cells of origin of the trigeminohypothalamic tract in the rat

Cells of Origin of theTrigeminohypothalamic Tract in the Rat

AMY MALICK2 AND RAMI BURSTEIN1,2*1Department of Anesthesia and Critical Care, Beth Israel Deaconess Medical Center,

Boston, Massachusetts 021152Department of Neurobiology and the Program in Neuroscience, Harvard Medical School,

Boston, Massachusetts 02115

ABSTRACTRecent studies have demonstrated that a large number of spinal cord neurons convey

somatosensory and visceral nociceptive information directly from cervical, lumbar, and sacralspinal cord segments to the hypothalamus. Because sensory information from head andorofacial structures is processed by all subnuclei of the trigeminal brainstem nuclear complex(TBNC) we hypothesized that all of them contain neurons that project directly to thehypothalamus. In the present study, we used the retrograde tracer Fluoro-Gold to examinethis hypothesis. Fluoro-Gold injections that filled most of the hypothalamus on one sidelabeled approximately 1,000 neurons (best case 5 1,048, mean 5 718 6 240) bilaterally (70%contralateral) within all trigeminal subnuclei and C1–2. Of these neurons, 86% weredistributed caudal to the obex (22% in C2, 22% in C1, 23% in subnucleus caudalis, and 18% inthe transition zone between subnuclei caudalis and interpolaris), and 14% rostral to the obex(6% in subnucleus interpolaris, 4% in subnucleus oralis, and 4% in subnucleus principalis).Caudal to the obex, most labeled neurons were found in laminae I–II and V and theparatrigeminal nucleus, and fewer neurons in laminae III–IV and X. The distribution ofretrogradely labeled neurons in TBNC gray matter areas that receive monosynaptic inputfrom trigeminal primary afferent fibers innervating extracranial orofacial structures (such asthe cornea, nose, tongue, teeth, lips, vibrissae, and skin) and intracranial structures (such asthe meninges and cerebral blood vessels) suggests that sensory and nociceptive informationoriginating in these tissues could be transferred to the hypothalamus directly by this pathway.J. Comp. Neurol. 400:125–144, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: hypothalamus; trigeminal; nociception; pain; autonomic; affective

Trigeminal pain is one of the most commonly citedsources of discomfort in humans. It is frequently caused byinjuries such as facial lacerations (Bakay and Glasauer,1980) and diseases such as trigeminal neuralgia (Kugel-berg and Lindblom, 1959), headache (Oleson et al., 1993),sinusitis (Saunte and Soyka, 1994), toothache (Sharav,1994), and temporomandibular joint pain syndrome (Sessleand Hu, 1991). Because organs such as the mouth, nose,and eyes serve functions that must be performed continu-ously for survival, pain that originates in these structuresis repeatedly aggravated and may therefore cause greatersuffering than pain originating in structures that are notused as frequently.

Clinically, symptoms associated with trigeminal painand suffering frequently include changes in behavioral,hormonal, and autonomic functions. Experimentally, simi-lar changes in endocrine, autonomic, and affective func-tions can be induced by noxious stimulation of structuresinnervated by the trigeminal nerve (Allison and Powis,

1971; Geppetti et al., 1988; Silver, 1992; Nordin andFagius, 1995), or by direct stimulation of areas in thetrigeminal brainstem nuclear complex (TBNC) that areknown to process trigeminal nociceptive information (Bere-iter and Gann, 1988a; McKitrick and Calaresu, 1988). Inaddition, innocuous stimulation of the cornea, nose, nasalmucosa, oral mucosa, tongue, teeth, lips, or facial skin caninduce respiratory, cardiovascular, and hormonal changesand can trigger behavioral responses such as tearing,

Grant sponsor: National Institutes of Health; Grant numbers: DE-10904and NS-35611–01; Grant sponsor: Department of Anesthesia at Beth IsraelDeaconess Medical Center; Grant sponsor: The Milton Fund; Grant spon-sor: The Boston Foundation; Grant sponsor: The Goldfarb family; Grantsponsor: The Fink family.

*Correspondence to: Rami Burstein, Ph.D. Department of Anesthesia,Harvard Institute of Medicine, Room 830, 77 Avenue Louis Pasteur, Boston,MA 02115. E-mail: [email protected]

Received 22 January 1998; Revised 18 June 1998; Accepted 19 July 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 400:125–144 (1998)

r 1998 WILEY-LISS, INC.

sneezing, chewing, swallowing, drinking, and rooting (Dub-ner et al., 1978; Fleming et al., 1982; Zeigler et al., 1984;Gann et al., 1985). Because many of these behavioral andphysiological responses can be altered by hypothalamicstimulation or ablation (Glickman and Schiff, 1967; Nautaand Haymaker, 1969; Waldbillig, 1975; Grossman et al.,1978; Morgan and Panksepp, 1980, 1981; Zeigler et al.,1984), they are believed to be partially mediated by thehypothalamus.

Accordingly, hypothalamic neurons exhibit changes intheir firing rates in response to stimulation of trigeminallyinnervated cephalic and orofacial structures. For example,noxious mechanical stimuli applied to the ear, nose, tongue,and tooth pulp have been shown to change the activity ofneurons located in the medial, lateral, anterior, and poste-rior hypothalamus (Cross and Green, 1959; Rudomin etal., 1965; Murakami et al., 1967; Morita et al., 1977;Kanosue et al., 1984). Noxious and innocuous thermalstimulation of facial structures alters the firing frequencyof anterior hypothalamic neurons (Murakami et al., 1967)and potentiates the release of adrenocorticotrophic hor-mone and catecholamines, along with the induction ofcardiovascular responses (Bereiter et al., 1990). Further-more, such potentiation of autonomic and endocrine func-tions has also been demonstrated following the directactivation of neurons in laminae I–II and V of subnucleuscaudalis (Bereiter and Gann, 1988b). Collectively, thesedata suggest that trigeminal sensory signals are processedby neurons in the TBNC and then transferred to thehypothalamus.

Theoretically, sensory information processed in theTBNC can reach the hypothalamus either via brainstemrelay nuclei that receive input from the TBNC and projectto the hypothalamus, or through direct trigeminohypotha-lamic projections. Brainstem candidates to relay sensoryinformation from the TBNC to the hypothalamus in the ratinclude the parabrachial nuclei (Saper and Loewy, 1980;Cechetto et al., 1985; Slugg and Light, 1994), the nucleusof the solitary tract (Ricardo and Koh, 1978; Menetrey andBasbaum, 1987), the caudal ventrolateral medulla (Saw-

chenko and Swanson, 1981; Lima and Coimbra, 1991), andthe periaqueductal gray matter (Beitz, 1982; Liu, 1983;Eberhart et al., 1985; Lima and Coimbra, 1989). Becauseof the large number of brainstem areas that receive inputfrom the TBNC and project to the hypothalamus, it hasbeen widely accepted that sensory trigeminal informationreaches the hypothalamus mainly via multisynaptic path-ways in the brainstem, thalamus, and cortex. Recently,however, several anatomical studies provided evidence fordirect projections of TBNC neurons to the hypothalamus:Ring and Ganchrow (1983) described degenerated fibers inthe hypothalamus following lesions of the trigeminal sub-nucleus caudalis (Vc); Burstein et al. (1990a), Carstens etal. (1990), and Li et al. (1997) showed retrogradely labeledneurons in the superficial layers of subnucleus caudalisfollowing injections of Fluoro-Gold (FG) to the hypothala-mus; and Iwata et al. (1992) found anterogradely labeledfibers in the posterior and lateral hypothalamus followinginjections of Phaseolus vulgaris-leucoagglutinin into themedullary dorsal horn. In none of these studies, however,were attempts made to study the origin of the trigeminalprojections to the hypothalamus from all TBNC nuclei andlaminae.

In the present study, we used the retrograde tracer FG tolabel as completely as possible the entire population ofsomatosensory trigeminal brainstem neurons that projectto all areas of the hypothalamus. In addition, we haveexamined trigeminal projections to rostral only and caudalonly hypothalamic areas because they contain neuronsthat play different roles in a variety of endocrine, auto-nomic, and behavioral functions. For example, nuclei ofthe rostral hypothalamus are involved in neurosecretorycontrol, regulation of circadian rhythms, and maintenanceof homeostatic functions (Swanson, 1987; Scammell et al.,1993; Saper, 1995; Simerly, 1995), whereas nuclei in thecaudal hypothalamus are involved in feeding behaviors(Bernardis, 1985), cortical arousal (Lin et al., 1989), andincreased cardiovascular tone (Waldrop et al., 1988; Par-ent, 1996). Each trigeminal brainstem subnucleus wasexamined separately because of the traditional notion that

Abbreviations

3V third ventricleA amygdalaac anterior commissureAH anterior hypothalamic areaArc arcuate hypothalamic nucleusC1 first cervical spinal segmentC2 second cervical spinal segmentCG central grayCo cochlear nucleicp cerebral peduncleCPu caudate putamenCu cuneate nucleusDMH dorsomedial hypothalamic nucleusf fornixGP globus pallidusGr gracile nucleusic internal capsuleicp inferior cerebellar peduncleLH lateral hypothalamic areaM mammillary bodyMG medial geniculate nucleusml medial lemniscusMoV motor trigeminal nucleusmtt mammillothalamic tractNTS nucleus of the solitary tractot optic tract

ox optic chiasmPaV paratrigeminal nucleusPe periventricular hypothalamic nucleiPO preoptic hypothalamic regionPH posterior hypothalamic areaPVN paraventricular hypothalamic nucleusRt reticular thalamic nucleusSCN suprachiasmatic nucleusscp superior cerebellar peduncleSe septal nucleism stria medullarisSN substantia nigraSON supraoptic nucleusSTh subthalamic nucleusstV spinal trigeminal tractTHT trigeminohypothalamic tractTM tuberomammillary nucleusXII hypoglossal nucleusVBC thalamic ventrobasal complexVc trigeminal subnucleus caudalisVi trigeminal subnucleus interpolarisVII facial nucleusVMH ventromedial hypothalamic nucleusVo trigeminal subnucleus oralisVp trigeminal subnucleus principalisZI zona incerta

126 A. MALICK AND R. BURSTEIN

different subnuclei play somewhat distinct roles in theprocessing of trigeminal somatosensory information. Forexample, laminae I–II subnucleus caudalis neurons in therat appear to process primarily nociceptive and thermal(Hutchison et al., 1997) information, whereas subnucleusoralis seems to process mainly oral and perioral informa-tion, and subnucleus principalis processes essentially non-nociceptive tactile information (reviewed in Willis, 1985;Sessle, 1987; Light, 1992; Cooper and Sessle, 1993).

MATERIALS AND METHODS

Male (16) and female (7) Sprague-Dawley rats (300–350g, Charles River Labs) were anesthetized with sodiumpentobarbital (60 mg/kg), supplemented with atropine(0.04 mg) to decrease bronchial secretion, and secured in astereotaxic frame (all protocols approved by the HarvardStanding Committee on Animals). In each case tracerinjections were made into the rostral hypothalamus (sevencases, four male 1 three female), caudal hypothalamus(six cases four male 1 two female), or both sites (six cases,four male 1 two female). Small holes were drilled in thefrontal and temporal bones on the left side to allowpenetration of a glass micropipette filled with FG into therostral and caudal hypothalamus, respectively. Glass mi-cropipettes (tip diameter 20–30 µm) were attached to a 5 µlHamilton microsyringe and a solution of 4% FG (in steriledistilled water) was deposited by pressure. Fluoro-Goldwas used as a retrograde tracer in this study because of itseffectiveness in labeling projecting neurons in the rat(Schmued and Fallon, 1986; Burstein et al., 1990a,b). Toinject the rostral hypothalamus while preventing FGspread to the thalamus, micropipettes were inserted at anangle of 29° from vertical in an anterior approach, and twoinjections (50 nl each) were made at two different mediolat-eral points (4.5 mm anterior to Bregma, 1.6 and 0.9 mmlateral to the midline, and 10.0 mm ventral to the dorsalsurface of the brain). To inject the caudal hypothalamuswhile preventing FG from spreading to the thalamus,amygdala, or brainstem, a double-angle approach wasused. Micropipettes were inserted into the caudal hypo-thalamus at an angle of 29° from vertical in an anteriorapproach and 60° from vertical in a temporal approach. Inthe caudal hypothalamus single injections (100 nl) weremade at 3 mm posterior to Bregma and 8.1 mm below thecortical penetration point. Because of the angled approach,the cortical penetration point was 8.9 mm lateral to themidline. If FG injections spread into the thalamus, zonaincerta, or midbrain, cases were discarded because theseareas have been shown in the rat to receive direct inputfrom the TBNC (Fukushima and Kerr, 1979; Peschanski,1984; Shammah-Lagnado et al., 1985; Carstens et al.,1990; Yoshida et al., 1991). Because of the angled ap-proaches to the hypothalamus, FG was found in theparietal cortex, lateral caudate putamen, medial caudateputamen, globus pallidus, and frontal cortex. In four cases,control injections (100 nl) were made into these regions.

The effectiveness of the injections was evaluated byexamining the parabrachial nuclei, the periaqueductalgray, the nucleus of the solitary tract, and the A1 neuronsof the caudal ventrolateral medulla for FG-labeled cellssince all of these areas are known to contain a largenumber of neurons that project to the hypothalamus(Ricardo and Koh, 1978; Saper and Loewy, 1980; Saw-chenko and Swanson, 1981; Eberhart et al., 1985).

Four to 7 days after FG injection, rats were anesthe-tized, intracardially perfused with 0.9% saline, and fixedwith low pH (6.5) 4% paraformaldehyde followed by highpH (9.2) 4% paraformaldehyde in phosphate buffer (Berodet al., 1981). The brain, brainstem, and upper cervicalspinal cord segments (C1–2) were removed, post-fixedovernight in the high pH paraformaldehyde solution, andthen transferred to a 20% sucrose solution (0.1 M phos-phate buffer, pH 7.4, at 4°C) for at least 24 hours. Brainareas containing the injection sites and brainstem andupper cervical spinal cord segments containing the retro-gradely labeled neurons were cut transversely on a freez-ing microtome at 100 and 40 µm, respectively. Two sets ofalternate sections were then mounted serially on gelatin-coated slides. One set was air-dried over night, cover-slipped with DPX (Fluka, Buchs, Switzerland), and exam-ined under darkfield illumination to delineate white andgray matter areas and under ultraviolet illumination todetermine the boundaries of the injection sites and thelocations of the retrogradely labeled neurons. The other setwas stained with thionin, coverslipped with Cytoseal(Fisher, Rockville, MD), and examined under brightfieldillumination for further identification of boundaries be-tween different subnuclei and gray matter laminae in thebrain, brainstem, and cervical segments. The cytoarchitec-tonic structures and boundaries of nuclei in the hypothala-mus and brainstem were identified and drawn according tothe atlases of Paxinos and Watson (1986) and Swanson(1987).

To determine whether rostrally located hypothalamicnuclei receive different TBNC input than caudally locatedhypothalamic nuclei, the hypothalamus was divided intorostral and caudal regions. The anatomical landmark forthis division was the anterior border of the arcuate nucleus.The rostral region (extending approximately 2.5 mm in theanterior-posterior plane) included the preoptic (PO), supra-optic (SON), suprachiasmatic (SCN), paraventricular(PVN) and periventricular (Pe) hypothalamic nuclei, theanterior hypothalamic area (AH), and the rostral parts ofthe lateral hypothalamic area (LH) and the ventromedial(VMH) and dorsomedial (DMH) nuclei. The caudal region(extending approximately 1.8 mm) included the arcuate(Arc), tuberomammillary (TM), and mammillary (M) hypo-thalamic nuclei, the posterior hypothalamic area (PH),and the caudal parts of the LH, VMH, and DMH.

Retrogradely labeled neurons in the TBNC and C1–2were observed with an epi-fluorescent illuminated micro-scope and mapped onto tissue section reconstructionsthrough a camera lucida drawing attachment. Neuronswere counted as FG positive if they included a nucleus,cytoplasm, and a part of their dendritic tree. In all cases,FG-labeled neurons were counted in alternate sections ofC2, C1, subnucleus caudalis (Vc), subnucleus interpolaris(Vi), subnucleus oralis (Vo), and subnucleus principalis(Vp). The boundaries between the different TBNC nucleiwere determined according to Jacquin and others (Gobel etal., 1981; Falls, 1984a,b; Jacquin et al., 1986b). Briefly, thepoint at which substantia gelatinosa is ‘‘displaced’’ andbecomes contiguous with the spinal tract of V was definedas the border between subnucleus caudalis and interpo-laris (Vc/Vi). This point was designated as 0.0. Subnucleuscaudalis was considered to extend caudally to approxi-mately the caudal end of the pyramidal decussation, apoint usually 2.0 mm caudal to the rostral border of Vc(approximately 25 sections counted). The transition zone

TRIGEMINOHYPOTHALAMIC TRACT 127

between Vc and Vi (i.e., sections that contain Vc dorsally,and Vi ventrally) extended between the 0.0 point and theobex (about 0.5–0.8 mm, about 10 sections). Subnucleusinterpolaris was considered to extend from the obex to therostral end of the inferior olivary complex, a point approxi-mately 2.0 mm rostral to the 0.0 point (about 19 sections).The paratrigeminal nucleus included the interstitial is-lands lying within the dorsal half of the spinal trigeminaltract, at the level of Vi and the rostralmost Vc/Vi transitionzone. Subnucleus oralis was considered to extend from thelevel of the caudal facial nucleus to the transition zonewith principalis (at the facial motor root level, 1.8 mm totallength, about 22 sections counted). Subnucleus principaliswas considered to extend from that point to the rostral endof the trigeminal complex (at the level of the parabrachialnucleus, 1.5 mm, about 18 sections counted).

The upper cervical spinal cord segments (C1–2) wereincluded in the data analysis because in the rat theyreceive direct input from trigeminal primary afferentfibers (Jacquin et al., 1983; Matesz, 1983; Marfurt andRajchert, 1991), and contain neurons that respond exclu-sively or preferentially to stimulation of organs innervatedby the trigeminal nerve (Strassman and Vos, 1993; Sugi-moto et al., 1994; Burstein et al., 1998). C1 segmentsusually extended from the caudal end of the pyramidaldecussation to the rostral end of the C2 dorsal rootlets(2.0–3.6 mm caudal to the 0.0 point, about 20 sections). C2segments usually extended 2.4 mm caudal to this point(3.6–6.0 mm caudal to the 0.0 point, about 30 sections).Since retrogradely labeled neurons in the intermediatezone, ventral horn, lateral spinal, and lateral cervicalnuclei in the dorsolateral funiculus, and the ventrolateralfuniculus of C1–2 were mapped and included in theanalysis of the spinohypothalamic tract (Burstein et al.,1990a), we mapped them in this study but did not countthem as THT neurons because these areas do not seem toreceive direct input from trigeminal primary afferentfibers or respond exclusively to trigeminal stimulation.

Counted neurons were assigned to laminae I–II, III–IV,V, and X. The borders between these laminae were basedon cytoarchitectonic characteristics of each lamina (Molan-der et al., 1989; Strassman and Vos, 1993) and theappearance of the white and gray matter under darkfieldillumination. In both C1–2 and Vc, laminae I–IV are easilyrecognized; the border between laminae II and III could beobserved by the poor myelination of the inner layer oflamina II in contrast to the rich myelination of lamina III.Lamina V, however, is better defined in C1–2 than in Vc. InC1–2, lamina V is comprised of the lateral reticulated areaventral to lamina IV. In Vc, the boundaries of lamina V arewell defined dorsally and laterally by the less reticulatedappearance of lamina IV, but poorly defined medially andventrally as it merges with the medullary reticular forma-tion. According to Nord and Kyler (1968), lamina V isconsidered equivalent to the lateral part of the medullaryreticular formation. However, since there is no obviousdifference between the lateral reticular formation and theregion immediately internal to it, namely, the intermedi-ate reticular formation, we have considered lamina V toextend 400 µm below the ventral border of lamina IV. Thisline should be considered conservative since neurons thatrespond exclusively to stimulation of organs innervated bythe trigeminal nerve have been recorded ventral andmedial to it (Nord and Kyler, 1968; Nagano et al., 1975;Villanueva et al., 1988; Hu, 1990) and because trigeminal

primary afferent fibers project directly to this area (Clarkeand Bowsher, 1962; Jacquin et al., 1982; Arvidsson andRice, 1991; Marfurt and Rajchert, 1991). In C1–2, laminaX was defined as the gray matter area encircling (300 µm)the central canal. In Vc, this gray matter area has givenway to the nucleus of the solitary tract.

To estimate the total number of THT neurons in theTBNC and C1–2 and to adjust for the possibility ofcounting labeled neurons more than once, correction fac-tors were applied to the numbers of counted neurons ofeach injection group (RC, RO, CA). Because labeled neu-rons were counted in 40-µm-thick alternate transversesections, three additional rats were injected with FG intothe rostral and caudal hypothalamus, the brainstem andC1–2 were cut horizontally, and the rostrocaudal extent oflabeled neurons in each gray matter area and each sub-nucleus was measured until the standard error of themean was less than 10%. Based on the thickness of thetransverse section (M) and the average rostrocaudal lengthof the labeled cell bodies (L), a correction factor (CF) wascalculated according to the first method of Abercrombie(1946): CF 5 M/(L1M). The most frequent error in thiscorrection factor method is that it assumes the nuclear (orcell soma) heights to be constant. However, this error canbe minimized if the thickness of the section exceeds thenuclear (or somal) height by a factor of more than 1.5(Clarke, 1992). Statistical analyses comparing the num-bers of neurons counted in the different injection groups,different laminae, and TBNC subnuclei were performedusing Student’s t-test (un-paired, two-tailed, P , 0.05considered significant).

RESULTS

Neurons labeled by injections of FG intoboth rostral and caudal hypothalamus

To examine the total population of neurons in the TBNCthat project to the hypothalamus, both the rostral andcaudal hypothalamus were injected with FG in six cases.In these cases, injections filled most of the hypothalamuson one side but did not spread to the thalamus, zonaincerta, or midbrain. The spread of these FG injectionswithin the hypothalamus is described in Table 1 (RC1–6),in which black circles denote the core of injection (usuallymarked by the development of necrosis) and white circlesdenote nuclei to which the FG spread without necrosis. Toprevent FG from spreading dorsally into the zona incerta,injections were centered in the ventral area of the rostralhypothalamus. Therefore, in five of six cases, FG did notfill the PVN. Photomicrographs of injections that filled therostral (A), middle (B) and caudal (C) hypothalamus areshown in Figure 1.

Following FG injection into the rostral and caudalhypothalamus, retrogradely labeled neurons were foundbilaterally in all subnuclei of the TBNC and in C1–2.Figure 1D–K shows photomicrographs of FG-labeled neu-rons in C2 (D), C1 (E), Vc (F-G), Vc/Vi (H), Vi (I), Vo (J), andVp (K). Reconstruction of the injections of FG and thelocations of retrogradely labeled neurons in RC1 arepresented in Figure 2.

In this case, 931 trigeminohypothalamic tract (THT)neurons were found in alternate sections of the examinedareas. The mean (n 5 6) numbers of THT neurons found ineach of the trigeminal subnuclei and within the different


gray matter laminae are shown in the histograms inFigure 3.

In the six cases in which FG was injected unilaterallyinto both the rostral and caudal hypothalamus, 931, 503,472, 568, 787, and 1,048 retrogradely labeled neuronswere counted in the alternate sections (mean 6 SD 5718 6 240). Approximately 70% of these neurons werelocated contralateral and 30% ipsilateral to the injectionsite. Along the rostrocaudal axis of the TBNC, approxi-mately 86% of the neurons were located caudal to the obex(within the 85 consecutive sections of C1–2, Vc, and Vc/vi)and 14% rostral to the obex (within the 59 consecutivesection of Vi, Vo, and Vp; Fig. 3A). Caudally located THTneurons were evenly distributed within C2 (22 6 7%), C1(22 6 3%), Vc (23 6 4%) and the Vc/Vi transition zone (18 66%). Rostrally located THT neurons were evenly distrib-uted within Vi (6 6 1%), Vo (5 6 1%) and Vp (4 6 1%).

By dividing the number of labeled neurons by thenumber of sections examined, the relative density oflabeling along the rostrocaudal TBNC was determined.The average number of labeled neurons per section wasabout 7.3 caudal to the obex and 0.6 rostral to the obex. InC2, C1, and Vc, where the laminar organization of thedorsal and medullary gray matter is still preserved, mostneurons were found within laminae I–II and V. Theselaminae contained approximately 24% (6 4) and 35% (6 6)of the entire population of THT neurons, respectively.Fewer neurons were found in laminae III–IV (4 6 1%) andX (5 6 1%). When each rostrocaudal level was examinedindividually, it was noted that the laminar distribution ofTHT neurons was slightly different in C1–2 than in Vc.Whereas in C2 and C1 significantly more neurons werelocated in lamina V (58 6 9 and 63 6 10%, respectively)than in laminae I–II (26 6 7 and 24 6 7%, respectively), inVc more neurons were found in laminae I–II (56 6 12%)than in lamina V (34 6 11%). The distribution of THTneurons in the different laminae of C2, C1, and Vc isillustrated in the histograms in Figure 3B. Labeled neu-rons located within the paratrigeminal nucleus were in-cluded in the Vc/Vi transition zone; they usually contrib-uted about 7% to the number of THT neurons counted inthis area. Since this region constitutes only 10% of theTBNC and C1–2, yet contains about 19% of the labeledneurons, it has more THT neurons per section than anyother region of the TBNC and C1–2.

To determine whether the THT differs in male vs. femalerats, we also compared the distributions and the numbersof THT neurons in the two female and four male rats of thisinjection group but found no significant differences (sinceno gender differences were found in the other injectiongroups, we did not separate them in the data analysis).

Neurons labeled by injections of FGinto the rostral hypothalamus

To examine the population of THT neurons that projectto the rostral hypothalamus, injections of FG were cen-tered in the rostral 2.5 mm of the hypothalamus, betweenthe preoptic nuclei and the posterior border of the anteriorhypothalamic nucleus, in seven cases. In these cases,injections filled a large portion of the rostral hypothalamuson one side but did not spread to the thalamus, zonaincerta, or midbrain (Table 1). As indicated in Table 1, formost cases it was difficult to prevent FG from spreading tothe arcuate nucleus. Nevertheless, the injection cores werealways restricted to the rostral nuclei. An example casewith FG injections that filled the rostral hypothalamus isillustrated in Figure 4A. Retrogradely labeled neuronswere found bilaterally in all subnuclei of the TBNC and inC1–2. Reconstructions of the locations of retrogradelylabeled neurons in RO2 are presented in Figure 4B. In thiscase, 672 THT neurons were found in alternate sections ofthe examined areas. The mean (n 5 7) numbers of THTneurons found in each of the trigeminal subnuclei andwithin the different gray matter laminae are shown in thehistograms in Figure 5.

In the seven cases in which FG was injected unilaterallyinto the rostral hypothalamus, 357, 672, 555, 466, 266,422, and 738 retrogradely labeled neurons were counted inthe alternate sections (mean 6 SD 5 495 6 169). Approxi-mately 62% of these neurons were located contralateraland 38% ipsilateral to the injection site. Following theserostral injections, the distribution of THT neurons alongthe rostrocaudal axis of the TBNC and within the differentlaminae of the gray matter was similar to their distribu-tion following the injections into both rostral and caudalareas.

Within the rostral injection group approximately 89% ofthe neurons were located caudal to the obex and 11%rostral to the obex (Fig. 5A). Caudally located THT neu-

TABLE 1. Hypothalamic Nuclei Injected with Fluoro-Gold1

Case

Rostral hypothalamic nuclei Caudal hypothalamic nucleiTotal no. ofTHT cellsPO SON AH SCN PVN Pe LH VMH DMH Arc LH VMH DMH PH TM M

RC1 s q q q q s q q q q q q q 931RC2 s q q s q q q s s s 503RC3 q q q q s s q q q q s q q s s 472RC4 q q s q q q q s q q s q q 568RC5 q q q s q q q s q q q q q q q 787RC6 q q q s q q q q q q q q q q q 1048RO1 s s q s s s q q s s 357RO2 q q q q s s q q s s s s 672RO3 q q q s s s q q s s 555RO4 q q q s s s q q s s 466RO5 q q q s s q q 266RO6 q q q q q s q s s 422RO7 q q q q s q q q s s s 738CA1 s q q q q s q q 249CA2 s s s q q q q s q q 406CA3 s s q q q q q q q q 392CA4 s s q q q q q q q 166CA5 s s s s q q q q q s q 515CA6 s s q q q q q q 412

1q, core of injection; s, spread of injection.


rons were evenly distributed within C2 (23 6 6%), C1(27 6 2%), Vc (24 6 2%) and Vc/Vi transition zone (15 67%), which included labeled neurons in the PaV. Thenumber of rostrally located THT neurons decreased from7 6 3% in Vi to 3 6 2% in Vo to 1 6 1% in Vp. In C2, C1, andVc, most neurons were found within laminae I–II and V.These laminae contained 27% (6 4) and 39% (6 8) of the

entire population of THT neurons, respectively. In compari-son, significantly fewer neurons were found in laminaeIII–IV (2 6 1%) and X (5 6 2%). This laminar distributionpattern of THT neurons was similar in C2 and C1, thoughnot in Vc. Whereas in C2 and C1 significantly moreneurons were located in lamina V (64 6 5%, respectively)than in laminae I–II (25 6 4%, respectively), in Vc more

Fig. 1. Photomicrographs showing Fluoro-Gold (FG) injection siteswithin the hypothalamus (A–C) and retrogradely labeled neurons ineach of the trigeminal brainstem subnuclei and C1–2 (D–K).A–C: Combined UV and brightfield illumination illustrating theappearance of FG in the rostral (A), middle (B), and caudal (C)hypothalamus. D–K: Ultraviolet illumination of FG labeled neuronsin lamina V of C2, ipsilaterally (D), and in lamina I of C1 (E), lamina V

of Vc (F), lamina I of Vc (G), lamina I of caudal Vi and rostral Vc (H),ventral Vi (I), lateral Vo (J), and Vp (K), all contralaterally. This figurewas created using Adobe Photoshop software on an Apple Power PC tocombine scanned slide images into a composite plate, and printed on aKodak dye sublimation printer. Only the brightness and sharpnesslevels were adjusted. Scale bars 5 800 µm for A–C, 200 µm for H,100 µm for D–G and I–K.


Fig. 2. Schematic drawings of FG injections in the rostral andcaudal hypothalamus (A) and the retrograde labeling they produced inthe TBNC and C1–2 (B) of case RC1. A: Drawing at top depicts mostanterior spread of injection, three drawings in the middle depict thecenter of injections in the rostral, middle and caudal hypothalamus,and drawing at bottom depicts most caudal spread of injection. Blackareas indicate core of injections, and gray areas depict the surround-ing spread of FG. B: Line drawings illustrating the locations of 931labeled neurons (filled circles) found in gray matter areas that receivemonosynaptic input from trigeminal primary afferent fibers and 514

labeled C1–2 neurons (open circles) found outside the central termina-tion field of the trigeminal nerve. The right side of all levels illustratedis contralateral to the injection. Note that FG injections did not spreadto the thalamus or midbrain, that most labeled neurons were foundcaudal to the obex, within laminae I–II and V, and that the onlyneurons counted as THT units are those located within the centraltermination field of the trigeminal nerve. (Labeled neurons outside theTBNC were not included in the reconstructions.) Scale bars 5 2 mm inA, 1 mm in B.

neurons were found in laminae I–II (56 6 10%) than inlamina V (38 6 10%). The distribution of THT neurons inthe different laminae of C2, C1 and Vc is illustrated in thehistograms in Figure 5B.

Neurons labeled by injections of FGinto the caudal hypothalamus

To examine the population of THT neurons that projectto the caudal hypothalamus, injections of FG were cen-tered in the caudal 1.8 mm of the hypothalamus, betweenthe anterior border of the arcuate nucleus and the poste-

rior border of the mammillary complex, in six cases. Inthese cases, injections filled most of the caudal hypothala-mus on one side but did not spread to the thalamus, zonaincerta, or midbrain (Table 1). As indicated in Table 1, thecore of all injections was restricted to the caudal hypothala-mus. However, it was difficult to prevent the spread of FGinto more rostral nuclei. In most cases, the posterior partof SON and LH contained some injection spread. In thisgroup, more THT neurons were counted in cases in whichFG spread rostrally to the LH. An example case with FGinjections that filled the caudal hypothalamus is illus-

Fig. 3. Histograms showing the mean (6 standard deviation)number of trigeminohypothalamic tract neurons found in the differenttrigeminal brainstem subnuclei and C1–2 (A), and their percentagesin the different laminae of C2, C1, and Vc (B) following injections of FGinto the rostral and caudal hypothalamus. Dark and light gray areas

in A illustrate the number of neurons found contralateral and ipsilat-eral to the injection, respectively. Note that in C1–2 more THTneurons are found in lamina V, and that in Vc more neurons are foundin laminae I–II.


Fig. 4. Schematic drawings of FG injections in the rostral hypo-thalamus (A) and the retrograde labeling they produced in the TBNCand C1–2 (B) of case RO1. A: Drawing at top depicts most anteriorspread of injection, three drawings in the middle depict the center ofinjections in the rostral and middle hypothalamus, and drawing atbottom depicts most caudal spread of injection. Black areas indicatecore of injections, and gray areas depict the surrounding spread of FG.B: Line drawings illustrating the locations of 672 labeled neurons(filled circles) found in gray matter areas that receive monosynaptic

input from trigeminal primary afferent fibers and 350 labeled C1–2neurons (open circles) found outside the central termination field ofthe trigeminal nerve. The right side of all illustrated levels iscontralateral to the injection. Note that the locations of THT neuronsin this case are similar to their locations in the case shown in Figure 2,and that the only neurons counted as THT units are those locatedwithin the central termination field of the trigeminal nerve. Forabbreviations, see list. Scale bars 5 2 mm in A, 1 mm in B.

trated in Figure 6A. Retrogradely labeled neurons werefound bilaterally in all subnuclei of the TBNC and in C1–2.Reconstructions of the locations of retrogradely labeledneurons in CA2 are presented in Figure 6B. In this case,406 THT neurons were found in alternate sections of theexamined areas. The mean (n 5 6) numbers of THTneurons found in each of the trigeminal subnuclei andwithin the different gray matter laminae are shown in thehistograms in Figure 7.

In the six cases in which FG was injected unilaterallyinto the caudal hypothalamus, 249, 406, 392, 166, 515, and412 retrogradely labeled neurons were counted in thealternate sections (358 6 127, mean 6 SD). Approximately

66% of these neurons were located contralateral and 34%ipsilateral to the injection site. Following these caudalinjections, the distribution of THT neurons along therostrocaudal axis of the TBNC was similar to their distri-bution following the rostral and caudal injections. Alongthe rostrocaudal axis of the TBNC, about 87% of theneurons were located caudal to the obex and 13% rostral tothe obex (Fig. 7A). In the posterior TBNC THT neuronswere evenly distributed within C2 (22 6 7%), C1 (29 6 8%),Vc (23 6 5%) and the Vc/Vi transition zone (13 6 5%),which included labeled neurons in the PaV. More anteriorTBNC THT neurons were evenly distributed within Vi(6 6 6%), Vo (4 6 4%), and Vp (3 6 1%). In C2, C1, and Vc,

Fig. 5. Histograms showing the mean (6 standard deviation)number of trigeminohypothalamic tract neurons found in the differenttrigeminal brainstem subnuclei and C1–2 (A), and their percentagesin the different laminae of C2, C1, and Vc (B) following injections of FG

into the rostral hypothalamus. Dark and light gray areas in Aillustrate the number of neurons found contralateral and ipsilateral tothe injection, respectively. Note the similarities between the distribu-tions of neurons in this and the previous group (summarized in Fig. 3).


Fig. 6. Schematic drawings of FG injections in the caudal hypo-thalamus (A) and the retrograde labeling they produced in the TBNCand C1–2 (B) of case CA1. A: The two drawings at top show no spreadof FG in the rostral hypothalamus, and the three drawings at bottomshow the center of injection in the caudal hypothalamus and the lackof FG spread in the midbrain. Black areas indicate core of injections,and gray areas depict the surrounding spread of FG. B: Line drawings

illustrating the locations of 406 labeled neurons (filled circles) found ingray matter areas that receive monosynaptic input from trigeminalprimary afferent fibers and 183 labeled C1–2 neurons (open circles)found outside the central termination field of the trigeminal nerve.The right side of all illustrated levels is contralateral to the injection.Note the locations of neurons in the paratrigeminal subnucleus. Forabbreviations, see list. Scale bars 5 2 mm in A, 1 mm in B.

where the laminar organization of the dorsal and medul-lary gray matter is still preserved, most neurons werefound within laminae I–II and V. However, their distribu-tion was different from the cases that received rostral onlyor rostral and caudal hypothalamic injections: there was asignificantly (P , 0.01) smaller number of THT neurons inlaminae I–II (15 6 9% of total neurons), and a significantly(P , 0.05) larger number in lamina V (49 6 8%). In C2,18 6 13% of the neurons were located in laminae I–II, and69 6 14% in lamina V; in C1, 10 6 8% of the neurons werelocated in laminae I–II, and 75 6 11% in lamina V; and inVc 33 6 12% of the neurons were located in laminae I–II,

and 55 6 9% in lamina V. The distribution of THT neuronsin the different laminae of C2, C1, and Vc is illustrated inthe histograms in Figure 7B.

Neurons labeled by control injectionsof FG into areas rostral and lateral

to the hypothalamus

In all cases, injections of FG into the hypothalamusspread along the injection tracts. Since the injection tractspassed through several cortical and telencephalic (CPu,GP, VP, LSN) areas shown to receive direct input from the

Fig. 7. Histograms showing the mean (6 standard deviation)number of trigeminohypothalamic tract neurons found in the differenttrigeminal brainstem subnuclei and C1–2 (A), and their percentagesin the different laminae of C2, C1, and Vc (B) following injections of FG

into the caudal hypothalamus. Dark and light gray areas in Aillustrate the number of neurons found contralateral and ipsilateral tothe injection, respectively. Note that unlike in the previous groups, Vccontained more labeled neurons in lamina V than in laminae I–II.


spinal cord (Cliffer et al., 1991), it is possible that someretrogradely labeled neurons in the TBNC and C1–2 wereneurons that project to these telencephalic or corticalareas but not through or to the hypothalamus. To test thispossibility, the glass micropipette was inserted along thesame tracts that were used to approach the hypothalamus,and FG was injected rostral (n 5 2) and lateral (n 5 2) tothe hypothalamus in four rats. Reconstructions of the twocontrol injections that produced the greatest number oflabeled neurons in the TBNC and C1–2 are shown inFigure 8.

Following control injections of FG along the rostral tract(Fig. 8A), 22 retrogradely labeled neurons were found inthe areas examined. This number represents 3% of thetotal number of THT neurons counted in the case in whichmaximal labeling was found (RO7, 738 cells), and 4% of thegroup’s mean (496 cells). Following control injections of FGalong the lateral tract (Fig. 8B), 77 retrogradely labeledneurons were found in the areas examined. This numberrepresents 15% of the total number of THT neuronscounted in the case in which maximal labeling was found(CA5, 515 cells), and 22% of the group’s mean (358 cells).The total number of neurons counted in the two controlgroups (99) represents approximately 14% of the meannumber of THT neurons counted in the six cases in whichinjections were made into both rostral and caudal hypo-thalamus.

Estimated numbers of THT neurons

In the case yielding the most labeled THT neuronsfollowing injections of FG into both rostral and caudalhypothalamus, 1,048 labeled cells were counted in alter-nate sections of C1–2 and the TBNC. By applying correc-tion factors to the number of counted neurons in eachsubnucleus and each lamina, we estimated that approxi-mately 1,429 neurons project directly from the TBNC tothe hypothalamus. Although minor differences were foundin the laminar distribution of THT neurons that project tothe rostral vs. caudal hypothalamus, no significant differ-ence was found between the total number of neurons thatproject to the two hypothalamic areas.

DISCUSSION

The present study extends previous observations oftrigeminohypothalamic connections by describing the loca-tions of neurons in all subnuclei of the TBNC that projectdirectly to rostral and caudal nuclei of the hypothalamususing the retrograde tracer FG.

Technical considerations

Fluoro-Gold is a weak base that can be taken up bynerve terminals, accumulated by lysosomes, and trans-ported to the cell body by passive diffusion (Wessendorf,1991). It was used in this study because it is a highlyeffective tracer for labeling the somata and proximaldendrites of long projecting neurons, because it is onlytransported retrogradely, even when injected in largevolumes (Schmued and Fallon, 1986), and because it wasused in the previous study of the projections from thespinal cord to the hypothalamus of the rat (Burstein et al.,1990a). Fluoro-Gold, however can also be taken up byaxons passing through, but not terminating in the injectedarea (Dado et al., 1990). Therefore, in interpreting ourresults, we assumed that the labeled neurons in the TBNC

are neurons whose axons pass through or terminate withinthe FG injection site. The three major implications of thisassumption for the present study are: 1) it is possible thatsome of the labeled neurons in the TBNC and C1–2 projectthrough the hypothalamus to more rostral telencephalicareas, 2) it is possible that injections of FG in the caudalhypothalamus labeled neurons whose axons pass throughthe caudal hypothalamus to its more rostral nuclei, and3) it is possible that injections of FG in the rostralhypothalamus labeled neurons whose axons reach therostral hypothalamus first and then curve posteriorly andissue collateral branches in the caudal hypothalamus.

Regarding the first implication, we determined themagnitude of the non-specific labeling in the TBNC andC1–2 (due to axons passing through the hypothalamus tomore rostral telencephalic areas) by injecting FG along thepipette tracts. The labeling in the TBNC and C1–2 follow-ing these injections suggests that about 14% of the cellsthat were counted as THT neurons may project to areasoutside the hypothalamus. Further support for thesedirect projections from the TBNC and C1–2 to more rostraltelencephalic regions in the rat was described recently in anumber of anatomical studies that used anterograde(Cliffer et al., 1991; Newman et al., 1996) and retrograde(Burstein and Giesler, 1989; Burstein and Potrebic, 1993)tracing techniques. If THT neurons do in fact projectthrough the hypothalamus to more rostral telencephalicareas, it is somewhat surprising because our electrophysi-ological studies have shown that almost none of the axons(of over 80 THT neurons) that reached the hypothalamusfrom subnucleus caudalis or C1–2 continued more ros-trally (unpublished observations). This finding is also truefor spinohypothalamic tract (SHT) neurons located in thecervical (Dado et al., 1994; Zhang et al., 1995; Kostarczyket al., 1997), lumbar (Burstein et al., 1991), and sacral(Katter et al., 1996) spinal cord.

Regarding the second and third implications, it is theo-retically possible that FG injections into the rostral hypo-thalamus would label THT neurons that project to rostralnuclei only, THT neurons that project to the rostralhypothalamus through its caudal area, and THT neuronsthat project to the rostral hypothalamus first and thencontinue caudally. Similarly, it is possible that FG injec-tions into the caudal hypothalamus would label THTneurons that project to caudal nuclei only, THT neuronsthat project to the rostral hypothalamus through itscaudal area, and THT neurons that approach the caudalhypothalamus from rostral to caudal. However, becauseprevious studies have shown that the axons of almost allspinohypothalamic and THT neurons approach the hypo-thalamus from its lateral border with the supraopticdecussation and internal capsule rather than through thecaudal nuclei (Burstein et al., 1991; Dado et al., 1994;Zhang et al., 1995; Kostarczyk et al., 1997; unpublishedobservations in our lab), it is likely that the injections ofFG in the caudal hypothalamus did not label many THTneurons that project to the rostral hypothalamus.

Locations of THT neuronsin the TBNC and C1–2

After FG injection into both the rostral and caudalhypothalamus, over 70% of the cells at the origin of theTHT were found caudal to the obex; in areas known toreceive direct input from primary afferent nociceptors(Light and Perl, 1979; Jacquin et al., 1986b; Sugiura et al.,


Fig. 8. Schematic drawings of control FG injections anterior (A)and lateral (B) to the hypothalamus, and the retrograde labeling theyproduced in the TBNC and C1–2 (C–D). A,C: Injections of FG in theorbitofrontal cortex and caudate-putamen labeled 22 neurons in graymatter areas that receive monosynaptic input from trigeminal pri-mary afferent fibers (filled circles) and 19 C1–2 neurons outside thecentral termination field of the trigeminal nerve (open circles).

B,D: Injections of FG in the parietal cortex, caudate-putamen, andglobus pallidus labeled 77 neurons in gray matter areas that receivemonosynaptic input from trigeminal primary afferent fibers (filledcircles) and 18 C1–2 neurons outside the central termination field ofthe trigeminal nerve (open circles). For abbreviations, see list. Scalebars 5 1 mm.

1986), to contain neurons that respond either preferen-tially or exclusively to noxious mechanical and thermalstimuli (Price et al., 1976; Hu, 1990; Renehan et al., 1986;Sessle, 1986; Hutchison et al., 1997; Burstein et al., 1998),and to contribute axons to other ascending pain pathways(Price et al., 1976; Hu et al., 1981; Burstein et al., 1987;Davis and Dostrovsky, 1988; Craig and Dostrovsky, 1991).Thus, a major role of the THT is presumably to conveynociceptive and thermal information to the hypothalamus.

In C1–2, approximately 85% of the neurons were foundin laminae I–II (25%) and V (60%). Because in the rat theseareas receive direct input from trigeminal primary affer-ent fibers (Matesz, 1983; Marfurt and Rajchert, 1991) andcontain neurons that respond to mechanical or thermalstimulation of the cornea (Lu et al., 1993), oral mucosa(Sugimoto et al., 1994), temporomandibular joint (Brotonet al., 1988; Hathaway et al., 1995), facial skin (Strassmanand Vos, 1993), and intracranial dura (Strassman et al.,1994a; Burstein et al., 1998), it is likely that THT C1–2neurons contribute significantly to the processing of noci-ceptive signals that arise in organs innervated by thetrigeminal nerve. Since neurons in lamina V of C1–2 canalso respond to innocuous stimulation (Abrahams et al.,1979; Chudler et al., 1991), and because about 4% of theTHT neurons in C1–2 were found in laminae III–IV, anarea known to receive input from low-threshold mechano-receptors (Hayashi, 1985; Jacquin et al., 1986b) andcontain neurons that respond mainly to low thresholdmechanical stimuli (Chudler et al., 1991), it is also possiblethat THT neurons in C1–2 contribute to the processing ofnon-nociceptive sensory information, although to a lesserextent.

Within the TBNC, subnucleus caudalis contained thelargest number of THT neurons (23 6 4% of the entirepopulation). These neurons were distributed in laminaeI–II (56 6 12% of Vc total), V (34 6 11%), and III–IV (10 64%). Traditionally, Vc has been considered as a TBNCregion that processes nociceptive information arising inoral and facial organs innervated by the trigeminal nerve(Dubner et al., 1978; Sessle, 1987; Light, 1992). Thisnotion has been supported by the similarities in thelaminar organization of Vc and the upper cervical dorsalhorn (Gobel et al., 1981), the direct input that laminae I–IIand V in Vc receive from trigeminal nociceptors (Jacquinet al., 1986b), and the responses of laminae I and V Vcneurons to noxious mechanical, chemical, or thermal stimu-lation of structures such as the cornea (Mosso and Kruger,1973; Nagano et al., 1975; Pozo and Cervero, 1993),cerebral vasculature (Davis and Dostrovsky, 1988; Strass-man et al., 1994a,b; Burstein et al., 1998), nasal mucosa(Anton et al., 1991), intraoral structures (Nord and Ross,1973; Amano et al., 1986; Carstens et al., 1995), temporo-mandibular joint (Broton et al., 1988), and facial skin(Price et al., 1976; Strassman and Vos, 1993; Hutchison etal., 1997; Burstein et al., 1998). As in C1–2, the distribu-tion of most THT neurons in laminae I–II and V of Vcstrongly supports a primarily nociceptive and thermal rolefor the THT. Nevertheless, the presence of some neurons inlaminae III–IV (10 6 4%), the preferential input of rapidlyconducting fibers with low-threshold receptive fields tothis gray matter area (Jacquin et al., 1986b; Miyoshi et al.,1994), and the almost exclusive activation of laminaeIII–IV neurons by low-threshold mechanical stimulation(Mosso and Kruger, 1973; Renehan et al., 1986) suggestagain that the THT may also process tactile information.

Approximately 18% of THT neurons were located in theVc/Vi transition zone. Although it was difficult to assignthese neurons to a specific lamina (because the laminarorganization of the medullary dorsal horn disappearshere), they were evenly distributed within the gray matterareas of rostral Vc and caudal Vi. Because of the relativepaucity of electrophysiological studies of neurons in themost rostral Vc and the most caudal Vi, it is difficult tospeculate on the response properties of these neurons andtheir role in conveying information to the hypothalamus.Nevertheless, neurons in this area seem to receive directafferent input from low- and high-threshold trigeminalmechanoreceptors (Jacquin et al., 1988) and respond tonoxious stimulation of the cornea, nasal mucosa, and oralstructures (Peppel and Anton, 1993; Pozo and Cervero,1993; Strassman and Vos, 1993; Meng et al., 1997),suggesting that their response properties are similar tothose of more caudally located Vc neurons. These proper-ties are somewhat different from those of the more anteriorVi neurons, which respond preferentially to low-thresholdstimuli such as vibrissae deflection (Jacquin et al., 1989a,b).Finally, some neurons in this complicated region werelocated in the dorsomedial and ventrolateral tips of thegray matter. These small regions were found to containneurons that respond to anesthetics (Strassman and Vos,1993) and therefore may be involved in non-somatosensoryfunctions such as cardiovascular and respiratory regula-tion. About 1% of all THT neurons were found within theparatrigeminal nucleus. These neurons may be involved inprocessing taste and gustatory information, as they re-ceive afferent input from oral and upper alimentary re-gions (Shigenaga et al., 1986; Altschuler et al., 1989), sendefferent projections to areas in the parabrachial complex,NTS, cortex, and thalamus that are associated with gusta-tion (Cechetto et al., 1985; Cechetto and Saper, 1987; Feiland Herbert, 1995), and are activated by stimulation of thetongue (Carstens et al., 1995).

Rostral to the obex, a small but consistent number ofTHT neurons were located in Vi (6 6 1%), Vo (5 6 1%) andVp (4 6 1%). Because neurons in Vi and Vp (excluding themost caudal and lateral region of Vi) receive most of theirsensory input from low-threshold mechanoreceptors (Jac-quin et al., 1986c, 1988; Miyoshi et al., 1994) and respondalmost exclusively to tactile stimulation of the face (Darian-Smith et al., 1963a,b; Nord and Kyler, 1968; Hayashi et al.,1984; Jacquin et al., 1986a, 1988, 1989b), their contribu-tions to the THT suggest again that tactile informationmay be conveyed to the hypothalamus through the THT.Alternatively, it is possible that these are among the fewnociceptive neurons found in Vi (Jacquin et al., 1989b).Regarding Vo, its neurons receive input from trigeminalprimary afferent fibers innervating vibrissae, skin, lingualmucosa, incisors, and jaws (Jacquin et al., 1993), andrespond mainly (although not exclusively) to noxious andinnocuous stimulation of intraoral structures such as theteeth, tongue, and oral mucosa (Darian-Smith et al.,1963a,b; Dallel et al., 1990; Raboisson et al., 1991).

In summary, the locations of THT neurons in the differ-ent trigeminal subnuclei and the different laminae suggestthat the hypothalamus receives direct nociceptive andtactile information originating in cutaneous, intraoral,and visceral organs innervated by the ophthalmic (cornea,dural blood vessels), maxillary (upper lip, vibrissae, nasalmucosa, temporomandibular joint), and mandibular (lower


lip and teeth, lingual and oral mucosa) branches of thetrigeminal nerve.

Projections to the rostraland caudal hypothalamus

The distribution of retrogradely labeled neurons in theTBNC following injections of FG into the rostral or caudalhypothalamus suggests that both projections originatefrom the same laminae of the gray matter and in the sametrigeminal subnuclei. As mentioned above, this suggeststhat nociceptive information originating in orofacial struc-tures is available to nuclei located in both the rostral andcaudal hypothalamus. Rostrally located nuclei such as thePVN, SON, SCN, PO, and anteroventral region are knownto play a role in 1) controlled release of hormones such asoxytocin, arginine-vasopressin and corticotropin-releasinghormone (CRH) through the PVN and SON, 2) regulationof circadian rhythms such as sleep through the SCN, and3) maintenance of homeostasis (e.g., thermoregulation,fluid balance) through preoptic and anteroventral neuronsnear the third ventricle (Swanson, 1987; Scammell et al.,1993; Saper, 1995; Simerly, 1995). Because noxious me-chanical and thermal stimulation of trigeminal organs arecapable of altering many of these functions, it is possiblethat the direct TBNC projections to the rostral hypothala-mus provide part of the afferent input necessary to initiatethese endocrine and autonomic responses. For example,noxious stimulation of the cornea and tooth pulp, or directactivation of nociceptive TBNC neurons cause the releaseof adrenocorticotropic hormone probably via the activationof CRH neurons in the PVN (Bereiter and Gann, 1988a;Bereiter et al., 1990). Hypothetically, noxious and innocu-ous thermal stimuli that activate thermosensitive laminaI neurons could be conveyed directly to thermoregulatoryregions in the hypothalamus and contribute to morecomplex autonomic responses. It could also be speculatedthat innocuous sensations such as dry lips and tonguemight activate low-threshold or wide dynamic range neu-rons that project to hypothalamic areas that regulateosmotic and volemic states and stimulate adjusting behav-iors such as thirst.

Caudally located nuclei such as the VMH, PH, TM, andSM and the mammillary body proper participate in theregulation of 1) feeding, drinking and licking behaviorsthrough the dorsomedial VMH (Bernardis, 1985) andposterior lateral (Norgren, 1970) hypothalamus, 2) corticalarousal and wakefulness through the tuberomammillaryhistaminergic nuclei (Lin et al., 1989; Saper, 1995; Sherinet al., 1996), 3) pseudo-affective emotional behaviors resem-bling aggressive responses such as biting, baring of teeth,and hissing through the ventrolateral VMH (Panksepp,1971; Kruk et al., 1983; Roeling et al., 1993); and4) modulation of cardiovascular and respiratory activitiesthrough the PH (Eldridge et al., 1981; DiMicco et al., 1986;Waldrop et al., 1988). Although it is common knowledgethat trigeminal pain is capable of reducing appetite,producing arousal, and altering cardiorespiratory tone,formal studies are lacking and the few clinical reportsfocus primarily on headache and migraine patients (Live-ing, 1873; Wolf, 1981; Drummond and Lance, 1984).

Comparisons with other ascendingsensory pathways

As mentioned above, most THT neurons were located inlaminae I–II, V, and X of C1–2 and laminae I–II and V of

Vc. These gray matter areas also contain neurons thatproject to the thalamus (Granum, 1986; Kemplay andWebster, 1986), nucleus of the solitary tract (Menetrey andBasbaum, 1987), parabrachial complex (Cechetto et al.,1985; Standaert et al., 1986; Feil and Herbert, 1995), andsuperior colliculus (Bruce et al., 1987). It is likely, there-fore, that similar sensory information is conveyed simulta-neously to multiple nuclei in the brainstem and thalamus.Theoretically, such information could be transferred byindividual neurons whose axons issue collateral branchesat multiple brainstem and diencephalic locations or byneurons whose axons project only to a single location.Demonstrations of the first option are found in two studies:the first describes low-threshold neurons in the trigeminalsubnucleus interpolaris whose axons issue collateralbranches in the superior colliculus, reticular formation,and thalamus (Jacquin et al., 1986a); and the seconddescribes nociceptive spinal cord neurons whose ascendingaxons issue collateral branches at multiple sites includingthe NTS, PB, superior colliculus, thalamus, and hypothala-mus (Kostarczyk et al., 1997). Support for the secondoption is found in studies showing differences in thedistributions of ascending spinal and trigeminal tractneurons. For example, most TBNC projections to theparabrachial region are ipsilateral (Cechetto et al., 1985),most TBNC projections to the thalamic nucleus submediusoriginate in Vc/Vi transition zone (Yoshida et al., 1991) andin lamina I at the level of Vc (Dado and Giesler, 1990), mosttrigeminal connections with motor areas such as thecerebellum and inferior olive seem to originate in Vi(Watson and Switzer, 1978; Feldman and Kruger, 1980),and most paratrigeminal neurons seem to project preferen-tially to the nucleus of the solitary tract (Craig and Burton,1981), parabrachial nuclei (Feil and Herbert, 1995), andcaudal hypothalamus. The projections of individual neu-rons to multiple brainstem nuclei that in turn project tothe hypothalamus (such as the trigemino-parabrachio-hypothalamic pathway; Bernard et al., 1996) establish anetwork of parallel pathways possibly ensuring that impor-tant sensory information will reach the hypothalamus.Because similar sensory signals are likely to be modulateddifferently on their way to the hypothalamus, it is alsopossible that each area transfers different information andtherefore serves different functions. The monosynapticpathway described in this study can theoretically transferless modulated information to the hypothalamus in thefastest way possible. These factors may be important forinitiating immediate responses to stimuli that threatensurvival.

ACKNOWLEDGMENTS

We thank Drs. A.M. Strassman, N.L. Chamberlin, C.B.Saper, and G.M. Bove for valuable comments, J.J. Fink foreditorial comments, T. Sequist and Y. Zhou for technicalsupport, and P.G. Moller for illustration assistance.

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cells of origin of the trigeminohypothalamic tract in the rat

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