Yasunobu Yasoshima

Neural substrates for conditioned taste aversion in the rat

Conditioned taste aversions (CTAs) are well known to be robust and long-lasting instances of learning induced by a single CS (taste)-US (malaise) pairing. CTA can be taken as a general model to search for neural mechanisms of learning and memory. In spite of extensive research on CTAs using a variety of approaches during the last three decades, the neural mechanisms of taste aversion learning still remain unsolved. In this article we propose a model of neural substrates of CTAs on the basis of our recent studies incorporating previous findings by other workers. Our studies mainly included experiments using ibotenic acid injections into various parts of the rat brain as a lesion technique, and c-fos immunohistochemistry in naive and CTA trained rats. CTAs were established by pairing the ingestion of saccharin (CS) with an ip injection of LiCl (US). Behavioral studies have shown that the parabrachial nucleus (PBN), medial thalamus, and basolateral nucleus of the amygdala are essential for both acquisition and retention of CTAs. C-fos studies suggested that association between gustatory CS and visceral US takes place in the PBN. The gustatory cortex (GC) may modify the strength of this association depending on the nature of the CS, viz., novel or familiar. The amygdala is indispensable for the expressions of CTAs. Tastes with hedonic values are stored in the GC in a long-term 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.

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.

Short-term and long-term excitability changes of the insular cortical neurons after the acquisition of taste aversion learning in behaving rats

Conditioned taste aversion, a long-lasting type of learning established after a single pairing of a novel taste and subsequent internal malaise, is an adaptive behavior to prevent animals from repeated intakes of poisonous substances. The present study was designed to identify the time-dependent excitability changes of cortical neurons to gustatory stimuli after the acquisition of conditioned taste aversion in freely behaving rats. Conditioned taste aversion to saccharin was established by an intraperitoneal injection of lithium chloride, a sickness-inducing agent, soon after an intraoral infusion of saccharin. Twenty minutes after the pairing, 25 (29%) of 86 rats showed aversive taste reactivities to saccharin, and 30 min after the pairing, all of the rats showed aversive behaviors to saccharin; these behavioral changes lasted throughout the test session (over 360 min). When unit activities were recorded from the insular cortex simultaneously with the behavioral test, 14 (11%) of 122 neurons showed a significant enhancement of excitability in response to saccharin, but not to other taste stimuli, after the acquisition of taste aversion. Eight of these 14 neurons showed a short-term enhancement: significant effects were detected only 30 min after the pairing. The remaining six neurons exhibited a long-term enhancement: the effects lasted over 360 min after the pairing. The existence of such short-term and long-term excitability changes suggests that the gustatory insular cortex is involved in different aspects of taste aversion learning.

Memory-dependent c-Fos expression in the nucleus accumbens and extended amygdala following the expression of a conditioned taste aversive in the rat

Retrieving the memory of a conditioned taste aversion involves multiple forebrain areas. Although the amygdala clearly plays a role in the expression of a conditioned taste aversion, critical regions, downstream from the amygdala remain to be defined. To this end, Fos immunoreactivity was used in the rat to explore forebrain structures associated with retrieval that have an anatomical relationship with the amygdala. The results showed that expression of a conditioned taste aversion to 0.5 M sucrose elicited neuronal activation in the nucleus accumbens and in a complex of structures collectively referred to as the extended amygdala. The posterior hypothalamus and parasubthalamic nucleus, which receive inputs from the extended amygdala, were also activated upon re-exposure to the sucrose conditioned stimulus. Fos immunoreactivity did not increase in these regions in response to an innately aversive tastant, quinine hydrochloride (conditioned stimulus control), nor to LiCl-induced visceral stimulation in unconditioned animals (unconditioned stimulus control). In addition, these regions did not respond to the sucrose conditioned stimulus in sham-conditioned animals. These results suggest that conditioned and innately aversive tastes are differentially processed in the forebrain circuitry that includes the nucleus accumbens and extended amygdala.

Decline of striatal dopamine release in parkin-deficient mice shown by ex vivo autoradiography.

Parkin is the causal gene of autosomal recessive juvenile parkinsonism (AR‐JP). Dopamine (DA) metabolism has been linked to Parkinsons disease (PD). To understand the pathogenesis of AR‐JP, we generated parkin‐deficient mice to assess the status of DA signaling pathway and examine DA release and DA receptor by ex vivo autoradiography. Ex vivo autoradiography using [11C]raclopride showed a clear decrease in endogenous DA release after methamphetamine challenge in parkin‐deficient mice. Furthermore, parkin deficiency was associated with considerable upregulation of DA (D1 and D2) receptor binding in vivo in the striatum and increased DA levels in the midbrain. Our results suggest that dopaminergic neurons could behave abnormally before neuronal death.

Differential activation of anterior and midline thalamic nuclei following retrieval of aversively motivated learning tasks

Two thalamic nuclear groups, the anterior thalamic nuclei (ATN) and midline and intralaminar thalamic complex (MITC) have connections to the prefrontal cortex, amygdala, hippocampus and accumbens that are important for learning and memory. However, the anatomical proximity between the ATN and MITC makes it difficult to reveal their roles in memory retrieval of aversive conditioned behavior. To address the issue, we explored the activation of the ATN and MITC, as represented by the expression of the immediate early gene c-fos, following either the retrieval of a conditioned taste aversion (CTA) induced by taste-LiCl pairing (visceral aversion) or of inhibitory avoidance (IA) induced by context-foot shock pairing (somatic aversion) in rats. The anterodorsal (AD) nucleus in the ATN was activated by foot shock and the recall of IA, but not by i.p. injection of LiCl or the recall of CTA. No significant elevation was observed in the other ATN following these treatments. Among nuclei of the MITC, the paraventricular thalamic nucleus (PVT) was activated by the delivery of shock or LiCl and by the recall of both CTA and IA, while the mediodorsal thalamus (MD) and central medial and intermediate thalamus (CM/IMD) were not. The innately aversive taste of quinine did not elevate c-fos expression in either the ATN or MITC. These results suggest that the PVT in the MITC is involved in the processing and retrieval of both taste-malaise and context-shock association tasks, while the AD in the ATN is involved in those of context-shock association only. The difference of the activity between the ATN and MITC demonstrates their functional and anatomical heterogeneity in neural substrates for aversive learning tasks.

Subthalamic Neurons Coordinate Basal Ganglia Function through Differential Neural Pathways

The subthalamic nucleus (STN) is a key component of basal ganglia circuitry that mediates a variety of motor functions. The STN neurons send glutamatergic projections to the output structures of basal ganglia, including the substantia nigra pars reticulata (SNr) and the entopeduncular nucleus, and also innervate the globus pallidus (GP). However, the mechanism by which the STN regulates motor functions in the neural circuitry is not fully understood. Here we performed conditional ablation of the STN neurons by using immunotoxin-mediated cell targeting. We then analyzed dopamine (DA)-mediated motor behavior and firing activity of the SNr and GP neurons. Ablation of the STN neurons increased spontaneous movement and reduced hyperactivity in response to DA stimulation. Ablation of these neurons modulated the pattern and rate of spontaneous firing of the SNr neurons, although it did not substantially affect spontaneous firing of the GP neurons. The ablation attenuated DA-induced suppression of the firing rate of the SNr neurons and inhibited DA-induced elevation of the rate of the GP neurons. In addition, pharmacological blockade of GP activation in response to DA stimulation inhibited the suppression of SNr activity and the resultant motor activation. These results suggest that the STN neurons suppress spontaneous behavior through their direct projection to the output neurons and that, in response to DA, they contribute to expression of behavior by acting on the output neurons mainly through the GP-mediated pathways. We conclude that the STN coordinates motor behavior through differential neural pathways depending on the state of DA transmission.

Effects of midazolam on the expression of conditioned taste aversion in rats

In conditioned taste aversion (CTA), the animals learn to avoid a taste substance (conditioned stimulus, CS) which was previously associated with visceral distress (unconditioned stimulus, US). The present study examined the effects of administration of midazolam (MDZ), a benzodiazepine agonist, after the acquisition of CTA on the expression of CTA. After ingestion of 0.5 M sucrose (CS) was paired with an intraperitoneal (i.p.) injection of 0.15 M LiCl (US), control rats showed strong CTA to the CS. However, a systemic injection of MDZ (1.5 mg/kg, i.p.) before the retention test prevented conditioned animals from rejecting the CS, but in the subsequent retention tests where the drug was not administrated, those animals again showed strong aversions to the CS. Aversive taste reactivity patterns to the intraorally infused sucrose and 0.3 M dl-alanine in the conditioned animals were also diminished by the similar injection of MDZ, but not by a serotonergic anxiolytic agent, buspirone (2.5 or 5.0 mg/kg, i.p.). General taste sensory deficit might not be induced by MDZ because the drug injection did not impair conditioned aversions to 0.2 M NaCl and 0.01 M HCl. Infusion of MDZ into the basolateral nucleus of the amygdala (BLA) also attenuated conditioned aversions to sucrose. These results suggest that systemic or intra-BLA administrations of MDZ impair the expression of CTA selectively to sweet-tasting substances, implying that a transient MDZ-induced CTA expression deficit is due to the enhancement of palatability of CSs with preferable tastes rather than general anxiolytic or amnesic effects of MDZ.