions of the PBlat impaired the acquisition of CTA, but those lesions made after the acquisition of CTA had no effect on the retention of this learning. The bilateral lesions of the PBmed abolished the acquisition and retention of CTA. The PBlat-lesioned rats showed normal taste preference behavior, but PBmed-lesioned rats showed impaired sensibility to taste stimuli. These results suggest that both the PBlat and PBmed are essential for the acquisition of taste aversion learning, but the PBlat is not necessary for retrieval of CTA.