THE JOURNAL OF COMPARATIVE NEUROLOGY 293499-523 (1990)

Projections of the Parabrachial Nucleus in the Pigeon (CoZumba Ziuia) J. MARTIN WILD, J.J.A. ARENDS, AND H. PHILIP ZEIGLER Department of Anatomy, Iiniversity of Auckland School of Medicine, Auckland, New Zealand (J.M.W.); Biopsychology Program, Hunter College (CUNY), New York, New York 10021 (J.J.A.A.,H.P.Z.)

ABSTRACT The ascending and descending projections of the parabrachial nuclear complex in the pigeon have been charted with autoradiographic and histochemical (WGA-HRP) techniques. The ascending projections originate from a group of subnuclei surrounding various components of the brachium conjunctivum, namely, the superficial lateral, dorsolateral, dorsomedial, and ventromedial subnuclei. The projections are predominantly ipsilateral and travel in the quintofrontal tract. They are primarily to the medial and lateral hypothalamus (including the periventricular nucleus and the strata cellulare internum and externum), certain dorsal thalamic nuclei, the nucleus of t,he pallial commissure, the bed nucleus of the stria terminalis, the ventral paleostriatum, the olfactory tubercle, the nucleus accumbens, and a dorsolateral nucleus of the posterior archistriatum. There are weaker or more diffuse projections to t,he rostra1 locus coeruleus (cell group A8), the compact portion of the pedunciilopontine tegmental nucleus, the central grey and intercollicular region, t,he ventral area of Tsai, the medial spiriform nucleus, the nucleus subrotundus, the anterior preoptic area, and the diagonal band of Broca. The parabrachial subnuclei have partially differential projections tn these targets, some of which also receive projections from the nucleus of the solitary tract (Arends, Wild? and Zeigler: J . Cornp. Neurol. 278:405-429, ’88).Most, of t,he targets, part,icularly those in the basal forebrain (viz., the periventricular nucleus and the strata celliilare internum and externum of the hypothalamus, the bed nucleus of the stria terminalis, and its lateral extension into the ventral paleostriatum, which may be comparable with the substantia innominata). have reciprocal connections with the parabrachial and solitary tract subnuclei and therefore may be said to compose parts of a “visceral forebrain system” analogous to that described in the rat (Van der Kooy et al: J . Comp. Neurol. 224:1-24, ’84). The descending projections to t,he lower brainstem arise in large part from a ventrolateral subnucleus that may be comparable with the KiillikerFuse nucleus of mammals. They are mainly to the ventrolateral medulla, nucleus ambiguus, and massively to the hypoglossal nucleus, particularly its tracheosyringeal portion. These projections are therefore likely to be importantly involved in the control of vocalization and respiration (Wild and Arends: Brain Kes. 407:191-794, ’87). Some of these results have been presented in abstract form (Wild, Arends, and Zeigler: SOC.NPurosci. Abst.

13308,’87). Key words: hirds, visceral system, quintofrontal, forebrain, hypothalamus, stria terminalis, Kolliker-Fuse, nucleus ambiguus, nucleus t,rartus solitarii

Accepted October 30, 1989. Address reprint requests to Dr. H.P. Zeigler, Biopsychology Program, Hunter College, 695 Park Ave., New York City, NY 10021.

0 1990 WILEY-LISS, INC.

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In mammals, the parabrachial nuclear complex (PB) is a major target of ascending projections from the nucleus of the solitary tract (nTS) (Ricardo and Koh, '78; Norgren, '78; Beckstead et al., '80; Mizuno e t al., '80). As such it represents a key relay for the transmission of general and (in nonprimates) special (gustatory) visceral afferent information to higher levels of the neuraxis as well as being an important source of input to autonomic regions of the lower brainstem and spinal cord (reviews: Mizuno et al., '80; Saper and Loewy, '80; Fulwiler and Saper, '84). In the pigeon, the parabrachial nuclear complex is also a major recipient of projections from nTS, and these projecttions are topographically organised both with respect to their origins within nTS subnuclei and their terminal fields within PB (Arends et al., '88). Thus, medial tier nTS subnuclei (Katz and Karten, '83) that receive gustatory and gastrointestinal inputs project upon dorsal and medial portions of PB, while lateral tier nTS subnuclei that receive cardiopulmonary input project upon lateral and ventrolateral PB. The organization of nTS projections to P B is therefore such as to produce a partial functional segregation within P B that can be correlated, to some extent, with the pattern of visceral inputs to nTS subnuclei. In the present study, we have delineated the projections of PB in the pigeon in order to determine the extent to which the partial functional segregation of visceral projec-

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archistriatum nucleus accumbens nucleus ambiguus ansa lenticularis dorsolatcral nuclcus of posterior archistriaturn area ventralis Tsai brachirim conjunctivum brachium conjunctivum afferentes bed nucleus of the stria terminalis cominissura anterior nuclcus centralis superior nucleus cuneatus ext.ernus nucleus dorsointermedius posterior thalami nucleus moturius dorsalis nervi vagi ectostriatum fasciculus diagonalis Brocae fasciculus longitudinalis medialis fasciculns prosencephali lateralis fasciculris uncinatus substantia grisea centralis hypothalamus nucleus intercollicularis nucleus int.rapeduncularis nucleus isthmo-opticus nucleus lateralis hypothalami nucleus lemnisci lateralis intermedius. pars caudalis nucleus lemnisci lateralis intermedius. pars rostralis nucleus lemnisci lateralis ventralis nucleus coeruleus lobus parolfactorius nucleus parasolitarius lateralis nucleus magnocellularis nucleus mesencephalicus lateralis, pars dorsalis nucleus motorius nervi trigemini neostriatum nucleus commissuralis pallii nucleus nervi oculomotorii nervus oculomotorius nervus Lrochlearis nervus trigeminus

tions to PR is maintained throughout its subsequent projections to higher and lower levels of the neuraxis. As in the previous study, injections of tritiated amino acids or wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP)were first made into P R to define a set of putative terminal fields within the brain (the spinal cord is excluded from this study). Injections of WGA-HRP were then made into these fields to verify and determine the origins of their afferent projections within various regions of PB. The results show that P B has both ascending and descending projections to a wide variety of structures which receive their afferents from partly separate PR subnuclei.

MATERIALS AND METHODS Subjects were male and female homing pigeons or white Carneaux. They were anesthetised with an intramuscular injection of either a mixture of ketamine (Ketalar, Parke Davis, 50 rng/kg) and xylazine (Rompun, Bayer, 5 mg/kg) or loco-Eyuithesin (2 ml/kg) and placed in a stereotaxic instrument with a pigeon head holder. Coordinates were obtained from the atlas of Karten and Hodos ('67). Pressure injections (20-40 nl) of either tritiated amino acids (an equalparts mixture of L-[2,3,4+?G3H]proline, spec. act. 130 mCi/ mmol and L-[3,4-3H] leucine, spec. act. 120 mCi/mmol each

nXlI nucleus nervi hypoglossi OM(T),OMtractus occipitomesencephalicus nucleus ovoidalis ov paleostriatum augmentatum PA nucleus parabrachialis PR sl pars superficialis lateralis d m pars dorsomedialis w m pars ventromedialis dl pars dorsolateralis vl pars ventrolateralis nucleus preopticus anterior POA paleostriatum primitivum PP nuclcus sensorius principalis nervi trigcmini PrV nucleus periventricularis magnocellularis I'VM tractus quintofrontalis QFT nucleus raphe K. nucleus dorsolateralis posterior thalami, pars rostralis rULI' nucleus reticularis parvoccllularis Kpc nucleus rotundus Kt nucleus tractus solitarii (nT)S nncleus subcoeruleus dorsalis SCd stratum cellulare cxternum SCE stratum cellulare internum SCI substantia gelatinosa Rolandi SG stratum griseum profundum SGP nucleus semilunaris SLu nucleus spiriformis lateralis SpL nucleus spiriformis medialis SpM nucleus subrotundus SRt thalamus dorsalis THAL lractus isthmo-opticus TI0 nucleus taeniae Tn tuberculum olfactonum '1'0 nucleus tegmenti pedunculopontinlls, pars compacta TPc tractus septomesencephalicus TSM nucleus e t tractus descendentis nervi trigemini TTD ventriculus v nucleus vestibularis descendens VeD paleostriatum ventrale vr nucleus vestibularis superior vs

PARABRACHIAL NUCLEUS PROJECTIONS IN PIGEONS

Fig. 1. Photomicrographs of transverse sections through t h e dorsolateral pons showing the location of the parabrachial nuclei a t caudal (A), middle (B), and rostra1 (C) levels (cresyl violet stain). sl, superficial

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lateral; dl, dorsolateral; vl, ventrolateral; dm, dorsomedial; vm, ventromedial parahrarhial nuclei. Calibration bars = 0.5 mm. See list of abbreviations.

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Fig. 2. A-N: Schematic transverse hemisections showing fiber (lines) and terminal labeling (dots) following an injection of tritiated amino acids into the dorsolateral pons centered on the dorsal and medial

parabrachial nuclei a t the level of the trochlear nerve (A). T h e weaker contralateral projections to the same regions as labeled ipsilaterally are not shown (see text, and the list of abbreviations).

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at. a final concentration of 40-50 pCi/pl) or WGA-HRP (Sigma) in Tris HC1 buffer, pH 8.6, were made via glass micropipett,es with tip diameters of 20-40 pm. In certain cases (e.g., olfactory tubercle) an injection was placed with the aid of prior electrophysiological recording of the depth or laterality of a structure, (e.g., base of the hemisphere). Following survival times of 24-72 hours, birds were deeply anesthetised and perfused through the carotids, those for autoradiography with saline followed by 10:; formalin, those for histochemistry with saline followed by 36' glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, plus 5 R sucrose. Following additional sucrose penetration, serial 50 fim frozen brain sections were collected in two series. For autoradiography, one in two sections was treated according to the method of Cowan et al. ('72), stored a t 4'C in the dark for 3-4 weeks, and then developed with Kodak D19 for 2 minutes at 18°C prior to being counterstained through the emulsion with cresyl violet. For histochemistry, both series of sections were treated with TMB according to the method of Mesulam ('78), and one was subsequently counterstained with either neutral red or thionin (Adams, '80). All material was examined with both light- and darkfield optics, and the projections and labeled cells were drawn with the aid of a camera lucida.

RESULTS Normal anatomy A comparison of the subnuclear organization of the parabrachial complex in the pigeon with that of mammals is complicated by certain differences in the distribution of fibers which constitute the superior cerebellar peduncle or brachium conjunctivum (BC). At isthmic levels RC is not a single, compact fiber bundle, but is split up into many efferent fascicles and a major cerebellar afferent tract (BCA) which has its origin in caudal thalamic and pretectal nuclei (Karten and Finger, '76; Clarke, '77). Thus the mammalian convention of subdividing the parabrachial complex into lateral or dorsal, and medial or ventral subnuclei with reference to a single, largely efferent RC (Meessen and Olszewski, '49; 'raber, '61; Fulwiler and Saper, '84; Kolesarova and Petrovicky, '87) is not appropriate for the pigeon. Instead we have chosen to define the subnuclei topographically in terms of the general location within the dorsolateral and lateral pons of cytoarchitecturally different cellular groupings. in combination with a consideration of the projections of the nucleus of the solitary tract to the region (Petrovicky, '66; Arends et al., '88). The topography of the subnuclei can best be appreciated following a description of the distribution of BC fibers at isthmic levels. Most caudally within the dorsolateral pons, RC is located between the dorsomedial aspect of the principal sensory trigeminal nucleus (PrV) and the fourth ventricle (Figs. lA, 6). Proceeding rostrally, as the pons separates from the cerebellum and is joined by the tecta, BC breaks up into a host of more medially situated, mostly contralaterally directed, ascending efferent fascicles and a more laterally situated afferent component (BCA) (Figs. lB, 6). At the level of the trochlear nerve most of the efferent fascicles of BC lie ventral to the occipitomesencephalic tract (OMT) and the locus coeruleus (LoC) (see Karten and Hodos, '67, A1.,50), whereas BCA forms a separate, relatively compact crescent of fibers located dorsolaterally. Further rostrally, at the

level of t.he isthmo-optic nucleus, BCA lies medially adjacent to t,he distinctive nucleus semilunaris (SLu) (Figs. 1 B,C, 6). On the basis of this description it is possible to define five relatively distinct parabrachial nuclear subdivisions. Medial PB subnuclei. Medial and ventromedial to RCA from rostral levels of PrV through isthmic levels is an area of diffusely scattered and clumped cells of various sizes. The area is traversed extensively by efferent BC fascicles, and fibers belonging in part to the radix of the mesencephalic trigeminal nucleus (MesV), in part to the occipitomesencephalic tract, and in part to the quintofrontal tract as this leaves the main sensory trigeminal nucleus and adjacent nuclei. On its medial aspect this area adjoins the locus coeruleus, which, in contrast t o the parabrachial nucleus, has many characteristically larger, more darkly staining, and frequently more densely packed cells. A definite border between the two regions is, however, difficult to draw. At isthmic levels there appear to be a t least two medial suhnuclei: a dorsornedial one (PBdm; Figs. l B , 6) composed of closely packed and relatively hasophilic cells situated medial to the dorsomedial and medial aspect of BCA; and a larger: poorly delineated ventrornedial one (PBvm; Figs. lB, 6) lying lateral to dorsal parts of the locus coeruleus and composed of scattered cells interspersed among fascicles belonging to the occipitomesencephalic tract. PBvm has no definite ventral border, but it tends to he separated from the more ventromedially situated nucleus subcoeruleus dorsalis (SCd) by contralat,erallydirected efferent BC fascicles. SCd also extends fiirt,her rostrally than does PBvm (see Fig. 6 in Kit,t and Brauth; '86). Lateral PB subnuclei. The superficial lateral subnucleus (PBsl) comprises a thin band of small, lightly staining cells situated lateral to the uncinate fasiculus, primarily at rostral levels of the main sensory trigeminal nucleus (Figs. lA, 6). PBsl is also present on the dorsum of BCA at isthmic levels, where it lies immediately ventral to the nucleus designated "LLd" in the atlas of Karten and Hodos ('67). The dorsolateral subnucleus (PBdl) is present mainly a t the level of the fourth cranial nerve root (Figs. lB, 6), where it occupies the region between the ventral border of BCA and the dorsal border of the rostral division of the intermediate nucleus of the lateral lemniscus (LLIr of Arends and Zeigler, '86, = VLV of Karten and Hodos, '67). It is composed of larger cells, frequently arranged as vertical columns interspersed with fibrous layers, some of which are parts of the rubrospinal tract (RST; Wild et al., '79). Dorsomedially PBdl merges with the lateral aspect of PBvm. The Iuentrolateral subnucleus (PRvI) is the most rostral nucleus of the complex. Its multipolar or fusiform neurons, oriented with their longer axis vertically, form a narrow band extending ventrally from PBdl almost down to the level of the ventral division of the lateral lemniscal nucleus (LLV of Arends and Zeigler, '86, and Wild, '87a) and wedged between the rostral intermediate nucleus of the lateral lemniscus (LLIr) and the rubrospinal tract medially, and the spinal lemniscus and rostral part of the trigeminal root laterally (Figs. lC, 6). Some of its cells arc medially over the dorsum of LLIr. PBvl is partly traversed by fibers of the lateral lemniscus. In each of the five subnuclei the cross-sectional area of the Nissl stained somas of 50 randomly selected cells was measured by tracing t,heir outlines at x 312.5 total magnification onto a digitising pad linked to a computer. The mean areas (mm2) of somas in the subnuclei were as follows: PBdm,

PARABRACHIAL NUCLEUS PROJECTIONS IN PIGEONS 68.04 (SE 2.84); PBvm, 101.12 (SE 6.12); PBsl, 42.63 (SE 4.35); PBd1, 128.18 (SE 7.19); P B d , 89.27 (SE 4.43). An ANOVA revealed an overall significant difference between these means (F, 4/245 = 38.85, P < 0.01), each of which differed significant.1~ from all others by at least P 0.05 (Newman-Keuls), except vm from vl. :c

Anterograde experiments Some anterograde data were available from several cases receiving P R injections of WGA-HRP in our previous study of projections of the nucleus of the solitary tract (Arends e t al., '88). However, most of the data presented here are derived from five additional cases, each of which received a 30-40 nl unilateral injection of tritiated amino acids centered on some part of the n P B complex and cut either in the transverse or sagittal plane. Although these injections were centered on different parts of the nuclear complex, there was considerable overlap of spread of the tracer in four of these. The projections in these four cases in which the injection covered the greater part of the complex, i.e., PBsl, PBdm, PBvm, and PBdl, were not sufficiently different from each other to warrant separate presentation. Figure 2 charts, therefore, as a typical example of a "dorsal" injection, the projections in a case where the injection was centered on PBdm and PBvm a t isthmic levels but with spread of the tracer to the lateral part of the locus coeruleus and the dorsolateral tegmentum, and to PBdl and PBsl. Also covered were the isthmo-optic and semilunar nuclei and the rostral portion of the main sensory trigeminal nucleus. Figure 3 presents photomicrographs of some of the terminal fields depicted schematically in Figure 2. Most of the projections resulting from dorsal PI3 injections were directed rostrally; and, except where otherwise noted, these were bilateral but with a strong ipsilateral predominance. Labeled fibers leave the site of injection (Fig. 2A) and gather within the quintofrontal tract, which is the major route of ascending projections from P B to forebrain areas. The isthmo-optic tract is also labeled, but since the trajectory and retinal termination of this tract is known (Cowan and Powell, '63), it is not considered further. At rostral pontine and caudal midbrain levels a contingent of labeled fibers courses dorsally from the quintofrontal tract to terminate in a rostral part of what Karten and Hodos designate the locus coeruleus (LoC; Fig. 2B). This region is now considered comparable with the A8 dopaminergic cell group of mammals (see Discussion, and Kitt and Brauth, '86). Scattered labeling is also present within the central grey (GCt) overlying the locus coeruleus and within the intercollicular region (ICo) medial to nucleus mesencephalicus lateralis, pars dorsalis (MLdj. Another contingent of labeled fibers crosses the midline in the decussation of HC; some fibers then ascend into the contralateral locus coeruleus, others group to form the contralateral quintofrontal tract. A little further rostrally at caudal midbrain levels terminal labeling is present within the ventral area of Tsai (AVT), the compact portion of the pedunculopontine tegmental nucleus ('I'Pc), and within the periaqueductal central grey (GCt) (Figs. 2C,D, 3A,B). A t the mesencephalic-diencephalic border there are terminations lateral to the lateral spiriform nucleus (SpL) (which presumably originate in the semilunar nucleus-Reiner e t al., '82), and within the meWithin the caudial spiriform nucleus (SpM) (Figs. 2E, X). dal diencephalon labeled fibers leave the quintofrontal tract

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to head in various directions: dorsally, ventromedially to cross to the opposite side via the tuberal region, and medially and dorsomedially t o terminate extensively throughout the strata cellulare internum and externum (SCI and SCE) of the hypothalamus (Figs. 2F,G, 3D,E). In addition a characteristic horizontal band of terminations is present at these levels within the dorsal thalamus just ventral to the nucleus dorsointermedius posterior (DIP), with a lighter terminal field dorsal to DIP (Figs. 2F, 3F). At levels of the nucleus ovoidalis (Ov) there is extensive labeling medial to the nucleus rotundus (Rt) and within the dorsal thalamic nuclei dorsolateralis posterior, pars rostralis (rL)LP)(Gamlin and Cohen, '86; Wild, '87c), and DIP (Figs. 2G,H, 3G). Labeling of medial thalamic nuclei is restricted to periventricular regions, but the nucleus subrotundus (SRt) ventral to nucleus ovoidalis is specifically labeled (Figs. 2G, 3H). More rostrally, diffuse labeling continues to be distributed throughout medial and lateral hypothalamic regions, but a small, concentrated patch of label is present bilaterally in the deepest part of the lateral hypothalamus (Figs. 2H, 31). Caudal to the level of the anterior commissure (CA) there is labeling within and around the periventricular nucleus (PVM) (Figs. 2I,J, 3J),and there is a patch of terminations, equally dense on either side, within the nucleus of the pallial commissure (nCPa) at levels straddling the anterior commissure (Figs. 2.J-L, 3K). More 1at.erally there is heavy labeling within the bed nucleus of the stria terminalis (RNST; Figs. 2J-I,, 31,) which extends laterally to the ventral paleostriatal region (VP-see Kitt and Brauth, '81) (Figs. 2K,L, 3M), and also to the region ventral to the occipito-mesencephalic tract a t the base of the hemisphere. There is also very light labeling within the paleostriatum primitivum (PP) (Fig. 2M), clearly distinguishable from labeling in the underlying the intrapeduncular nucleus (INP). Labeled fibers continue laterally and caudally from the VP region across the dorsal part of the archistriatum finally to terminate specifically within a cytoarchitecturally distinct nucleus a t the dorsolateral edge of the caudal archistriatum (Karten and Hodos, '67; A4.5-6.0) (Figs. 2EH, 3N). We name this nucleus the dorsolateral nucleus of the posterior archistriatum (Apdl), but note that its dorsal border with the overlying neostriatum is not well defined. In addition to the labeling of this archistriatal nucleus there is lighter laheling of the nucleus taeniae (Tn). The labeling within the bed nucleus of the stria terminalis is continuous rostrally with labeling around the base of the ventricle throughout the entire rostral extent of the nucleus accrimbens (Ac) (Figs. 2M,N, 30,P). Additional, more diffuse labeling is found within the anterior preoptic nucleus, within the area of the diagonal band of Rroca (FDR) ventromedial to Ac, and within the olfactory tubercle as far rostral as the rostral extent of Ac (Fig. 2M,N). All these results were replicated to a variable extent in a series of cases receiving 20-40 nl injections of WGA-HRP into different regions of P B or covering large areas of the dorsolateral pons. These cases also provided data on the afferent projections t.o PB, some of which are detailed below (see Reciprocal Projections). Descending projections from four of the tritiated amino acid injections were very light and are not shown in Figure 2. There is sparse labeling throughout the ventrolateral medulla which extends dorsomedially through the parvocellu1ar reticular nucleus (Rpc) to the dorsal vagal complex, where it, lies lateral to the lateral tier subnuclei of the

Fig. 3. A-P: Darkfield photomicrographs depicting examples of ipsilateral terminal labeling throughout the brain rostral to the injection of tritiated amino acids into the parabrachial nuclei, which is shown in Figure 2A. A AVT lateral to the third nerve (NIII); B: GCt between the cerebral aqueduct (Aq) medially and the white matter (wm) laterally; C: SpM; D: SCT adjacent to the third ventricle (3vj; E: SCE dorsomedial to the heavily labeled quintofrontal tract (QFT);F: labeling surrounding the caudal pole of DIP; G rDLP; H: SRt dorsal to OM; I: the ventral LHy: the micrograph is tilted counterclockwise slightly and the unlabeled optic tract is to the right; J: PVM adjacent to the third ventricle (3v);K bilaterally in nCPa dorsal to the third ventricle (3v); L: BNST overlying OM; M:VP lateral to BNST shown in L; N: Apdl; 0 Ac adjacent to the ventral tip of the ventricle (v) of the hemisphere; P: Ac a t i t s most rostral extent. Calibration bars = 200 fim.

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PARABRACHIAL NUCLEUS PROJECTIONS IN PIGEONS

Fig. 6 . Darkfield photomicrographs depicting ipsilateral terminal labeling in the ventrolateral medulla (A and B) and in the hypoglossal nucleus (C) following an injection of tritiated amino acids covering PBvl (see text). Calibration bars = 200 ,urn.

nucleus of the solitary tract and medial to the dorsal motor nucleus of the vagus (DMNX). I t does not encroach on the vagal nuclei themselves. In the fifth case the tritiated amino acid injection in PB was angled laterally into PBvl from a dorsomedial position so as to avoid the more dorsally situated PB nuclei. Although it was centered on PBv1, there was in fact dorsal spread so as t o include PBvm, PBdl, and the medial parts of the locus coeruleus. The rostra1 projections from this injection site were similar to, although less extensive than, those described above. The descending projections, however, were heavier than and differenl from those of the dorsal injection sites and were very similar to those previously described briefly by us following injections of WGA-HRP centered on PBvl (Wild and Arends, '87). Figure 4 presents a full charting of the descending projections from a case where the WGA-HRP injection was entirely confined to PBvl (Fig. 4Aj, and pholomicrographs of corresponding terminal fields resulting from the fifth tritiated amino acid injection are presented in Figure 5. As before, the projections were bilateral but with a strong ipsilateral predominance. Labeled fibers descend through the ventrolateral pons and upper medulla in a compact bundle somewhat ventral t o the descending tract of the trigeminal nerve (TTD) (Fig. 4B,Cj. From the level of the descend-

Fig. 4. A-H: Schematic sections depicting fiber (lines) and terminal (small dots) labeling caudal to an injection of WGA-HRP into the ventrolateral parabrachial nucleus (vl). Larger dots represent retrogradely labeled neurons. See list of abbreviations.

ing vestibular nucleus caudally, retrogradely labeled cells and presumed terminations are found amongst the labeled fibers throughout the subtrigeminal nucleus (ST) and the full extent of the nucleus ambigrius (Am) (Figs. 4D-G, 5A,R). Labeled fibers continue dorsomedially from Am to terminate densely within the hypoglossal nucleus (Figs. 4EG, 5C) which contains cells innervating both the tongue and the syrinx (Wild and Zeigler, 80; Wild and Arends, '87). The terminal field within nXII has a characteristic dorsomedial extension into the neuropil. There was clearly no labeling of the dorsal motor nucleus of the vagus but there was very light labeling of the commissural portion of the nucleus of the solitary tract. At the spinomedullary junction retrogradely labeled cells and terminations are found in a caudal ext,ension of nucleus ambiguus situated horizontally between the dorsal and ventral horns (Fig. 4H). There were no ascending projections visible in this WGAHKP case, although retrogradely labeled neurons were observed in the dorsomedial subnucleus (DM) of the intercollicnlar nucleus (ICo) of the midbrain (see \?'ild and Arends, '87).

Retrograde experiments Because certain structures receive projections from both the nucleus of the solitary tract (nTS) and parabrachial nuclei, several of the retrograde cases from our study of nTS projections (Arends et al., '88) also supplied data for the present strid?;. In a further 38 birds injections of WGA-HRP were made into various sites to verify the origins of the orthograde projections within P B and t o determine the extent to which these projections originate within specific PB subnuclei. Figure 6 plots the location of cells retrogradely labeled from most of the putative PB targets onto a series of standard transverse sections equally spaced t,hroiighout the rostrocaudal extent of PR. They were drawn from normal, Nissl stained material and correspond approximately to the levels A1.OO, A1.25, A1.50, and A1.75 in the atlas of Karten and Hodos ('67). For each of the putative targets the pattern of retrograde labeling in PB was confirmed in a t least two cases and is described briefly below; an example of this labeling for most of the injection sites is shown schematically in Figure 6; and selected photomicro-

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graphs are presented in Figure 7. It should be noted that all injections in rostral targets which resulted in the retrograde labeling of P B neurons also labeled neurons in the locus coeruleus, and some, but not all, injections also labeled neurons in nucleus subcoeruleus dorsalis. For the sake of clarity these are not shown in Figure 6. Ovactory tubercle (TO) (n = 3, e.g., 8812). Besides TO, these injections invariably involved structures lying dorsal to TO, such as ventral parts of the paleostriatum. In case 8812 the injection was centered on caudal regions of TO and overlapped with an area of diffuse anterograde labeling which is depicted in Figure 2N.Retrograde labeling in PB was very extensive, with labeled neurons in PBsl, PBdm, Pbvm, and PBdl down to the dorsal aspect of the rostral intermediate nucleus of the lateral lemniscus (LLIr). Diagonal band of Broca (FDB) (n 2, e.g., 8629). This injection was centered on the diagonal band of Broca (FDB) between the quintofrontal tract and the medial preoptic nucleus but spread caudally to invade rostrodorsal regions of the thalamus. Retrograde labeling within P B was extensive but was particularly concentrated in PRdl. Archistriatum (Apdl) (n 4, e.g., 8558 and 8368). In two cases the injection was largely confined to Apdl with only minimal spread to the overlying neostriatum or more ventral archistriatum. In these, labeled cells were found predominantly in the medial part of PBvm and to a lesser extent in PBdl. A small cluster of' labeled cells was also found in the thalamus in nucleus subrotundus ventral to nucleus ovoidalis (not shown). In another case (8368),which received a massive injection covering large parts of the caudolateral hemisphere, retrogradely labeled cells in P B were much more numerous and were distributed throughout all subnuclei except PBvl (Fig. 7A). .Vucleus taeniae (Tn) (n 2, example not shown). Small injections fairly well confined to this nucleus labeled cells in locus coeruleus but not in PB. Nucleus accumbens (Ac) (n = 2, e.g., 8567). This was a small injection at the base of the ventricle, anterior to the level of the anterior commissure. It labeled a small number of cells in PBdl. Bed nucleus of the stria terminalis (BNST) (n 3, e.g., 8613). Injections in this region have a natural tendency to be restricted to the base of the ventricle because of the underlying occipitomesencephalic tract which limits more ventral spread. A few labeled cells were found in PBsl and PBdm, but most were located within PBdl dorsal to the rostral intermediate nucleus of the lateral lemniscus (Fig. ~

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33).

Ventral paleostriatum (VP) (n = 2, e.g., 8810). These injections were aimed at the more caudal regions of VP which were shown to receive anterograde projections from P B injections, i.e., lateral to the bed nucleus of the stria terminalis, overlyiag the occipito-mesencephalic tract, at the level of the anterior commissure. Heavily labeled cells were found specifically in PBdm. P e r i v e n t r i c u l a r nucleus (PVM) (n = 3, e.g., 8606). Despite the fact that each of these injections covered dorsornedial regions of the anterior diencephalon, none of them labeled many cells in P B and what cells were labeled were lightly labeled. Nucleus of the pallial commissure (nCPa) (n = 4, e.g., 8719). This injection was centered a t the level of the anterior commissure and covered nCPa bilaterally. There was minimal spread below the commissure, but the medial

septa1 nucleus (SM) was substantially involved. (The anterograde tracing experiments did not reveal a projection to SM, however.) Numerous labeled neurons were located in PRsl at caudal nuclear levels, and clustered in PBdm more rostrally. More scattered neurons also occurred in PRvm and PBdl. HUpotha1amu.s (Hy) (n = 4, e.g., 8753). These injections filled both medial and lateral parts of mid-regions of the hypothalamus, including rostral parts of the stratum cellulare internum (SCI), and consistently produced massive retrograde labeling in PR which was heavily concentrated medial to BCA in PBdm, PBvm, and rostral parts of PBdl (Fig. .in).Fewer labeled cells were also found in PBsl. Injections in more caudal regions ofSC1 produced only a few lightly laheled cells in PBsl. Stralum cellulare externum (SCE) (n = 2, e.g., 8729). These injections were centered more caudally and laterally in the hypothalamus, but the pattern of retrograde labeling in PR was similar to that produced by more rostromedial injections. Dorsal thalamus (n - 6, e.g., 8744). All these injections produced a consistent pattern of retrograde labeling in more dorsal parts of PB (Fig. 7C). In case 8744 the injection was centered on the nucleus dorsolateralis anterior, pars medialis (DLM) at A 6 2 5 and labeled cells largely confined to PBsl and PBdm. Nucleus spiriformis medialis (SpM) (n = 2, example not shown). These injections produced anterograde axonal labeling in BCA (Karten and Finger, '76), but retrogradely labeled few PB neurons which were confined to PRdm. Central grey (GCt) (n = 5, e.g., 8806). Injections confined to the periaqueductal GCt at the level of the oculomotor nerve proved difficult to make and produced lightly labeled cells in PBdm and PBdl, hut cells in the locus coeruleus and the nucleus subcoeruleus dorsalis were both more numerous and more densely labeled. Ventral a r e a of Tsai (AVT) (n = 2, e.g., not shown). Although both these injections covered the nucleus, there was spread more rostrally into caudal levels of the hypothalamus. They produced extensive labeling primarily of' PBdl. Nucleus ambiguus (Am) (n = 3, e.g., 8254). This injection was angled laterally into the ventrolateral medulla rostral to t,he obex so as to cover the nucleus amhiguus and the subtrigeminal region. Labeled cells in P B were entirely rest,ricted to PRvl (Fig. 7E). In one other case a few labeled cells were also found in PBsl. H y p o g l o s s a l n u c l e u s ( n X I I ) (n = 3, e . g . , 8668). The placement of this small injection into the tracheosyringeal portion of the hypoglossal nucleus was electrophysiologically controlled (see Wild and Arends, '87) and

Fig. 6. Standardised schematic representation of the 1ocat.ion of retrogradely labeled neurons (dots) in the parabrachial nuclei following injections of WGA-HRP into the sites shown in hlack a t left. Each row is a single exemplary case (see text) with the labeled neurons charted a t four rostrocaudal levels through t h e nuclear complex the most caudal level is in the second column, and the most rostral level is in the fifth. T h e top row is a schematic outline of the parabrachial nuclei and adjacent structures: the dotted lines represent efferent BC fascicles as determined by an injection of WGA-HRP into the deep cerebellar nuclei (courtesy of J.J.A. Arends).

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Fig. 7. Darkfield photomicrographs showing retrogradely labeled the dorsal thalamus (C); the hypothalamus (D); the ventrolateral neurons in the parahrachial nuclei in a selection of cases with WGA- medulla (E); the hypoglossal nucleus (F). Orthograde labeling is also HRP ipjections in the caudolateral hemisphere (Apdl) (A); BNST (B); apparent, particularly in A, B, and D. Calibration bars = 200 Wm.

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resulted in labeled cells confined to PBvl (Fig. 7F); some of these arched dorsally over the rostral intermediate nucleus of the lateral lemniscus. Nucleus of the tractus solitarius/dorsal motor nucleus ofthe vagua (nTS/DMNX) (n = 8, no example shown in Fig. 6). Most of these injections were used to define nTS eff'erents (Arends e t al., '88); but since they frequently were not confined to nTS and involved parts of nXII in particular, it is not clear whether the retrogradely labeled cells found in these cases (predominantly in PBvl and a few scattered in PBm) project to the vagal complex. around it, to nXII or to all three regions.

Reciprocal labeling Afferent projections to PB arising in caiidal brainstem structures have been documented by Arends et al. ('88) (see also Fig. 4). In the present study injections of WGA-HRP into PB showed that many midbrain and forebrain structures which received ascending projections from PB were also the source of descending projections to the region of PR. Figure 8 plots the location of retrogradely labeled cells in a case receiving an injection of WGA-HRP centered on the dorsal and medial P B subnuclei. Labeled cells were found in the olfactory tubercle, the anterior preoptic nucleus, the bed nucleus of the stria terminalis and the ventral paleostriatum, a few in the nucleus of the pallial commissure, the periventricular nucleus (PVM), the dorsal diencephalon extending from PVM to the bed nucleus of the stria terminalis, the lateral hypothalamic nucleus, the strata cellulare internum and externum, the ventral area of Tsai, the midbrain central grey, the compact portion of the pedunculopontine tegmental nucleus, and the raphe. Labeled cells were also found in the tectum, but these in all probability are the source of a well-known projection to the isthmo-optic nucleus (Cowan and Powell, '63) which was also covered by the injection. Injections of WGA-HRP into these regions (see Retrograde Labeling above) confirmed that they did provide projections which had terminal fields in PB, mostly within PBdm and PBvm. The precise topography of these projections, however, is beyond the scope of this paper, and has been partly described by Rerk ('87).

DISCUSSION Definition of the parabrachial nucleus and its projections: Methodological and interpretive problems Our initial description of parabrachial nucleus in the pigeon was based upon an analysis of the projections of the nucleus of the solitary tract (nTS) (Arends et al., '88). In the present study we have additionally used topographic and cytoarchitectural criteria to define a minimal number of parabrachial subnuclei, and this description has served as a starting point for an experimental study of PB projections in the pigeon. These projections are illustrated schematically in parasagittal view in Figure 9. Analysis of the retrograde data indicates that the parabrachial subnuclei are the origins of projections which may be distinguished to a variable extent from those of neighboring nuclei such as the locus coeruleus (LoC) and the nucleus subcoeruleus dorsalis (SCd), and from each other. The problem of differentiating the projections of P B from those of neighboring nuclei is particularly acute with respect to LoC.

The retrograde data showed that every injection of WGAHKP into a rostral target which labeled PB neurons also labeled LoC neurons at the same rostrocaudal levels. This suggests that, as in mammals (Jones and Moore, '77; Saper and Loewy, '80; van der Kooy et al., '84), the projections of PI3 and LoC have several targets in common, although the telencephalic projections of LoC are considerably more extensive than those of P B (cf. Kitt and Brauth, '86). Indeed, some of the present retrograde data suggest that the projections of LoC are even more extensive than indicated by Kitt and Rrauth ('86). The projections of PB also partly overlap those of SCd, but certain differences in the pattern of these projections add hodological support to the topographical distinction between SCd and P B in the pigeon. SCd (and rostral parts of LOG)were postulated by Kitt and Brauth ('86) to he comparable with the mammalian A8 dopaminergic cell group, on the basis of their histochemistry and their pattern of projections upon the telencephalon, including the striatum. (The avian PB region does not appear to contain catecholamine containing neurons, however: Dube and Parent, '81; Shiosaka et al., '81; Wild, unpublished observations). SCd efferents were traced by Kitt and Rrauth ('86) to rostral LoC, ventral area of Tsai, the periaqueductal grey (GCt), hypothalamus and thalamus (sparse except for the nucleus subrotundus), the ventral paleostriatum, and nucleus accumbens, all regions which were found to receive projections from PB in the present study. However, the two nuclei have other, quite different projections. Kitt and Rrauth ('86) found projections from SCd, which partly ascend via the ansa lenticularis (AL), to the dorsal archistriatum (Ad) and parts of the paleostriatum, the lohus parolfactorius (LPO) in particular. In the present study AL did not appear to carry ascending projections from the region of our PB injections and we found no evidence of terminations within LPO or Ad, despite the fact that fibers traverse Ad to terminate within the dorsolateral nucleus of the posterior archistriatum (Apdl), a nucleus not found to receive SCd projections by Kitt and Brauth ('86). Furthermore, the projections from PB to the hypothalamus and thalamus seem much heavier and more extensive than the projections from SCd (cf Kitt and Brauth, '86). Considering now the projections of the individual PB subnuclei, those of the subnuclei situated more dorsally, viz., sl, dm, vm and dl, are clearly distinct from those of vl, the single subnucleus situated most ventrally, in that the dorsally situated group provided virtually all the ascending projections while vl provided most of the descending projections. A few sl neurons, however, may project to the ventrolateral medula (present results) or to the spinal cord (Cabot et al., '82). Within the dorsal group itself, however, the subnuclei cannot completely be differentiated on the basis of their projections to different targets using the present techniques. That is, although injections in certain targets (e.g., the ventral paleostriatum) do give rise to labeled cells confined to a single subnucleus (e.g., PBdm), most injections label cells in more than one subnucleus. This could reflect several possibilities: 1)our subnuclear parcellation does not accurately reflect the organizational complexity of the sources of the differential projections; 2) different neurons in the same parabrachial subnucleus project to different targets; 3) single neurons project to more than one target by avonal collateralisation (as was suspected

PARABRACHIAL NUCLEUS PROdECTIONS IN PIGEONS for the rostrally projecting nTS neurons-Arends et al., '88); 4)uptake hy damaged fibers passing through a more caudal injection site-eg, hypothalamus-to a more rostral or dorsal target.

Functional considerations:Topographical segregation of visceral function within the viscerosensory system of the pigeon Our previous study of efferents of the nucleus of the solitary tract (nTS) (Arends et al., '88) revealed a partial functional segregation of nTS projections to PB which could be correlated with the pattern of visceral inputs to nTS subnuclei, as delineated earlier by Katz and Karten ('83). The gustatory and gastrointestinal nTS subnuclei (with a single major exception-see Arends et al., '88) project upon the dorsal and medial portions of PB; the cardiovascular and pulmonary nTS subnuclei project upon ventrolateral PB, but, on the basis of retrograde tracing experiments, probably also project upon more dorsal PB regions. One aim of the present study was to clarify the extent to which this partial topographic segregation is preserved in the projections from P B to higher levels of the viscerosensory system. The present data have shown that dorsal and medial PI3 subnuclei are the origins of ascending projections to higher levels of the neuraxis, including the hypothalamus and ventral telencephalon, and that the ventrolateral P B subnucleus is the major origin of projections to lower levels of the neuraxis. Thus, the efferent projections of PI3 subnuclei are correlated to a significant extent with the differential afferent projections to those subnuclei arising in nTS (Arends et al., '88),and thereby indicate a substantial degree of functional differentiation within the viscerosensory system as a whole. Basically, the pattern of connectivity within this system suggests that gustatory and gastrointestinal inputs are channeled through dorsal and medial parabrachial regions to the forebrain, while cardiovascular and pulmonary inputs are channeled through the ventrolateral parabrachial subnucleus to the medulla. However, the probability of some cardiovascular and pulmonary input gaining access to more rostral brain regions via ascending projections arising in more dorsal parabrachial areas such as PRdm and PBdl adds a measure of hodological and functional complexity to this basic distinction. Unfortunately, for most aspects of visceral function, including taste, there are few physiological data which might help to confirm or even suggest a functional role for some of the structures found to receive parabrachial projections in the present study, and such physiological data as are available are difficult to interpret in the present conlext. For example, modulation of cardiovascular and pulmonary activity by electrical brain stimulation in the pigeon has frequently been reported (e.g., Macdonald and Cohen, '73),but the loci from which these effects can be obtained include regions such as the hypothalamus, dorsolateral pons, and ventrolateral and dorsomedial medulla, which may be either the origins or recipients of PI3 and/or nTS projections. The effects have usually been interpreted in the context of descending autonomic control systems (Mcdonald and Cohen, '73); but, given the present data, it, is not clear whether the observed cardiovascular or pulmonary activation from some of these loci reflects stimulation of afferent or efferent pathways, or both. Given the absence of definitive physiological correlates for the observed projections, speculation as to the possible

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function of some of them is briefly mentioned. Firstly, with regard to taste, it can be noted that the archistriatal subnucleus, Apdl, lies adjacent to the olfactory recipient piriform cortex (Reiner and Karten, '85). Apdl receives a projection from dorsal and medial parabrachial areas (present results), which in turn receive a projection from the nTS gustatory suhnricleus, mVal (Dubbeldam, '84; Ganchrow et al., '86; Arends et al., '88). Perhaps these facts could imply a common chemoreceptive zone in the caudolateral hemisphere. Interestingly, when the piriform cortex is surgically disconnected from the rest of the hemisphere, a procedure which is also likely to remove the P B input to Apdl, chicks show a deficiency in monitoring an amino acid deficient diet (Firman and Kuenzel, '88). Secondly, with regard to the PR projections to the dorsal thalamus, some of the recipient nuclei such as the nucleus dorsointermedius posterior (DIP) and the rostral subdivision of the nucleus dorsolateralis posterior (rDLP) have subsequent projections to extensive regions of the telencephalon, including the paleostriatum and neostriaturn, amongst others (Wild, '87b). It is possible, therefore, that this set, of projections is similar to that in mammals from P B lo the telencephalon via the thalamic intralaminar nuclei, and t,herefore could mediate ascending reticular activating influences as suggested for this pathway in mammals (Saper and Loewy, '80). Thirdly: we have previously pointed out the proximity of the nucleus of the pallial commissure (nCPa) to the subseptal (subfornical) organ (Arends et al., '88). This, together with the fact that nCPa receives projections from both nTS and PB (Arends et al., '88; present study), could perhaps suggest the involvement of nCPa in thirst mechanisms. And fourthly, the fact that P B projects to the medial spiriform nucleus (SpM), but not to the cerebellum, suggests that PB has only indirect connections with the cerebellum in the pigeon (Karten and Finger, '76). This projection could transmit either somatosensory and/or viscerosensory information derived from cranial or peripheral nerve inputs to the dorsolateral medulla (Katz and Karten, '83; Arends et al., '84; '88; Wild, '89).

Descending parabrachial projections and the control of vocalization and respiration The ventrolateral subnucleus of the parabrachial nuclear complex is the nexus of a remarkable set of connections. In addition to inputs from the ventrolateral medulla, nucleus ambiguus, and the dorsomedial subnucleus of the intercollicular nucleus (ICo-DM), it is the primary target of an nTS subnucleus (1Ps) receiving a dense and specific primary afferent input from the lung (Katz and Karten, '83; Wild and Arends, '87). Its outputs include projections back to the ventrolateral medulla, including the nucleus ambiguus, massively to the hypoglossal nucleus (which is largely tracheosyringeal), and minimally to the commissural subnucleus of nTS.I t does not appear to project to the spinal cord, however (see Cabot et al., '82, and Webster and Steeves, '88, for negative evidence). With the exception of a spinal projection, the efferent connections of PBvl in the pigeon seem directly comparable with some of those of the mammalian Kolliker-Fuse nucleus (K-F), which also projects to the ventrolateral medulla (Fulwiler and Saper, '84), including the nucleus ambiguus, and has been implicated in the control of lower brainstem respiratory related nuclei (Denavit-Saubii. and Riche, '77; Riche

N

Fig. 8. Caudal (top left) to rostral (bottom right) series of schematic hemisections depicting the location of retrogradely labeled new rons (dots) rostral t o an injection of WGA-HRP into the dorsolateral

pons centered on the dorsal and medial parabrachial nuclei (top). The locations of labeled neurons caudal to such an injection may be found in Arends et al. ('88), and see Figure 4. See list of abbreviations.

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Fig. 9. Schematic parasagittal section showing the projections of the parabrachial nuclei. Dashed lines indicate that unequivocal retrograde confirmation of the projection is lacking. See list of abbreviations.

et al., ’79; Bystrzycka, ’80; Rye et al., ’88).In the rat the trajectory of the K-F projections has been defined as the ventrolateral branch of Probst’s tract (Rye et al., ’88),which is similar to the course of the PBvl projections in the pigeon. J T view ~ of the nuclei innervated in both rat and pigeon, this ventrolateral pathway may also be involved in vocal control. The PBvl projections in the pigeon may also be analogous to those of the “pre-trigeminal nucleus of the dorsal tegmental area (DTAM)” in the clawed frog, in which animal a vocalization pathway has also been delineated (Wetzel et al., ’85). DTAM projects to laryngeal motoneurons involved in mate calls, and on topographical and hodological grounds these authors have likewise postulated that DTAM in the frog could be similar to the K-F nucleus of mammals (cf. Wild and Arends, ’87). In Schmidt’s (’76) neural model of calling behavior in frogs, DTAM is postulated as the vocal or expiratory phase generator, a designation perhaps equally applicable to PBvl of the pigeon. If this is its role, then it is possible that the pulmonary input to PRvl via nTS subnucleus 1Ps is inhibitory during inspiration. Although the “control center” for vocalization in nonsongbirds such as pigeon and chicken has hitherto been ronsidered to be the intercollicular subnucleus dorsomedialis (ICo-DM) (review in Seller, ’80)which projects to both PBvl

and nXII (Wild and Arends, ’87),the input to nXII supplied by P B d is considerably greater than that supplied by DM (Wild and Arends, ’87).In any case, the observation that the avian syrinx is innervated by nXII, and that syringeal activity is present during both vocalization and respiration (Youngren et al., ’74; Vicario and Nottebohm, ’88), suggests a prominent role for PBvl in the control of these interactive functions.

Comparative considerations The general organization of PB projections in pigeon and rat. Although the parabrachial projections have been the focus of attention in a range of different mammalian species, the most comprehensive reviews of t,he subject are those for the rat (Saper and Loewy, ’80; Fulwiler and Saper, ’84) which will be used as a basis for comparison with our findings in the pigeon. I t is immediately clear from an inspection of Saper and Loewy’s (’80) summary figures (Fig. 7) and the present Figure 9 that there are basic similarities in the pattern of PB projections in the two species, with major ascending projections t o the thalamus, hypothalamus, and other parts of the basal forebrain such as the bed nucleus of the stria terminalis, and descending projections to the ventrolateral medulla. The viscerosensory projections

PARABRACHIAL NUCLEUS PROJECTIONS IN PIGEONS in diverse vertebrate species thus have fundamental features in common, presumably reflecting similarly basic homeostatic mechanisms. It is equally clear, however, that there are significant species differences in the pattern of PB projections, which may in part reflect the individual species' requirements for the regulation of vegetative functions: 1) The medial parabrachial subnucleus in the rat projects to prefrontal, infralimbic, septo-olfactory, and insular cerebral cortices, regions for which there are no readily apparent equivalences in the pigeon. 2) The projections to the amygdala in the rat are more extensive than those to the archistriatum in the pigeon where t,hey appear to be limited to the single posterolaterally situated nucleus, Apdl. 3 ) In contrast with the rat, there are few, if any, ascending projections arising from the ventrolateral subnucleus in the pigeon, the subnucleus we have considered on other grounds to be comparable with the K-F nucleus (see above). 4)There appear to he no direct projections to the cerebellum in the pigeon, unlike the rat. Indirect projections via the medial spiriform nucleus have been mentioned above. 5) Unlike the case in the rat, the descending projections in the pigeon arise in large part only from the ventrolateral (vl) subnucleus. Although these projections seem to be analogous to some of t,hose from the K-F nucleus, in the rat there are other descending projections arising in both medial and lateral P B subnuclei. These differences cannot easily be explained by the lack of correspondence (i.e., non-homology) between similarly named subnuclei in the two species. For instance, the subnuclei we have called dorsomedial (dm) and ventromedial (vm) may well include components which in the rat or other mammals would be considered lateral in relation to the brachium conjunctivum. Nevertheless, neither dm nor vm, nor either of the other two subnuclei situated dorsal to vl, appear to give rise to a significant descending projection in the pigeon. Viscerosensory projections in the pigeon and the concept oJa oisceral forebrain. Van der Kooy et al. ('84) have proposed that, on the basis of reciprocal connections with viscerosensory relay nuclei (nTS, PB), a group of cortical and subcortical structures in the rat together comprise a visceral forebrain system. These structures include the medial and lateral prefrontal cortex, paraventricular, arcuate and posterolateral hypothalamic nuclei, bed nucleus of the stria terminalis, and central nucleus of the amygdala. Similar connections in other mammals (e.g., Price and Amaral, '81; Shipley, '82; Schwaber et al., '82; Yasui et al., '85; Willet et al., '86, '87) lend credence to the generality of this concept which is currently receiving substantial functional support (e.g., Cechetto and Saper, '87; Ruggiero et al., '87). Several studies have shown that the pigeon also exhibits a set of reciprocal connections between certain forebrain structures and the dorsal vagal and parabrachial nuclear complexes (Berk and Finkelstein, '83; Berk, '87; Arends et al., '88; present results: cf. Figs. 2 and 8). However, the extent. to which the structures are comparable in the two classes is presently unclear. For example, although a prefrontal "cortex" has been proposed for the pigeon on biochemical and behavioral grounds (Divac, '79; Mogensen and Divac, '82; Divac and Mogensen, '85; Divac et al., '85; see also Reiner, '861, the region so nominated (the posterodorsolateral neostriatum-PDL) does not meet t,he essential defining criterion for mammalian prefrontal cortex, namely, innervation by a thalamic nucleus which could be considered comparable with the mediodorsal. In fact, not only has

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a such an avian nucleus not yet been identified, but also there is no evidence that PDL receives a projection from any thalamic nucleus in birds. Furthermore, PDL does not give rise to any long descending projections, unlike the mammalian prefrontal cortex (Van der Kooy et al., '84). We suggest, therefore, that if there is a visceral forebrain in birds it is unlikely to include a component comparable with mammalian prefrontal cortex. Similarly, the equivalence of any avian nuclear region to the central nucleus of the amygdala in mammals is distinctly problematic. Such a region must presumably be sought within the archistriatum since this structure includes nuclear components considered comparable with the mammalian amygdala (Zeier and Karten, '71). But the archistriatal nucleus Apdl, which was found to receive a dense projection from PB in the present study, seems an unlikely candidate a) on topographical grounds, and b) because it neither receives a projection from nTS nor gives rise to descending projections upon lower visceromotor nuclei (cf. Schwaber et al., '82; Rerk, '87; Arends et al., '88). Instead it appears t,o project to the bed nucleus of the stria terminalis (BNST: Zeier and Karten, '71; Wild, Arends, and Zeigler, unpublished data), resembling in this and other respects certain components of the posterior basolateral nuclear complex of the mammalian amygdala (de Olmos et al., '85). which also receive parabrachial projections (Saper and Loewy, '80). In the absence of any other obvious candidates within the amygdala proper, it can be noted that recent conceptions of the organisation of t,he mammalian amygdala have emphasized the continuity of the central nucleus of the amygdala with more medial cell fields composing the lateral parts of the bed nucleus of the stria terminalis (BSI'L) and the sublenticular portion of the suhstantia innominata (SLSI) (de Olmos et al., '85). In fact, all these structures are characterized by reciprocal connections with the parabrachial nuclei (Schwaher et al., '82) and by a similar pattern of immunohistochemical reactivity (see de Olmos et al., '85; Alheid and Heimer, '88). In the pigeon parabrachial injections of WGAHHP also produce anterograde and retrograde labeling in the bed nucleus of the stria terminalis, labeling which extends laterally to a marked degree into an area defined by Kitt and Rrauth ('81) as the ventral paleostriatum (VP). On the basis of its connections, this area was suggested by Kitt and Brauth ('81, ~ 1 5 6 5 to ) be comparable with the "mammalian ventral pallidum (or substantia innominata)." In the context of recent mammalian terminology, the juxtaposition of these two terms is unfortunate, since the term "substantia innominata" is recommended for only the sublenticular grey (i.e., the SLSI), as distinct from the more rostra1 subcommissural region, which is considered a true component of the ventral pallidum (Heimer et al., '85; Alheid and Heimer, '88). Nevertheless, the present data, and those of Berk ('87),support t,he idea that the area overlying the occipitomesencephalic tract between the bed nucleus of the stria terminalis (BNST) medially and the amygdala laterally, and ventral to caudal regions of the paleostriatum, is comparable with thc SLSJ of mammals. It is still unclear a t this stage whether there is an avian equivalent of the mammalian central amygdaloid nucleus, but immunohistochemical studies of markers such as angiotensin I1 and cholecystokinin, which are characteristic of BNST, SLSI, and the central amygdaloid nucleus in mammals (Alheid and Heimer, '88), might help to resolve the issue in the pigeon.

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Two of the three remaining components of the mammalian visceral forebrain appear to have well-defined counterparts in the pigeon brain. Thus, the periventricular nucleus PVM of the pigeon is comparable with the paraventricular nucleus of mammals in terms of its efierents (Cabot et al., ’82; Berk and Finkelstein, ’83),afferents (Arends et al., ’88),’ and immunocytochemistry (Berk el al., ’82). And the stratum cellulare externum (SCE) of the avian hypothalamus seems comparable on topographic and hodological grounds with the posterolateral hypothalamus of the rat (Van der Kooy et al., ’84; Berk, ’87; present results). As shown by Rerk and Finkelstein (’83) and Berk (’87),these basal forebrain structures have descending projections with trajectories throughout the brainstem and terminal fields within the parabrachial and dorsal vagal nuclei very similar to those in mammals. Since we have shown that these structures also receive reciprocal projections from nTS and P B (Arends et al., ’88; present results), it seems that a good case can be made for the presence of a visceral forebrain system in the pigeon which includes PVM, SCI and SCE, BNST, and its lateral extension into the ventral paleostriatal region. We would also suggest that the region known as the nucleus accumbens (Ac) in the pigeon (Karten and Hodos, ’67) be included, as well as the olfactory tubercle, and perhaps the nucleus of the pallial commissure, since these, too, have reciprocal P B connections. It is recognised, however, that the area designated “nucleus accumbens” in birds may in fact not be comparable with the mammalian nucleus accumbens and is more likely to be a rostra1 extension of the bed nucleus of the stria terminalis (Reiner et al., ’83; Wild, ’87b). In addition, also on the grounds of reciprocal P B connections, extensive dorsal areas of the diencephalon which are coextensive with the course of the hypothalamic branch oft he occipitomesencephalic tract (HOM: Zeier and Karten, ’71) could be included, but it is not clear with what mammalian structures they should be considered comparable. Van der Kooy et al. (’84) hypothesized that the visceral forebrain functions to override reflex or homeostatic mechanisms at the brainstem level, particularly during periods of strcss Or emotional activity. Tn the present context it seems more neutral simply to think in terms of a modulatory role of the forebrain on autonomic function (Berk, ’87). It seems unlikely to us, however, that such modulation would be effected, a t the level of nTS, directly on subnuclei in receipt of primary afferent input from specific visceral organs, because most of this input is to subnuclei which do not project to the forebrain (Arends e t al., ’88) and which receive only limited afferents from the forebrain (Berk, ’87). Whether or to what extent “visceral forebrain” structures in the pigeon do have such modulatory effects is presently unknown. The present findings have provided the anatomical foundations for studies designed to answer this and many other questions relating to visceral function in birds.

ACKNOWLEDGMENTS Supported by grants to J.M.W. from the Medical Research Council of New Zealand, the Kiwi Lottery Board of ’In the present study, although PVM was clearly anterogradely labeled by tracer injections in PB, WGA-HRP injections covering PVM only lightly labeled a few P B neurons retrogradely (see Results). We have no satisfactory explanation of this apparent discrepancy, but note that the PB projection lo PVM, like the nTS projection to PVM (Arends e t al., ’88), is rather diffuse and not confined within the nuclear boundaries. Perhaps this is not conducive to optimal uptake for retrograde transport.

Control, and the Auckland University Research Committee; and by NSF grant 85-07374, NIMH grant MH-08366, and Research Scientist Award MH-00320 to H.P.Z. We gratefully acknowledge the assistance of Mrs. Agnes Reilly and Mrs. S.M. Braan-Stroo in the preparation of histological materials.

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