Preprocholecystokinin mRNA-expressing neurons in the rat parabrachial nucleus: Subnuclear...

Preprocholecystokinin mRNA-ExpressingNeurons in the Rat Parabrachial Nucleus:

Subnuclear Localization, EfferentProjection, and Expression

of Nociceptive-Related IntracellularSignaling Substances

OLA HERMANSON,1 DAN LARHAMMAR,2 AND ANDERS BLOMQVIST1*1Department of Biomedicine and Surgery, Division of Cell Biology, Faculty of Health

Sciences, University of Linkoping, S-581 85 Linkoping, Sweden2Department of Neuroscience, Unit of Pharmacology, Uppsala University,

S-751 24 Uppsala, Sweden

ABSTRACTThe pontine parabrachial nucleus (PB) is a major target for ascending fibers from

nociresponsive dorsal horn neurons. Several different neuropeptides have been identified inthe PB. By using double-labeling methods that combine in situ hybridization histochemistrywith retrograde tract tracing and immunohistochemistry, we have examined the subnuclearlocalization of preprocholecystokinin mRNA (ppCCK)-containing neurons, investigated theirefferent projection, and analyzed their expression of intracellular signaling substances thatmay be of importance for nociceptive processing. The results show that neurons containingppCCK are preferentially localized to the superior lateral subnucleus (PBsl), whereas othersubnuclei, such as the dorsal lateral, external lateral, central lateral, and ventral lateralsubnuclei, and the Kolliker-Fuse nucleus, contain only moderate to small numbers of suchneurons. Injections of the retrograde tracer cholera toxin subunit b into the ventromedialhypothalamus demonstrated that ppCCK-containing neurons in PBsl were projection neu-rons. Following nociceptive stimulation, the ppCCK-containing neurons expressed FOSprotein as well as phosphorylated cyclic AMP-responsive element-binding protein (CREB). Inaddition, Ca21/calmodulin-dependent kinase II (CaMKII) was heavily and rather selectivelyexpressed in PBsl and was co-localized to ppCCK-containing neurons. These observationsshow that nociceptive stimuli activate a cholecystokinin pathway from the parabrachialnucleus to the ventromedial hypothalamus that may be important for homeostatic responsesto tissue damage, and point to a putative intracellular route for Ca21-mediated FOStranscription via CaMKII and CREB for the regulation of ppCCK transcription. J. Comp.Neurol. 400:255–270, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: FOS; cyclic AMP-response element-binding protein (CREB); Ca21/calmodulin-

dependent kinase II (CaM kinase II); ventromedial hypothalamic nucleus; in situ

hybridization

Anatomical and electrophysiological studies have demon-strated that the pontine parabrachial nucleus (PB) is amajor target for nociceptive-responsive neurons in thesuperficial layer (lamina I) of the spinal and medullarydorsal horn (Wiberg and Blomqvist, 1984; Cechetto et al.,1985; Hylden et al., 1986; Blomqvist et al., 1989; Light etal., 1993; Jasmin et al., 1994; Bernard et al., 1995; Feil andHerbert, 1995; Craig, 1995), in addition to its input from

Grant sponsor: Swedish Medical Research Council; Grant number: 7879;Grant sponsor: Swedish Brain Foundation.

Ola Hermanson’s present address: CMM West, Department of Medicine,USCD, 9500 Gilman Drive, La Jolla, CA 92093–0648.

*Correspondence to: Anders Blomqvist, Division of Cell Biology, Facultyof Health Sciences, University of Linkoping, S-581 85 Linkoping, Sweden.E-mail: [email protected]

Received 31 January 1997; Revised 12 June 1998; Accepted 19 June 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 400:255–270 (1998)

r 1998 WILEY-LISS, INC.

autonomic-related areas in the lower brainstem (Herbertet al., 1990). In accordance with these observations, it hasbeen shown both in anesthetized (e.g., Bernard and Bes-son, 1990; Bernard et al., 1994) and awake (Hermanson etal., 1992; Blomqvist et al., 1994; Hermanson and Blom-qvist, 1996, 1997a,c; Hermanson et al., 1998) animals thatcells in PB are influenced by noxious stimuli. The neuronalcell bodies in the PB of the rat can be grouped into differentsubnuclei by their appearance in Nissl staining, theirneurochemical content, and their efferent projections, andeach subnucleus seems to be involved in distinct sets ofautonomic mechanisms (cf. Fulwiler and Saper, 1984). Thespinal input terminates predominantly in the dorsal partof the lateral PB (Cechetto et al., 1985; Bernard et al.,1995; Feil and Herbert, 1995). By using FOS immunohisto-chemistry, we recently demonstrated in the awake rat thatmany neurons in this part of the PB, particularly thedorsal lateral (PBdl), and superior lateral (PBsl) subnu-clei, are activated by mechanical and chemical noxiousstimuli, and that to a major extent FOS expression inthese subnuclei is elicited by ascending nociceptive infor-mation from the spinal cord (Hermanson and Blomqvist,1996).

The noxious stimuli-evoked FOS in parabrachial neu-rons may be involved in transcriptional control of severaldifferent genes (cf. Morgan and Curran, 1991). Recently,we demonstrated that noxious stimuli-evoked FOS ex-pressed in the PBdl was co-localized with preprodynorphinmRNA (ppDYN), and that FOS induction in this sub-nucleus was associated with an increased ppDYN expres-sion (Hermanson et al., 1998), observations that areconsistent with a role for FOS in the regulation of dynor-phin transcription (Lucas et al., 1993). However, since thePBsl does not express preprodynorphin mRNA eitherduring basal conditions or after noxious stimulation (Her-manson et al., 1998), the noxious stimuli-evoked FOS inthe PBsl must be involved in some other transcriptionalevent(s).

Previous immunohistochemical studies have shown thathypothalamic-projecting neurons in the PBsl contain cho-lecystokinin-like immunoreactivity (Kubota et al., 1983;Zaborszky et al., 1984; Fulwiler and Saper, 1985), andwork in cell lines has suggested that FOS influencescholecystokinin mRNA transcription (Monstein, 1993a,b).Taken together, these observations point to the possibilitythat noxious stimuli-evoked FOS in the PBsl may berelated to cholecystokinin-producing neurons, and if so,noxious stimuli could influence cholecystokinin-dependentmechanisms in the hypothalamus through these neurons.

In the present study, a series of experiments was under-taken to characterize the cholecystokininergic neurons inthe PB and their relation to noxious stimuli-evoked activ-ity. By using in situ hybridization for preprocholecystoki-nin mRNA (ppCCK) we have examined the subnucleardistribution of cholecystokinin-expressing neurons. Withthe combined use of retrograde tract tracing and in situhybridization we have investigated whether the ppCCK-containing neurons are hypothalamic-projecting neurons.By using double labeling for in situ hybridization andimmunohistochemistry, we have investigated whether theppCCK-containing neurons express noxious stimuli-induced FOS. Finally, we have analyzed the expression ofputative intracellular messengers that may influence FOSand/or ppCCK transcription, such as phosphorylated cyclicAMP-response element-binding protein (pCREB) and Ca21/calmodulin-dependent kinase II (CaMKII). Preliminary

results have been reported elsewhere (Hermanson et al.,1995).

MATERIALS AND METHODS

All observations were made in adult male Sprague-Dawley rats (250–400 g; B&K Universal, Sweden). Naiveanimals were used for the localization of ppCCK-express-ing neurons and CaMKII immunoreactivity. Other ani-mals were subjected to stereotaxic injection of retrogradetracer into the hypothalamus, or to ether anesthesia withor without subsequent noxious stimulation by formalininjection into the skin. All experimental procedures wereapproved by the Animal Care and Use Committee at theUniversity of Linkoping.

Hypothalamic injections

The rats were anesthetized with 6% chloral hydrate (0.5ml/100 g) and placed in a stereotaxic frame. Pressureinjections of 0.15–0.20 µl of 1% cholera toxin subunit b(CTb; Sigma, St. Louis, MO) were made with a Hamiltonsyringe into the medial hypothalamus, with the injectioncenter in the area of the ventromedial hypothalamicnucleus. The animals were allowed to survive for 48 hours(Ericson and Blomqvist, 1988).

FOS expression

The nociceptive stimulation and control procedures havebeen described in detail elsewhere (Hermanson and Blom-qvist, 1996). For the present experiments, the rats wereplaced in a quiet room with moderate lighting for 3 hours.During a brief (,90 seconds) ether anesthesia, they re-ceived an injection of 100 µl of 5% formalin into onehindpaw. They were then allowed to survive for 60–90minutes, time points previously shown to result in maxi-mal FOS labeling in the PB (Hermanson and Blomqvist,1996).

Expression of phosphorylated CREB

Experimental and control animals were placed in sepa-rate rooms for 3 hours as above. During a brief etheranesthesia, the experimental animals received an injec-tion of 100 µl of 5% formalin into one hindpaw. The controlanimals were anesthetized in the same way, but were notgiven any formalin injection. After 20 (n 5 2) or 30 minutes(n 5 4), the animals were killed as described below (seeTissue preparation). These survival times were chosen toinvolve the second phase of formalin-induced pain (Tjølsenet al., 1992), and to minimize the interference of thehandling of the animals and the ether anesthesia on CREBphosphorylation (cf. Thompson et al., 1995; Kovacs andSawchenko, 1996). Care was taken that the procedurespreceding the perfusion (including the ether anesthesia)did not exceed 2 minutes to avoid the effect of theseprocedures on the expression of phosphorylated CREB(pCREB; Ginty et al., 1993). Care was also taken that theformalin-injected and control animals were treated inexactly the same way during the entire experiment.

Tissue preparation

The animals were anesthetized with ether (60–90 sec-onds) before they received an overdose of sodium pentobar-bital (120 mg/kg; Apoteksbolaget, Sweden) injected intothe liver. The animals were then immediately perfusedtranscardially with 100–200 ml of 0.9% NaCl followed by400–600 ml of 4% paraformaldehyde in 0.1 M phosphate-

256 O. HERMANSON ET AL.

buffered saline (PBS, pH 7.4). The brain and the lumbarspinal cord were removed, stored at 4°C overnight infixative to which 20% sucrose had been added, cut on afreezing microtome at 20 (brainstem) or 40 µm (spinal cordand hypothalamus), and collected in 1-in-4 series in sterilePBS (for immunohistochemistry or double labeling) or in4% paraformaldehyde (for in situ hybridization or thioninstaining).

Immunohistochemistry

Sections intended for immunohistochemistry or for im-munohistochemistry in combination with in situ hybridiza-tion were first incubated free-floating in sterile low-saltPBS containing bovine serum albumin (BSA, 1%; Sigma),heparin (500 IU/ml; Kabi Pharmacia, Sweden), RNaseinhibitor (RNasin, 20 U/ml; Promega, Madison, WI), andprimary antibody (rabbit anti-c-FOS [0.1 µg/ml; SantaCruz Biotechnology, Santa Cruz, CA]; rabbit anti-pCREB[1 µg/ml; Upstate Biotechnology, Lake Placid, NY; thisantibody detects CREB phosphorylated at Ser-133, anevent necessary for the transcriptional activity of CREB;Ginty et al., 1993]; mouse anti-Ca21/calmodulin-depen-dent kinase IIalpha [2 µg/ml; Boehringer-Mannheim,Mannheim, Germany; Erondu and Kennedy, 1985]; goatanti-cholera toxin subunit B [0.1 µg/ml; List, Campbell,CA]) for 24 hours at room temperature (single labeling) orfor 72 hours at 4°C (double labeling). All the primaryantisera are well characterized. The antiserum againstFOS was raised in animals immunized with a peptidecorresponding to amino acids 3–16 of the conserved N-terminal region of FOS (Sambucetti and Curran, 1986).This region is not contained in any known FOS-relatedantigen, and the antibody displays no cross-reactivity withsuch antigens. The antiserum against pCREB was raisedin animals immunized with a 14-amino acid phosphory-lated peptide corresponding to amino acids 123–136 ofCREB (Ginty et al., 1993), and affinity purified againstunphosphorylated CREB; this antibody detects phosphory-lated but not unphosphorylated CREB (Ginty et al., 1993;see also Ji and Rupp, 1997). Cross-reactivity against theCREB-related proteins cyclicAMP-response element modu-lator tau (CREMt) and activation transcription factor 1(ATF-1), which contain the same epitope as pCREB (Leeand Masson, 1993), has been demonstrated (Hummler etal., 1994); however, basal expression of CREMt mRNA isvery low in the PB (Lamprecht and Dudai, 1995), andATF-1 expression in the brainstem has not yet beeninvestigated. The anti-Ca21/calmodulin-dependent kinaseII alpha antiserum was obtained from a hybridoma cellline following immunization with the purified protein; thespecificity of this antiserum and procedural controls forimmunohistochemistry have been carefully documented(Erondu and Kennedy, 1985). The cholera toxin B antise-rum was obtained from animals immunized with cholerage-noid. It has been used extensively for tract-tracing studies,and labels only the exogenous protein (Luppi et al., 1990;Hermanson et al., 1994).

After the primary antibody solution, the sections wereincubated in sterile PBS containing BSA (1%), heparin(500 IU/ml) and secondary antibody (swine anti-rabbit,rabbit anti-mouse, or rabbit anti-goat IgG; all 1:100;Dakopatts, Sweden) followed by peroxidase anti-peroxi-dase (PAP) complex (PAP rabbit, PAP mouse, or PAP goat;all 1:150; Dakopatts) in PBS with BSA (1%) and heparin(500 IU/ml), for 1 hour each (room temperature). Sectionsused for single labeling were processed in 0.035% diamino-

benzidine tetrahydrochloride (DAB; Sigma), 2.5% ammo-nium nickel sulphate, and 0.02% H2O2 in sodium acetatebuffer (NaAc, pH 6.0) for 5–8 minutes. Sections used fordouble labeling were soaked in 1% cobalt acetate in NaAcfor 10 minutes, rinsed thoroughly, and then processed inDAB (0.035%), ammonium chloride (0.04%), glucose (0.2%),and glucose oxidase (0.07 U/ml) in NaAc at 4°C for 4–18hours. Subsequently, the sections were rinsed with NaAcand PBS and stored in 4% paraformaldehyde before beingmounted on poly-L-lysine-coated slides.

In situ hybridization

The ppCCK probe was prepared from a 0.535 kb cDNAinsert (Deschenes et al., 1984; a generous gift from Dr.Jack E. Dixon) subcloned into a Bluescript KS1 plasmid,and linearized with Xho for antisense and Xba for senseprobes. Probe synthesis and labeling were performedusing T7 polymerase (antisense) or T3 polymerase (sense)and 35S-labeled uridine 58-(a-thio) triphosphate (Amer-sham, Buckinghamshire, UK) as previously described(Simmons et al., 1989; Hermanson et al., 1994). The probewas purified on a Sephadex G-50 column (Kabi Pharmacia,Uppsala, Sweden).

The slides were treated with 4% paraformaldehyde in0.1 M PBS for 30 minutes and incubated with 0.001%proteinase K in a solution of 0.05 M ethylenediaminetetra-acetate (EDTA) and 0.1 M Tris (pH 8.0) for 30 minutes at37°C before hybridization. Control sections were in addi-tion treated with 0.002% RNase A for 30 minutes at 37°Cbefore hybridization. The hybridization solution consistedof tRNA (0.5 mg/ml), dithiothreitol 0.1 M), formamide(50%), dextran sulfate (10%), Denhardt’s solution (2%),NaCl (0.3 M), Tris (10 mM), EDTA (1 mM), and probe at afinal concentration of 5 3 106 cpm/ml. The hybridizationsolution was applied on coverslips, and the slides weregently placed on top of the coverslips, turned, and sealedwith DPX. The hybridization was then performed for 20hours (single labeling) or 48 hours (double labeling; Her-manson et al., 1994) at 58°C.

After the hybridization, the slides were placed in a 43standard saline citrate (SSC) buffer for 20–40 minutesunder agitation to remove the coverslips, and then rinsedfour times in 43 SSC, before incubation in 0.002% RNaseA in 0.5 M NaCl, 10 mM Tris, and 1 mM EDTA for 30minutes at 37°C. Subsequently, the slides were rinsed in aseries of SSC buffers (23 SSC, 23 SSC, 13 SSC, 0.53SSC) before being heated to 75°C in 0.13 SSC for 30minutes. Dithiothreitol was added to all buffers. Afterdehydration and defatting, the slides were dipped inphotographic emulsion (Kodak NTB2, diluted 1:1 in dis-tilled water), stored at 220°C for 6–10 days, developed inKodak D-19, fixed, rinsed, and coverslipped.

Analysis

Sections were analyzed under darkfield and brightfieldillumination at 60–3003 magnification. The location oflabeled neurons was plotted with a camera lucida. Cytoar-chitectonic boundaries were drawn from adjacent thionin-stained sections and superimposed on the plots usingtransversely cut capillaries as landmarks. Delineation ofthe subnuclear groups in the PB followed Fulwiler andSaper (1984); however, the dorsal lateral subnucleus wasconsidered to extend 50–100 µm more rostrally thandescribed by Fulwiler and Saper (1984; see Hermansonand Blomqvist, 1996; Hermanson et al., 1998). A grainaccumulation in the emulsion layer .10 times background

CCK NEURONS IN RAT PARABRACHIAL NUCLEUS 257

labeling (,0.5 grains/100 µm2) within an area .80 µm2

was considered to represent a ppCCK-expressing neuron1.In these experiments background was measured over themedial parabrachial nucleus, which does not display CCK-immunoreactive neurons (Kubota et al., 1983; Zaborszkyet al., 1984; Fulwiler and Saper, 1985) and which in thepresent experiment was devoid of grain accumulationssuggestive of a specific ppCCK expression. Neurons display-ing distinct immunohistochemical reaction product in thenucleus (FOS and pCREB) or the cytoplasm (CTb andCaMKII) were considered specifically labeled. To excludenonspecifically stained profiles, the illumination of themicroscopic field and the drawing paper was adjusted sothat only profiles that displayed a labeling intensity abovebackground level (determined by examining the centralnucleus of the inferior colliculus) were seen, and only suchprofiles were counted. However, in most cases, identifica-tion of immunolabeled neurons was unequivocal sincethere was negligible background staining (see Figs. 4F, 6F,8F). Immunohistochemically stained neurons that alsodisplayed an accumulation of silver grains .10 timesbackground (,0.5 grains/100 µm2) in the overlaying emul-sion layer were identified as double-labeled, provided thatthe silver grains were distributed in a way that corre-sponded to the shape of the underlying profile, as dis-played by the cytoplasmic immunohistochemical marker(i.e., CTb and CaMKII; see Figs. 4E, 8C,D) or surroundedan immunohistochemically stained nucleus (FOS andpCREB; see Figs. 5C–E, 6C,D). Quantitative analysis ofthe proportion of single- and double-labeled neurons wascarried out on sections through the superior lateral para-brachial subnucleus (PBsl)2, to which an overwhelmingmajority of the ppCCK-expressing neurons were localized(see Results).

RESULTS

Control hybridization

Hybridization with sense probes or RNase pretreatmentfollowed by hybridization with antisense probes did notresult in any specific hybridization signal (data not shown).

Distribution of ppCCK-expressingneurons in PB

The distribution of ppCCK-expressing neurons was ex-amined in 10 animals. A selective staining pattern wasseen. Thus, a very heavy ppCCK expression was consis-tently found in the superior lateral subnucleus (PBsl; Figs.1, 2B,C). Significant, but much sparser labeling, was alsoseen in PBdl, particularly in its rostral part adjacent toPBsl (Fig. 2C), and in the ventral lateral subnucleus (Fig.2D–G). The external lateral, the central lateral (PBcl), andthe Kolliker-Fuse subnuclei displayed small numbers oflightly labeled neurons (Fig. 2).

Retrograde labelingof ppCCK-expressing neurons

After injections of CTb (n 5 5) into the hypothalamusthat involved the ventromedial hypothalamic nucleus, inaddition to several other hypothalamic cell groups (Fig. 3),dense retrograde labeling was seen in PBsl (Fig. 4A),predominantly on the ipsilateral side, being consistentwith data from anterograde and retrograde tract tracingstudies (e.g., Saper and Loewy, 1980; Fulwiler and Saper,1984, 1985; Bester et al., 1997b). Other parabrachialsubnuclei that contained CTb-labeled neurons includedthe extreme lateral subnucleus, PBdl, and PBcl (Fig. 4A).

In situ hybridization for ppCCK on sections processedfor CTb immunohistochemistry (n 5 2; Fig. 4B–F) revealedthat hypothalamic-projecting PBsl neurons expressed pp-CCK (Fig. 4C,D). Other parabrachial subnuclei showedonly a few double-labeled neurons (Fig. 4E). Quantitativeanalysis performed on two sections through the PBsl ineach of two animals showed that 76% and 86%, respec-tively, of the retrogradely labeled neurons also expressed

1PB neurons vary between 10 and 22 µm in diameter (Fulwiler and Saper,1984), corresponding to cross-sectional areas of 80–380 µm2.

2The sectioning strategy yielded at least 2 sections in each series thatwere cut through the PBsl. Thus, cell counts are based on analysis of 2sections in each animal.

Fig. 1. Adjacent transverse sections through the parabrachialnucleus. A: Stained with thionin. B: In situ hybridization for preprocho-lecystokinin mRNA (ppCCK). Dorsal is upward, medial is to the left.Note the heavy expression of ppCCK in the superior lateral sub-nucleus (SL), which in the thionin stain is distinguished from the

adjacent dorsal lateral subnucleus (DL) by its somewhat largerneurons. Arrows in A and B point to the same capillary. CL, centrallateral subnucleus; scp, superior cerebellar peduncle. Scale bar 5100 µm.


Fig. 2. A–H: Camera lucida drawings of transverse sections throughthe parabrachial nucleus, hybridized for preprocholecystokinin mRNA(ppCCK). The sections are spaced 140 µm apart. (A) is rostral. Dorsalis upward, and medial is to the left. Note the dense ppCCK expression(dots) in the superior lateral subnucleus (SL). CL, central lateral

subnucleus; DL, dorsal lateral subnucleus; EL, external lateral sub-nucleus; IL, internal lateral subnucleus; K-F, Kolliker-Fuse nucleus;me 5, mesencephalic trigeminal tract; scp, superior cerebellar pe-duncle; VL, ventral lateral subnucleus. Scale bar 5 250 µm.

ppCCK, and that 66% and 70%, respectively, of the ppCCK-expressing neurons were retrogradely labeled from thehypothalamus.

Noxious stimuli-induced FOSimmunoreactivity in ppCCK-

expressing neurons

Formalin injection into one hindpaw (n 5 10) resulted inthe same FOS labeling pattern in PB (Fig. 5A), as has beendescribed previously (Hermanson and Blomqvist, 1996).Thus, after 60–90 minutes of survival many FOS-labeledneurons were seen in PBsl, PBdl, and PBcl on both sides ofthe midaxis, but with a contralateral predominance inPBdl and PBsl.

Immunohistochemical staining for FOS combined within situ hybridization for ppCCK (Fig. 5B–F) was performedin four cases. Consistently, many double-labeled neuronswere present in PBsl (Fig. 5C–E). Few or no double-labeledneurons were seen in other parts of PB (Fig. 5F). The FOSexpression in PBsl was almost exclusively localized toppCCK-containing neurons; thus, cell counts showed thatin each of the four rats, 97–98% of all FOS-labeled neuronsin PBsl expressed ppCCK. However, not all ppCCK-containing neurons expressed FOS (Fig. 5C–E); out of thetotal population of ppCCK-expressing neurons in PBsl, anaverage of 46 6 6% (SEM) were FOS labeled.

Noxious stimuli-evoked phosphorylatedCREB-like immunoreactivityin ppCCK-expressing neurons

Immunohistochemical staining with an antibody againstpCREB resulted in labeled nuclei of neurons in severalregions of PB. The intensity of the labeling varied acrosscontrol animals (n 5 6), and weak to moderate labelingwas seen in several PB subnuclei including PBsl. Through-out, however, the pCREB-labeling was more intense inanimals subjected to formalin injection (Fig. 6A) (n 5 6)than in controls. A detailed account of the findings onpCREB expression in PB subnuclei other than PBsl isgiven in a separate report (Hermanson and Blomqvist,1997a).

Quantitative analysis of pCREB labeling in PBsl showedthat the number of labeled neurons in the formalin-injected animals in average was more than twice that inthe control animals (71 6 14 [SEM] labeled neurons persection vs. 32 6 15), with the largest differences betweenexperimental animals and controls seen after the longer ofthe two survival periods. Double-labeling (Fig. 6B–F) intwo noxiously stimulated animals revealed that 41% and83%, respectively, of the ppCCK-labeled neurons in PBslalso expressed pCREB-like immunoreactivity, and that71% and 75% of the pCREB-expressing neurons also

Fig. 3. Drawings showing extent of reaction product followinginjection of cholera toxin subunit b (CTb) into the ventromedialhypothalamus. 3v, third ventricle; AD, anterodorsal nucleus of thethalamus; AHN, anterior hypothalamic nucleus; AM, anteromedialnucleus of the thalamus; AMd, anteromedial nucleus, dorsal part;AMv, anteromedial nucleus, ventral part; ARH, arcuate nucleus; AV,anteroventral nucleus of the thalamus; DMH, dorsomedial hypotha-

lamic nucleus; f, fornix; ic, internal capsule; LHA, lateral hypotha-lamic area; ME, median eminence; ot, optic tract; PVH, paraventricu-lar hypothalamic nucleus; RE, nucleus reuniens; RT, reticular thalamicnucleus; sm, stria medullaris; VAL; ventral anterior-lateral complex ofthe thalamus; VMH ventromedial hypothalamic nucleus; ZI, zonaincerta. Scale bar 5 1 mm.


Fig. 4. Hypothalamic-projecting, preprocholecystokinin mRNA(pp-CCK)-expressing neurons in the parabrachial nucleus. A: Retrogradelabeling in the ipsilateral parabrachial nucleus following the injectionof cholera toxin subunit B (CTb) shown in Figure 3. Dorsal is upward,and medial is to the left. CL, central lateral subnucleus; DL, dorsallateral subnucleus; scp, superior cerebellar peduncle; SL, superiorlateral subnucleus. B: Darkfield view of the corresponding areashowing the ppCCK expression in a section (different from that shownin A) that also was processed for CTb immunoreactivity. Note thestrong autoradiographic signal over PBsl and the low level of back-ground labeling; cf. the single-labeled section shown in Figure 1B. Dueto a refraction artefact in the myelin, the superior cerebellar peduncle(scp) is discernable. C: Brightfield view of the superior lateral sub-nucleus, taken from a section adjacent to that shown in B, demonstrat-ing double-labeling for retrogradely transported CTb and ppCCK.

Retrogradely labeled neurons contain immunohistochemical reactionproduct in the cytoplasm. Neurons expressing ppCCK are covered bysilver grains in the emulsion layer. Arrows point at examples ofdouble-labeled neurons. D: Double-labeled neurons (arrowheads) inthe superior lateral subnucleus, taken from the labeled area shown inB. Note accumulation of silver grains over retrogradely labeledprofiles. E: Retrograde labeling in the central lateral subnucleus,taken from the same section as shown in C. One double-labeled profileis seen (arrowhead); note the correspondence between the grainaccumulation and the cytoplasmic staining. The other CTb-immunore-active profiles shown are ppCCK negative. F: ppCCK mRNA-expressing profiles (open arrows) in the central nucleus of the inferiorcolliculus, taken from the same section as shown in C and E. Noteabsence of immunohistochemical background staining. Scale bars 5100 µm in A and B, 50 µm in C, 25 µm in D–F.


Fig. 5. FOS expression in preprocholecystokinin mRNA (ppCCK)-expressing neurons. A: Noxious stimuli-induced FOS in the superiorlateral subnucleus (SL) of the parabrachial nucleus. Dorsal is upward,and medial is to the left. scp, superior cerebellar peduncle. B: Darkfieldview of the corresponding area showing the ppCCK expression in asection (different from that shown in A) that also was processed forFOS. Note the strong and specific autoradiographic signal over PBsl,similar to that seen following processing for ppCCK only (cf. Fig. 1B).C–E: High-power brightfield micrographs, showing double labeling forFOS and ppCCK in the superior lateral subnucleus, taken from twodifferent cases. Arrowheads point at double-labeled neurons, and openarrows point at single-labeled FOS- or ppCCK-positive neurons. Notethat FOS, detected by immunohistochemistry, is localized to the

nucleus, whereas ppCCK mRNA, represented by silver grains in theemulsion layer, is preferentially distributed in the area surroundingthe nucleus, being consistent with its cytoplasmic localization. Grainsover the nucleus probably represent ppCCK in the cytoplasm coveringthe nucleus. Thus, in double-labeled neurons, the FOS-labeled nucleusis surrounded by silver grains. F: Part of the dorsal lateral subnucleus,demonstrating single-labeled FOS-immunoreactive neurons (open ar-rows). Note absence of autoradiographic labeling around these pro-files. Accumulation of silver grains at lower left represents single-labeled ppCCK-expressing profiles. Note the low autoradiographicbackground signal (see also unlabeled areas in C–E.). Scale bars 5 100µm in A and B, 15 µm in E–F. C and D are at the same magnificationas E.


Fig. 6. CREB expression in preprocholecystokinin mRNA(ppCCK)-expressing neurons. A: Expression of phosphorylated CREB in theparabrachial nucleus after noxious stimulation. Dorsal is upward, andmedial is to the left. scp, superior cerebellar peduncle; SL, superiorlateral subnucleus. B: Darkfield view of the corresponding areashowing the ppCCK expression in a section (different from that shownin A) that also was processed for phosphorylated CREB. Note thestrong and specific autoradiographic signal over PBsl (cf. Fig. 1).C: Brightfield micrograph of neurons in the superior lateral sub-nucleus double-labeled for phosphorylated CREB and ppCCK, takenfrom the labeled area shown in B. Double-labeled neurons (arrow-heads) are characterized by an immunohistochemical staining of thenucleus and an accumulation of silver grains over the surroundingcytoplasm. Characteristically, silver grains have accumulated alongthe rim of the nucleus (small arrows in neuron indicated withdouble-arrowheads; cf. also Fig. 5). D: A solitary double-labeled neuron

(arrowhead) in the central lateral subnucleus in a section adjacent tothat shown in B and C. Several single-labeled pCREB-immunoreac-tive nuclei are also seen. Note the unequivocal identification of thedouble-labeled neuron, which displays an accumulation of silvergrains surrounding the nucleus not present around the other nuclei.Note also the very low autoradiographic background. E: ppCCK-negative pCREB-immunoreactive profiles (open arrows) in the centrallateral subnucleus, present in the same section as that shown in B andC. Note the absence of autoradiographic signal around the immunoposi-tive nuclei. Note also the very low autoradiographic background. F:ppCCK-expressing profiles (open arrows) in the central nucleus of theinferior colliculus, present in the same sections as that shown in B, C,and E. Note the absence of nonspecific immunohistochemical staining.Also note the low autoradiographic background (star). Scale bar 5 100µm in A and B, 15 µm in C–F.


expressed ppCCK (Fig. 6C). In contrast, few double-labeled neurons were seen in other parts of PB (Fig. 6D,E),except for the ventral lateral subnucleus where a smallnumber of such neurons was found.

CaMKII-like immunoreactivityin ppCCK-expressing neurons

Staining for CaMKII immunoreactivity (n 5 8) resultedin a selective staining pattern in PB. Thus, dense cell bodylabeling was seen in PBsl (Fig. 7A,B). Some labeling wasalso seen in PBdl (Figs. 7A,B, 8A), whereas other parts ofPB, as well as most of the pontine brainstem, with theexception of the pedunculopontine nucleus (Fig. 7A,B) andthe substantia nigra, displayed little or no CaMKII immu-noreactivity. Analysis of sagittal sections revealed thatdendrites or dendrite-like profiles of PBsl neurons reachedseveral neighboring regions, including PBdl (Fig. 7C).

Double labeling for ppCCK (Fig. 8B-F) in two animalsshowed that 78% and 85%, respectively, of the ppCCK-labeled neurons also expressed CaMKII-like immunoreac-tivity, suggesting a high degree of co-localization betweenCaMKII and ppCCK (Fig. 8C,D). On the other hand,examination of the CaMKII-labeled population for ppCCKshowed that 34% and 31% of the CaMKII-labeled neuronsexpressed ppCCK. However, the animals used for CaMKIIand ppCCK double-labeling displayed a 25% lower count ofppCCK-labeled neurons in PBsl than the animals used forthe other double-labeling experiments (36 ppCCK cells/section in the CaMKII/ppCCK double-labeled sections vs.49 ppCCK cells/section in the FOS/ppCCK and pCREB/ppCCK double-labeled sections), and it is possible that theproportion of double-labeled neurons was underesti-mated3. Very few double-labeled neurons were detected inother parts of PB (Fig. 8E).

DISCUSSION

This study demonstrates a heavy ppCCK expressionamong neurons in a specific subnucleus of PB, the PBsl,and shows that many of the ppCCK-expressing neurons inthis subnucleus project to the hypothalamus. These obser-vations confirm and extend the findings in previous immu-nohistochemical studies showing CCK-like immunoreactiv-ity in hypothalamic-projecting neurons in PB (Kubota etal., 1983; Zaborszky et al., 1984; Fulwiler and Saper, 1985;Schumacher et al., 1991). Furthermore, the present find-ings demonstrate that noxious stimulation evokes FOS-and increases pCREB-like immunoreactivity in ppCCK-containing neurons in PBsl and that such neurons expressCaMKII immunoreactivity. These observations are allnovel and point to putative intracellular mechanismsthrough which noxious stimuli may regulate CCK tran-scription and influence hypothalamic activity.

Technical considerations

The method for the combined immunochemical andhybridization histochemical localizations has been re-ported and evaluated previously (Hermanson et al., 1994),but some technical issues need to be discussed here also.For tract tracing we utilized immunohistochemical

3It is conceivable that the lower number of ppCCK-labeled neuronsreflected the fact that these animals were naive animals, whereas thoseused for double-labeling with FOS and phosphoCREB had been noxiouslystimulated.

Fig. 7. Ca21/calmodulin dependent kinase II (CaMKII) immunore-activity in the parabrachial nucleus. A: Sagittal section through theupper pons stained with thionin. Dorsal is upward, and rostral is tothe left. B: Adjacent section showing immunoreactivity for CaMKII.Note dense CaMKII labeling in the superior lateral subnucleus (SL).The labeled area at lower left is in the pedunculopontine tegmentalnucleus. Boxed area, shown at higher magnification in C, encompassespart of the dorsal lateral subnucleus (DL). CL, central lateral sub-nucleus; scp, superior cerebellar peduncle. C: Sagittal section athigher magnification showing CaMKII-labeled dendrites (arrows)extending into the dorsal lateral subnucleus (right). Scale bars 5 500µm in A and B, 20 µm in C.


Fig. 8. Ca21/calmodulin dependent kinase II (CaMKII) immunore-activity in preprocholecystokinin mRNA (ppCCK)-expressing neuronsin the parabrachial nucleus. A: Low-power transverse section showingCaMKII immunoreactivity. Dorsal is upward, and medial is to the left.CL, central lateral subnucleus; DL, dorsal lateral subnucleus; scp,superior cerebellar peduncle; SL, superior lateral subnucleus.B: Darkfield view of the corresponding area showing the ppCCKexpression in a section also processed for CaMKII immunoreactivity,which is discernible as a grayish background shade. Note that thedistribution of the CaMKII-expressing neurons is similar to that ofppCCK-expressing neurons. C,D: Pairs of micrographs showing double-labeled neurons (arrowheads) in the superior lateral subnucleus,taken from the labeled area shown in B. C was focused on the emulsionlayer to demonstrate the autoradiographic in situ hybridizationsignal, and D was focused on the tissue level to show the immunohisto-chemical reaction product. Double-labeled neurons (arrowheads) dis-

play CaMKII immunoreactivity in the cytoplasm and an accumulationof silver grains in the overlaying emulsion layer. Note the closecorrespondence between the distribution of the silver grains and thecytoplasmic immunocytochemical labeling, with few or no silvergrains present over the CaMKII-negative nucleus (best seen inneurons cut through the nuclear plane, indicated by arrowheads in D.A few CaMKII-positive but ppCCK-negative neurons are also seen(open arrows). Asterisks show grain accumulations indicating single-labeled ppCCK-positive neurons (note absence of immunohistochemi-cal staining in D. E: Single-labeled CaMKII-positive neurons in thedorsal lateral nucleus, seen in the same section as the neurons shownin C and D. Note the very low autoradiographic background (see alsounlabeled area at lower left in C. F: ppCCK-expressing profiles (openarrows) in the central nucleus of the inferior colliculus, present in thesame section as shown in B–E. Note absence of immunohistochemicalbackground staining. Scale bar 5 100 µm in A and B, 15 µm in C–F.


detection of retrogradely transported choleragenoid, whichis a very sensitive method (Ericson and Blomqvist, 1988),well suited for the use in studies of gene expression inselect neuronal populations. Originally utilizing monoclo-nal antibodies, we obtained a Golgi-like retrograde label-ing (Ericson and Blomqvist, 1988); with the commerciallyavailable polyclonal antiserum employed in the presentstudy, a more granular reaction product is produced (seealso Luppi et al., 1990). However, retrogradely labeledneurons are still unequivocally identified, since no or littlebackground staining is produced (Fig. 4F). The use of long35S-labeled riboprobes for in situ hybridization histochem-istry also results in a very high sensitivity with thepossibility of detecting as little as 2–10 target molecules/cell (Simmons et al., 1989), but carries some technicallimitations. Because of the energy profile of the b-emissionfrom 35S-isotopes (Brady and Finlan, 1990), radiolabeledprofiles located in the lower parts of the section may giverise to only weak autoradiographic staining in the overlay-ing emulsion layer. This could result in an underestima-tion of the number of double-labeled profiles. On the otherhand, the possibility also exists that an accumulation ofsilver grains resulting from an unstained profile in thetissue is falsely associated with a more deeply located,immunopositive structure. While this is rather unlikely forcells immunolabeled for cytoplasmic antigens, since theidentification of such cells as double labeled is assisted by adistribution of the silver grains corresponding to the shapeof the underlying profile (e.g., Figs. 4E, 8C,D), it presents amore significant problem with nuclear markers, such asFOS and pCREB, particularly in regions containing denselypacked neurons. However, in most cases the correspon-dence between the two markers was unequivocal, with anaccumulation of silver grains surrounding the immunoposi-tive cell nucleus (Figs. 5C–E, 6C,D). Nevertheless, themethodological considerations imply that the figures onthe proportions of single- and double-labeled neuronsobtained should be interpreted with caution and regardedas estimates rather than precise calculations. For one ofthe populations a comparison can be made with resultsobtained using a different method. In the study by Ful-wiler and Saper (1985), which combined retrograde trac-ing with fluorescent dyes and immunofluorescence forCCK, 80–90% of the PBsl neurons that projected to theventromedial hypothalamus were CCK positive. Thesefigures are similar to those obtained in the present study(76% and 86%, respectively).

It is of considerable interest that almost all noxiousstimuli-evoked FOS was seen in ppCCK-expressing neu-rons. As is discussed below, this co-localization indicatesthat FOS in PBsl may regulate CCK transcription. Thefinding that about half of the ppCCK-expressing neuronsdid not display FOS does not exclude the possibility thatFOS may be a transcription factor in these cells as well,because the FOS labeling may vary depending on stimulusintensity and localization (Hermanson and Blomqvist,1996, 1997c). Thus, it is conceivable that more ppCCKneurons could display FOS after other noxious stimulationprocedures. However, the possibility remains that someppCCK neurons do not produce FOS after noxious stimula-tion. Since the production of FOS may be regulated byCREB, it is of interest in this context to note that theexpression of pCREB varied considerably across animals.This variation, which also was evident in the double-labeling material, in which 41% and 83% of the ppCCK

neurons expressed pCREB, may reflect differences in thedegree of CREB phosphorylation between animals (Herde-gen et al., 1994).

Since phosphorylation of CREB has been investigatedonly recently with immunohistochemical methods (Kovacsand Sawchenko, 1996; Hermanson and Blomqvist, 1997a;Houpt, 1997; Ji and Rupp, 1997), the significance of thelabeling seen in the noxiously stimulated and controlanimals deserves some comments. Despite the variabilityacross animals seen in the present study, all formalin-injected animals showed increased pCREB immunoreactiv-ity compared with their controls. Accordingly, these obser-vations show a formalin-induced phosphorylation of CREBin PB, similar to what was recently demonstrated in therat spinal cord (Ji and Rupp, 1997). Phosphorylation ofCREB is a rapid process that starts within a few minutesafter stimulation (Thompson et al., 1995; Ji and Rupp,1997). Since the second phase of pain following formalininjection begins after about 15–20 minutes (Tjølsen et al.,1992), the increased pCREB expression seen after thesurvival times used in this study (20–30 minutes) shouldmainly reflect this stimulus. However, in the study by Jiand Rupp (1997) pCREB expression was already seen inthe lumbar spinal dorsal horn 10 minutes after formalininjection into the paw, suggesting that the short first phaseof pain that is present during a few minutes immediatelyafter the formalin injection could also have influenced thepCREB expression seen in the present study. BecauseCREB phosphorylation is very sensitive to sensory stimuli,control and experimental animals were handled in exactlythe same way to minimize uncontrolled variables. How-ever, the origin of the basal pCREB expression seen in thecontrol animals remains obscure. It is possible that thispCREB was induced by the handling of animals or thebrief ether anesthesia administered, although the timesfor killing of the animals were chosen to minimize sucheffects. In the paraventricular hypothalamic nucleus, induc-tion of pCREB expression was maximal between 5 and 15minutes after ether anesthesia and had ceased 30 minutesafter stress (Kovacs and Sawchenko, 1996). Thus, in thepresent study the effects due to handling or ether anesthe-sia should be attenuated, particularly following the longertime points used. It is also of interest to note that Ji andRupp (1997) reported that a short halothane anesthesiagiven immediately before the formalin injection reducedpCREB expression in the spinal cord. Accordingly, most ofthe pCREB immunoreactivity seen in the control animalsmay reflect normally occurring CREB phosphorylation.Such an assumption would be consistent with the interani-mal variation observed, and with the role of the PB inautonomic regulation (Fulwiler and Saper, 1984).

Nociceptive signaling in PBsl

This is the first demonstration of FOS in ppCCK-expressing neurons. The gene encoding ppCCK displays aso-called AP-1/CRE-like site in its regulatory sequence(Haun and Dixon, 1990; Monstein, 1993a), and it has beenshown in neuroblastoma cells that FOS can bind to thissite and possibly regulate ppCCK transcription (Monsteinet al., 1992; Monstein, 1993a,b). Taken together with thesedata, the present findings suggest that noxious stimuli-evoked FOS may influence ppCCK transcription in PBslneurons. However, since the noxious stimuli-evoked FOS-immunoreactive neurons in other parts of PB did notexpress ppCCK, FOS probably regulates the transcription


of other genes in these neurons. For example, we haverecently shown that noxious stimuli-evoked FOS is co-localized with preprodynorphin mRNA in PBdl (Herman-son et al., 1998), being consistent with a role of FOS indynorphin transcription (Lucas et al., 1993; Hunter et al.,1995). On the other hand, FOS does not seem to regulatepreproenkephalin transcription in PB, since FOS is gener-ally not localized to enkephalin-expressing neurons (Her-manson and Blomqvist, 1997a,b).

A rise in intracellular Ca21 levels has been shown toactivate FOS in PC12 cells (Thompson et al., 1995), andthis activation is mediated by CaMKII and a subsequentphosphorylation of CREB (Sheng et al., 1991), which bindsto a AP-1/CRE-like site on the c-fos gene (Greenberg et al.,1992). The present findings suggest that this intracellularpathway may also exist in the central nervous system.Studies in hippocampal neurons have demonstrated aCa21-dependent CREB phosphorylation (Deisseroth et al.,1996), but this seems to involve nuclear Ca21/calmodulin-dependent protein kinase IV (Bito et al., 1996).

The regulation of CREB activity is very complex (Leeand Masson, 1993), and several kinases including CaMKIIhave been shown to phosphorylate CREB (Gonzalez andMontminy, 1989; Sheng et al., 1991; Enslen et al., 1994).This study shows a dense CaMKII expression in PBsl andadjacent parts of PBdl (Fig. 8A), and it is therefore likelythat CaMKII is involved in CREB/FOS activation in thesesubnuclei. However, since pCREB and FOS can be de-tected in several parabrachial subnuclei that do not ex-press CaMKII (Hermanson and Blomqvist, 1996,1997a,c,d), the phosphorylation of CREB and induction ofFOS in these subnuclei must involve other kinases. Fur-thermore, since CaMKII is present in the cytoplasm andCREB is localized to the nucleus, phosphorylation ofCREB by CaMKII should involve some intermediate signal-ing molecule.

Previous studies on the distribution of CaMKII haveshown a high expression in several forebrain regions(Erondu and Kennedy, 1985; Benson et al., 1992; Ochiishiet al., 1994; Hallbeck et al., 1996), but little expression inmost brainstem areas. In accord with the present immuno-histochemical findings, Benson et al. (1992) reportedCaMKIIalpha gene expression in neurons in the parabra-chial nucleus and the subpeduncular tegmental nucleus(corresponding to our pedunculopontine nucleus), andErondu and Kennedy found CaMKII-immunoreactive fi-bers in the substantia nigra. The possible expression ofother Ca21/calmodulin-dependent protein kinases thanCaMKII in the PB is presently investigated in this labora-tory. Preliminary observations suggest that CaMKIV isdensely expressed in several PB subnuclei that also ex-press pCREB (Hermanson and Blomqvist, 1997d), but notin PBsl, pointing to a subnuclear selectivity in kinaseexpression.

There are several signal substances in the spinal affer-ents to PB that may evoke a Ca21 rise and a subsequentFOS transcription. For example, it has been demonstratedthat substance P-like immunoreactivity (SP) is present inspinal afferents to PB (Blomqvist and Mackerlova, 1995),and it has been shown that the preprotachykinin mRNAexpression in spinoparabrachial neurons increases afternoxious stimulation (Noguchi and Ruda, 1992). SubstanceP receptor immunoreactivity and NK-1 (the substance Preceptor) binding sites are present in the dorsal parts ofPB (Dam et al., 1990; Takeda et al., 1995). It has also been

shown that neurons in the dorsal PB express NK-1 recep-tor mRNA (Maeno et al., 1993). Since activation of theNK-1 receptor elicits an increase in postsynaptic Ca21-levels and c-fos transcription (Oury-Donat et al., 1994),noxious stimuli-evoked FOS in PBsl may be elicited by SP.

However, the anatomical connection between the spinalfibers and the PBsl neurons remains to be clarified. Spinalfibers terminate primarily in PBdl, whereas PBsl onlyreceives a small number of such fibers (Feil and Herbert,1995). In the present study, we show using CaMKII-immunohistochemistry that PBsl dendrites extend intoPBdl (Fig. 7C), and it is possible that these dendrites aretargeted by spinoparabrachial fibers. Electron microscopicinvestigations have demonstrated synaptic contacts be-tween terminals of ascending spinal fibers and dendrites ofhypothalamic-projecting parabrachial neurons (Ericsonand Blomqvist, 1988), but it is not known whether suchdendrites belong to PBsl neurons. It should be pointed outthat the CaMKII-stained processes seen in PBdl shouldnot be axons, since CaMKII has only been demonstrated incell bodies, dendrites, and axon terminals (Kelly, 1991;Hanson and Schulman, 1992; Hallbeck et al., 1996).

Functional considerations

Consistent with previous anatomical (Wiberg and Blom-qvist, 1984; Wiberg et al., 1987; Ericson and Blomqvist,1988; Blomqvist et al., 1989; Ma et al., 1989; Hermansonand Blomqvist, 1996; Bester et al., 1997a; Hermanson etal., 1998) and electrophysiological observations (Bernardet al., 1994; Bester et al., 1995), this study suggests thatnoxious information from the spinal cord activates hypotha-lamic-projecting PB neurons. Specifically, we show thatthe CCK-containing neuronal population of PBsl, whichprojects solely to VMH (Fulwiler and Saper, 1985; Schu-macher et al., 1991), is influenced by nociceptive stimula-tion. CCK in VMH is almost exclusively derived from PBsl(Zaborszky et al., 1984). Several roles for CCK in VMHhave been discussed. For example, CCK in VMH has longbeen associated with satiety (cf. Willis et al., 1986), whichmay be elicited by an increase in blood glucose (Levine andMorley, 1981) as a result of an increase in blood glucagon(Frohman and Bernardis, 1971; Borg et al., 1995). Such aneffect is probably the result of an elevation of sympatheticactivity (Bray, 1993; Yoshimatsu et al., 1992) via projec-tions from VMH to the periaqueductal gray matter (Saperet al., 1976; Krieger et al., 1979; Canteras et al., 1994). Theeffect of CCK in VMH has been mimicked by electricalstimulation of PB (Ino et al., 1987; Takaki et al., 1990);intriguingly, noxious stimulation of the hindpaw has beenshown to evoke increased levels of blood glucagon in theanesthetized rat (Kurosawa et al., 1994). In addition,stimulation of VMH increases glucose uptake by skeletalmuscles (Minokoshi et al., 1994). Thus, a modulation of theactivity of ppCCK-expressing neurons in PB may affect theexcitability in neuronal circuits of VMH that regulateblood glucose metabolism. Hence, it is possible that nox-ious stimuli-evoked events in PBsl neurons influenceglucose homeostasis as part of an integrated response totissue damage.

It has recently been shown that administration of the obgene product leptin, which results in reduced food intake(Halaas et al., 1995), induces FOS in PBsl neurons (Elm-quist et al., 1997). Although it is not yet known whetherthe leptin-sensitive neurons in PBsl express CCK andbelong to the same population as those that are activated


by nociceptive stimuli, the finding by Elmquist et al. (1997)raises the intriguing possibility that PBsl neurons inte-grate nociceptive and humoral information of importancefor feeding behavior and satiety. It is interesting in thiscontext to note that PBsl neurons are also activated bysomatic and visceral inflammation (Lanteri-Minet et al.,1993, 1994), conditions known to induce anorexia (Plata-Salaman, 1996) The activation of PBsl neurons seenfollowing inflammation could not only be the result of thestimulation of peripheral nociceptors at the site of inflam-mation, but could also be exerted, directly or indirectly, bycirculating inflammatory mediators. The latter possibilityis suggested by the strong immediate-early gene expres-sion of PBsl neurons seen bilaterally following a unilateralmonoarthritis (Lanteri-Minet et al., 1994), which differsfrom the predominantly contralateral activation seen afteracute nociceptive stimulation (Hermanson and Blomqvist,1996).

In conclusion, the results of the present study add to ourunderstanding of how nociceptive stimuli influence auto-nomic functions. The parabrachial nucleus seems to be akey relay for such interactions. Observations from this andother laboratories have demonstrated that noxious infor-mation from different body parts reaches the parabrachialsubnuclei in topographically distinct patterns (Cechetto etal., 1985; Slugg and Light, 1994; Feil and Herbert, 1995;Hermanson and Blomqvist, 1996, 1997d). Each of theparabrachial subnuclei in turn subserves mutually differ-ent functions through their distinct efferent connections(Fulwiler and Saper, 1984), and seems to do so by the use ofspecific sets of inter- and intracellular signaling mecha-nisms (e.g., Blomqvist et al., 1994; Chamberlin and Saper,1995; Herbert and Flugge, 1995; Guthmann et al., 1996;1997; Hermanson and Blomqvist, 1997a,b,d; Hermansonet al., 1998; present study). This topographic and neuro-chemical specificity, then, may serve as a substrate for afinely tuned homeostatic response to impending or estab-lished tissue damage.

ACKNOWLEDGMENTS

We thank Martin Hallbeck and Ludmila Mackerlova forhelp with the illustrations.

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