INTERNATIONAL SYMPOSIUM ON OLFACTION AND TASTE

Functional Organization of the RodentParabrachial Nucleus

Takashi Yamamoto,a Motohide Takemura,b Tadashi Inui,c

Kunio Torii,d Naohiro Maeda,e Makoto Ohmoto,e

Ichiro Matsumoto,e and Keiko Abee

aKio University, Faculty of Health Sciences, Nara, JapanbOsaka University Graduate School of Dentistry, Osaka, Japan

cOsaka University Graduate School of Human Sciences, Osaka, JapandInstitute of Life Sciences, Ajinomoto Co., Inc., Kawasaki, Japan

eGraduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan

The rodent parabrachial nucleus (PBN) is not merely a sensory relay station but alsoplays an important role in integrating various ascending and descending inputs togetherwith plastic changes of neuronal responses after learning and experience. The limbic andreward 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. Toexplore how the rat PBN is functionally organized, we detected activation patterns ofneurons mainly by means of c-fos immunohistochemistry to show neuronal activationin different situations of ingestive behavior. The expression pattern was different undernutritionally replete and deficient conditions, perceptually new and familiar conditions,and learned and unlearned conditions. As for the possible functions, the rostral part ofthe external lateral subnucleus is related to general visceral inputs; the caudal part of theexternal lateral subnucleus, aversive behavior; the dorsal lateral subnucleus, ingestivebehavior; and the central medial subnucleus, taste of NaCl. Because several genes werelocalized in specific subnuclei, we are trying to correlate the gene expressions withpossible functional significance.

Key words: rat; parabrachial nucleus; subnuclei; functional organization; Fos-like im-munoreactivity; gene expression; conditioned taste aversion; ingestive behavior

Multiple Functions of theParabrachial Nucleus

Several previous studies have suggested thatthe parabrachial nucleus (PBN) is related notonly to taste but also to other functions includ-ing cardiovascular, respiratory, and gastroin-testinal regulation; processing of hemodynamicsignals; control of hormone release; and noci-ceptive processing. Microelectrical stimulation

Address for correspondence: Takashi Yamamoto, Faculty of HealthScience, Kio University, 4-2-2 Umami-naka, Kohryo, Kitakatsuragi,Nara 635-0832, Japan. Voice: +81-745-54-1601; fax: +81-745-54-1600.ta.yamamoto@kio.ac.jp

within the PBN elicits somatic and autonomicresponses depending on the site stimulated.1,2

The anatomical correlates of these functions,however, have not yet been precisely examined.

The PBN receives taste and gastrointestinalafferent information, which is integrated andfurther influenced by centrifugal inputs fromthe higher central nervous system to ultimatelyinfluence feeding-related behaviors. As shownin Figure 1, the PBN consists of several subnu-clei. To understand more about the functionalroles (or behavioral correlates) of different sub-nuclei of the PBN in ingestive behavior, we useddifferent techniques, such as electrophysiologi-cal unit recording, immunohistochemistry for

International Symposium on Olfaction and Taste: Ann. N.Y. Acad. Sci. 1170: 378–382 (2009).doi: 10.1111/j.1749-6632.2009.03883.x C© 2009 New York Academy of Sciences.

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Yamamoto et al.: Organization of PBN 379

Figure 1. Subnuclei of the rat PBN. cl, central lateral; cm, central medial; dl, dorsallateral; el, external lateral; em, external medial; il, internal lateral; KF, Koelliker–Fuse; vl,ventral lateral; vm, ventral medial; BC, brachium conjunctivum; LC, locus coeruleus; Me5,mesencephalic nucleus of the trigeminal nerve.

FOS (polyclonal anti–c-fos antibody, 1:5000,#sc-52; Santa Cruz Biotechnology, Santa Cruz,CA), and more recently gene expression anal-ysis using the DNA microarray technologies inrats under different experimental paradigms.

Novel Versus Familiar Tastes

We divided rats into two groups. One groupwas familiarized with 0.3 M NaCl by exposureto the solution twice a day until the amountof intake was stabilized. The other group hadno experience with 0.3 M NaCl. On the testday, rats of the novel and familiar groups werepresented with 10 mL of 0.3 M NaCl and theirbrains were removed 2 h later. FOS expres-sion was detected exclusively in the dorsal lat-eral (DL) subnucleus and central medial (CM)subnucleus. As shown in Figure 2, FOS ex-pression was enhanced greatly in the DL af-ter familiarization, and this enhancement wasreduced after making lesions of the corticalgustatory area (CGA) with ibotenic acid. FOSexpression in the CM was reduced after fa-miliarization in rats with or without CGA.

Figure 2. Mean (±SEM, n = 5) number of FOSneurons in the central medial (cm) subnucleus anddorsal lateral (dl) subnucleus of the PBN to ingestionof 5 mL of 0.3 M NaCl. Novel, rats drank the solu-tion for the first time; familiar, rats drank the solutionafter familiarization; CGAx, rats drank the familiarsolution after lesions of the cortical gustatory area(CGA). ∗P < 0.05.

Thus, the familiarity-related plasticity in theDL but not the CM was dependent on theCGA. These findings suggest that familiarityis related to modulation of activity in the DL,which is implicated in pleasant ingestion,3 andthe CM, which is the putative main recipientzone of NaCl taste.3,4 The DL activation may

380 Annals of the New York Academy of Sciences

be at least partly controlled by the CGA,5 butthe CM activation is maintained after corticalablation.

Nutritionally Replete VersusDeficient Conditions

L-Lysine is one of the essential amino acids.Animals deficient in lysine suffer from severeillness and seek lysine. Deficient rats prefer andselectively ingest lysine on the basis of its taste asa cue signal for its detection. Although healthycontrol rats do not prefer lysine solution, wetrained them to ingest 0.2 M lysine through re-peated exposure. For a week before the trainingsession, control (lysine replete) rats ate ordinaryfood pellets and drank water, whereas experi-mental (lysine deficient) rats also ate ordinaryfood pellets but were familiarized with 0.2 Mlysine. In the training session, we replaced or-dinary food pellets with a lysine-free diet for15 days with 0.2 M lysine and water for controlrats but with only water for experimental rats,and we gave 0.2 M lysine solution to rats inboth groups on the test day. The deficient ratsshowed strong FOS expression in the DL andthe rostral part of the external lateral (EL) sub-nucleus to the lysine solution (Fig. 3). Becauselysine-deficient rats prefer lysine solution6

and show enhanced vagal responses to lysineapplied to the hepatoportal region,7 the DLand the rostral part of the EL may be related topreferential intake3 and visceral information,3

respectively.

Highly Versus Poorly NutritiousSubstances

We performed this experiment to examineany possible difference of FOS expression pat-tern among the subnuclei of the PBN in re-sponse to visceral stimulation with highly orpoorly nutritious chemicals. Each rat receivedan aliquot (7.5 mL) of stimulus liquid througha polyethylene tube by introducing it to the

Figure 3. Mean (±SEM, n = 4) number of FOSneurons in the rostral part of the external lateral (el)subnucleus and dorsal lateral (dl) subnucleus of thePBN to ingestion of 5 mL of 0.2 M L-lysine (Lys). Lysine-deficient rats exhibited significantly (P < 0.05) moreneurons both in the el and dl.

stomach from the mouth, bypassing gustatorystimulation. Highly nutritious chemical stimuliwere 0.5 M glucose, 0.5 M maltose, and 20%Polycose, and poorly nutritious chemical stimuliwere 0.5 M lactose, 0.5 M ethanol, and 0.15 MLiCl. FOS neurons were found mostly in theDL and the rostral part of the EL in the PBN;more precisely, as shown in Figure 4, glucose,maltose, and Polycose showed stronger FOSexpression in the DL than in the EL, whereasthe reverse was true for poorly nutritious lac-tose, ethanol, and LiCl. These results suggestthat viscerally or nutritiously acceptable stimuliinduce stronger activation of neurons in the DLrather than in the rostral part of the EL.

Hedonic Shift from Positiveto Negative

Conditioned taste aversion (CTA) is a ro-bust form of classical conditioning in which an-imals reject a taste (conditioned stimulus [CS])when its initial exposure is followed by an aver-sive internal symptom induced by a toxic sub-stance (unconditioned stimulus). The PBN is

Yamamoto et al.: Organization of PBN 381

Figure 4. The proportion of mean number of FOSneurons between the dorsal lateral (dl) subnucleusand rostral part of the external lateral (el) subnu-cleus in response to intragastric infusions of highly nu-tritious substances (glucose, maltose, and Polycose)and poorly nutritious substances (lactose, ethanol,and LiCl).

a key brain structure significantly involved inthe acquisition of CTA according to lesion–behavior studies, including a recent study show-ing that blockade of c-fos gene expression in thePBN impairs CTA acquisition.8

CTA to NaCl solution resulted in enhancedPBN neuronal responses in anesthetized rats,and this enhancement failed to occur af-ter decerebration at the supracollicular level.9

Also, the CTA-induced enhanced neuronalactivities to NaCl were observed only inamiloride-sensitive NaCl-best units,9 a subset ofNaCl-best units that exhibits sodium responsessubstantially reduced by amiloride, an epithe-lial sodium channel blocker.

FOS neurons after reexposure to the NaClCS were specifically detected10 in the CM, theputative main recipient zone of gustatory in-formation of NaCl,3,4 and the caudal part ofthe EL, where activity increases in responseto the aversive tastants.3 The increased FOSexpression was not observed after decerebra-tion.10 The results indicate that neurons in theCM and EL respond to aversively conditionedtaste and that the increased activation dur-ing CTA expression requires inputs from theforebrain.

TABLE 1. Functional Significance of the Subnucleiof the PBN

Subnuclei Function

External lateral (rostral part) General visceralExternal lateral (caudal part) Hedonically negative

AversiveDorsal lateral Hedonically positive

IngestiveFamiliar

Central medial Sodium taste

Functional Neuroanatomy

The aforementioned elevated FOS expres-sion in the CM could lead to increased salienceor magnitude of the perceived taste of the CS(NaCl). Neurons in the EL respond well to aver-sive taste stimuli, such as quinine or HCl.3,11

The significantly increased FOS expression inthe caudal part of the EL after CTA to NaClsuggests that taste information of conditionedas well as innate aversive stimuli is representedin this area. Although not shown by direct evi-dence, elevated activation in the EL after aver-sive learning might be derived from the basolat-eral nucleus of the amygdala, which is activatedby reexposure to the CS after the acquisition ofCTA.12

Searches of functional roles of the PBNin taste-dependent ingestive behavior havemethodological limitations because of the par-ticipation of a variety of orofacial sensory andmotor functions and postingestive factors. De-spite such limitations, as shown in Table 1, gus-tatory and gastrointestinal acceptability may berepresented in the DL, NaCl taste excites neu-rons in the CM, and aversive feeling may berepresented in the EL.

To explore more about regional differenceswithin the PBN, we are carrying out DNAmicroarray analysis of total RNA samples ex-tracted from the PBN. We selected nearly 30genes whose between-tissue differences werelarge in expression intensity, and we analyzedtheir expression patterns in the PBN by in situ

hybridization. Some genes were exclusively

382 Annals of the New York Academy of Sciences

localized in particular subnuclei, indicating in-volvement of particular peptides, such as pro-dynorphin in the DL and calcitonin gene–related peptide in the EL and external medialsubnucleus. DNA array analysis is a promisingtool in searching new substances specifically in-volved in quality-specific taste responses andin various aspects of taste-dependent ingestivebehavior.

Acknowledgments

This work was partly supported by the In-ternational Glutamate Technical Committeegrant and Ajinomoto Co., Inc.

Conflicts of Interest

The authors declare no conflicts of interest.

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