THE JOURNAL OF COMPARATIVE NEUROLOGY 368:45-56 (1996)

Subnuclear Localization of FOS-Like Immunoreactivity in the Rat Parabrachial

Nucleus After Nociceptive Stimulation

OLA HERMANSON AND ANDERS BLOMQVIST Department of Cell Biology, Faculty of Health Sciences, University of Linkoping,

S-581 85 Linkoping, Sweden

ABSTRACT The effect of noxious stimulation on the expression of FOS-like immunoreactivity (FOS-LI)

in neurons of the parabrachial nucleus (PB) was studied in awake, freely moving rats. In one series of experiments, the rats were subjected to noxious mechanical stimulation (pinch) of either the nape of the neck or the base of the tail for 20 seconds every 5 minutes for 90 minutes, and then they were killed by transcardial perfusion after 45-210 minutes. Control animals received innocuous mechanical stimulation (brush) of the tail. Noxious stimuli resulted in FOS-LI in neurons in the dorsal part of the lateral PB, with heavy labeling in the superior lateral (PBsl) and the dorsal lateral (PBdl) subnuclei. FOS-LI was also elicited in the central lateral subnucleus (PBcl) and, although much more sparsely, in the external lateral subnucleus and the Kolliker-Fuse nucleus. Tail and neck stimulation resulted in similar labeling patterns, but more neurons, particularly in PBsl, expressed FOS-LI after pinch of the tail than of the neck.

In another series of experiments, rats received injection of 5% formalin into one hindpaw. After 75-90 minutes, FOS-LI was seen in the same parts of PB as after noxious mechanical stimulation. The heaviest labeling was seen on the side contralateral to the injection side, with statistically significant (P < 0.05) side differences present in PBsl and PBdl.

In a third series of experiments, rats were hemisected at low cervical-upper thoracic segments, allowed 2 weeks to recover, and then given formalin injections in both hindpaws. Significantly more neurons were FOS-labeled in PBdl, PBsl, and PBcl on the side contralateral to the hemisection than on the ipsilateral side.

These observations are discussed in relation to the organization of the spinal afferent input and the efferent connections of PB. It is concluded that the FOS-LI expression in PBdl and PBsl and probably also in PBcl, to a large extent, is evoked by the ascending spinal nociceptive input to PB. Because these subnuclei project to several hypothalamic regions, it is suggested that neurons in PB that express FOS after noxious mechanical and chemical stimulation primarily are involved in autonomic and homeostatic responses to behavioral situations that involve tissue-damaging stimuli.

Indexing terms: immediate-early gene, c-fos, immunohistochemistry, pain, Kolliker-Fuse nucleus

c) 1996 Wiley-Liss, Inc.

The parabrachial nucleus (PB) in the dorsolateral pons is an important structure for a variety of autonomic and homeostatic mechanisms and, for example, has been associ- ated with cardiovascular regulation (Mraovitch et al., 1982; Hubbard et al., 1987; Ward, 1988), gustatory functions (Norgren and Leonard, 1973; Li et al., 1994; Swank and Bernstein, 1994), respiration (Smith et al., 1989; Lara et al., 19941, body fluid balance (Ohman and Johnson, 1986; Kobashi et al., 1993), sleep (Saito et al., 19771, and analge- sia (Girardot et al., 1987; Haws et al., 1989; Chiang et al., 1994). The PB has been suggested to be involved also in nociceptive mechanisms, because it is a major target for

ascending projections from nociceptive-responsive neurons in the superficial layer (lamina I) of the spinal and medul- lary dorsal horn (Wiberg and Blomqvist, 1984; Hylden et al., 1986; Light et al., 1987; Wiberg et al., 1987; Craig, 1995). The lamina I fibers terminate primarily in the dorsal parts of the lateral PB (Cechetto et al., 1985; Blomqvist et al., 1989; Slugg and Light, 1994; Craig, 1995). In accor-

Accepted November 25,1995 Address reprint requests to A. Blomqvist, Department of Cell Biology,

Faculty of Health Sciences, University of Linkoping, S-581 85 Linkoping, Sweden.

o 1996 WILEY-LISS, INC.

46 0. HERMANSON AND A. BLOMQVIST

minutes from the end of the stimulation, the animals were killed by transcardial perfusion (see below).

Formalin injection Sixteen rats received injection of 100 p1 5% formalin

(26-30°C; Dallel et al., 1995) into the back of one hindpaw during a brief ( < 90 seconds) ether anesthesia and were then allowed to survive for 30 (n = 1),45 (n = l), 60 (n = 31, 75 (n = 4), 90 (n = 3), 180 (n = 2), or 480 (n = 2) minutes.

Spinal cord hemisection followed by formalin injection

Eight rats were subjected to spinal cord hemisection at lower cervical segments. The animals were anesthetized with chloral hydrate (0.3 gikg) and, following dorsal lami- nectomy at lower cervical-upper thoracic levels and removal of the dura, one side of the spinal cord was transected with a scalpel blade. Between 15-17 days after the hemisection, the animals received bilateral injections of 5% formalin (100 p1; 26-30°C) into the back of both hindpaws and were killed by perfusion after 75-90 minutes. Two hemisected animals served as controls and did not receive formalin injections.

Additional controls 1) Noxious pinch of the skin elicited defensive behaviors,

such as biting directed at the artery clamp. To check whether biting resulted in FOS-like immunoreactivity (FOS- LI), three animals were given food and water after having been deprived for 16-20 hours and were then killed after 1 hour. 2) Because defensive behaviors in rats may involve ultrasonic (22-28 kHz) vocalizations (Bandler and Depau- lis, 1991; Macedonia and Evans, 1993), and because ultra- sound can elicit FOS-LI (Friauf, 1992; Pierson and Snyder- Keller, 19941, three animals were subjected to ultrasonic stimulation (25 kHz) for 20 seconds every 5 minutes (n = 2) or continuously (n = 1) for 90 minutes and were then allowed to survive for 90 minutes. 3) To evaluate the effect of ether anesthesia on the FOS expression (Watts, 1991) in the formalin injection experiment, five animals were briefly anesthetized but were not injected with formalin, and they were killed immediately (n = 2) or after 75 (n = 2) or 120 minutes (n = 1).

Tissue preparation At the end of the survival period, the animals were given

an overdose of sodium pentobarbital (120 mg/kg, i.p.) and were perfused transcardially within 10 minutes with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brain and spinal cord were removed, stored in a 20% sucrose-fixative solution for 24-48 hours, and cut transversely at 40 pm on a freezing microtome. The sections from the first 27 mechanically stimulated rats were processed with a FOS antiserum obtained from Cambridge Biochemicals (sheep anti-c-FOS; OA-11-823; 0.18 pgiml). The sections from the other animals were processed with a FOS antiserum obtained from Oncogene (rabbit anti-c- FOS; PC05; 0.10 pgiml). The sections were incubated in primary antibody solution [0.2% bovine serum albumin and 3.3% normal serum (Dakopatts) in 0.1 M phosphate buffer, pH 7.41 overnight at room temperature under constant agitation. Bound primary antibody was visualized with secondary antibody (rabbit anti-sheep, 1:30, or swine anti- rabbit, 1:30; Dakopatts) and peroxidase-antiperoxidase (PAP) complex (PAP-goat, 1:120, or PAP-rabbit, 1:120;

dance with the anatomical data, electrophysiological stud- ies have shown that neurons in the lateral part of PB are responsive to nociceptive stimulation (Bernard and Besson, 1990; Hayashi and Tabata, 1990; Slugg and Light, 1990).

Immunohistochemical demonstration of the protoonco- gene protein FOS has been shown to be a useful anatomical tool for the demonstration of synaptic activation of neurons (Bullitt, 1990; Morgan and Curran, 1991; Luckman et al., 1994). In the present study, we have used this method to localize neurons in PB that are activated by peripheral noxious stimulation. The purpose was to provide knowledge on the subnuclear distribution of PB neurons that express FOS consequent to acute nociceptive stimulation. Such knowledge is important for our understanding of the functional role of the nociceptive input to PB, because the different nuclear groups in PB have different efferent connections (Fulwiler and Saper, 1984). Awake, freely moving animals were subjected to pinching of the skin at the nape of the neck or the base of the tail or were given formalin injections into one hindpaw to evoke FOS expres- sion in PB. Furthermore, rats were given formalin injec- tions into both hindpaws subsequent to spinal cord hemisec- tion to clarify whether the FOS expression was a result of ascending nociceptive input to PB. A preliminary account of the findings has been given in abstract form (Hermanson et al., 1992).

MATERIALS AND METHODS Male Sprague-Dawley rats (B & K Universal, Stockholm,

Sweden; 250-450 g) were used. All experimental proce- dures were approved by the Animal Care and Use Commit- tee at the University of Linkoping.

Noxious mechanical stimulation Forty-two rats were divided randomly into 14 groups,

with three rats in each group. Each group consisted of two rats that were subjected to noxious stimulation and one rat that served as a control. The three rats were placed in separate cages for 2-4 hours in a quiet, dusky room. All experiments were initiated after an additional 30 minutes of habituation of the animals to the experimenter. Noxious stimuli were delivered with an artery clamp (Dieffenbach BH 27; Aesculap, Federal Republic Germany; 650-750 g; the pinch was mildly painful when tested on the experiment- er’s skin) placed on the base of the tail in one rat and on the nape of the neck in the second rat (Fleischmann and Urca, 1988) for 20 seconds every 5 minutes during 90 minutes. For a control of nonspecific effects, we used innocuous cutaneous stimulation (brush) of the base of the tail. At 45 (n = 6), 60 (n = 12), 75 (n = 91, 90 (n = 9), or 210 (n = 6)

cl, PBcl CnF dl, PBdl el, PBel il KF me5 PB SCP sl, PBsl vl

Abbreviations

central lateral parabrachial subnucleus nucleus cuneiformis dorsal lateral parabrachial subnucleus external lateral parabrachial subnucleus internal lateral parabrachial subnucleus Kolliker-Fuse nucleus mesencephalic trigeminal tract parabrachial nucleus superior cerebellar peduncle superior lateral parabrachial subnucleus ventral lateral parabrachial subnucleus

NOXIOUS-EVOKED FOS IN PARABRACHIAL NUCLEUS 47

Dakopatts) followed by peroxidase processing with 0.035% 3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydro- gen peroxide with 2.5% ammonium nickel sulphate in 0.1 M sodium acetate buffer, pH 6.0, for 5-8 minutes. Adjacent sections were stained with thionin. Specificity of the immu- nohistochemical labeling was checked by preabsorption of the diluted primary antiserum with 1 pg/ml of the s-14-c- fos peptide (Oncogene) for 4 hours.

Data analysis The distribution of labeled neurons in the parabrachial

area was plotted with a camera lucida. The numbers of labeled neurons in different subnuclei were quantified on every second section with a microcomputer image analysis system (MCID, Imaging Research, Canada) connected to a light microscope (Nikon Optiphot-2). Cell counts of labeled neurons also included the ventrolateral periaqueductal grey matter (PAGvl), the cuneiform nucleus (CnF) and the locus coeruleus (LC). Sections were analyzed with a x 4 plan- apochromatic objective (numerical aperture, 0.2) at a final magnification of X570. All illumination and camera set- tings were fixed for each group of animals (experimental and control) to yield accurate comparisons between ani- mals. Across groups, background density level was varied (between 128 and 181; scale 0-255) to exclude counting of unspecifically stained profiles. Nuclear groups were identi- fied in adjacent thionin sections (Fig. 1). Nomenclature of the region of the PB followed Paxinos and Watson (19861, whereas the subdivisions of PB were delineated according to Fulwiler and Saper (1984), with the following two exceptions: The extreme lateral nucleus could not be delin- eated accurately in all animals, and, due to the close proximity to the lateral lemniscus (where FOS was ex- pressed also in several control animals), the extreme lateral subnucleus was not included in the quantitative analysis. The dorsal lateral subnucleus was considered to extend approximately 50-100 km more rostrally than described by Fulwiler and Saper (1984). Statistical analyses were per- formed on animals that displayed maximal labeling in PB, i.e., 60-75 minutes after the end of the mechanical stimula- tion and 75-90 minutes after the formalin injection (see below). Differences in cell counts between the animals that were subjected to noxious pinch of the neck or tail and those subjected to innocuous cutaneous stimulation (five groups with a total of 15 rats) were analyzed statistically by using a paired t test. A paired t test was also used for statistical analysis of side differences in animals subjected to unilat- eral formalin injection (n = 7) of the hindpaw and in animals subjected to spinal cord hemisection (n = 6) followed by formalin injections into both hindpaws.

RESULTS Antisera sensitivity and specificity

A similar labeling pattern, with staining of the cell nucleus (Sambucetti and Curran, 19861, was obtained with both primary antisera. However, the Oncogene antiserum yielded a more distinct labeling than the Cambridge antise- rum; therefore, cell counts on the number of PB neurons that expressed FOS-LI after noxious mechanical stimula- tion of the tail and neck were restricted to the brains that had been processed with the Oncogene antiserum, whereas the cases processed with the Cambridge antibody were used for qualitative analysis. Preadsorption of the diluted antise-

rum with 1 pg/ml of the s-14-c-fos peptide totally abolished the labeling.

Noxious mechanical stimulation Pinching of the nape of the neck or the base of the tail

evoked FOS-LI in neurons bilaterally in lamina I of the dorsal horn in the upper cervical and sacral spinal cord segments, respectively (Fig. 2A,B). In the upper cervical segments, the labeled neurons were present in the lateral part of the dorsal horn (Fig. 2A) and in the sacral segments throughout its mediolateral extent (Fig. 2B), in line with the general principles for the somatotopic representation in the dorsal horn (Molander and Grant, 1985; Nyberg and Blomqvist, 1985). After noxious stimulation of the tail, labeled neurons were also present in deeper laminae of the dorsal horn, but the numbers of labeled neurons were much smaller than in lamina 1-11. Zero or only single FOS-LI neurons were present in the dorsal horn of the rats that received innocuous cutaneous stimulation, irrespective of the survival time.

Labeled neurons were present bilaterally in the lateral PB in noxiously stimulated rats. The labeling pattern was largely symmetrical on the two sides, consistent with the placement of the noxious stimuli on midline skin areas. Large numbers of labeled neurons were seen 45-90 min- utes after the end of the noxious stimulation, with maximal labeling present after 60-75 minutes (Figs. 3A,B, 4, 5A). After 210 minutes, only small numbers of labeled cells were found.

A dense cluster of labeled neurons was present in the dorsal part of the lateral PB (Fig. 3A,B) and encompassed the superior and dorsal subnuclei (PBsl and PBdl, respec- tively; Figs. 4, 5 ) . This cluster extended about 300-400 pm in the rostrocaudal direction, with the rostra1 end located 150-200 pm anterior to the level of separation between the inferior colliculus and the pons. A significant but scattered labeling was seen in the central lateral subnucleus (PBcl; Figs. 4, 5A). Sparse labeling was present in the Kolliker- Fuse nucleus (KF) and in the external lateral subnucleus (PBel; Figs. 4, 5A). The medial PB (PBm), the internal lateral subnucleus (PBil), and the ventral lateral sub- nucleus (PBvl) contained zero or only single labeled neu- rons. The labeling pattern was the same in both groups of noxiously stimulated rats (Figs. 3A,B, 4), but the numbers of labeled neurons, particularly in PBsl, were larger in animals that were pinched in the tail (Fig. 5A). Rats that received innocuous cutaneous stimulation displayed zero or only single FOS-LI neurons in PB, except for two rats, where small numbers of labeled neurons were present in PBsl and PBcl.

After noxious mechanical stimulation, FOS-LI was also present in structures adjacent to PB, such as the LC, the CnF, and the caudal PAGv1. However, significant numbers of labeled neurons were seen in these nuclei also after innocuous cutaneous stimulation (data not shown).

Formalin injections Injection of 5% formalin into one hindpaw resulted in

FOS-LI neurons in the ipsilateral lamina I of the dorsal horn after 30-minute survival. The heaviest labeling was found 75-90 minutes after formalin injection (Fig. 2 0 , and, at this survival time, small numbers of labeled neurons were also present in the deeper laminae of the spinal gray matter. After 180 minutes, single labeled neurons were also

48 0. HERMANSON AND A. BLOMQVIST

Fig. 1. Light micrographs of thionin-stained, transverse, 40-wm-thick sections through the parabra- chial region showing the subnuclear organization a t different rostrocaudal levels. A is 140 wm rostra1 to the level of separation between the inferior colliculus and the pons. B is at the level of separation. C and D are 140 and 300 pm caudal to the level of separation, respectively. For abbreviations, see list. Scale bar = 100 wm.

present contralateral to the injection site. After 480 min- utes, almost all labeling in the spinal cord had disappeared.

Scattered labeled neurons were seen in PB 45 minutes after the formalin injection. The heaviest labeling was found after 75-90 minutes and had the same distribution as that seen after noxious mechanical stimulation (Figs. 3C-F, 6). With longer survival periods, the numbers of labeled neurons were smaller, and, after 480 minutes, only single labeled neurons were detected in PB.

Quantitative analysis of the numbers of labeled neurons in PB at the time of maximal labeling (75-90 minutes) revealed that the largest numbers of labeled neurons were present on the side contralateral to the injection site (Fig.

5B). The side differences were statistically significant (P < 0.05) for PBsl and PBdl. A large but statistically not significant difference was found also for PBcl, whereas no consistent pattern was found for PBel and KF (Fig. 5B).

In the formalin-injected rats, FOS-LI was also present in LC, CnF, and PAGvl. No significant side differences were found in these areas.

FOS labeling following spinal cord hemisection

The pattern of FOS-labeling in the spinal dorsal horn following formalin injection into both hindpaws in rats with spinal cord hemisections (Fig. 7) was the same as in rats

NOXIOUS-EVOKED FOS IN PARABRACHIAL NUCLEUS 49

- Fig. 2. Light micrographs showing FOS-like immunoreactivity (FOS-

LI) in the dorsal horn of the upper cervical spinal cord (A), the sacral spinal cord (B), and the lumbar spinal cord (C ) 60-75 minutes after nociceptive stimulation of the nape of the neck, the base of the tail, and the ipsilateral hindpaw, respectively. Scale bar = 100 pm.

with intact spinal cords. Also, in PB, the labeling pattern was similar to that seen in the other stimulation experi- ments. Labeled neurons were present on both sides, but with a predominance for the side contralateral to the hemisection (Fig. 8). Statistically significant (P < 0.05) side differences regarding the numbers of labeled neurons were found for PBsl, PBdl, and PBcl, but not for PBel or KF (Fig. 5C).

Control experiments In control animals that were subjected to spinal cord

hemisection but that did not receive formalin injections, only single labeled neurons were detected in the lumbar spinal cord and the PB area. Animals that had been subjected to a brief ether anesthesia without any subse- quent mechanical or chemical stimulus also displayed only single labeled neurons in the spinal cord and brainstem.

After ultrasonic stimulation at 25 kHz, single FOS-LI neurons were seen in PBcl and PBel. However, many labeled neurons were present in other areas, e.g., the inferior colliculus, CnF, and PAGv1, as previously demon- strated (Friauf, 1992; Pierson and Snyder-Keller, 1994).

Refeeding after food deprivation resulted in FOS-LI in PBel (Li et al., 1994). Small numbers of labeled neurons were also detected in PBcl.

DISCUSSION The present study demonstrates that noxious stimula-

tion in awake rats evokes FOS-LI in PB. FOS-expressing neurons were located in the lateral PB and were found preferentially in PBsl, PBdl, and PBcl. Noxious stimulation of one hindpaw resulted in FOS-LI preferentially on the side contralateral to the injection site, and hemisection of the cervicothoracic spinal cord prior to bilateral hindpaw stimulation resulted in an attenuation of FOS-LI in PB on the hemisected side.

Correlation of the FOS expression with the ascending spinal nociceptive input to PB The FOS expression in the lateral part of PB and its

contralateral predominance following unilateral hindpaw stimulation is in line with the general organization of the ascending spinal input (Cechetto et al., 1985; Blomqvist et al., 1989; Sluggand Light, 1994; Craig, 1995) and may seem to indicate that the FOS expression in PB following periph- eral noxious stimulation is a consequence of activation of spinoparabrachial fibers. Thus, nociceptive-responsive spi- nal cord neurons that project to PB have been demon- strated (Hylden et al., 1986; Light et al., 1987, 1993; Menetrey and de Pommery, 1991; Noguchi and Ruda, 1992; Ding et al., 1994; Jasmin et al., 1994; Wang et al., 19941, and neurons in the lateral part of PB that are activated by noxious stimuli have been recorded (Bernard and Besson, 1990; Slugg and Light, 1990; Bernard et al., 1994). The anterograde tracing study by Feil and Herbert (1995) demonstrated that the superficial dorsal horn of lumbar segments projects preferentially to the contralateral PBdl and adjacent lateral portions of PBcl; in the present study, the same PB regions contained many FOS-LI neurons after noxious hindpaw stimulation.

However, several discrepancies exist between the termi- nation pattern of the spinal fibers and the distribution of FOS-LI. Thus, the heaviest labeling was seen in PBsl (see also Lanteri-Minet et al., 19941, but this subnucleus was shown by Feil and Herbert (1995) to be devoid of a direct spinoparabrachial projection. Bernard et al. (1995) re- ported that PBsl received spinal afferent fibers, in apparent contrast to the findings by Feil and Herbert (19951, but their delineation of PBsl differed from that used by Feil and Herbert (1995) and by us and clearly encompassed part of the PBdl. Our delineation of PBsl followed Fulwiler and Saper (1984; see also Herbert et al., 1990; Bernard et al., 1994; Slugg and Light, 1994); this delineation is supported

Fig. 3. A,B: Light micrographs showing FOS-LI neurons in the superior lateral parabrachial subnucleus 60 minutes after noxious pinch of the neck (A) and tail (B). The sections are from the same rostrocaudal level as Figure 1A. C-F: FOS-LI in the parabrachial nucleus (PB) 75 minutes after formalin injection into the contralateral hindpaw. The rostrocaudal levels correspond to Figure 1A-D. For abbreviations, see list. Scale bar = 100 km.

NOXIOUS-EVOKED FOS IN PARABRACHIAL NUCLEUS 51

A

D U

Fig, 4. Schematic drawings of coronal sections through the PB ordered from rostra1 (A,A') to caudal (D,D') showing the distribution of FOS-LI neurons (dots) 60 minutes after noxious pinch of the neck (A-D) and tail (A'-D'). For abbreviations, see list.

52 0. HERMANSON AND A. BLOMQVJST

Number of FOS-expressing neurons in parabrachial subnuclei at the time of maximal labeling. Cell counts were done on every second section. The numbers in A represent the numbers of labeled neurons on one side. A 60-75 minutes after noxious mechanical stimulation of the neck (hatched bars) and tail (open bars), and innocuous cutaneous stimula- tion of the tail (solid bars). Animals were divided into five groups with three rats (neck, tail, and control) in each group; thus, each bar represents five animals. A paired t test was used to calculate values of probability (asterisks indicate P < 0.05 for noxious stimuli vs control). B: 75-90 minutes after formalin injection into the contralateral (hatched bars) and ipsilateral (open bars) hindpaw in = 7). C: Contra- lateral (hatched bars) and ipsilateral (open bars) to a cervical hemisec- tion (n = 6) 75-90 minutes following formalin injections into both hindpaws. Differences between the two sides were statistically analyzed with a paired t-test (asterisks indicate P < 0.05). Although the average number of FOS-like neurons was larger in the experimental series shown in C than in that shown in B, this difference was not statistically significant (Mann-Whitney; P = 0.4). For abbreviations, see list.

Schematic drawings of coronal sections through the PB 75 minutes after formalin injection into the contralateral hindpaw (for details, cf. Fig. 4). For abbreviations, see list.

53 NOXIOUS-EVOKED FOS IN PARABRACHIAL NUCLEUS

LE 2 LE 5

L E 8 LE 10

Fig. 7. Drawings of lower cervical or upper thoracic spinal cord sections showing the extent of the lesions (solid areas) in the rats (LE 2, LE 5, LE 6, LE 7, LE 8, and LE 10) that were subsequently noxiously stimulated.

by the specific projection of PBsl to the ventromedial hypothalamic nucleus (VMH; Fulwiler and Saper, 1984; Hermanson and Blomqvist, unpublished observations) and its very high density of cholecystokinin-containing neurons (Zaborszky et al., 1984; Fulwiler and Saper, 1985; Herman- son et al., 1995). From the illustrations in the paper by Bernard et al. (19951, it appears that the area correspond- ing to PBdl in the present study received labeled terminals, whereas the PBsl proper was unlabeled.

Intracellular staining studies have shown that many PB neurons have dendritic trees that cross the subnuclear boundaries and extend into several other subnuclei (Luo et al., 1990; Herbert and Bellintani-Guardia, 1995). Accord- ingly, although the dendritic extent of the neurons in PBsl is not yet known, it is possible that PBsl neurons have dendrites that extend into the terminal domain of the ascending spinal fibers and, thereby, receive ascending nociceptive information. The possibility that such an input to PBsl may exist is suggested by the attenuated labeling observed after unilateral hemisection of the spinal cord (Fig. 5C); at the same time, this set of experiments also suggests that the noxiously evoked activation of PB neu- rons can take place via alternative routes, because a substan- tial FOS expression was elicited despite the fact that the ascending ipsilateral spinal input had been interrupted. However, midline crossing of spinal ascending fibers at levels above the hemisection (Mehler et al., 1960) or the presence of projections between the two PBs (Saper and Loewy, 1980; Fulwiler and Saper, 1984; Krukoff et al., 1992) should also be considered.

In the present study, more FOS-LI was consistently elicited after pinch of the tail than of the neck (Fig. 5A). Also, after chemical and mechanical stimuli, the FOS-LI differed, because, in general, more parabrachial neurons expressed FOS-LI after chemical stimulation than after mechanical stimulation (Fig. 5A-C). Such differences may reflect differences in the intensity of the stimuli, but they may also reflect differences in the central representation of the different skin areas. It is interesting to note that anterograde tracing studies have suggested that the spinal input to PB is topographically organized, in that different rostrocaudal regions of the spinal dorsal horn preferentially project to different PB subnuclei (Feil and Herbert, 1995). For example, the superficial laminae of the lumbar spinal dorsal horn, which are the terminal site of peripheral fibers from the hindpaw (Molander and Grant, 19851, project densely to PBdl and the outer portion of PBcl, whereas the corresponding part of the upper cervical segments that receives input from the nape of the neck projects only sparsely to these PB regions (Feil and Herbert, 1995). It is conceivable that such differences in the projection patterns account for some of the differences in FOS labeling follow- ing stimulation of different body parts. Thus, the different magnitude of the input from the upper cervical and lumbar segments to the PBdl and PBcl could explain the differences in FOS-LI in the these nuclei as well as in PBsl (see above) following noxious stimuli of the neck and tail, respectively.

Several regions that have been implicated in nociceptive mechanisms did not display significant FOS-LI. For ex- ample, electrophysiological studies have reported that neu- rons in PBel are nociceptive responsive (Bernard et al., 19941, and PBel has been suggested to be a major relay for nociceptive information to the amygdala (Bernard and Besson, 19901, which is the main efferent target of PBel neurons (Fulwiler and Saper, 1984; Bernard et al., 1991). Yet, in this and in other studies of noxiously evoked FOS expression in unanesthetized animals (Lanteri-Minet et al., 1994), the labeling in PBel was sparse. FOS labeling has been shown in PBel after noxious stimulation in anesthe- tized animals (Bullitt, 1990; Herdegen et al., 1991). How- ever, it is well established that PBel receives dense input from the nucleus of the solitary tract (Berkley and Scofield, 1990; Herbert et al., 1990); thus, it is likely that the FOS expression seen in anesthetized animals is due to afferent vagal activity elicited by eardiovascular effects of the anes- thesia, particularly because anesthesia alone (Krukoff et al., 1992; Lee and Beitz, 1993; Rocha et al., 1994) as well as vagal stimulation (Kobashi et al., 1993; Chan and Saw- chenko, 1994) and vasomotor changes (Murphy et al., 1994) result in dense FOS labeling in PBel. Furthermore, few dorsal horn fibers terminate in PBel. Feil and Herbert (1995) found that the superficial lamina of the trigeminal nucleus caudalis and of the upper cervical segments project densely only to a small ventral portion of PBel (PBelv), and these authors saw virtually no terminal labeling in PBel following tracer injection into the lumbar dorsal horn. Feil and Herbert (1995) found terminal labeling lateral to PBel in the so-called lateral crescent area (Chamberlin and Saper, 1992). Bernard et al. (1995) also found terminal labeling in the lateral crescent area, but they ascribe part of this terminal field to the outer portion of PBel. Interest- ingly, the few FOS-labeled cells that were observed in PBel in the present study tended to be located along its lateral border (Figs. 4B’, 6B-D).

It has been suggested that the discrepancy between the lack of substantial spinal afferent projection to PBel and the electrophysiological reports on nociceptive activation of

54 0. HERMANSON AND A. BLOMQVIST

Fig. 8. FOS-LI in the PB elicited by formalin injection into both hindpaws. A is contralateral and B is ipsilateral to a preceding cervical hemisection (rat LE 10 in Fig. 7). Note the heavier labeling contralateral to the hemisected side. scp, Superior cerebellar peduncle. Scale bar = 100 Fm.

PBel neurons could be explained by the presence of a nociceptive spinal afferent input to PBel dendrites located within the termination field of the spinal fibers (Feil and Herbert, 1995; Saper, 1995). This is unlikely considering the sparse FOS expression seen in PBel following periph- eral nociceptive stimulation. Thus, the present observa- tions do not support an important role for PBel neurons in nociceptive processing.

However, the possibility that putative nociceptive signals to PBel, in contrast to those signals mediated by solitary tract neurons, involve transcription factors other than FOS must be considered. In situ hybridisation studies (Herman- son et al., 1992; Blomqvist et al., 1994) have shown that PBel neurons are enkephalinergic, supporting previous immunohistochemical evidence that neurons in this part of PB contain enkephalin (Finley et al., 1981). Because studies on the mechanisms of preproenkephalin (ppENK) gene expression have indicated that FOS may not be a crucial factor in ppENK transcriptional mechanisms (cf. Borsook et al., 1994; Johnston and Morris, 1994), as was initially proposed (Sonnenberg et al., 19891, it is possible that the enkephalinergic PBel neurons do not express FOS follow- ing noxious stimulation.

The possibility that FOS may not be involved in the ppENK transcription may also explain the very sparse FOS expression in KF and PBil, the two other major enkephalin- ergic subnuclei in PB (Hermanson et al., 1992; Blomqvist et al., 1994; Hermanson and Blomqvist, 1995). Both KF and PBil receive afferent input from the spinal cord, although the density of the spinal input to KF is controversial (cf. Slugg and Light, 1994; Bernard et al., 1995; Feil and Herbert, 1995). We have previously demonstrated an in- creased ppENK mRNA expression in KF following mechani- cal nociceptive stimulation, suggesting that nociceptive signals activate KF neurons (Blomqvist et al., 1994). It is conceivable that this activation, at least for the majority of the neurons, is coupled to other transcription factors, such as cyclic AMP response element-binding protein (CREB; Borsook et al., 1994; Hermanson, unpublished observa- tions).

Functional considerations The three subnuclei that contained the vast majority of

the FOS-labeled neurons in the present study (PBsl, PBdl, and PBcl) all project to the hypothalamus; thus, these nuclei provide a possible route by which noxiously evoked activity may influence hypothalamic mechanisms (Wiberg and Blomqvist, 1984; Ma et al., 1989; Hermanson et al., 1992). PBsl projects primarily to VMH (Fulwiler and Saper, 1984), which has been implicated in a variety of functions, such as defensive and sexual behavior and feeding (Swan- son, 1987). It is of interest that PBsl neurons that project to VMH contain the opiate antagonist cholecystokinin (Za- borszky et al., 1984; Fulwiler and Saper, 19851, suggesting that cholecystokinin transmission in the hypothalamus may be influenced by nociceptive stimuli.

The main target for PBdl and PBcl is the median preoptic hypothalamic nucleus (Berk and Finkelstein, 1981; Saper and Levisohn, 1983; Fulwiler and Saper, 1984; Lind and Swanson, 19841, and PBdl and PBcl may provide a route by which nociceptive stimuli influence cardiovascular mecha- nisms. PBdl and PBcl as well as PBsl also project to the paraventricular hypothalamic nucleus (Berk and Finkel- stein, 1981; Fulwiler and Saper, 1984; Slugg and Light, 19911, which is a key region for central autonomic regula- tion. These data suggest that neurons in PB that express FOS after noxious mechanical and chemical stimulation are involved primarily in autonomic and homeostatic responses to behavioral situations that involve tissue-damaging stimuli.

ACKNOWLEDGMENTS We thank Ludmila Mackerlova for helping with the

illustrations. This study was supported by grants from the Swedish Medical Research Council (project 78791, the Magn. Bergvall Foundation, the Clas Groschinsky Memo- rial Fund, the Lars Hierta Memorial Fund, the h e Wiberg Foundation, and the County Council of Ostergotland.

NOXIOUS-EVOKED FOS IN PARABRACHIAL NUCLEUS 55

LITERATURE CITED Bandler, R., and A. Depaulis (1991) Midbrain periaqueductal gray control of

defensive behavior in the cat and the rat. In A. Depaulis and R. Bandler (eds): The Midbrain Periaqueductal Gray Matter. New York: Plenum Press, pp. 175-198.

Berk, M.L., and J.A. Finkelstein (1981) Afferent projections to the preoptic area and hypothalamic regions in the rat brain. Neuroscience 6:1601- 1624.

Berkley, K.J.. and S.L. Scofield (1990) Relays from the spinal cord and solitary nucleus through the parabrachial nucleus to the forebrain in the cat. Brain Res. 529:333-338.

Bernard, J.F., and J.M. Besson (1990) The spino(trigemino)pontoamygda- loid pathway: Electrophysiological evidence for an involvement in pain processes. J. Neurophysiol. 633.473490.

Bernard, J.F., J. Carroue, and J.M. Besson (1991) Efferent projections from thc external parahrachial area to the forebrain: A Phaseolus vulgaris leucoagglutinin study in the rat. Neurosci. Lett. 122:257-260.

Bernard, J.F., G.F. Huang, and J.M. Besson (1994) The parabrachial area: Electrophysiological evidence for an involvement in visceral nociceptive processes. J. Neurophysiol. 71: 16461660.

Bernard, J.F., R. Dallel, P. Raboisson, L. Villanueva, and D. Le Bars (1995) Organization of the efferent projections from the spinal cervical enlarge- ment to the parabrachial area and periaqueductal gray: A PHA-L study in the rat. J. Comp. Neurol. 353:480-505.

Blomqvist, A., W. Ma, and K.J. Berkley (1989) Spinal input to the parabra- chial nucleus in the cat. Brain Res. 480:29-36.

Blomqvist, A,, 0. Hermanson, H. Ericson, and D. Larhammar (1994) Activation of a bulhospinal opioidergic projection hy pain stimuli in the awake rat. Neuroreport 5461464.

Borsook, D., C. Konradi, 0. Falkowski, M. Comb, and S.E. Hyman (19941 Molecular mechanisms of stress-induced proenkephalin gene regulation: CREB interacts with the proenkephalin gene in the mouse hypothala- mus and is phosphorylated in response to hyperosmolar stress. Mol. Endocrinol. 8240-248.

Bullitt, E. (1990) Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. J. Comp. Neurol. 29651 7-530.

Cechetto, D.F., D.G. Standaert, and C.B. Saper (1985) Spinal and trigeminal dorsal horn projections to the parahrachial nucleus in the rat. J. Comp. Neurol. 240:153-160.

Chamberlin, N.L., and C.B. Saper (1992) Topographic organization of cardiovascular responses to electrical and glutamate microstimulation of the parabrachial nucleus in the rat. J. Comp. Neurol. 326:245-262.

Chan, R.K.W., and P.E. Sawchenko (1994) Spatially and temporally differen- tiated patterns of c-fos expression in hrainstem catecholaminergic cell groups induced by cardiovascular challenges in the rat. J. Comp. Neurol. 348:433460.

Chiang, C.Y., J.W. Hu, and B.J. Sessle (1994) Parahrachial area and nucleus raphe magnus-induced modulation of nociceptive and nonnociceptive trigeminal subnucleus caudalis neurons activated by cutaneous or deep inputs. J. Neurophysiol. 71:2430-2445.

Craig, A.D. (1995) Distribution of Brainstem Projections from Spinal Lamina I Neurons in the Cat and the Monkey. J. Comp. Neurol. 361:225-248.

Dallel, R., P. Rahoisson, P. Clavelou, M. Saade, and A. Woda (1995) Evidence for a peripheral origin of the tonic nociceptive response to subcutaneous formalin. Pain 61:11-16.

Ding, Y.-Q., B.-Z. gin, J.3. Li, and N. Mizuno (1994) Induction of c-fos-like protein in the spinoparahrachial tract-neurons locating within the sacral parasympathetic nucleus in the rat. Brain Res. 659:283-286.

Feil, K., and H. Herbert (1995) Topographic organization of spinal and trigeminal somatosensory pathways to the rat parabrachial and Kolliker- Fuse nuclei. J. Comp. Neurol. 353:506-528.

Finley, J.C.W., J.L. Maderdrut, and P. Petrusz (1981) Theimmunocytochemi- cal localization ofenkephalin in the central nervous system of the rat. J. Comp. Neurol. 198:541-565.

Fleischmann. A,, and G. Urca (1988) Different endogenous analgesia sys- tems are activated hy noxious stimulation of different body regions. Brain Res. 45549-57.

Friauf, E. (1992) Tonotopic order in the adult and developing auditory system of the rat as shown by c-fos immunocytochemistry. Eur. J. Neurosci. 4:798-812.

Fulwiler, C.E., and C.B. Saper (1984) Subnuclear organization of the efferent connections of the parahrachial nucleus in the rat. Brain Res. Rev. 7.929-259.

Fulwiler, C.E., and C.B. Saper (1985) Cholecystokinin-immunoreactive innervation of the ventromedial hypothalamus in the rat: Possible substrate for autonomic regulation of feeding. Neurosci. Lett. 53:289- 296.

Girardot, M.-N., T.J. Brennan, M.E. Martindale, and R.D. Foreman (1987) Effects of stimulating the subcoeruleus-parabrachial region on the nonnoxious and noxious responses of T1-Ti spinothalamic tract neurons in the primate. Brain Res. 409:19-30.

Haws, C.M., A.M. Williamson, and H.L. Fields (1989) Putative nociceptive modulatory neurons in the dorsolateral pontomesencephalic reticular formation. Brain Res. 483:272-282.

Hayashi, H., and T. Tabata (1990) Pulpal and cutaneous inputs to somatosen- sory neurons in the parahrachial area of the cat. Brain Res. 511: 177-179.

Herbert, H., and B. Bellintani-Guardia (1995) Morphology and dendritic domains of neurons in the lateral parabrachial nucleus of the rat. J. Comp. Neurol. 354t377-394.

Herbert, H., M.M. Moga, and C.B. Saper (1990) Connections of the parabra- chial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat. J. Comp. Neurol. 293:540-580.

Herdegen, T., J.D. Leah, T. Walker, B. Basler, R. Bravo, and M. Zimmer- mann (1991) Noxious stimulation induces molecular genetic events in neurons of the central nervous system: Expression of immediate-early gene encoded proteins. In M.R. Bond, J.E. Charlton, and C.J. Woolf (eds): Proceedings of the Sixth World Congress on Pain. New York: Elsevier, pp. 325-330.

Hermanson, O., and A. Blomqvist (1995) Enkephalinergic and catecholamin- ergic neurons constitute separate populations in the rat Kolliker- FuselA7 region. Neurosci. Lett. 190:57-60.

Hermanson, O., H. Ericson, D. Larhammar, and A. Blomqvist (1992) Expression of FOS-like immunoreactivity and preproenkephalin mRNA in the rat parabrachial nucleus after nociceptive mechanical stimulation of the skin. SOC. Neurosci. Ahstr. 18:832.

Hermanson, O., M. Hallheck, D. Larhammar, and A. Blomqvist (1995) Preprocholecystokinin mRNA expressing neurons in the rat parahra- chial nucleus. SOC. Neurosci. Abstr. 21:893.

Hubbard, J.W., R.A. Buchholz, T.K. Keeton, and M.A. Nathan (1987) Parahrachial lesions increase plasma norepinephrine concentration, plasma renin activity and enhance baroreflex sensitivity in the conscious rat. Brain Res. 421:226-234.

Hylden, J.L.K., H. Hayashi, R. Dubner, and G.J. Bennett (19861 Physiology and morphology ofthe lamina I spinomesencephalic projection. J. Comp. Neurol. 24 7: 505-5 15.

Jasmin, L., H. Wang, K. Tarczy-Hornoch, J.D. Levine, and A.I. Bashaum (1994) Differential effects of morphine on noxious stimulus-evoked FOS-like immunoreactivity in subpopulations of spinoparabrachial neu- rons. J. Neurosci. 14:7252-7260.

Johnston, H.M., and B.J. Morris (1994) Induction of c-fos gene expression is not responsible for increased proenkephalin mRNA levels in the hippo- campal dentate gyrus following NMDA stimulation. Mol. Brain Res. 25:147-150.

Kobashi, M., H. Ichikawa, T. Sugimoto, and A. Adachi (1993) Response of neurons in the solitary tract nucleus, area postrema and lateral parabra- chid nucleus to gastric load of hypertonic saline. Neurosci. Lett. 158:47-50.

Krukoff, T.L., T.L. Morton, K.H. Harris, and J.H. Jhamandas (1992) Expression of c-fos protein in rat brain elicited hy electrical stimulation of the pontine parahrachial nucleus. J. Neurosci. 123582-3590.

Lantbri-Minet, M., J. Weil-Fugazza, J. de Pommery, and D. Menetrey (1994) Hindbrain structures involved in pain processing as revealed by the expression of c-Fos and other immediate early gene proteins. Neurosci- ence 58:287-298.

Lara, J.P., M.J. Parkes, L. Silva-Carvhalo, P. Izzo, M.S. Dawid-Milner, and K.M. Spyer (1994) Cardiovascular and respiratory effects of stimulation of cell bodies of the parabrachial nuclei in the anaesthetized rat. J. Physiol. (London) 477:321-329.

Lee, J.H., and A.J. Beitz (1993) The distribution of brain-stem and spinal cord nuclei associated with different frequencies of electroacupuncture analgesia. Pain 5211-28.

Li, B.-H., A.C. Spector, and N.E. Rowland (1994) Reversalofdexfenfluramine- induced anorexia and c-FOSlc-JUN expression by lesion in the lateral parahrachial nucleus. Brain Res. 640:255-267.

Light, A.R., E. Casale, and M. Sedivec (1987) The physiology and anatomy of spinal laminae I and I1 neurons antidromically activated by stimulation in the parabrachial region of the midbrain and pons. In F.F. Schmidt, G.-H. Schaihle, and C. Vahle-Hinz (eds): Fine Afferent Nerve Fibers and Pain. Weinheim: VCH, pp. 347-356.

56

Light, A.R., M.J. Sedivec, E.J. Casale, and S.L. Jones (1993) Physiological and morphological characteristics of spinal neurons projecting to the parabrachial region of the cat. Snmatosens. Motor Res. 10:309-325.

Lind, R.W., and L.W. Swanson (1984) Evidence for corticotropin releasing factor and leu-enkephalin in the neural projection from the lateral parabrachial nucleus to the median preoptic nucleus: A retrograde transport, immunohistochemical double labeling study in the rat. Brain Res. 321217-224.

Luckman, S.M., R.E.J. Dyball, and G. Leng (1994) Induction of c-fos expression in hypothalamic magnocellular neurons requires synaptic activation and not simply increased spike activity. J. Neurosci. 14.4825- 4830.

Luo, P., J. Li, and Z. Rao (1990) A morphological analysis of neurons in the lateral parabrachial nucleus: An intracellular horseradish peroxidase study in the rat. Neurosci. Lett. 112:133-136.

Ma, W., A. Blomqvist, and K.J. Berkley (1989) Spino-diencephalic relays through the parabrachial nucleus in the cat. Brain Res. 480.37-50.

Macedonia, J.M., and C.S. Evans (1993) Variation among mammalian alarm call systems and the problem of meaning in animal signals. Ethology 93: 177-197.

Mehler, W.R., M.E. Feferman, and W.J.H. Nauta (1960) Ascending axon degeneration following anterolateral cordotomy. An experimental study in the monkey. Brain 83:718-750.

Menetrey, D., and J. de Pommery (1991) Origins of spinal ascending pathways that reach central areas involved in visceroception and viscero- nociception in the rat. Eur. J. Neurosci. 3:249-259.

Molander, C., and G. Grant (1985) Cutaneous projections from the rat hindlimb foot to the substantia gelatinosa of the spinal cord studied by transganglionic transport of WGA-HRP conjugate. J. Comp. Neurol. 237:476484.

Morgan, J.I., and T. Curran (1991) Stimulus-transcription coupling in the nervous system: Involvement of the inducible proto-oncogenes fos and jun. Annu. Rev. Neurosci. 14:421-451.

Mraovitch, S., M. Kumada, and D.J. Reis (1982) Role of the nucleus parabrachialis in cardiovascular regulation in cat. Brain Res. 232:57-75.

Murphy, A.Z., M. Ennis, M.T. Shipley, and M.M. Behbehani (1994) Direction- ally specific changes in arterial pressure induce differential patterns of Fos expression in discrete areas of the rat brainstem: A double-labeling study for Fos and catecbolamines. J. Comp. Neurol. 34936-50.

Noguchi, K., and M.A. Ruda (1992) Gene regulation in an ascending nociceptive pathway: Inflammation-induced increase in preprotachyki- nin mRNA in rat lamina I spinal projection neurons. J. Neurosci. 122563-2572.

Norgren, R., and C.M. Leonard (1973) Ascending central gustatory path- ways. J . Comp. Neurol. 150:217-238.

Nyberg, G., and A. Blomqvist (1985) The somatotopic organization of forelimb cutaneous nerves in the brachial dorsal horn: An anatomical study in the cat. J. Comp. Neurol. 24228-39.

Ohman, L.E., and A.K. Johnson (1986) Lesions in lateral parabrachial nucleus enhance drinking to angiotensin I1 and isoproterenol. Am. J. Physiol. 251:R504-R509.

Paxinos, G., and C. Watson (1986) The Rat Brain in Stereotaxic Coordinates. Sydney: Academic Press.

Pierson, M., and A. Snyder-Keller (1994) Development of frequency-selective domains in inferior colliculus of normal and neonatally noise-exposed rats. Brain Res. 636.55-67.

0. HERMANSON AND A. BLOMQVIST

Rocha, M.J.A., E. Friauf, and H. Herbert (1994) Effects of cardiovascular and osmotic stimuli on c-fos expression in the parabrachial nucleus of anaesthetized rats. SOC. Neurosci. Abstr. 20:298.

Saito, H., K. Sakai, and M. Jouvet (1977) Discharge patterns of the nucleus parabrachialis lateralis neurons of the cat during sleeping and waking. Brain Res. 134:59-72.

Sambucetti, L.C., and T. Curran (1986) The Fos protein complex is associated with DNA in isolated nuclei and hinds to DNA cellulose. Science 234: 14 17-14 19.

Saper, C.B. (1995) The spinoparabrachial pathway: Shedding new light on an old path. J. Comp. Neurol. 353:477479.

Saper, C.B., and D. Levisohn (1983) Afferent connections of the median preoptic nucleus in the rat: Anatomical evidence for a cardiovascular integrative mechanism in the anteroventral third ventricular (AV3V) region. Brain Res. 288:21-31.

Saper, C.B., and AD. Loewy (1980) Efferent connections of the parabrachial nucleus in the rat. Brain Res. 197:291-317.

Slugg, R.M., and A.R. Light (1990) Physiology and morphology of nocirecep- tive and thermoreceptive parabrachial neurons. Soc. Neurosci. Abstr. 16:1144.

Slugg, R.M., and A.R. Light (1991) Efferent projections from the lateral parabrachial nucleus as demonstrated by PHA-L. SOC. Neurosci. Abstr. 17:1204.

Slugg, R.M., and A.R. Light (1994) Spinal cord and trigeminal projections to the pontine parabrachial region in the rat as demonstrated with Phuseo- lus uulguris leucoagglutinin. J. Comp. Neurol. 339.49-61.

Smith, J.C., D.E. Morrison, H.H. Ellenberger, M.R. Otto, and J.L. Feldman (1989) Brainstem projections to the major respiratory neuron popula- tions in the medulla of the cat. J. Comp. Neurol. 281:69-96.

Sonnenberg, J.L., F.J. Rauscher 111, J.I. Morgan, and T. Curran (1989) Regulation of proenkephalin by Fos and Jun. Science 46:1622-1625.

Swank, M.W., and I.L. Bernstein (1994) c-FOS induction in response to a conditioned stimulus after single trial taste aversion learning. Brain Res. 636.202-208.

Swanson, L.W. (1987) The hypothalamus. Jn A. Bjorklund, T. Hokfelt, and L.W. Swanson (eds): Handbook of Chemical Neuroanatomy, Vol. 5: Integrated Systems of the CNS, Part I . Amsterdam: Elsevier, pp. 1-124.

Wang, L.G., H.M. Li, and J.S. Li (1994) Formalin induced FOS-like immunoreactive neurons in the trigeminal spinal caudal subnucleus project to contralateral parabrachial nucleus in the rat. Brain Res. 649:62-70.

Ward, D.G. (1988) Stimulation of the parabrachial nuclei with monosodium glutamate increases arterial pressure. Brain Res. 462383-390.

Watts, A.G. (1991) Ether anesthesia differentially affeds the content of prepro-corticotropin-releasing hormone, prepro-neurotensinineurome- din N and prepro-enkephalin mRNAs in the hypothalamic paraventricu- lar nucleus of the rat. Brain Res. 544:353-357.

Wiherg, M., and A. Blomqvist (1984) The spinomesencephalic tract in the cat: Its cells of origin and termination pattern as demonstrated by the intraaxonal transport method. Brain Res. 291.1-18.

Wiberg, M., J. Westman, and A. Blomqvist (1987) Somatosensory projection to the mesencephalon: An anatomical study in the monkey. J. Comp. Neurol. 264:92-117.

Zaborszky, L., M.C. Bcinfeld, M. Palkovits, and L. Heimer (1984) Brainstem projection to the hypothalamic ventromedial nucleus in the rat: A CCK-containing long ascending pathway. Brain Res. 303:225-23 1.