THE JOURNAL OF COMPARATIVE NEUROLOGY 448:53101 (2002)

Brainstem Projections to Midline and Intralaminar Thalamic Nuclei of The Rat

KARL E. KROUT, REBECCA E. BELZER, AND ARTHUR D. LOEWY* Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT The projections from the brainstem to the midline and intralaminar thalamic nuclei were examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin -subunit (CTb) were made in each of the intralaminar nuclei of the dorsal thalamus: the lateral parafascicular, medial parafascicular, central lateral, paracentral, oval paracentral, and central medial nuclei; in the midline thalamic nucleithe paraventricular, intermediodorsal, mediodorsal, paratenial, rhomboid, reuniens, and submedius nuclei; and, in the anteroventral, parvicellular part of the ventral posterior, and caudal ventral medial nuclei. The retrograde cell body labeling pattern within the brainstem nuclei was then analyzed. Nearly every thalamic site received a projection from the deep mesencephalic reticular, pedunculopontine tegmental, dorsal raphe, median raphe, laterodorsal tegmental, and locus coeruleus nuclei. Most intralaminar thalamic sites were also innervated by unique combinations of medullary and pontine reticular formation nuclei such as the subnucleus reticularis dorsalis, gigantocellular, dorsal paragigantocellular, lateral, parvicellular, caudal pontine, ventral pontine, and oral pontine reticular nuclei; the dorsomedial tegmental, subpeduncular tegmental, and ventral tegmental areas; and, the central tegmental eld. In addition, most intralaminar injections resulted in retrograde cell body labeling in the substantia nigra, nucleus Darkschewitsch, interstitial nucleus of Cajal, and cuneiform nucleus. Details concerning the pathways from the spinal trigeminal, nucleus tractus solitarius, raphe magnus, raphe pallidus, and the rostral and caudal linear raphe nuclei to subsets of midline and intralaminar thalamic sites are discussed in the text. The discussion focuses on brainstem thalamic pathways that are likely involved in arousal, somatosensory, and visceral functions. J. Comp. Neurol. 448:53101, 2002. 2002 Wiley-Liss, Inc.
Indexing terms: attention; autonomic nervous system; cerebral cortex; nociception; sleep; vigilance

The midline and intralaminar thalamic nuclei have been called nonspecic relay nuclei, because they were thought to project to vast areas of the cerebral cortex (e.g., Lorente de No, 1938; Morison and Dempsey, 1942; Schei bel and Scheibel, 1972; Jones and Leavitt, 1974). However, this idea has been refuted because each of the midline and intralaminar thalamic nuclei have been demonstrated to project to limited and specic cortical regions, as well as to subregions of the basal ganglia and amygdala (e.g., Berendse and Groenewegen, 1990, 1991; Turner and Herkenham, 1991; Yasui et al., 1991; for review, see Bentivoglio et al., 1991; Groenewegen and Berendse, 1994). These forebrain circuits affect behavioral or cognitive responses and may be modulated by ascending afferent information arising from the brainstem and spinal cord (Alexander et al., 1990; Groenewegen et al., 1990, 1999).
2002 WILEY-LISS, INC.

A classic example of this brainstem regulation of forebrain activity was demonstrated by Moruzzi and Magoun in 1949. They found that electrical stimulation of the brainstem reticular formation caused desynchronization of the cortical electroencephalogram (EEG). They suggested that these profound cerebral cortical changes were

Grant sponsor: National Institute of Heart, Lung, and Blood of the National Institute of Health; Grant number: HL-25449. *Correspondence to: Arthur D. Loewy, Department of Anatomy and Neurobiology, Box 8108, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110. E-mail: loewya@pcg.wustl.edu Received 29 November 2001; Revised 28 January 2002; Accepted 31 January 2002 DOI 10.1002/cne.10236 Published online the week of April 29, 2002 in Wiley InterScience (www. interscience.wiley.com).

54 due to activation of a group of neurons lying in the core of the reticular formation and proposed that this ascending reticular activating system (ARAS) exerted a major inuence on sleep, wakefulness, and arousal (for review, see Gottesmann, 1999; Vincent, 2000). Subsequent anatomic and physiological studies have shown that the pathways responsible for these effects originate from the brainstem reticular formation and terminate within the midline and intralaminar thalamic nuclei (e.g., Morison and Dempsey, 1942; Hunter and Jasper, 1949; Nauta and Kuypers, 1958; Scheibel and Scheibel, 1958; Bowsher, 1975). In the 50 years since this discovery, the exact cells of origin that innervate the individual midline and intralaminar thalamic nuclei have not yet been determined. Numerous anterograde studies have examined the brainstem inputs to the thalamus (e.g., Peschanski and

K.E. KROUT ET AL. Besson, 1984c; Jones and Yang, 1985; Vertes et al., 1986, 1999; Hallanger and Wainer, 1988; Bernard et al., 1990; Vertes, 1991), but none of them have systematically dened the full extent of brainstem sites which innervate the midline and intralaminar thalamic nuclei. Similarly, several retrograde studies have been published which described the brainstem inputs to these thalamic targets, but these either relied on injections which encompassed multiple thalamic nuclei or limited the scope of their investigation to only one or two thalamic nuclei (e.g., Hallanger et al., 1987; Cornwall and Phillipson, 1988; Groenewegen, 1988; Carstens et al., 1990; Niimi et al., 1990; Yoshida et al., 1992; Bolton et al., 1993; Newman and Ginsberg, 1994). Therefore, a mapping of the complete brainstem inputs to individual midline and intralaminar thalamic nuclei has not yet been described and would be

Abbreviations A5 AD AM AP APT AV CL CLiR CM CnF Cu Dk DmTg,DMTg DPGi DpMe DR DRc DRd DRv DRvl ECIC f fr Gi GiA Gr IC IMD INC IP IRt LC LD LDTg LGN LH LHb LM LP lPF LRt MD MdD MdV MG MHb MM mPF MR NTS OPC PAG PaR PB PBP A5 area anterodorsal thalamic nucleus anteromedial thalamic nucleus area postrema anterior pretectal nucleus anteroventral thalamic nucleus central lateral thalamic nucleus caudal linear nucleus of the raphe central medial thalamic nucleus cuneiform nucleus cuneate nucleus Nucleus of Darkschewitsch dorsomedial tegmental area dorsal paragigantocellular nucleus deep mesencephalic reticular nucleus dorsal raphe nucleus dorsal raphe nucleus, caudal part dorsal raphe nucleus, dorsal part dorsal raphe nucleus, ventral part dorsal raphe nucleus, ventrolateral part external cortex of the inferior colliculus fornix fasciculus retroexus gigantocellular reticular nucleus gigantocellular reticular nucleus, alpha part gracile nucleus inferior colliculus intermediodorsal thalamic nucleus interstitial nucleus of Cajal interpeduncular nucleus intermediate reticular nucleus locus coeruleus laterodorsal thalamic nucleus laterodorsal tegmental nucleus lateral geniculate nucleus lateral hypothalamic area lateral habenular nucleus lateral mammillary nucleus lateral posterior thalamic nucleus lateral parafascicular thalamic nucleus lateral reticular nucleus mediodorsal thalamic nucleus medullary reticular nucleus, dorsal part medullary reticular nucleus, ventral part medial geniculate nucleus medial habenular nucleus medial mammillary nucleus medial parafascicular thalamic nucleus median raphe nucleus nucleus of the solitary tract oval paracentral thalamic nucleus periaqueductal gray matter pararubral nucleus parabrachial nucleus parabrachial pigmented nucleus pc PC PC-c PC-r PCRt PnC PnO PnV Po PPTg Pr Pr5 PRC PT PVA PVP PVT Re Rh RIP RLiR RMg RN RPall RRF Rt RVLM SC scp sm SM SNpc SNpr Sp5 Sp5C Sp5I Sp5O SPTg SRD SubC SuM TMv VA Ve VL VM VMc VP VPM VPpc VTA xscp ZI posterior commissure paracentral thalamic nucleus paracentral thalamic nucleus, caudal part paracentral thalamic nucleus, rostral part parvicellular reticular nucleus pontine reticular nucleus, caudal part pontine reticular nucleus, oral part pontine reticular nucleus, ventral part posterior thalamic nuclear group pedunculopontine tegmental nucleus prepositus nucleus principal sensory trigeminal nucleus precommissural nucleus paratenial thalamic nucleus paraventricular thalamic nucleus, anterior paraventricular thalamic nucleus, posterior paraventricular thalamic nucleus, middle reuniens thalamic nucleus rhomboid thalamic nucleus raphe interpositus nucleus rostral linear nucleus of the raphe raphe magnus nucleus red nucleus raphe pallidus nucleus retrorubral eld reticular thalamic nucleus rostral ventrolateral medulla superior colliculus superior cerebellar peduncle stria medullaris of the thalamus submedius thalamic nucleus substantia nigra, compact part substantia nigra, reticular part spinal trigeminal nucleus spinal trigeminal nucleus, caudal part spinal trigeminal nucleus, interpolar part spinal trigeminal nucleus, oral part subpeduncular tegmental nucleus dorsal reticular subnucleus (Subnucleus reticularis dorsalis) subcoeruleus nucleus supramammillary nucleus tuberomammillary nucleus, ventral ventral anterior thalamic nucleus vestibular nuclei ventrolateral thalamic nucleus ventromedial thalamic nucleus ventromedial thalamic nucleus, caudal part ventral posterior thalamic nucleus ventral posteromedial thalamic nucleus ventral posterior thalamic nucleus, parvicellular part ventral tegmental area decussation of the superior cerebellar peduncle zona incerta

BRAINSTEM-THALAMIC CONNECTIONS particularly useful in terms of dening which regions of the brainstem could possibly inuence the basal ganglia and cerebral cortex. In the present study, we made highly restricted injections of the retrograde tracer cholera toxin -subunit (CTb) in each midline or intralaminar thalamic nucleus and provided maps of the retrograde cell body labeling throughout the entire brainstem. These data will complement earlier reports in this series on the thalamic afferents from the periaqueductal gray (Krout and Loewy, 2000b), parabrachial nucleus (Krout and Loewy, 2000a), and superior colliculus (Krout et al., 2001).

55 goat anti-CTb solution (List) made in 0.1 M Na2HPO4 buffer containing 0.1% sodium azide and 0.3% Triton X-100 (Sigma, St. Louis, MO) (pH 7.4) for 2 days. Sections were washed in KPBS (0.02 M potassium phosphate buffer; pH 7.4), incubated in biotinylated donkey anti-goat antibodies (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA) for 3 hours, washed, and moved into ABC solution (Vector Laboratories, Burlingame, CA) for 1 hour. CTblabeled neurons were visualized with diaminobenzidine (DAB; Sigma Fast; Sigma; 1 tablet/15 ml of distilled water), mounted on gelatin-coated slides, and dried. The tissues were gold-intensied (modied from Kitt et al., 1994; see Krout et al., 1998, for details), counterstained with 0.6% thionin (Fischer, Fair Lawn, NJ), and cover-slipped with DPX mounting medium (BDH Limited, Poole, UK). The distribution of the retrogradely labeled cells within the brainstem was mapped by using an X-Y plotting system (MDplot ver. 3.3, AccuStage, St. Paul, MN). The purpose of this study was to provide a qualitative map of the inputs from the entire brainstem to the midline and intralaminar thalamus. Thus, the semiquantitative data in Table 1 are designed to highlight the pattern innervation and not present absolute numbers of neurons. Even for cases with the same injection target, small variations in injection site placement, injection volume, and uptake and transport of the CTb result in a slight variation in the number of retrogradely labeled cells. In addition, although it did not appear to be a problem in the present study, CTb can be taken up by bers of passage (for further discussion, see Luppi et al., 1990; Chen and Aston-Jones, 1995; Ruigrok et al., 1995; Krout and Loewy, 2000a,b; Krout et al., 2001). As an approximation, the number of labeled neurons in each brainstem nucleus was translated into a ve point scale: 0 1 cells none ( ), 25 cells light ( ), 6 15 cells moderate ( ), 16 25 cells dense ( ), 26 or more cells very dense ( ). The average density of CTb labeling from all cases with a particular injection target is given in Table 1. The pattern of brainstem3thalamic innervation is further illustrated in retrograde cell body labeling maps from representative cases that were transposed onto standardized drawings of the brainstem (modied from Paxinos and Watson, 1997) with the aid of a camera lucida (Figs. 4 20). The research described in this report was reviewed and approved by the Washington University School of Medicine Animal Care and Biological Safety Committees and conformed to NIH guidelines.

MATERIALS AND METHODS

Stereotaxic injections of CTb (List Biological, Inc., Campbell, CA, 1% solution made in distilled water) were made with glass micropipettes in thalamic nuclei of male Sprague-Dawley rats (n 85; 200 400 g; Simonsen Laboratories, Gilroy, CA) by using coordinates derived from the rat atlas of Paxinos and Watson (1997). Iontophoretic ejections were made by using 7 A on/off pulses delivered from a Midguard precision current source (Stoelting, Wood Dale, IL) for 15 minutes. Five to 9 days later, these rats were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and pseudorabies virus was injected into the stellate ganglion. After 5 additional days (10 14 days after the CTb injections), the animals were reanesthetized and perfused transcardially with 0.9% saline solution followed by 500 ml of 4% paraformaldehyde solution (pH 7.4). The brains were stored for at least 2 days in xative before being cut in the transverse plane at 50 m on a freezing microtome. The sections were collected in 0.1 M Na2HPO4 buffer containing 0.1% sodium azide (pH 7.4). As determined by immunohistochemical methods (see Krout and Loewy, 2000b, for details), some of the animals ( 30%) showed no signs of viral infection. The pattern of CTb-positive cells in animals with viral infections was virtually identical to those animals that had no infection. The results of the viral experiments will be reported in a separate communication. The 85 cases used in the present account were selected from material used in previous reports (Krout and Loewy, 2000a,b; Krout et al., 2001) on the basis that their injection sites were restricted mainly to one thalamic nucleus. Injection sites were drawn by using a camera lucida, scanned into a computer, and traced in CorelDraw8 (Corel Corp., Ottawa, Ontario). The targets included the central lateral (n 4), central medial (n 7), lateral parafascicular (n 4), medial parafascicular (n 4), oval paracentral (n 4), paracentral (n 6), anteroventral (n 3), intermediodorsal (n 5), mediodorsal (n 3), paratenial (n 5), rhomboid (n 4), reuniens (n 6), submedius (n 5), caudal ventromedial (n 4), and parvicellular part of the ventral posteromedial (n 4) thalamic nuclei, as well as three levels of the paraventricular thalamic nucleus: anterior (n 6), middle (n 5), and posterior (n 6). The thalamic nuclei were delineated according to anatomic descriptions published by Berendse and Groenewegen (1990) and Paxinos and Watson (1997). Several examples of injection sites are shown in Figure 1. A one-in-ve series of transverse sections through the entire brainstem from these cases was processed immunohistochemically by the avidin-biotin complex (ABC) method for visualization of CTb retrogradely labeled neurons. First, sections were placed in a 1:40,000 solution of

RESULTS
Because the results of the present experiments are extensively detailed in Table 1 and in Figures 220, this section will present only an overview of the results. However, these gures present the data throughout the entire brainstem from a representative case of each thalamic injection target. In addition, several examples of retrograde cell body labeling are presented in Figures 2 and 3. Finally, the thalamic afferents from other brainstem sites, viz., periaqueductal gray matter, parabrachial nucleus, and superior colliculus, have been described previously (Krout and Loewy, 2000a,b; Krout et al., 2001). CTb injections were made in 18 different thalamic sites. Each injection site was composed of two areas: the core

56

TABLE 1. Summary of Retrograde Cell Body Labeling in the Brainstem1 Thalamic Nuclei Intralaminar IPF mPF OPC PC-c PC-r PVA PVT PVP IMD MD PT Rh Re SM AV VPoc VMc Midline Other

CI

CM

Reticular formation Medullary MDd MDv SRD Gi GiA DPGi LPGi Pontine IRt PCRt PnC PnV DmTg PnO SubC Mescencephalic SPTg DpMe RRF VTA PPTg CTF Raphe system RMg ROb RPall RIP DRc DRd DRv DRvl MR CLiR RLiR PmR Other nuclei Medullary Sp5C Sp5I RVLM Cu Gr NTS Ve Pr Sp5O Pontine A5 NI Pr5 LC Bar LDTg Mesencephalic CnF DTg PL IP PBP SNpr SNpc Dk INC , none; , light; , moderate; , dense; , more than 26 cells. For abbreviations, see list.

K.E. KROUT ET AL.

Data represented on a ve-point scale for the number of labeled neurons:

BRAINSTEM-THALAMIC CONNECTIONS

57

Fig. 1. A series of photomicrographs showing the extent of the cholera toxin -subunit injection site in the anterior paraventricular thalamic nucleus (PVA; case 4312) (A), rostral paracentral thalamic nucleus (PC; case 3907) (B), middle paraventricular thalamic nucleus (PVT; case 4318) (C), and, intermediodorsal thalamic nucleus (IMD; case 3912) (D). For abbreviations, see list. Scale bar 1 mm.

region where neurons were lled with tracer, and a halo region where CTb was found only in the neuropil. The injection targets were divided into the intralaminar thalamic nuclei central lateral (CL, Fig. 4), central medial (CM, Fig. 5), lateral parafascicular (lPF, Fig. 6), medial parafascicular (mPF, Fig. 7), oval paracentral (OPC, Fig. 8), and paracentral nuclei (PC, Fig. 9), the midline thalamic nucleianterior paraventricular (PVA, Fig. 10), middle paraventricular (PVT, Fig. 11), posterior paraventricular (PVP, Fig. 12), intermediodorsal (IMD, Fig. 13), mediodorsal (MD, Fig. 14), paratenial (PT, Fig. 15), reuniens (Re, Fig. 16), rhomboid (Rh, Fig. 17), and submedius nuclei (SM, Fig. 18), and other thalamic nuclei anteroventral (AV), parvicellular part of the ventral posteromedial (VPpc, Fig. 19), and caudal ventromedial nuclei (VMc, Fig. 20). Some of these cases were used in previous publications (Krout and Loewy, 2000a,b; Krout et al., 2001).

Intralaminar thalamic nuclei

All intralaminar nucleus cases contained a dense to very dense concentration of CTb-positive neurons in the deep mesencephalic reticular nucleus. Labeling in other reticular formation sites was more varied, but, in general, fewer cells were identied after PC injections and more

after lPF and mPF injections. In the raphe system, only mPF injections resulted in a moderate number of labeled cells in the raphe pallidus nucleus. The dorsal raphe nucleus was labeled in every intralaminar experiment, and the median raphe nucleus contained a moderate number of labeled neurons in the CL, CM, lPF, and mPF experiments. Every intralaminar target resulted in retrograde labeling in the substantia nigra and the locus coeruleus nucleus. The laterodorsal tegmental nucleus, dorsal tegmental nucleus, and the cuneiform nucleus were most heavily labeled after lPF, mPF, CM, and CL injections. Injections in the mPF and lPF resulted in CTb-positive neurons in a cell-poor area sandwiched between the pars compacta of the substantia nigra and the medial lemniscus (Figs. 6B, 7B). This region has been identied by Paxinos et al. (1999) as the parabrachial pigmented nucleus. In addition, a few cells were scattered in the nucleus of the solitary tract (NTS), vestibular nuclei, and prepositus nucleus, particularly after lPF injections. Several differences were found in the pattern of labeling between PC cases that had injections in the caudal PC (at the level where the OPC is still present; n 2) and those with injections in the middle or rostral part of PC (n 4).

58

K.E. KROUT ET AL.

Fig. 2. Photomicrographs of a case that had cholera toxin -subunit injected into the lateral parafascicular thalamic nucleus (case 3311). A: A section through the midbrain where the outlined region is enlarged in B and shows ipsilateral retrograde cell body

labeling in the deep mesencephalic nucleus. C: A section through the lower brainstem where the outlined region is enlarged in D and shows a contralateral retrograde cell body labeling in the dorsal paragigantocellular nucleus. Scale bars 1 mm in A,C, 100 m in B,D.

In addition to the dense, specic projection from the internal lateral parabrachial subnucleus to the caudal portion of PC (see Krout and Loewy, 2000a, for discussion), caudal PC injections resulted in more retrograde labeling in several other brainstem sites (see Table 1). For exam-

ple, the dorsomedial tegmental area, pedunculopontine tegmental area, oral pontine reticular nucleus, and the pars reticulata portion of the substantia nigra each had more labeled cells after caudal PC injections than after more rostral PC injection sites.

BRAINSTEM-THALAMIC CONNECTIONS

59

Fig. 3. A: Photomicrograph of a case that had cholera toxin -subunit (CTb) injected into the reuniens thalamic nucleus (case 4466), illustrating retrograde cell body labeling in the contralateral pedunculopontine tegmental nucleus. B: Greater detail of the outlined region in A. C: Photomicrograph of a case that had CTb injected into

the parvicellular portion of the ventral posterior thalamic nucleus (case 3211) illustrating contralateral retrograde cell body labeling in the caudal part of the spinal trigeminal nucleus. D: Greater detail of the outlined region in C. Scale bars 1 mm in A,C, 100 m in B,D.

Midline thalamic nuclei

Few medullary or pontine reticular areas were labeled after midline thalamic injections. One exception was the Rh experiments. In these cases, a large number

of labeled cells were found in the gigantocellular reticular, caudal pontine reticular, and dorsomedial tegmental nuclei. More robust labeling was identied at the level of the midbrain, where the majority of midline

Fig. 4. A: CTb injection site in the central lateral thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 2227. (Note: In Figures 4-20, data are plotted on standardized transverse sections of the brainstem that were modied from Paxinos and

Watson, 1997.) In levels B-G, one triangle represents three labeled neurons. In levels H-Q, one dot represents one labeled neuron. Approximate bregma levels are given on the lower right-hand corner of each section. For abbreviations, see list.

Figure 4

(Continued)

Fig. 5. A: Cholera toxin -subunit injection site in the central medial thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 3886. For abbreviations, see list.

Figure 5

(Continued)

Fig. 6. A: Cholera toxin -subunit injection site in the lateral parafascicular thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 3313. For abbreviations, see list.

Figure 6

(Continued)

Fig. 7. A: Cholera toxin -subunit injection site in the medial parafascicular thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 3665. For abbreviations, see list.

Figure 7

(Continued)

Fig. 8. A: Cholera toxin -subunit injection site in the oval paracentral thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 3207. For abbreviations, see list.

Figure 8

(Continued)

Fig. 9. A: Cholera toxin -subunit injection site in the paracentral thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 3907. For abbreviations, see list.

Figure 9

(Continued)

Fig. 10. A: Cholera toxin -subunit injection site in the anterior paraventricular thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 2332. For abbreviations, see list.

Figure 10

(Continued)

Fig. 11. A: Cholera toxin -subunit injection site in the middle paraventricular thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 2530. For abbreviations, see list.

Figure 11

(Continued)

Fig. 12. A: Cholera toxin -subunit injection site in the posterior paraventricular thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 2653. For abbreviations, see list.

Figure 12

(Continued)

Fig. 13. A: Cholera toxin -subunit injection site in the intermediodorsal thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 2515. For abbreviations, see list.

Figure 13

(Continued)

Fig. 14. A: Cholera toxin -subunit injection site in the mediodorsal thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 2516. For abbreviations, see list.

Figure 14

(Continued)

Fig. 15. A: Cholera toxin -subunit injection site in the paratenial thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 2483. For abbreviations, see list.

Figure 15

(Continued)

Fig. 16. A: Cholera toxin -subunit injection site in the reuniens thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 3005. For abbreviations, see list.

Figure 16

(Continued)

Fig. 17. A: Cholera toxin -subunit injection site in the rhomboid thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 3048. For abbreviations, see list.

Figure 17

(Continued)

Fig. 18. A: Cholera toxin -subunit injection site in the submedius thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 2999. For abbreviations, see list.

Figure 18

(Continued)

Fig. 19. A: Cholera toxin -subunit injection site in the parvicellular part of the ventral posteromedial thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 3211. For abbreviations, see list.

Figure 19

(Continued)

Fig. 20. A: Cholera toxin -subunit injection site in the caudal ventromedial thalamic nucleus. BQ: Pattern of retrograde cell body labeling from case 2930. For abbreviations, see list.

Figure 20

(Continued)

94 thalamic targets resulted in CTb transport to the deep mesencephalic reticular and pedunculopontine tegmental nuclei. The pattern of labeling in the raphe system was largely similar to that of the intralaminar thalamic nuclei, namely, numerous cells in the dorsal raphe nucleus. In particular, the almost exclusive brainstem projection to the PT was from the caudal and dorsal portions of the dorsal raphe nucleus (in addition to a moderate concentration of cells in its ventral and ventrolateral portions) (Fig. 15). Raphe pallidus neurons were labeled after injections in the PVT system. The median raphe nucleus was labeled after CTb injections in every midline thalamic site. In addition, the MD experiments resulted in a dense concentration of labeled neurons in the caudal and rostral linear raphe nuclei. A few CTb-positive neurons in the spinal trigeminal nucleus were observed in the Re and SM cases. NTS labeling was found predominately in the medial and commissural NTS subnuclei after CTb injections in all portions of the PVT as well as after Rh and Re injections. Every midline site resulted in labeling in the locus coeruleus and laterodorsal tegmental nuclei; and, all midline thalamic sites (except for the PT) received a projection from the cuneiform nucleus. The parabrachial pigmented nucleus was densely labeled after PVP (Fig. 12B) and Re (Fig. 16B) injections and moderately labeled in the MD (Fig. 14B) cases. The only dense substantia nigra labeling was found in its pars reticulata after Rh injections.

K.E. KROUT ET AL. Loewy, 2000a,b; Krout et al., 2001). These allowed us to obtain sample sizes of three to seven cases for each thalamic target to make a detailed analysis of the distribution of the brainstem cells that project to each midline and intralaminar thalamic nucleus. The discussion will be organized in two parts: (1) a comparison of the present data with previous anterograde and retrograde tracing investigations; and (2) a functional interpretation of these brainstem inputs based on the cerebral cortical targets of the thalamic nuclei (Berendse and Groenewegen, 1991; Groenewegen and Berendse, 1994; Moga et al., 1995).

Comparison to previous studies

Reticular formation. Few studies have examined the ascending projections of individual nuclei of the reticular formation in the rat. Most anterograde tracer studies were inconclusive, because the injections used in these investigations involved multiple reticular formation nuclei (e.g., Zemlan et al., 1984; Jones and Yang, 1985; Vertes et al., 1986). Although they conrm that there is a signicant projection from the brainstem reticular formation to the midline and intralaminar thalamic nuclei, few point-topoint details can be obtained from these reports. For example, anterograde injections involving the dorsal gigantocellular, gigantocellular, and the alpha part of the gigantocellular reticular nuclei indicated that these areas project to almost all midline and intralaminar thalamic sites except the PVT, SM, MD, and PT (Jones and Yang, 1985; also Vertes et al., 1986). These ndings were conrmed by the present retrograde results. However, the size of injections used in these anterograde studies do not allow for the analysis of more subtle distinctions, such as the very dense projection from the dorsal gigantocellular reticular nucleus to the lPF (Fig. 6) or that Rh receives inputs predominantly from the gigantocellular reticular nucleus but not surrounding reticular regions (Fig. 17). One of the exceptions to this trend of large reticular formation injection sites is a study by Villanueva et al. (1998) where small injections of Phaseolus vulgaris leucoagglutin in (PHA-L) were made in the subnucleus reticularis dorsalis (SRD) and cuneate nucleus (also Bernard et al., 1990). The complete retrograde mapping of the inputs to individual midline and intralaminar thalamic nuclei provided in the present report was extremely consistent with this anterograde tracer study. Dense concentrations of labeled cells were found in the SRD and cuneate nucleus after lPF and VMc injections, whereas moderate to light labeling was observed in the mPF, OPC, PC, Re, Rh, and VPpc cases. Another study made restricted injections of [3H]leucine in the mesencephalic or pontine reticular formation and examined the ascending projections (Vertes and Martin, 1988). The results of injections in the caudal pontine and oral pontine reticular formation were very similar to each other and matched almost exactly the pattern of innervation demonstrated by the CTb method used here. These similarities include not only a dense projection from both reticular formation areas to the lPF and mPF (among other sites), but also parallel our nding that PVA and PVP, but not PVT, receive a slight projection from the caudal pontine reticular nucleus. The deep mesencephalic reticular nucleus projects heavily to all intralaminar thalamic nuclei, including sites that were not heavily innervated by the caudal or oral

Other thalamic nuclei

A simple progression in the overall number of CTbpositive cells was observed among these three nuclei: VMc had the most labeled cells, followed by VPpc, with very few brainstem neurons projecting to the AV. Most reticular formation areas had at least moderate labeling after VMc injections (Fig. 20), but only the parvicellular reticular nucleus, deep mesencephalic reticular nucleus, pedunculopontine tegmental nucleus, and central tegmental eld were at least moderately labeled in the VPpc cases (Fig. 19). Each of the VPpc cases resulted in a dense concentration of labeled neurons along the dorsolateral limit of the caudal part of the spinal trigeminal nucleus and, more rostrally, in the principal sensory trigeminal nucleus (Fig. 19 J,K,P,Q). A few labeled neurons were found in the caudal spinal and principal sensory portions of the trigeminal nucleus in the VMc experiments. These same two targets (VMc and VPpc) also resulted in cuneiform nucleus, laterodorsal tegmental nucleus, locus coeruleus, A5 region, NTS, and cuneate nucleus labeling. Finally, the pars reticulata portion of the substantia nigra was found to contain a very dense concentration of CTb-positive neurons after VMc injections (Fig. 20BD).

DISCUSSION
The present results demonstrate that each of the midline and intralaminar thalamic nuclei receives input from a selective set of brainstem nuclei. This study took advantage of a collection of over 400 rat brains in which CTb was iontophoresed into the thalamus. From this library, we chose 85 key cases with injections centered in a single midline or intralaminar thalamic nucleus that had minimal involvement of adjacent cell groups (see Krout and

BRAINSTEM-THALAMIC CONNECTIONS pontine reticular nuclei, such as the OPC and PC. In addition, most midline thalamic sites, including the PVT system, IMD, Rh, and Re are also densely innervated. These results correlate well with anterograde tracing experiments (Vertes and Martin, 1988) with a single exception: our data demonstrate a moderate projection from the deep mesencephalic reticular nucleus to MD, whereas Vertes and Martin (1988) showed no innervation of this cell group. This discrepancy is likely due to (1) slight spread of our CTb injection sites into either the PVT system or CL, both of which had a dense innervation from the deep mesencephalic reticular nucleus; or (2) differences in the demarcation of the border between CL and MD, which can be particularly difcult at rostral levels of the thalamus. Substantia nigra. Substantia nigra3thalamic projections have been investigated by several groups (e.g., Deniau and Chevalier, 1992; Nishimura et al., 1997; Sakai et al., 1998; Groenewegen et al., 1999; Tsumori et al., 2000). The anterograde data presented in these reports suggest a dense nigrothalamic projection to the lPF, mPF, CL, OPC, VMc, and Rh. The retrograde data reported here agree with these studies. In addition to these general patterns of innervation, the projections of subcomponents of the substantia nigra to specic midline and intralaminar thalamic sites have been conrmed. For example, the CL receives inputs predominately from the reticular, but not the compact, part of substantia nigra (Fig. 1; Deniau and Chevalier, 1992). In addition, both Tsumori et al. (2000) and Deniau and Chevalier (1992) demonstrated a projection from the caudal dorsolateral portion of the reticular part of substantia nigra to the OPC (although these studies refer to the OPC as either the ventrolateral [Tsumori] or rostroventral [Deniau] parafascicular thalamic nucleus). Our data conrm these ndings (see Fig. 8C,D). One prominent discrepancy can be noted in the projection from the substantia nigra to the MD. Anterograde tracer studies using PHA-L demonstrated a projection from the substantia nigra to the lateral and medial segments of the MD (e.g., Deniau and Chevalier, 1992; Groenewegen et al., 1999). Our data showed no retrogradely labeled cells in substantia nigra after MD injections. These CTb injections were located in the rostral half of MD and either excluded or only slightly involved its lateral segment; however, each injection encompassed the medial segment. One possible explanation for the lack of labeling is that the nigral projection to MD innervates the caudal portion of MD more heavily than its rostral portion (Deniau and Chevalier, 1992; Groenewegen et al., 1999) and the CTb injections in the rostral half of the MD may have limited uptake of the tracer by nigrothalamic bers. Nucleus Darkschewitsch. To date, only one study has investigated the thalamic projections of the nucleus Darkschewitsch with anterograde tracing methods in the rat (Krout and Loewy, 2000b). This report and the present data both showed that the predominate thalamic targets are the lPF and CL. Other innervated sites included the VMc, Re, and Rh. In addition, both reports demonstrated only a very slight projection from the nucleus Darkschewitsch to the OPC, PC, CM, and PVP. The anterograde map presented in the report by Krout and Loewy (2000b) correlates well with the present CTb data. Pedunculopontine tegmental nucleus. As shown here, the pedunculopontine tegmental nucleus innervates

95 all midline and intralaminar thalamic sites, except for the PT. Anterograde PHA-L tracer experiments also showed that this nucleus has widespread projections to these thalamic nuclei (Hallanger et al., 1987). Although these experiments did not result in anterogradely labeled axonal terminals in the lPF, subsequent anterograde tracing studies, using biotinylated dextran amine, demonstrated that the pedunculopontine tegmental nucleus projects densely to the lPF (Erro et al., 1999). Retrorubral eld. The retrorubral eld projects to the same midline and intralaminar thalamic nuclei as the pedunculopontine tegmental nucleus, but the projection is weaker. This nding is comparable to the extensive anterograde labeling found in the midline and intralaminar thalamic nuclei after the retrorubral eld was injected with PHA-L (Hallanger and Wainer, 1988). Their report suggested that the retrorubral eld projects to the PC; however, we found that CTb injections centered in this thalamic site (n 6) did not result in any labeled neurons in the retrorubral eld. Thus, it is likely that their PHA-L injections into the retrorubral eld spread into adjacent structures such as the deep mesencephalic reticular nucleus or the rostral end of the pedunculopontine tegmental nucleus, because both regions contained retrogradely labeled cells after CTb injections in the PC. Cuneiform nucleus. A limited number of studies have examined the projection from the cuneiform nucleus to the thalamus (Edwards and de Olmos, 1976; Hallanger and Wainer, 1988). Our data largely agrees with these reports; namely, a moderate to dense projection to the PVT system, CL, CM, mPF, and Rh. However, a few discrepancies were noted. First, Hallanger and Wainer (1988) showed that the Re and lPF were devoid of any labeled bers (their Fig. 5D), but CTb injections in these two thalamic targets resulted in moderate retrograde cell body labeling in the cuneiform nucleus. The reason for this difference is unclear. Also, Hallanger and Wainer (1988) showed many labeled terminals in the PC (their Fig. 5D,E); but, in the six PC cases examined here, only one case had any CTbpositive cells in the cuneiform nucleus. One explanation for this discrepancy may be in the placement of the anterograde injection site and the denition of the cuneiform nucleus used here. As can be seen in Figure 9G, after a CTb injection in the PC, a few CTb-labeled neurons are found just ventral and lateral to the cuneiform nucleus. This region appears to be included in the PHA-L injection site shown by Hallanger and Wainer (1988, their Fig. 5I). Thus the anterogradely labeled axonal terminals in the PC may originate from neurons that lie just beyond the borders of the cuneiform nucleus. Raphe nuclei. The thalamic targets of the dorsal raphe and median raphe nuclei have been extensively mapped by using small injections of PHA-L (Vertes, 1991; Vertes et al., 1999; Krout and Loewy, 2000b). Similar to the present retrograde tracer data, Vertes (1991) used an anterograde tracer and showed that virtually all midline and intralaminar nuclei receive a projection from the dorsal raphe nucleus. In addition, both studies showed that the PC and OPC receive input from the rostral, but not the caudal portions of the dorsal raphe nucleus. The retrograde tracer data reported here demonstrated that most thalamic sites also receive inputs from the median raphe, and these data largely conrm earlier anterograde work (Vertes et al., 1999). One small point of disagreement, however, was the projection to the PC: our data point to a

96 weak projection to this thalamic site, whereas Vertes et al. (1999) indicated a robust projection arising from the median raphe nucleus. The source of this discrepancy is unclear. Anterograde tracer studies have suggested that neurons in more caudal raphe nuclei, such as the raphe magnus nucleus project to the PVT (e.g., Peschanski and Besson, 1984b; Hermann et al., 1996). Our ndings conrm these results, but also indicate that a moderate projection arises from the raphe pallidus nucleus. Laterodorsal tegmental nucleus. The laterodorsal tegmental nucleus projects to every midline/intralaminar thalamic site except for the OPC (see Fig. 10 of Krout and Loewy, 2000a, for a photomicrograph of the OPC). This widespread projection correlates well with the extensive thalamic terminal labeling seen after a PHA-L injection into laterodorsal tegmental nucleus (Satoh and Fibiger, 1986). However, this anterograde tracer study also showed few labeled terminals in the PVT system, particularly in the PVA, whereas CTb injections at every rostrocaudal site throughout the PVT system consistently resulted in dense retrograde labeling in the laterodorsal tegmental nucleus. Because a few terminals do exist in the PVT, this nding may be indicative of the sensitivity of the CTb method. Alternatively, it may indicate that a large number of laterodorsal tegmental neurons have a limited number of terminals in the PVT. A5 and C1-3 catecholamine cell groups. Several reports have suggested that the PVT receives adrenergic and noradrenergic inputs from C1-3 regions and the A5 region, respectively (Byrum and Guyenet, 1987; Phillipson and Bohn, 1994). Although we did not examine the neurochemical properties of neurons in these areas, we were unable to identify retrogradely labeled cells in the vicinity of either the C1-3 or A5 cell groups. The explanation for these discrepancies almost certainly lies in technical differences between these reports and the present data. For the C1-3 neurons, Phillipson and Bohn (1994) used several techniques that, as compared with the current study, signicantly altered both the number of neurons labeled and their identication as adrenergic: (1) different tracers (Fluoro-Gold or uorescent microspheres vs. CTb); (2) multiple injections in the PVT (5 6 per animal vs. 1 per animal); and (3) double-labeling techniques with antibodies against phenylethanolamine N-methyltransferase (PNMT). For the A5 region, the anterograde tracing data from Byrum and Guyenet (1987) demonstrated a projection to the posterior but not middle or anterior regions of the PVT (i.e., PVP, but not PVA or PVT). In addition, their experiments using the retrograde transport of latex microspheres indicated only a small projection from the A5 region to the PVT. This nding suggests that the input from the A5 region to the PVT system may either be very small or originate predominately from non-noradrenergic neurons. Vestibular nuclei. As shown by anterograde tracing studies, the major thalamic target of the vestibular nuclei is the lPF (Shiroyama et al., 1995, 1999; Lai et al., 2000). In addition, these reports demonstrate a vestibular termination in the OPC, MD, Rh, mPF, CL, and VMc. Our results conrmed that the lPF receives the densest input from the vestibular nuclei of any thalamic site studied. However, CTb injections in the MD and OPC did not result in consistent labeling in the vestibular nuclei. The lack of labeling in the present MD experiments was likely the result of the rostrally positioned injection sites, be-

K.E. KROUT ET AL. cause anterior MD areas contained few anterogradely labeled axonal terminations (see above) (Shiroyama et al., 1999; Lai et al., 2000). The reason for the lack of CTbpositive neurons after OPC injections is not clear. Other, less robust projections from the vestibular nuclei, such as those to the Rh, mPF, CL, and VMc, were each conrmed by our retrograde tracing data. Nucleus of the solitary tract. Anterograde axonal tracing methods have been used to show that the main midline/intralaminar target of the NTS is the PVT system, including its posterior, middle, and anterior subdivisions (Ricardo and Koh, 1978; Ter Horst and Streeand, 1994; Ruggiero et al., 1998). These reports have also indicated that a few scattered terminal bers were present in the Re, CM, Rh, lPF, mPF, VMc, and OPC. Our data agree with these ndings both in terms of location and intensity of innervation. However, at least one difference was noted. In the VPpc experiments, NTS neurons were labeled. Earlier anterograde tracing studies failed to nd terminal axonal labeling in this region (Ricardo and Koh, 1978; Ter Horst and Streeand, 1994; Ruggiero et al., 1998). However, Ruggiero et al. (1998) found dense terminal labeling ventral to the fasciculus retroexus and identied this region as the ventral sector of the parafascicular nucleus. This area may correspond to the region we have dened here as the VPpc.

Functional considerations
For much of past half-century, the midline and intralaminar thalamic nuclei were thought to be a nonspecic relay center that globally activated the cerebral cortex in response to signals from the brainstem reticular formation (e.g., Lorente de No, 1938; Moruzzi and Ma goun, 1949; Jones and Leavitt, 1974). This concept has changed. Neuroanatomic studies have now shown that the efferent projections from these thalamic nuclei to the cerebral cortex, striatum, and amygdala are highly specic (Berendse and Groenewegen, 1990, 1991; Turner and Herkenham, 1991; Groenewegen and Berendse, 1994; Moga et al., 1995). More recent hypotheses propose a role for brainstem neurons not only in arousal and sleep/ wakefulness cycles, but also in attention, homeostasis, and emotion (e.g., Berendse and Groenewegen, 1990, 1991; Dostrovsky and Guilbaud, 1990; Powell et al., 1990; Grunwerg and Krauthamer, 1992; Groenewegen and Berendse, 1994; Gottesmann, 1999). Because more than 50 brainstem nuclei provide inputs to the midline and intralaminar thalamic nuclei, a comprehensive discussion of each of these sites is beyond the scope of this study. However, the present report, along with the others in this series (Krout and Loewy, 2000a,b; Krout et al., 2001), support the idea that brainstem nuclei contribute to specialized forebrain pathways by means of specic projections to the midline and intralaminar thalamic nuclei. To focus attention on several major issues, this discussion will concentrate on arousal, motor, somatosensory, and visceral systems. Although functional interpretations can be deduced from the anatomic connections, there is a paucity of physiological observations regarding brainstem3thalamic3 forebrain circuits. In addition, because the concept of the nonspecic arousal system had dominated the thinking in this eld, the midline and intralaminar thalamic nuclei were often considered as a single functional unit with the

BRAINSTEM-THALAMIC CONNECTIONS same function and connectivity. This intellectual bias has inuenced physiological experiments that were designed to dissect the role(s) of individual thalamic nuclei in arousal, cognition, and other higher-level brain activities, such as memory (but see Mair, 1994; Van Der Werf et al., 1999, 2000; Jeljeli et al., 2000). Arousal systems. Several lines of evidence have implicated the medial thalamus and reticular formation in modulating arousal. Electrical stimulation of the intralaminar thalamus in anaesthetized animals produces EEG desynchronization (Morison and Dempsey, 1942; Hunter and Jasper, 1949), an effect duplicated by electrical stimulation of the reticular formation (Moruzzi and Magoun, 1949). This effect has been shown more directly by studies which show that the reticular formation can inuence the cerebral cortex and other forebrain regions through connections with the intralaminar thalamus (Steriade, 1996, 1999; Erro et al., 1999). In humans, PET studies demonstrated that activation of both the reticular formation and intralaminar nuclei occurs during tasks requiring arousal/attention (Kinomura et al., 1996). In addition, thalamic relay cells change their activity, depending on whether the animal is awake or asleep (Glenn and Steriade, 1982; Steriade and Glenn, 1982; Steriade, 1999). Unfortunately, most of these studies have relied on techniques that stimulate bers of passage or that simply correlate brainstem and cortical activity without showing a causal relationship. Because the present study has shown that midline and intralaminar thalamic nuclei receive robust inputs from nonreticular areas, other brainstem cell groups, besides the reticular formation, may also be important in thalamic control of arousal. Three examples can be cited. First, the locus coeruleus provides a noradrenergic input to thalamus and cerebral cortex (Jones and Moore, 1977; Loughlin et al., 1982; Peschanski and Besson, 1984a), and the discharge rate of these neurons increases in response to novel stimuli and decreases during sleep (Aston-Jones and Bloom, 1981; Foote et al., 1983). Because lesions of this region do not affect the sleep-wake cycle (Jones and Moore, 1977), the locus coeruleus has been proposed to play a role in the maintenance, but not initiation, of arousal. Second, dorsal raphe neurons display similar properties to those seen in the locus coeruleus: they decrease their ring rate during slow wave sleep. However, lesions of the dorsal raphe nucleus produce a different effect than locus coeruleus lesions, viz., they cause insomnia (Lydic et al., 1987; for review see Robbins et al., 1998). Third, other cell groups important in maintaining normal transitions in the level of arousal include the laterodorsal tegmental nucleus and the pedunculopontine nucleus that provide cholinergic inputs to the thalamus and brainstem and have been implicated in the switch from slow-wave to rapid eye movement sleep (Edley and Graybiel, 1983; Rye et al., 1987, 1988; Steriade et al., 1990; Bolton et al., 1993). In the present study, we have demonstrated that the deep mesencephalic reticular, laterodorsal tegmental, pedunculopontine tegmental, dorsal raphe, and locus coeruleus nuclei project to every midline and intralaminar thalamic site (Fig. 21). Overlapping distributions of labeled neurons in the deep mesencephalic reticular nucleus were observed after the various thalamic injections. However, the present experimental design did not examine multiple thalamic sites with double-tracer techniques; thus, it is not known whether subsets of thalamic nuclei

97 receive inputs from single deep mesencephalic reticular nucleus neurons or whether there is a segregation of thalamically projecting neurons in this region. This issue is important because, if it is shown that single brainstem neurons innervate multiple thalamic sites, then it could be predicted that these cells would inuence multiple areas of the cerebral cortex. This possibility may be particularly relevant, because the deep mesencephalic reticular and pedunculopontine tegmental nuclei are the brainstem regions that were stimulated by Moruzzi and Magoun (1949) to produce EEG desynchronization. Changes in arousal modify the responsiveness of thalamic neurons to stimuli (Glenn and Steriade, 1982; Steriade and Glenn, 1982). For example, stimulation in the area of the cholinergic neurons of the pedunculopontine tegmental and laterodorsal tegmental nuclei during sleep potentiated the response of thalamic neurons to somatic stimulation (Pare and Steriade, 1990; Steriade, 1996). Also, ascending inputs to the intralaminar nuclei may play a role in modulating information ow in the cerebral cortex, because stimulation of the mesencephalic reticular formation facilitated oscillation between two visual areas (Munk et al., 1996). Thus, the level of brainstem activation may affect thalamocortical, thalamostriatal, or even corticocortical transmission. Motor systems. Several midline and intralaminar thalamic sites, particularly the lPF and CL, may play a key role in movement. Their contribution to motor activity can be deduced from their anatomic connectivity as well as by lesion and stimulation studies. The lPF projects not only to motor-related areas of cerebral cortex and striatum but also to the subthalamic nucleus (Berendse and Groenewegen, 1990, 1991; Mouroux and Feger, 1993). Because stimulation of the lPF inhibited movement in freeroaming rats (Mileikovsky et al., 1994), we have previously suggested that this lPF3subthalamic circuit may be related to stopping ongoing behavior and allowing the initiation of new motor programs (Krout et al., 2001). In addition to these efferent projections, the lPF receives inputs from motor related brainstem sites, including the substantia nigra, nucleus Darkschewitsch, superior colliculus, cuneiform nucleus, vestibular nuclei, and dorsal column nuclei (see Table 1) (Krout and Loewy, 2000b; Lai et al., 2000; Krout et al., 2001). However, the lPF also receives projections from a wide range of other brainstem areas that have been associated with other modalities such as autonomic function (e.g., rostral ventrolateral medulla and NTS), somatosensory function (e.g., spinal trigeminal nucleus), and some sites that integrate these and other functions (e.g., periaqueductal gray matter and SRD) (Roy et al., 1992; Behbehani, 1995; Krout and Loewy, 2000b). These inputs suggest that many different types of information affect motor control at this level of the neuraxis. The CL has also been associated with motor control. For example, bilateral lesions of CL result in decits in a specialized motor task called the rotorod test but not in the other motor tasks (Jeljeli et al., 2000). This decit was interpreted to be related to disruption of a cerebellar3 CL pathway and not to a generalized decrease in attention or arousal. However, this interpretation is complicated by the fact that CL also receives dense projections from brainstem structures such as the superior colliculus, periaqueductal gray matter, cuneiform nucleus, nucleus Darkschewitsch, and several other nuclei (see Table 1) (Yamasaki

98

K.E. KROUT ET AL.

Fig. 21. Summary gure illustrating a subset of arousal-related brainstem nuclei that innervate almost every midline and intralaminar thalamic nucleus. Thalamic nuclei situated on the midline and

the caudal thalamic nuclei also receive visceral-related inputs. These data suggest that convergent arousal/visceral information may affect large regions of cerebral cortex (top panel). For abbreviations, see list.

BRAINSTEM-THALAMIC CONNECTIONS and Krauthamer, 1990; Grunwerg and Krauthamer, 1992; Krout and Loewy, 2000b; Krout et al., 2001). It is unclear how these multiple sites may contribute to altering motor command signals at both the cortical and striatal levels. Somatosensation and nociception. Midline and intralaminar thalamic nuclei neurons respond to innocuous somatic stimuli, noxious stimuli, or, in some cases, both (e.g., Reyes-Vazquez et al., 1989; Dostrovsky and Guilbaud, 1990). These medial thalamic neurons have receptive elds that encompass large areas of the body, whereas principal thalamic relay neurons respond to stimuli applied to small regions of the contralateral body (e.g., Willis, 1985; Monconduit et al., 1999). This nding suggests a fundamental difference in the functions of these two pathways. Somatic information reaches the thalamus by means of several neural circuits. Anatomic studies have shown a direct projection from the dorsal horn of the spinal cord to all intralaminar and most midline thalamic sites and this ascending system likely transmits somatosensory and visceral information (Cliffer et al., 1991). Here, we have demonstrated that only a few thalamic regions may receive somatic inputs from the spinal trigeminal nucleus; these include the VPpc, VMc, lPF, mPF, and OPC. Other studies have suggested that somatosensory/nociceptive information may also reach the thalamus by indirect means, for example, by means of relays in the parabrachial nucleus (Bester et al., 1999; Krout and Loewy, 2000a) or brainstem reticular nuclei (Peschanski and Besson, 1984c; Bernard et al., 1990; Roy et al., 1992; Villanueva et al., 1998). Whatever the circuits involved, the large receptive eld sizes found in midline/intralaminar thalamic neurons suggest that this system is not required for localizing stimuli but may be responsible for modulating orienting or arousal responses (Orem et al., 1973; Maldonado and Schlag, 1984; Schlag-Rey and Schlag, 1984; Merker and Schlag, 1985; Grunwerg and Krauthamer, 1992). Visceral systems. The present study has demonstrated that the NTS is linked to the CM, lPF, mPF, OPC, PVT system, Rh, Re, VPpc, and VMc. In addition, visceralrelated parabrachial subnuclei also project to these same sites (see Krout and Loewy, 2000a). Based on the efferent terminations of these thalamic nuclei, these data suggest that visceral information could reach medial prefrontal, anterior cingulate, motor, perirhinal, and insular cortices (see Fig. 21), as well as other forebrain structures such as the amygdala, ventral subiculum, and striatum (Herkenham, 1979; Arbuthnott et al., 1990; Berendse and Groenewegen, 1990, 1991; Groenewegen et al., 1990; Turner and Herkenham, 1991; Moga et al., 1995). These ndings suggest that the hypothesis proposed by Neafsey (1990) that the primary viscerosensory area is only the insular cortex (see also Cechetto and Saper, 1987; Allen et al., 1991) should be broadened, because visceral sensory information may be distributed to many more cortical sites than had been previously postulated.

99 impinge on a single thalamic nucleus and, perhaps, on single neurons. This concept is supported by anatomic and physiological data that show that the thalamic nuclei receive convergent inputs (e.g., Reyes-Vazquez et al., 1989; Grunwerg and Krauthamer, 1990, 1992; Groenewegen et al., 1999). In addition, many of the raphe system or reticular formation neurons that project to the thalamus are likely to be polysensory (see Mason, 2001). Thus the function of the midline and intralaminar thalamic nuclei is likely to be best understood holistically, as part of an integrative system modulating subtle changes in behavior or attention in response to a wide range of somatosensory and visceral signals.

ACKNOWLEDGMENTS
We thank Alex Field and Xay Van Nguyen for their excellent technical assistance.

LITERATURE CITED
Alexander GE, Crutcher MD, DeLong MR. 1990. Basal gangliathalamocortical circuits: parallel substrates for motor, oculomotor, prefrontal and limbic functions. Prog Brain Res 85:119 146. Allen GV, Saper CB, Hurley KM, Cechetto DF. 1991. Organization of visceral and limbic connections in the insular cortex of the rat. J Comp Neurol 311:116. Arbuthnott GW, MacLeod NK, Maxwell DJ, Wright AK. 1990. Distribution and synaptic contacts of the cortical terminals arising from neurons in the rat ventromedial thalamic nucleus. Neuroscience 38:47 60. Aston-Jones G, Bloom FE. 1981. Norepinephrine-containing locus coeruleus neurons in behaving rats exhibit pronounced responses to nonnoxious environmental stimuli. J Neurosci 1:887900. Behbehani MM. 1995. Functional characteristics of the midbrain periaqueductal gray. Prog Neurobiol 46:575 605. Bentivoglio M, Balercia G, Kruger L. 1991. The specicity of the nonspecic thalamus: the midline nuclei. Prog Brain Res 87:53 80. Berendse HW, Groenewegen HJ. 1990. Organization of the thalamostriatal projections in the rat, with special emphasis on the ventral striatum. J Comp Neurol 299:187228. Berendse HW, Groenewegen HJ. 1991. Restricted cortical termination elds of the midline and intralaminar thalamic nuclei in the rat. Neuroscience 42:73102. Bernard JF, Villanueva L, Carroue J, Le Bars D. 1990. Efferent projections from the subnucleus reticularis dorsalis (SRD): a Phaseolus vulgaris leucoagglutinin study in the rat. Neurosci Lett 116:257262. Bester H, Bourgeais L, Villanueva L, Besson JM, Bernard JF. 1999. Differential projections to the intralaminar and gustatory thalamus from the parabrachial area: a PHA-L study in the rat. J Comp Neurol 405:421 429. Bolton RF, Cornwall J, Phillipson OT. 1993. Collateral axons of cholinergic pontine neurones projecting to midline, mediodorsal and parafascicular thalamic nuclei in the rat. J Chem Neuroanat 6:101114. Bowsher D. 1975. Diencephalic projections from the midbrain reticular formation. Brain Res 95:211220. Byrum CE, Guyenet PG. 1987. Afferent and efferent connections of the A5 noradrenergic cell group in the rat. J Comp Neurol 261:529 542. Carstens E, Leah J, Lechner J, Zimmermann M. 1990. Demonstration of extensive brainstem projections to medial and lateral thalamus and hypothalamus in the rat. Neuroscience 35:609 626. Cechetto DF, Saper CB. 1987. Evidence for a viscerotopic sensory representation in the cortex and thalamus in the rat. J Comp Neurol 262: 27 45. Chen S, Aston-Jones G. 1995. Evidence that cholera toxin B subunit (CTb) can be avidly taken up and transported by bers of passage. Brain Res 674:107111. Cliffer KD, Burstein R, Giesler GJ Jr. 1991. Distributions of spinothalamic, spinohypothalamic, and spinotelencephalic bers revealed by anterograde transport of PHA-L in rats. J Neurosci 11:852 868. Cornwall J, Phillipson OT. 1988. Afferent projections to the parafascicular

CONCLUSION
Many of the studies on the physiological properties of the midline and intralaminar properties have concentrated on only one or a few modalities, such as nociception. However, the large number of brainstem nuclei labeled after CTb injections into each thalamic site suggest that many different types of sensory or feedback information

100
thalamic nucleus of the rat, as shown by the retrograde transport of wheat germ agglutinin. Brain Res Bull 20:139 150. Deniau JM, Chevalier G. 1992. The lamellar organization of the rat substantia nigra pars reticulata: distribution of projection neurons. Neuroscience 46:361377. Dostrovsky JO, Guilbaud G. 1990. Nociceptive responses in medial thalamus of the normal and arthritic rat. Pain 40:93104. Edley SM, Graybiel AM. 1983. The afferent and efferent connections of the feline nucleus tegmenti pedunculopontinus, pars compacta. J Comp Neurol 217:187215. Edwards SB, de Olmos JS. 1976. Autoradiographic studies of the projections of the midbrain reticular formation: ascending projections of nucleus cuneiformis. J Comp Neurol 165:417 431. Erro E, Lanciego JL, Gimenez-Amaya JM. 1999. Relationships between thalamostriatal neurons and pedunculopontine projections to the thalamus: a neuroanatomical tract-tracing study in the rat. Exp Brain Res 127:162170. Foote SL, Bloom FE, Aston-Jones G. 1983. Nucleus locus ceruleus: new evidence of anatomical and physiological specicity. Physiol Rev 63: 844 914. Glenn LL, Steriade M. 1982. Discharge rate and excitability of cortically projecting intralaminar thalamic neurons during waking and sleep states. J Neurosci 2:13871404. Gottesmann C. 1999. The neurophysiology of sleep and waking: intracerebral connections, functioning and ascending inuences of the medulla oblongata. Prog Neurobiol 59:154. Groenewegen HJ. 1988. Organization of the afferent connections of the mediodorsal thalamic nucleus in the rat, related to the mediodorsalprefrontal topography. Neuroscience 24:379 431. Groenewegen HJ, Berendse HW. 1994. The specicity of the nonspecic midline and intralaminar thalamic nuclei. Trends Neurosci 17:5257. Groenewegen HJ, Berendse HW, Wolters JG, Lohman AH. 1990. The anatomical relationship of the prefrontal cortex with the striatopallidal system, the thalamus and the amygdala: evidence for a parallel organization. Prog Brain Res 85:95116. Groenewegen HJ, Galis-de Graaf Y, Smeets WJ. 1999. Integration and segregation of limbic cortico-striatal loops at the thalamic level: an experimental tracing study in rats. J Chem Neuroanat 16:167185. Grunwerg BS, Krauthamer GM. 1990. Vibrissa-responsive neurons of the superior colliculus that project to the intralaminar thalamus of the rat. Neurosci Lett 111:2327. Grunwerg BS, Krauthamer GM. 1992. Sensory responses of intralaminar thalamic neurons activated by the superior colliculus. Exp Brain Res 88:541550. Hallanger AE, Wainer BH. 1988. Ascending projections from the pedunculopontine tegmental nucleus and the adjacent mesopontine tegmentum in the rat. J Comp Neurol 274:483515. Hallanger AE, Levey AI, Lee HJ, Rye DB, Wainer BH. 1987. The origins of cholinergic and other subcortical afferents to the thalamus in the rat. J Comp Neurol 262:105124. Herkenham M. 1979. The afferent and efferent connections of the ventromedial thalamic nucleus in the rat. J Comp Neurol 183:487517. Hermann DM, Luppi PH, Peyron C, Hinckel P, Jouvet M. 1996. Forebrain projections of the rostral nucleus raphe magnus shown by iontophoretic application of cholera toxin b in rats. Neurosci Lett 216:151154. Hunter J, Jasper HH. 1949. Effects of thalamic stimulation in unanaesthetised animals EEG. Clin Neuorphysiol 1:305324. Jeljeli M, Strazielle C, Caston J, Lalonde R. 2000. Effects of centrolateral or medial thalamic lesions on motor coordination and spatial orientation in rats. Neurosci Res 38:155164. Jones EG, Leavitt RY. 1974. Retrograde axonal transport and the demonstration of non-specic projections to the cerebral cortex and striatum from thalamic intralaminar nuclei in the rat, cat and monkey. J Comp Neurol 154:349 377. Jones BE, Moore RY. 1977. Ascending projections of the locus coeruleus in the rat. II. Autoradiographic study. Brain Res 127:2553. Jones BE, Yang TZ. 1985. The efferent projections from the reticular formation and the locus coeruleus studied by anterograde and retrograde axonal transport in the rat. J Comp Neurol 242:56 92. Kinomura S, Larsson J, Gulyas B, Roland PE. 1996. Activation by attention of the human reticular formation and thalamic intralaminar nuclei. Science 271:512515. Kitt CA, Hohmann C, Coyle JT, Price DL. 1994. Cholinergic innervation of mouse forebrain structures. J Comp Neurol 341:117129.

K.E. KROUT ET AL.

Krout KE, Loewy AD. 2000a. Parabrachial nucleus projections to midline and intralaminar thalamic nuclei of the rat. J Comp Neurol 428:475 494. Krout KE, Loewy AD. 2000b. Periaqueductal gray matter projections to midline and intralaminar thalamic nuclei of the rat. J Comp Neurol 424:111141. Krout KE, Jansen AS, Loewy AD. 1998. Periaqueductal gray matter projection to the parabrachial nucleus in rat. J Comp Neurol 401:437 454. Krout KE, Loewy AD, Westby GW, Redgrave P. 2001. Superior colliculus projections to midline and intralaminar thalamic nuclei of the rat. J Comp Neurol 431:198 216. Lai H, Tsumori T, Shiroyama T, Yokota S, Nakano K, Yasui Y. 2000. Morphological evidence for a vestibulo-thalamo-striatal pathway via the parafascicular nucleus in the rat. Brain Res 872:208 214. Lorente de No R. 1938. Cerebral cortex: architecture, intracortical connec tions, motor projections. In: Fulton J, editor. Physiology of the nervous system. London: Oxford University Press. p 291340. Loughlin SE, Foote SL, Fallon JH. 1982. Locus coeruleus projections to cortex: topography, morphology and collateralization. Brain Res Bull 9:287294. Luppi PH, Fort P, Jouvet M. 1990. Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identication of retrogradely labeled neurons. Brain Res 534:209 224. Lydic R, McCarley RW, Hobson JA. 1987. Serotonin neurons and sleep. I. Long term recordings of dorsal raphe discharge frequency and PGO waves. Arch Ital Biol 125:317343. Mair RG. 1994. On the role of thalamic pathology in diencephalic amnesia. Rev Neurosci 5:105140. Maldonado HM, Schlag J. 1984. Unit activity related to head and eye movements in central thalamus of cats. Exp Neurol 86:359 378. Mason P. 2001. Contributions of the medullary raphe and ventromedial reticular region to pain modulation and other homeostatic functions. Annu Rev Neurosci 24:737777. Merker B, Schlag J. 1985. Role of intralaminar thalamus in gaze mechanisms: evidence from electrical stimulation and ber-sparing lesions in cat. Exp Brain Res 59:388 394. Mileikovsky B, Verevkina SV, Nozdrachev AD. 1994. Effects of stimulation of the frontoparietal cortex and parafascicular nucleus on locomotion in rats. Physiol Behav 55:267271. Moga MM, Weis RP, Moore RY. 1995. Efferent projections of the paraventricular thalamic nucleus in the rat. J Comp Neurol 359:221238. Monconduit L, Bourgeais L, Bernard JF, Le Bars D, Villanueva L. 1999. Ventromedial thalamic neurons convey nociceptive signals from the whole body surface to the dorsolateral neocortex. J Neurosci 19:9063 9072. Morison RS, Dempsey EW. 1942. A study of thalamocortical relations. Am J Physiol 139:410 416. Moruzzi G, Magoun HW. 1949. Brain stem reticular formation and activation of the EEG. Electroencephalogr Clin Neurophysiol 1:445 473. Mouroux M, Feger J. 1993. Evidence that the parafascicular projection to the subthalamic nucleus is glutamatergic. Neuroreport 4:613 615. Munk MH, Roelfsema PR, Konig P, Engel AK, Singer W. 1996. Role of reticular activation in the modulation of intracortical synchronization. Science 272:271274. Nauta WJH, Kuypers HGJ. 1958. Some ascending pathways in the brainstem reticular formation. In: Jasper HH et al, editors. Reticular formation of the brain. Henry Ford Hospital Symposium. Boston: Little Brown. p 330. Neafsey EJ. 1990. Prefrontal cortical control of the autonomic nervous system: anatomical and physiological observations. Prog Brain Res 85:147165. Newman DB, Ginsberg CY. 1994. Brainstem reticular nuclei that project to the thalamus in rats: a retrograde tracer study. Brain Behav Evol 44:139. Niimi K, Kusunose M, Ono K, Yanagihara M. 1990. Brainstem afferents to the intralaminar nuclei of the cat thalamus studied by the horseradish peroxidase method. J Hirnforsch 31:107122. Nishimura Y, Takada M, Mizuno N. 1997. Topographic distribution and collateral projections of the two major populations of nigrothalamic neurons. A retrograde labeling study in the rat. Neurosci Res 28:19. Orem J, Schlag-Rey M, Schlag J. 1973. Unilateral visual neglect and thalamic intralaminar lesions in the cat. Exp Neurol 40:784 797.

BRAINSTEM-THALAMIC CONNECTIONS
Pare D, Steriade M. 1990. Brain cholinergic systems. Steriade M, Biesold D, editors. Oxford: Oxford University Press. p 337354. Paxinos G, Watson C. 1997. The rat brain in stereotaxic coordinates. 3rd ed. San Diego: Academic Press. Paxinos G, Carrive P, Wang H, Wang P-Y. 1999. Chemoarchitechtonic atlas of the rat brainstem. San Diego: Academic Press. Peschanski M, Besson JM. 1984a. Coerulear projections to the lateral diencephalon in the rat. An anatomical study using wheat-germ agglutinin conjugated to HRP. Neurosci Lett 46:329 334. Peschanski M, Besson JM. 1984b. Diencephalic connections of the raphe nuclei of the rat brainstem: an anatomical study with reference to the somatosensory system. J Comp Neurol 224:509 534. Peschanski M, Besson JM. 1984c. A spino-reticulo-thalamic pathway in the rat: an anatomical study with reference to pain transmission. Neuroscience 12:165178. Phillipson OT, Bohn MC. 1994. C1-3 adrenergic medullary neurones project to the paraventricular thalamic nucleus in the rat. Neurosci Lett 176:6770. Powell DA, Watson KL, Buchanan SL. 1990. Neuronal activity in the mediodorsal and intralaminar nuclei of the dorsal thalamus during classical heart rate conditioning. Brain Res 532:211221. Reyes-Vazquez C, Prieto-Gomez B, Dafny N. 1989. Noxious and nonnoxious responses in the medial thalamus of the rat. Neurol Res 11: 177180. Ricardo JA, Koh ET. 1978. Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat. Brain Res 153:126. Robbins TW, Granon S, Muir JL, Durantou F, Harrison A, Everitt BJ. 1998. Neural systems underlying arousal and attention Implications for drug abuse. Ann N Y Acad Sci 846:222237. Roy JC, Bing Z, Villanueva L, Le Bars D. 1992. Convergence of visceral and somatic inputs onto subnucleus reticularis dorsalis neurones in the rat medulla. J Physiol Lond 458:235246. Ruggiero DA, Anwar S, Kim J, Glickstein SB. 1998. Visceral afferent pathways to the thalamus and olfactory tubercle: behavioral implications. Brain Res 799:159 171. Ruigrok TJ, TeuneTM, van der Burg J, Sabel-Goedknegt H. 1995. A retrograde double-labeling technique for light microscopy. A combination of axonal transport of cholera toxin B-subunit and a gold-lectin conjugate. J Neurosci Methods 61:127138. Rye DB, Saper CB, Lee HJ, Wainer B H. 1987. Pedunculopontine tegmental nucleus of the rat: cytoarchitecture, cytochemistry, and some extrapyramidal connections of the mesopontine tegmentum. J Comp Neurol 259:483528. Rye DB, Lee HJ, Saper CB, Wainer BH. 1988. Medullary and spinal efferents of the pedunculopontine tegmental nucleus and adjacent mesopontine tegmentum in the rat. J Comp Neurol 269:315341. Sakai ST, Grofova I, Bruce K. 1998. Nigrothalamic projections and nigrothalamocortical pathway to the medial agranular cortex in the rat: single- and double-labeling light and electron microscopic studies. J Comp Neurol 391:506 525. Satoh K, Fibiger HC. 1986. Cholinergic neurons of the laterodorsal tegmental nucleus: efferent and afferent connections. J Comp Neurol 253:277302. Scheibel ME, Scheibel AB. 1958. Structural substrates of integrative patterns in the brainstem reticular core. In: Jasper HH et al, editors. Reticular formation of the brain. Henry Ford Hospital Symposium. Boston: Little Brown. p 3155. Scheibel ME, Scheibel AB. 1972. Input-output relations of the thalamic nonspecic system. Brain Behav Evol 6:332358. Schlag-Rey M, Schlag J. 1984. Visuomotor functions of central thalamus in monkey. I. Unit activity related to spontaneous eye movements. J Neurophysiol 51:1149 1174.

101
Shiroyama T, Kayahara T, Yasui Y, Nomura J, Nakano K. 1995. The vestibular nuclei of the rat project to the lateral part of the thalamic parafascicular nucleus. centromedian nucleus in primates. Brain Res 704:130 134. Shiroyama T, Kayahara T, Yasui Y, Nomura J, Nakano K. 1999. Projections of the vestibular nuclei to the thalamus in the rat: a Phaseolus vulgaris leucoagglutinin study. J Comp Neurol 407:318 332. Steriade M. 1996. Arousal: revisiting the reticular activating system. Science 272:225226. Steriade M. 1999. Brainstem activation of thalamocortical systems. Brain Res Bull 50:391392. Steriade M, Glenn LL. 1982. Neocortical and caudate projections of intralaminar thalamic neurons and their synaptic excitation from midbrain reticular core. J Neurophysiol 48:352371. Steriade M, Datta S, Pare D, Oakson G, Curro Dossi RC. 1990. Neuronal activities in brain-stem cholinergic nuclei related to tonic activation processes in thalamocortical systems. J Neurosci 10:25412559. Ter Horst GJ, Streeand C. 1994. Ascending projections of the solitary tract nucleus. In: Barraco IR, editor. Nucleus of the solitary tract. Boca Raton: CRC Press. p 93103. Tsumori T, Yokota S, Lai H, Yasui Y. 2000. Monosynaptic and disynaptic projections from the substantia nigra pars reticulata to the parafascicular thalamic nucleus in the rat. Brain Res 858:429 435. Turner BH, Herkenham M. 1991. Thalamoamygdaloid projections in the rat: a test of the amygdalas role in sensory processing. J Comp Neurol 313:295325. Van Der Werf YD, Weerts JG, Jolles J, Witter MP, Lindeboom J, Scheltens P. 1999. Neuropsychological correlates of a right unilateral lacunar thalamic infarction. J Neurol Neurosurg Psychiatry 66:36 42. Van der Werf YD, Witter MP, Uylings HB, Jolles J. 2000. Neuropsychology of infarctions in the thalamus: a review. Neuropsychologia 38:613 627. Vertes RP. 1991. A PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat. J Comp Neurol 313:643 668. Vertes RP, Martin GF. 1988. Autoradiographic analysis of ascending projections from the pontine and mesencephalic reticular formation and the median raphe nucleus in the rat. J Comp Neurol 275:511541. Vertes RP, Fortin WJ, Crane AM. 1999. Projections of the median raphe nucleus in the rat. J Comp Neurol 407:555582. Vertes RP, Martin GF, Waltzer R. 1986. An autoradiographic analysis of ascending projections from the medullary reticular formation in the rat. Neuroscience 19:873 898. Villanueva L, Desbois C, Le Bars D, Bernard JF. 1998. Organization of diencephalic projections from the medullary subnucleus reticularis dorsalis and the adjacent cuneate nucleus: a retrograde and anterograde tracer study in the rat. J Comp Neurol 390:133160. Vincent SR. 2000. The ascending reticular activating system: from aminergic neurons to nitric oxide. J Chem Neuroanat 18:2330. Willis WD. 1985. Nociceptive pathways: anatomy and physiology of nociceptive ascending pathways. Philos Trans R Soc Lond B Biol Sci 308: 253270. Yamasaki DS, Krauthamer GM. 1990. Somatosensory neurons projecting from the superior colliculus to the intralaminar thalamus in the rat. Brain Res 523:188 194. Yasui Y, Saper CB, Cechetto DF. 1991. Calcitonin gene-related peptide (CGRP) immunoreactive projections from the thalamus to the striatum and amygdala in the rat. J Comp Neurol 308:293310. Yoshida A, Dostrovsky JO, Chiang CY. 1992. The afferent and efferent connections of the nucleus submedius in the rat. J Comp Neurol 324: 115133. Zemlan FP, Behbehani MM, Beckstead RM. 1984. Ascending and descending projections from nucleus reticularis magnocellularis and nucleus reticularis gigantocellularis: an autoradiographic and horseradish peroxidase study in the rat. Brain Res 292:207220.