Tadashi Inui

Functional organization of the rodent parabrachial nucleus.

The rodent parabrachial nucleus (PBN) is not merely a sensory relay station but also plays an important role in integrating various ascending and descending inputs together with plastic changes of neuronal responses after learning and experience. The limbic and reward systems receive ingestion‐related information via the cortical areas in primates, whereas in rodents the information is sent to these systems mostly via the PBN. To explore how the rat PBN is functionally organized, we detected activation patterns of neurons mainly by means of c‐fos immunohistochemistry to show neuronal activation in different situations of ingestive behavior. The expression pattern was different under nutritionally replete and deficient conditions, perceptually new and familiar conditions, and learned and unlearned conditions. As for the possible functions, the rostral part of the external lateral subnucleus is related to general visceral inputs; the caudal part of the external lateral subnucleus, aversive behavior; the dorsal lateral subnucleus, ingestive behavior; and the central medial subnucleus, taste of NaCl. Because several genes were localized in specific subnuclei, we are trying to correlate the gene expressions with possible functional significance.

Cannabinoid in the nucleus accumbens enhances the intake of palatable solution.

It has been suggested that the activation of cannabinoid receptors in the nucleus accumbens shell facilitates feeding behavior. However, it remains unclear whether cannabinoid ligands enhance the palatability of food to facilitate feeding. In this study, we examined whether microinjecting an endogenous cannabinoid agonist, anandamide into the nucleus accumbens shell would affect the intake of water, sweet or bitter solutions. Microinjections of anandamide into the nucleus accumbens shell selectively increased the intake of saccharin (sweet) solution, but had no effect on the intake of water nor quinine (bitter) solution. These results suggest that activation of the cannabinoid receptors in the nucleus accumbens shell selectively facilitates the intake of palatable foods and solutions.

Effects of brain lesions on taste-potentiated odor aversion in rats

Rats failed to acquire aversions to odor stimulus, which was followed 30 min later by an unconditioned stimulus (US). However, when the odor stimulus was accompanied by a taste stimulus, they acquired odor aversions as well as taste aversions. In this phenomenon, referred to as a taste-potentiated odor aversion, lesions of the amygdala disrupted both taste and odor aversions, whereas lesions of the parvicellular part of ventroposteromedial thalamic nucleus (VPMpc) or insular cortex (IC) disrupted taste aversion but attenuated only odor aversion. These results suggest that both taste and odor stimuli are associated with US in the amygdala and that taste inputs delivered to the amygdala through the IC and/or VPMpc play an important role in potentiation of odor aversion.

The brain mapping of the retrieval of conditioned taste aversion memory using manganese-enhanced magnetic resonance imaging in rats.

Manganese-enhanced MRI (MEMRI) is a newly developed noninvasive imaging technique of brain activities. The signal intensity of MEMRI reflects cumulative activities of the neurons. To validate the use of MEMRI technique to investigate the neural mechanisms of learning and memory, we tried to map brain areas involved in the retrieval of conditioned taste aversion (CTA) memory. CTAs were established to saccharin (conditioned stimulus: CS) by pairing its ingestion with an i.p. injection of LiCl (unconditioned stimulus: US). LiCl solutions (as a robust aversion chemical) of 0.15 M were injected i.p. 15 min after drinking the saccharine solution (CS). After the two times conditionings, these rats showed a robust aversion to the saccharine solution (CS). Rats of the control group were injected saline i.p. instead of LiCl solutions. The MRI signal intensities at the gustatory cortex (GC), the core subregion of the nucleus accumbens (NAcC), the shell subregion of the nucleus accumbens (NAcSh), the ventral pallidum (VP), the central nucleus of amygdala (CeA), the lateral hypothalamus (LH), and the basolateral nucleus of amygdala (BLA) of the conditioned group were higher than those of the control group. There were no significant differences between the conditioned and the control groups in the intensities for other regions, such as the striatum area, motor cortex, cingulate cortex, interstitial nucleus of the posterior limb of the anterior commissure and hippocampus. These indicate that the GC, NAcC, NAcSh, VP, CeA, LH and BLA have important roles in the memory retrieval of CTA.

The role of the ventral pallidum GABAergic system in conditioned taste aversion: Effects of microinjections of a GABAA receptor antagonist on taste palatability of a conditioned stimulus

When subjects receive a taste stimulus (conditioned stimulus, CS) that is paired with malaise, they acquire conditioned taste aversion (CTA). It is thought that the taste CS changes from appetitive to aversive after acquisition of CTA. Previous studies have suggested that the ventral pallidum (VP) is involved in the hedonics of taste stimuli, therefore the present study investigated whether the VP is a neural substrate for the shift in preference of the CS after CTA acquisition. In the first experiment, CTA-learned rats received microinjections of the GABA(A) receptor antagonist bicuculline into the VP just before presentation of the CS (saccharin or quinine) in a single-bottle test. The bicuculline-injected rats showed higher intake of the saccharin CS than the vehicle-injected rats. To test whether these results were due to a change in taste preference for the CS, in the second experiment, we examined the effects of bicuculline on the affective aspects of the saccharin CS using a taste reactivity test, which is a useful tool for evaluating taste palatability. The bicuculline-injected rats showed higher appetitive and lower aversive responses to the saccharin CS than the vehicle-injected group. These results suggest that the higher saccharin intake observed in the first experiment was at least partly due to the bicuculline injection, which changed the perceived palatability of the taste CS (saccharin) from aversive to appetitive. The GABAergic system in the VP may play an important role in hedonic-based ingestive behaviors after CTA.

GABAergic transmission in the rat ventral pallidum mediates a saccharin palatability shift in conditioned taste aversion.

We previously found that the blockade of γ‐aminobutyric acid (GABA)A receptors in the ventral pallidum (VP) alters the taste palatability of a conditioned stimulus (CS) from aversive to ingestive after the establishment of conditioned taste aversion (CTA). Because these results suggest that GABAergic transmission in the VP mediates decreased palatability of the taste in CTA, the present study aimed to examine the effects of taste stimulation on the extracellular release of GABA in the VP using in vivo microdialysis. Initially, rats received a paired presentation of 5 mm saccharin or 0.3 mm quinine solution with an intraperitoneal injection of 0.15 m lithium chloride (S‐CTA and Q‐CTA groups) or saline (S‐control and Q‐control groups). After conditioning, microdialysis was carried out before, during and after the presentation of the CS via an intra‐oral cannula. We measured the latency of the first aversive orofacial responses to the CS as behavioral indices. In the S‐CTA group, which rapidly rejected the CS (within 100 s), the GABA efflux was significantly increased (147%) and was maintained for 2 h. On the other hand, the S‐control group expressed no aversive responses and showed no significant alterations in GABA efflux. Although the Q‐CTA group immediately expressed aversive responses to the CS (within 30 s), GABA release was not changed by presentation of the CS, which was similar in the Q‐control group. These findings suggest that the palatability shift from ingestive to aversive in conditioned aversion to saccharin, but not quinine, is mediated by the change in GABAergic transmission in the VP.

Activation of projective neurons from the nucleus accumbens to ventral pallidum by a learned aversive taste stimulus in rats: a manganese-enhanced magnetic resonance imaging study.

Conditioned taste aversion (CTA) causes a palatability shift of a taste stimulus (conditioned stimulus, CS) from ingestive to aversive. We previously found that the ventral pallidum (VP) mediates the palatability shift in CTA. Because the VP receives major projections from the nucleus accumbens (NAc), we examined whether the presentation of CS activates the NAc-VP projective neurons after the establishment of CTA, using a manganese-enhanced magnetic resonance imaging technique. Rats were implanted with a guide cannula in the NAc and an intraoral cannula. After the surgery, they received a pairing of 5 mM saccharin solution with an i.p. injection of 0.15 M lithium chloride (CTA group) or saline (sham group). Two days after the conditioning, rats were microinjected with manganese chloride (MnCl2) into the NAc. Thirty minutes later, the rats were presented with saccharin (CTA-CS and sham-CS groups) or water (CTA-DW and sham-DW groups) via the intraoral cannula. Only the CTA-CS group showed a robust aversion to the CS. At 1 and 2 h after the MnCl2 injection, T1-weighted MR images were acquired using an 11.7 T MRI. Imaging analysis showed that significantly more manganese moved toward the VP in the CTA-CS group than in the other groups. These results indicate that the conditioned aversive taste enhanced the activities of the projective NAc-VP neurons and suggest specific involvement of the NAc-VP pathway in the rejection of CS in retrieval of CTA.

Spatial differences in molecular characteristics of the pontine parabrachial nucleus

Neurons in the pontine parabrachial nucleus (PBN) transduce signals for the general visceral sensory, somatic sensory, gustatory, and autonomic nervous systems, and the various PBN neurons that perform these functions are intermingled. In this study, we analyzed PBN gene expression profiles in male Wistar rats and obtained data on gene expression in the PBN and the principal sensory nucleus of the trigeminal nerve (Pr5). Using these data in combination with in situ hybridization analyses, we identified genes that showed higher expression in the PBN than in Pr5. Our findings indicate that expression patterns in the PBN were different for different genes: Fxyd6, syt5, and plxnc1 were expressed in many neuron populations in the PBN, while the expression patterns of calcr and asb4 were restricted to the central lateral subnucleus and waist area. Furthermore, calcr and asb4 expression patterns were distinct from those of neurotransmitters/neuropeptides such as neurotensin and calcitonin gene-related peptides. Satb2 was specifically expressed in the waist area, which is essential for gustation. In-depth analysis of spatial distribution in the PBN enabled classification of the genes into seven characteristic spatial expression patterns. Expression signatures differed significantly in the subnuclei of the rostral half, mediodorsal half, and ventrolateral third of the PBN, indicating a correlation between the spatial arrangement of the subnuclei and the molecular characteristics of the corresponding neurons. Thus, our results provide valuable information regarding the molecular features and neurotransmission mechanisms of PBN neurons that transmit specific types of information.

Delta-opioid receptor blockade in the ventral pallidum increases perceived palatability and consumption of saccharin solution in rats.

The ventral pallidum (VP) is involved in ingestive behaviour. It receives dense GABAergic projections from the nucleus accumbens. GABAergic terminals in the VP co-express enkephalin, an endogenous ligand of delta-opioid receptors. The role of the delta-opioid receptors in the VP in the context of ingestive behaviour remains unclear, in contrast to the well-understood involvement of the mu-opioid receptors. We used the single-bottle test to examine the effects of VP microinjections of the delta-opioid receptor antagonist naltrindole on consumption of a saccharin solution. Naltrindole injections significantly increased the intake of saccharin, but not water, during a 2-h test session. We also investigated perceived palatability of saccharin using a taste reactivity test. The drug treatments increased ingestive responses to intraorally infused saccharin. Further experimentation explored the role of VP delta-opioid receptors in behavioural responses to saccharin that were previously paired with malaise upon the retrieval of conditioned taste aversion (CTA). Naltrindole-injected rats exhibited longer latency for the first occurrence of aversive responses than vehicle-injected control rats. However, there was no between-group difference in total aversive responses. These results suggest that naltrindole injections into the VP induce an enhancement of perceived palatability of a normally preferred saccharin solution, and thereby facilitate consumption of the solution. On the other hand, delayed aversive responses to the conditioned aversive saccharin suggest that the delta-opioid receptors in the VP mediate the initiation of aversive taste reactivity responses to the conditioned stimulus upon CTA retrieval.

Activation of efferents from the basolateral amygdala during the retrieval of conditioned taste aversion.

The basolateral amygdala (BLA) is critical in the retrieval of conditioned taste aversion (CTA). Although BLA neurons have axonal connections with several brain regions, it is unclear which efferent pathways are functional in CTA. The present study investigated the involvement of efferents from BLA in CTA retrieval with manganese (Mn(2+))-enhanced magnetic resonance imaging (MEMRI). Rats receiving intraoral saccharin infusion paired with intraperitoneal administration of lithium chloride (LiCl) were presented with saccharin (C-S and BC-S groups) or water (C-W group) on the test day. The BC-S group was administered with LiCl 15 min before saccharin presentation on the conditioning day (backward conditioning, BC). Another two groups were injected with saline (S-S and S-W groups) instead of LiCl. On the test day, 50 nL of 40-mM manganese chloride (MnCl2) was injected into BLA before the intraoral fluid infusion. Using MRI, we analyzed Mn(2+) movements, which indicated the activation of efferent neurons. The C-S group showed the highest activities in several efferents from BLA. Of them, the activities of the efferents to the nucleus accumbens core (NAcC), the anterior part of the bed nucleus of the stria terminalis (aBNST), and the central amygdala (CeA) were larger in the C-S group than in the Q group, which was presented with a normally aversive quinine solution. Although rats equivalently rejected conditioned aversive saccharin and quinine, the aversive responses in the C-S group, and not the Q group, were due to CTA retrieval. Therefore, our results indicated that BLA efferents to NAcC, aBNST, and CeA were specifically activated during CTA retrieval, suggesting that these efferents are key components in the neural mechanisms of CTA.