Afferent connections of the parvocellular reticular formation: a horseradish peroxidase study in the rat.

0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd ( 1992 IBRO

Neuroscience Vol. 50, No. 2, pp. 403-425. 1992 Printed in Great Britain

A F F E R E N T C O N N E C T I O N S OF THE P A R V O C E L L U L A R RETICULAR FORMATION: A HORSERADISH P E R O X I D A S E S T U D Y IN THE R A T S. J. SHAMMAH-LAGNADO,*+ M. S. M. O. COSTA+ and J. A. RICARDO*b'I'~ *Department of Physiology and Biophysics, Institute of Biomedical Sciences of the University of Sao Paulo, 05508 Sao Paulo, SP, Brazil :~Department of Morphology, Federal University of Rio Grande do Norte, 59072 Natal, RN, Brazil A~tract--The afferent connections of the parvocellular reticular formation were systematically investigated in the rat with the aid of retrograde and anterograde horseradish peroxidase tracer techniques. The results indicate that the parvocellular reticular formation receives its main input from several territories of the cerebral cortex (namely the first motor, primary somatosensory and granular insular areas), districts of the reticular formation (including its contralateral counterpart, the intermediate reticular nucleus, the nucleus of Probst's bundle, the dorsal paragigantocellular nucleus, the :t part of the gigantocellular reticular nucleus, the dorsal and ventral reticular nuclei of the medulla, and the mesencephalic reticular formation), the supratrigeminal nucleus and the deep cerebellar nuclei. Moderate to substantial input to the parvocellular reticular formation appears to come from the central amygdaloid nucleus, the parvocellular division of the red nucleus, and the orofacial and gustatory sensory cell groups (comprising the mesencephalic, principal and spinal trigeminal nuclei, and the rostral part of the nucleus of the solitary tract), whereas many other structures, including the substantia innominata, the field H 2 of Forel, hypothalamic nuclei, the superior colliculus, the substantia nigra pars reticulata, the retrorubral field and the parabrachial complex, seem to represent relatively modest additional input sources. Some of these projections appear to be topographically distributed within the parvocellular reticular formation. From the present results it appears that the parvocellular reticular formation receives afferents from a restricted group of sensory structures. This finding calls into question the traditional characterization of the parvocellular reticular formation as an intermediate link between the sensory nuclei of the cranial nerves and the medial magnocellular reticular districts, identified as the effector components of the reticular apparatus. Some of the possible physiological correlates of the fiber connections of the parvocellular reticular formation in the context of oral motor behaviors, autonomic regulations, respiratory phenomena and sleelz~waking mechanisms are briefly discussed.

The parvocellular reticular f o r m a t i o n ( R F p ) occupies the dorsolateral third of the p o n t o m e d u l l a r y reticular core. merging caudally, at the level of the area postrema, with the dorsal reticular nucleus of the medulla (for cytoarchitectonic delineation, see Refs 69, 79, 117). The R F p has been implicated in several functional d o m a i n s t h a t include oral m o t o r behaviors, ~6"29'v2~°8cardiovascular regulation, 23 respiratory p h e n o m e n a 3'76 and paradoxical sleep mechanisms. 93 To date, the afferent c o n n e c t i o n s of the R F p have not been examined in a single comprehensive study. Several subcortical projections to the R F p have been indicated in the rat with the retrograde horseradish peroxidase ( H R P ) technique by Mehler, 69 in his search for a n a n a t o m i c a l substrate of m o t i o n induced vomiting. O t h e r retrograde reports have focused on a particular source o f input to the tTo whom correspondence should be addressed. Abbreviations: cRFp, parvocellular reticular formation, caudal part; HRP, horseradish peroxidase; PHA-L, Phaseolus vulgaris-leucoagglutinin; RFp, parvocellular reticular formation; rRFp, parvocellular reticular formation, rostral part; WGA HRP, horseradish peroxidase conjugated with wheatgerm agglutinin. 403

RFp, such as the central amygdaloid nucleus, j~3 p a r a b r a c h i a l complex, ~° mesencephalic trigeminal nucleus 91 a n d deep cerebellar nuclei. 8~ Cortical efferents from s e n s o r i m o t o r areas were traced to the R F p with degenerative 6°'6~66~17 and axoplasmic transport techniques, 66'~3°'t3~a n d recently, the c o n t r i b u t i o n of various cortical districts was examined in a t h o r o u g h a n t e r o g r a d e report on corticoreticular p a t h w a y s in the rat. v5 Subcortical inputs to the R F p have been suggested in a n t e r o g r a d e tracing studies of the efferent c o n n e c t i o n s of several structures, including the h y p o t h a l a m u s , 41'5°'5L64 superior colliculus, ~ red nucleus 47 and b r a i n stem reticular nuclei. ~4~ Finally, in electrophysiological e x p e r i m e n t s , 32"v7"~°4 it was s h o w n t h a t R F p units are responsive to different kinds of peripheral stimuli, and, in a n a t o m i c a l investigations 9'J°'68"69,vS'83"84"91"n15 projections from sensory cell groups to the R F p have been identified. F r o m the a f o r e m e n t i o n e d studies, it appears that structures in m a n y of the m a j o r subdivisions of the b r a i n m a y influence the RFp. However, the overall set of structures is not known, the relative density of the various projections is unclear, and the issue of possible t o p o g r a p h i c o r g a n i z a t i o n has not been resolved.

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S, J. SHAMMAH-LAGNADOe t aL

The present report represents a systematic attempt to provide a comprehensive picture of the R F p afferents in the rat, using combined retrograde and anterograde tracing techniques. Some of these results have been published previously in abstract form. 95 EXPERIMENTAL PROCEDURES

experimental procedures adopted in the anterograde series were basically similar to the ones described for the retrograde group. It should be noted, however, that only WGA HRP was employed for anterograde tracing, since it appears to be superior to free HRP for this purpose. "~' Moreover, in the anterograde cases the survival period varied from 24 to 48 h. The tissue sections were microscopically examined under both brightfield and darkfield illumination for the presence and location of either retrogradely labeled perikarya or anterogradely transported label. The injection sites and the distribution of transported label were charted onto projection drawings of selected Nissl-stained sections. In order to provide a rough quantitative impression of the relative density of labeled neurons among the afferent structures, cell counts were made in the representative experiments of the retrograde series. The cytoarchitectonic parcellation and nomenclature adhere basically to the rat brain atlas of Paxinos and Watson. 79

The present study is based on retrograde and anterograde experimental series carried out on adult Wistar female rats ranging from 170 to 210 g in weight. All of the surgical procedures were performed under anesthesia with chloral hydrate (Merck, 400 mg/kg, i.p.). In the retrograde experiments, unilateral deposits of either free HRP (22 animals) or HRP conjugated with wheatgerm agglutinin (WGA-HRP) (10 rats) were placed stereotaxically in the RFp. In order to obtain restricted injection sites, the tracers were delivered by microelectrophoresis from glass micropipettes (internal tip diameter 10-14 #m) filled RESULTS with either a 13% solution of HRP (Sigma, type VI) in Tris-HCl buffer at pH 8.6 or a 5% solution of WGA-HRP (Sigma, Triticum vulgaris, peroxidase type VI) in distilled Anatomy of the parvocellular reticular formation water. A positive constant current of 0.5-1 pA (in the The R F p forms an elliptical cylinder in the dorsoHRP cases) or of 0.2-0.4#A (in the WGA-HRP group) lateral tegmentum (see Refs 69, 79, 117). It extends was passed through the solution for either 4-9 min (in the HRP series) or 45 s to 2min (in the WGA-HRP exper- from the caudal pole of the m o t o r trigeminal nucleus iments). Upon completion of the injection, the pipette was to the spino-medullary junction, where it fuses with left in situ for an additional 15-30 min. After a survival the dorsal reticular nucleus of the medulla. The period varying from 48 to 72 h the animals were deeply anesthetized and perfused transcardially with a solution of R F p is bounded laterally by the principal sensory and spinal trigeminal nuclei, medially by the inter1.0% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) followed by a solution of 10% mediate reticular nucleus, and dorsally by the sucrose in 0.1 M phosphate buffer according to the provestibular nuclei and the nucleus of the solitary tract; cedure described by Mesulam. 7°The brains and spinal cords its indefinite ventral edge is assumed to abut the were removed, stored at 4°C in the sucrose buffer solution motor nucleus of the facial nerve and the ambiguus for periods ranging from 2 h to three days, and cut on a freezing microtome in the frontal plane at 40/zm. All nucleus. Primarily, the R F p is composed of small of the brain sections and every fifth spinal cord section cells densely arranged, but its rostral part (rRFp), were processed according to the tetramethylbenzidine tech- co-extensive with the genu of the facial nerve, nique. 7°After being mounted on chrome alum-coated slides, displays many large cells. The r R F p has also been the sections were lightly counterstained for Nissl substance referred to as the ~ part of the RFp. 79 In the present with 1% Neutral Red at pH 4.8. A total of 65 rats were used in the anterograde exper- study, the remainder (caudal) R F p was designated iments. In this series, many of the structures in which the cRFp. Embedded in the latter district lies retrogradely labeled neurons had been consistently found a distinct cell group, intensely stained, the linear after our RFp injections received microelectrophoretic nucleus of the medutla. 33'79 deposits of WGA-HRP: cortical districts (n =21), the substantia innominata (n = 4), the central amygdaloid nuRetrograde labeling experiments cleus (n = 2), the zona incerta and fields of Forel (n = 2), the superior colliculus (n =2), the central gray substance The present results are based on the observations (n = 2), deep eerebellar nuclei (n = 5), and secondary sensory cell groups (n = 27). Because the entire brain is often made in eight rats (six H R P experiments and two processed in our studies, some of these cases have already W G A - H R P cases) whose injection sites are shown in been utilized in previous reports. Fig. 1. In these animals the tracer deposits were Multiple micropipette penetrations were made in cortcentered in the r R F p (R-9, R-14, R-42 and R-45), the ical and cerebellar territories. The surgical exposure of c R F p (R-16, R-20 and R-25) or encompassed both the rostroventral district of the first motor and primary somatosensory cortical areas was accomplished after eye territories (R-26). The overall distribution of the retrograde neuronal labeling was basically similar in enucleation. In the case of these two cortical districts and of the granular insular cortex, an angulated approach all of these experiments. A representative case, R-9, was used in order to introduce the pipette perpendicularly is described in detail in the following paragraphs. The to the surface of the injected areas, thereby avoiding leakage of marker along a pipette track in other territories few differences noted between the r R F p and c R F p of the cerebral cortex. For injections in the dorsal column cases will be pointed out separately below. A sumnuclei and in the caudal portion of the nucleus of the mary of our retrograde observations is presented in solitary tract, the dorsal surface of the lower medulla was Table 1. exposed in the atlanto-occipital space. The iontophoretic parameters used in the anterograde studies ranged from Case R-9 0.3/~A for 3 min through a micropipette with an internal tip diameter of 15-18/zm, to 1/~A for 8min through a In this case the H R P deposit was located in the micropipette with an internal tip diameter of 27 pm. The caudal part of the r R F p and involved peripherally

Parvocellular reticular formation afferents the intermediate reticular nucleus (Figs 1, 2R,S, 3A). T h e retrograde labeling p a t t e r n observed in R-9 is illustrated in Fig. 2. Telencephalon Cerebral cortex. A b o u t 2 5 % of all of the labeled n e u r o n s n o t e d in this case were in the cerebral cortex. The m a r k e d perikarya, m o s t of which were situated contralaterally in layer V, were f o u n d in rostral cortical areas. The largest n u m b e r of retrogradely labeled cells appeared in the first m o t o r area (see Ref. 74) (Fig. 2 A - C ) and in the primary s o m a t o s e n s o r y cortex ~24 (Fig. 2 B - G ) ; the m a r k e d n e u r o n s were c o n c e n t r a t e d in the rostroventral

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district o f each of these two cortical fields. A moderate n u m b e r of labeled perikarya were seen in the g r a n u l a r insular area, 22 a general visceral sensory cortex (Fig. 2E, G). Additionally, a very modest retrograde labeling was detected in the secondary s o m a t o s e n s o r y area (Fig. 2E, G) a n d in the rostral extent of the posterior a g r a n u l a r insular area (as delineated by Krettek a n d Price 59) (Fig. 2F). N o evidence of retrograde t r a n s p o r t was f o u n d in any o t h e r cortical territory. Other structures. A m o d e r a t e n u m b e r of m a r k e d cells were observed ipsilaterally in the central amygdaloid nucleus, mainly in its medial part (Fig. 2G, H). Fewer, faintly labeled, perikarya were

Abbreviations used in the .figures a AC ac acd aid aip aiv BC bst c cg cp cu cvl cV db DBC dg dh dr Fx G g ga gp gri Gv gv HI H2 ha hi hp ia IC in ip ir iV kf la lc li 11 lot lr mdl meV mf

ambiguus nucleus anterior commissure accumbens nucleus dorsal division of the anterior cingulate area dorsal agranular insular area posterior agranular insular area ventral agranular insular area brachium conjunctivum bed nucleus of the stria terminalis central nucleus of the amygdaloid complex central gray substance caudate-putamen cuneiform nucleus caudoventrolateral reticular nucleus spinal trigeminal nucleus, caudal part nucleus of the diagonal band of Broca decussation of the brachium conjunctivum dorsal paragigantocellular reticular nucleus "dorsolateral hump" region dorsal raphe nucleus fornix genu of the facial nerve gigantocellular reticular nucleus gigantocellular reticular nucleus, e part globus pallidus granular insular area ventral tegmental nucleus of Gudden gigantocellular reticular nucleus, ventral part Forel's field H i Forel's field H 2 anterior hypothalamic nucleus lateral hypothalamic area paraventricular nucleus of the hypothalamus anterior interpositus nucleus of the cerebellum internal capsule interpeduncular nucleus posterior interpositus nucleus of the cerebellum intermediate reticular nucleus spinal trigeminal nucleus, interpolar part K611ikerFuse nucleus nuclei of the lateral lemniscus locus coeruleus linear nucleus of the medulla lateral nucleus of the cerebellum, large-celled division nucleus of the lateral olfactory tract lateral reticular nucleus dorsolateral protuberance of the medial nucleus of the cerebellum mesencephalic trigeminal nucleus motor nucleus of the facial nerve

ML mm mr mrf MT MI mV ot oV P pb pbi pf pn pnc pno pnv pr prcm pVdm pVvl rd rfp rm rp rrf rv rvl s sbc sc sid siv SM SmI Smll snr SO st sV tv v vi vl vs zi VII XII

medial lemniscus middle part of the medial nucleus of the cerebellum median raphe nucleus mesencephalic reticular formation mammillothalamic tract first motor area motor trigeminal nucleus olfactory tubercle spinal trigeminal nucleus, oral part pyramid parabrachial area parabigeminal nucleus parafascicular nucleus of the thalamus pontine nuclei caudal pontine reticular nucleus oral pontine reticular nucleus ventral pontine reticular nucleus nucleus of Probst's bundle medial precentral area principal sensory trigeminal nucleus, dorsomedial part principal sensory trigeminal nucleus, ventrolateral part dorsal reticular nucleus of the medulla parvocellular reticular nucleus red nucleus, magnocellular part red nucleus, parvocellular part retrorubral field ventral reticular nucleus of the medulla rostroventrolateral reticular nucleus subthalamic nucleus subcoeruleus nucleus superior colliculus substantia innominata, dorsal division substantia innominata, ventral division stria medullaris of the thalamus primary somatosensory area secondary somatosensory area substantia nigra, pars reticulata optic nerve layer of the superior colliculus nucleus of the solitary tract supratrigeminal nucleus thalamic ventral complex medial vestibular nucleus descending vestibular nucleus lateral vestibular nucleus superior vestibular nucleus zona incerta facial nerve hypoglossal nucleus

S.J. SHAMMAH-LAGNADOet al.

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R-9

R-14

R-16

R- 42

R- 2 0

R-45

R-25

Fig. 1. Drawings of the injection sites in the eight cases in which the tracer deposit was centered in the RFp. rRFp cases: R-9 (HRP), R-14 (HRP), R-42 (HRP) and R-45 (HRP). cRFp cases : R-16 (HRP), R-20 (HRP) and R-25 (WGA-HRP). The case R-26 (WGA-HRP) encompassed rRFp and cRFp districts. The black areas represent the center of the injection sites, whereas hatching denotes regions of less dense marker deposit. Photomicrographs of cases R-9, R-45 and R-20 are shown in Fig. 3A-C~ located in the ipsilateral substantia innominata, chiefly in its dorsal division37'38 (Fig. 2G), and only sporadic marked cells appeared in the lateral part of the ipsilateral bed nucleus of the stria terminalis (Fig. 2F).

Diencephalon Subthalamic region. A small group of labeled neurons was seen ipsilaterally in the Field H2 of

Forel at the level of the caudal portion of the subthalamic nucleus (Fig. 21), Hypothalamus. A modest number of marked perikarya were noticed in the ipsilateral hypothalamus (Fig. 2H, I). Most of these labeled cells were located in the lateral hypothalamus, particularly in its posterolateral region in continuity with the labeling observed in the field H 2 of Forel. The remaining ones were seen in the parvocellular division of the

Parvocellular reticular tbrmation afferents

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Table 1. Summary of the present retrograde and anterograde observations Region that was retrogradely labeled and/or explored in our anterograde series Telencephalon Cerebral cortex First motor* Primary somatosensory* Secondary somatosensory Granular insular area* Posterior agranular insular area Prefrontal, medial division* Retrosplenial* Primary auditory* Primary visual* Bed nucleus of the stria terminalis Substantia innominata* Central amygdaloid nucleus* Diencephalon Subthalamic region, field H 2 of Forel* Lateral hypothalamic area Hypothalamic paraventricular nucleus Midbrain and isthmus region Superior colliculus, intermediate layer* Central gray substance* Mesencephalic reticular formation Red nucleus, parvocellular part Substantia nigra, pars reticulata Retrorubral field Dorsal raphe nucleus Mesencephalic trigeminal nucleus Parabrachial complex Pons Supratrigeminal nucleus Principal sensory trigeminal nucleus, dorsomedial part* Subcoeruleus nucleus Pontine reticular formation, magnocellular part Cerebellum Lateral nucleus "Dorsolateral hump" Lateral nucleus/"dorsolateral hump"* Posterior interpositus nucleus Medial nucleus* Medulla oblongata Parvocellular reticular formation* Intermediate reticular nucleus Dorsal paragigantocellular nucleus Gigantocellular reticular nucleus, principal part Gigantocellular reticular nucleus, ~ part Gigantocellular reticular nucleus, ventral part Nucleus of Probst's bundle Dorsal reticular nucleus Ventral reticular nucleus Rostroventrolateral reticular nucleus Caudoventrolateral reticular nucleus Spinal trigeminal nucleus* Medial vestibular nucleus* Descending vestibular nucleus* Nucleus of the solitary tract* Dorsal column nuclei Spinal cord, cervical segments

Density of the projection to the RFp

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The relative density of the projection to the RFp is indicated by the following symbols: + + + , high; + + , moderate; + , low; + / - , the existence of a low-density projection was suggested by the retrograde data, but could not be confirmed in anterograde experiments. Contralateral projections are indicated in parentheses. (?) The actual density of retrograde and anterograde labeling is uncertain because of the proximity of the injection site. The structures marked with an asterisk are those which were injected in our anterograde experimental series.

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Fig. 2(A-G). Caption on p. 410. paraventricular nucleus of the hypothalamus, and occasionally in the ipsilateral dorsomedial hypothalamic nucleus. Midbrain and isthmus region Mesencephalic reticular formation. This district, also termed deep mesencephalic nucleus, 79 displayed a moderate amount of HRP-positive perikarya (approximately 3% of the total number of the marked neurons observed in R-9); they were distributed on both sides of the brainstem with a contralateral predominance (Fig. 2J, K). Only rarely were labeled cells found bilaterally in the cuneiform and the pedunculopontine tegrnental nuclei.

Mesencephafic trigeminal nucleus. This structure contained ipsilaterally, throughout its rostrocaudal extent, a relatively high proportion of marked perikarya (Fig. 2L-O). Superior colliculus. A moderate collection of labeled neurons were seen contralaterally in the superior colliculus, mainly in the "flank" of the stratum griseum intermediate, sublayer b (see Bickford and Hall 13) (Fig. 2J, K). Other structures. A modest retrograde labeling was noticed bilaterally in the central gray substance (Fig. 2J-M), the dorsal raphe nucleus (Fig. 2L) and the parabrachial complex, in both its medial and lateral divisions, as well as in the K611iker-Fuse

Parvocellular reticular formation afferents

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l Fig. 2(H-P). Caption on p. 410.

nucleus (Fig. 2N, O), and ipsilaterally in the substantia nigra, mainly in the dorsolateral portion of its pars reticulata and in the adjoining retrorubral field (Fig. 2J, K). Very few marked cells were present contralaterally in the parvocellular district NSC 50/2

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of the red nucleus; as will be detailed below, the number of retrogradely labeled neurons counted in the latter structure varied appreciably in our R F p cases according to the location of the tracer deposit.

410


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Fig. 2(Q-Z). Fig. 2. Chartings of selected frontal sections showing the distribution of retrogradely labeled neurons in rat R-9, representative of the rRFp cases. Each dot represents one labeled neuron. In R and S the black areas represent the center of the injection sites, whereas hatching denotes regions of less dense marker deposit. A photomicrograph of the R-9 injection site is shown in Fig. 3A:


Pons Supratrigeminal nucleus. The largest number of labeled pontine perikarya were found in the supratrigeminal nucleus, which contributed approximately 6% of all of the marked neurons noticed in R-9; this labeling was distributed bilaterally, with an ipsilateral predominance (Fig. 20). Principal sensory trigeminal nucleus. A retrograde labeling of moderate density was noticed bilaterally, with some ipsilateral predominance, in the dorsomedial part of the principal sensory trigeminal nucleus v9 (Fig. 20, P). Pontine reticular formation. A modest amount of retrogradely marked cells were distributed bilaterally in the portion of the lateral pontine tegmentum that surrounds the motor trigeminal nucleus, referred to as the subcoeruleus and the intertrigeminal regions 79 (Fig. 20). Very few HRP-positive perikarya were observed bilaterally in the oral, caudal and ventral pontine reticular nuclei (Fig. 2N-P). Cerebellum A conspicuous neuronal labeling (about 9% of all of the marked perikarya observed in R-9) was noticed in the deep cerebellar nuclei (for terminology, see Ref. 57). The most heavily labeled districts were the "dorsolateral hump" (Figs 2P-S, 3F), chiefly ipsilaterally, and the dorsolateral protuberance of the medial nucleus (Figs 2R, S, 3G), contralaterally. A moderate number of marked neurons were found in the large-celled division of the lateral nucleus (Fig. 2P, Q); they were bilaterally distributed with a strong ipsilateral predominance. Retrogradely labeled cells were also seen in the ipsilateral posterior interpositus nucleus, mainly

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in the lateral portion of its large-celled division (Fig. 2R, S).

Medulla oblongata Reticular structures. A considerable proportion (about 30%) of all of the marked perikarya observed in R-9 were distributed bilaterally in bulbar reticular structures. Most of these labeled neurons were located in a dorsolateral strip that encompasses the RFp, the intermediate reticular nucleus and the nucleus of Probst's bundle (Figs 2P Y, 3D). A stillappreciable retrograde labeling was found in the dorsal paragigantocellular nucleus, the ~ part of the gigantocellular reticular nucleus and the dorsal and ventral reticular nuclei of the medulla (Fig. 2Q V, X, Y). Some marked cells, in addition, appeared scattered in the principal and ventral parts of the gigantocellular reticular nucleus, as well as in the ventrolateral medulla oblongata, including the districts of the latter territory that have been called rostro- and caudoventrolateral reticular nuclei (see Ref. 89) (Fig. 2Q X). Secondary sensory cell groups. A substantial neuronal labeling was seen bilaterally in the spinal trigeminal nucleus and, chiefly ipsilaterally, in the nucleus of the solitary tract. In the spinal trigeminal nucleus, the marked cells were found mainly in the oral part, clustered in its dorsal third (Fig. 2Q T); some HRP-positive perikarya were also detected in the interpolar part and more rarely in the caudal part of the spinal trigeminal nucleus (Fig. 2 U W , Y). Most of the labeled cells observed in the nucleus of the solitary tract were located in the rostral, orogustatory division, particularly in its ventrolateral sector (Figs 2T, U, 3E). The rather modest retrograde labeling found in the caudal, interoceptive division of

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Fig. 3. Brightfield photomicrographs of RFp injection sites (A~2) and of retrogradely labeled neurons (D~3). (A) Case R-9 (HRP); (B) case R-45 (HRP); (C) case R-20 (HRP); (D) nucleus of Probst's bundle; (E) nucleus of the solitary tract; (F) "dorsolateral hump" region; (G) dorsolateral protuberance of the medial nucleus of the cerebellum. Scale bar in A = 1 mm for A~C; scale bar in D = 20/~m for D~3.

412


that nucleus appeared mainly in its medial part, being noteworthy in the presence of vividly marked perikarya medially bordering the solitary tract at the level of the area postrema (Fig. 2V-Y). The number and distribution of labeled neurons in the nucleus of the solitary tract differed markedly in the present experiments, according to the location of the injection sites. Retrogradely marked cells were noted bilaterally in the vestibular complex, mainly in the medial and inferior nuclei and, to a lesser degree, in the superior and lateral nuclei (Fig. 2P-V). No retrograde labeling was seen in the cochlear or the dorsal column nuclei. Spinal cord

A few marked perikarya were detected on both sides of the cervical segments of the spinal cord, mainly in laminae IV, VII and VIII (Fig. 2Z). Other cases

Basically similar observations were made in the other r R F p cases, with one noted exception. In R-42 and R-45 (Figs 1, 3B), whose injection sites were small and almost confined to the rRFp, as well as in R-20, a c R F p case, which like R-42 and R-45 did not encroach into the intermediate reticular nucleus (Figs 1, 3C), only a scanty retrograde labeling was found in the caudal part of the nucleus of the solitary tract. This observation, also supported by the present anterograde data (see next section), strongly suggests that most of the retrograde labeling seen in the caudal part of the nucleus of the solitary tract in R-9 is attributable to the peripheral involvement of the intermediate reticular nucleus noted in that case. Even if such involvement may have also contributed to the retrograde labeling observed in the contralateral intermediate reticular nucleus in R-9 (the commissural reticular connections are known to be profuse), it should be noted that t h i s reticular nucleus still exhibited a substantial number of marked cells in the above-mentioned more restricted R F p cases. Some differences in the retrograde labeling pattern were found between r R F p and c R F p cases. Thus, while a prominent collection of marked neurons were seen in the nucleus of Probst's bundle and in the rostral division of the nucleus of the solitary tract following r R F p injections, only a few labeled cells appeared in these territories in the e R F p experiments. Conversely, the proportion of HRP-positive perikarya noticed in the medial district of the central amygdaloid nucleus and the parvocellular part of the red nucleus seemed to be higher in the cRFp cases. It should be pointed out that the latter nucleus contained a very expressive number of labeled neurons in those R F p cases (not shown in Fig. 1) whose injection sites substantially included the dorsal reticular nucleus of the medulla. In one rat (R-16) the tracer deposit, centered in the cRFp, also spread into the linear nucleus of the medulla (Fig. 1). I n this case,

the retrograde labeling present in the "dorsolateral hump" was particularly dense. Anterograde labeling experiments

In order to further substantiate some of the sources of input to the R F p suggested by our retrograde data, and also in an attempt to study the pathways and terminal fields of these RFp afferents, injections of W G A - H R P were made into many brain territories. Since physiological data indicate that the RFp may represent a site of convergence of peripheral information and cortical projections (see Ref. 14), numerous secondary sensory cell groups and cortical districts were systematically explored in the present anterograde experiments. Photomicrographs illustrating the location of representative injection sites in the diverse territories which were investigated in our anterograde experiments are shown in Fig. 4. A summary of the present anterograde observations can be found in Table 1. Cerebral cortex. The cortical injections were placed in either the first motor area, the primary somatosensory cortex, the granular insular district, the medial division of the prefrontal cortex (extensively involving the medial precentral and the dorsal anterior cingulate territories59), the temporal (mainly the primary auditory area), the retrosplenial (its granular and agranular subdivisions) or the occipital (chiefly the primary visual area) districts. 79 None of these tracer deposits encroached upon the underlying white matter. Among the aforementioned cortical territories, only the first motor, the primary somatosensory and the granular insular areas gave rise to labeled fibers that, after coursing through the internal capsule, cerebral peduncle and pyramid, distributed themselves to the R F p bilaterally with a strong contralateral predominance; the crossing of the midline occurred basically at medullary levels. Six rats received injections in the first motor cortex. The W G A - H R P deposit was located in either the rostroventral extent (n = 1) (Fig. 4A), the rostral half (sparing, however, the rostroventral sector) (n = 4) (Fig. 4B) or the caudal portion of the primary motor area (n = 1). The present observations suggest the existence of some topographic arrangement in the corticoreticular projection under consideration. Thus, in the rostroventral case, a dense, though patchy, terminal field was noticed throughout the R F p (Fig. 5B). In the remaining rostral cases, a rather modest labeling was observed in a restricted ventral sector of the RFp, along the anteroposterior extent of this structure, while a substantial anterograde transport appeared, at the level of the area postrema, in the dorsal reticular nucleus of the medulla. In all of the aforementioned rostral cases, a light anterograde labeling also marked the linear nucleus of the medulla. In agreement with the retrograde data reported in the preceding section, no unequivocal evidence of anterograde transport was found in the R F p in the caudal case.


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J

M Fig. 4. Brightfield photomicrographs of WGA-HRP deposits in cases that are representative of the various territories that were explored in our anterograde series. (A, B) First motor cortex; (C) primary somatosensory cortex; (D) granular insular area; (E) medial division of the prefrontal cortex; (F) primary auditory cortex; (G) retrospleuial cortex; (H) primary visual cortex; (I) substantia innominata; (J) central amygdaloid nucleus; (K) superior colliculus; (L) subthalamic region; (M) medial nucleus of the cerebellum; (N) lateral nucleus of the cerebellum/"dorsolateral hump" region; (O) central gray substance; (P) principal sensory trigeminal nucleus; (Q) spinal trigeminal nucleus, oral part; (R) medial vestibular nucleus: (S) descending vestibular nucleus; (T,U) nucleus of the solitary tract; (V) dorsal column nuclei. Scale bar = 1 ram.

In four animals large tracer deposits were placed in the rostral portion of the primary somatosensory area (Fig. 4C). In all of these cases the entire anteroposterior extent of the R F p was peppered with dust-like anterograde transport. Injections of W G A H R P practically confined to the granular insular cortex were obtained in two rats (Fig. 4D). Labeled fibers in these cases distributed themselves chiefly to the dorsal sector of the c R F p (Fig. 5A).

Substantia innominata. In one case a W G A - H R P deposit involved both the dorsal and ventral divisions of the substantia innominata and also infringed slightly upon the central amygdaloid nucleus. In two other cases the injections were centered in the dorsal division of the substantia innominata and did not spread into the central amygdaloid nucleus. In the latter two cases an appreciable W G A - H R P leakage was noticed along the pipette track in the caudal portion of the primary somatosensory cortex

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Fig. 5. Darkfield photomicrographs showing anterograde labeling in the RFp after WGA-HRP injections in the granular insular area (A), rostrolateral district of the first motor area (B), substantia innominata (C), medial nucleus of the cerebellum (D), "dorsolateral hump" region/lateral nucleus of the cerebellum (E), medial vestibular nucleus (F). Scale bar = I00 ttm. and in the globus pallidus (Fig. 4I); it should be remembered, however, that none of these territories showed retrogradely marked cells following tracer deposits in the RFp. In each of the three rats under consideration, dust-like anterograde transport was observed mainly in the dorsal portion of the ipsilateral cRFp (Fig. 5C). Central amygdaloid nucleus. In two animals the injection sites were located in the central amygdaloid nucleus with no involvement of the substantia innominata (Fig. 4J). In each of these cases, a small bundle of labeled amygdalofugal fibers emerged from the densely innervated parabrachial area and, traveling ventrolaterally between the principal sensory and the motor trigeminal nuclei, reached the ipsilateral RFp. In agreement with the retrograde data previously described, the cRFp appeared to be marked more prominently than the rRFp. Subthalamic region. One rat received a large W G A - H R P deposit centered in the field H 2 of Forel but encompassing also the subthalamic nucleus, the zona incerta, the field H~ of Forel, and, peripherally, part of the lateral hypothalamus; a conspicuous leakage of the marker was present along the pipette track in the thalamus (Fig. 4L). Issuing from the injection site, descending labeled fibers surrounded the dorsal and ventrolateral margins of the magno-

cellular division of the red nucleus and further caudally, in the pontine tegmentum, coursed in the subcoeruleus and the intertrigeminal districts. This contingent led to a terminal field in the ipsilateral RFp that marked primarily the cRFp. A very light anterograde transport was also present in the contralateral cRFp. Superior colliculus. In two cases the injection sites were located in the lateral third of the superior colliculus and involved its superficial and deep layers without infringing upon the mesencephalic reticular formation (Fig. 4K). The labeled tectofugal fibers, after crossing the midline, incorporated themselves into the predorsal bundle and gave rise to a light anterograde transport in the contralateral RFp. In these cases, a moderate labeling was also noticed contralaterally in the linear nucleus of the medulla. It should be pointed out that, although the tectoreticular projections, taken as a whole, did not seem to be very conspicuous, the magnocellular reticular nuclei were appreciably more marked than the RFp. Central gray substance. In two animals massive tracer deposits were placed in the periaqueductal central gray substance. In one of these cases, the injection site encroached slightly on the adjacent mesencephalic reticular formation (Fig. 40). In each

Parvocellular reticular formation afferents of these rats, a moderately dense terminal field was seen, predominantly ipsilaterally, in a restricted ventral sector of the bulbar tegmentum that included the ambiguus nucleus and the adjacent intermediate reticular nucleus. Deep cerebellar nuclei. Two rats received large injections centered in the medial cerebellar nucleus; these W G A - H R P deposits also substantially involved the cerebellar cortex, and, peripherally, the most medial part of the anterior interpositus nucleus (Fig. 4M). Issuing from the injection site, labeled fibers crossed the midline and incorporated themselves into the hook bundle of Russell. Part of this fiber contingent passed along the medial border of the inferior cerebellar peduncle and turned ventrally toward the contralateral RFp. A substantial dust-like anterograde labeling filled the r R F p (Fig. 5D). This labeling became gradually more sparse along the rostrocaudal dimension of the cRFp, and disappeared just caudal to the level of the linear nucleus of the medulla In two cases the W G A - H R P injections involved the "dorsolateral hump", the lateral cerebellar nucleus, the overlying cerebellar cortex, and, to a lesser degree, the lateral portion of the anterior interpositus nucleus (Fig. 4N). Emerging from the injection site, labeled cerebellar fibers ran medially and collected in the lateral half of the brachium conjunctivum. Part of this contingent, stemming from the brachium conjunctivum, gave rise to a conspicuous ipsilateral descending bundle that coursed ventrolaterally and distributed itself in the RFp. The latter structure exhibited anterograde labeling throughout its whole extent, but two focuses of denser innervation were noticed, one encompassing the r R F p (Fig. 5E), and the other, the linear nucleus of the medulla. Contralaterally, a substantial anterograde transport was observed in the latter nucleus, but the R F p itself appeared very lightly marked. Secondary sensory cell groups. In two animals the tracer deposits, placed in the principal sensory trigeminal nucleus, did not intrude significantly into the parabrachial area or the supratrigeminal nucleus. In one of these cases the injection site involved the caudal two-thirds of the principal sensory trigeminal nucleus (Fig. 4P), and, in the other, it also included the rostral extent of the oral spinal trigeminal nucleus. Similar observations were made in these two experiments. A rather modest anterograde labeling was found bilaterally in the r R F p and, ipsilaterally, in the cRFp. Six rats received injections in different districts of the spinal trigeminal nucleus. The injection sites were practically confined to the oral part in three cases (Fig. 4Q) and to the interpolar part in two other animals; in the remaining rat the tracer deposit massively involved the interpolar part and the caudal district of the oral part. In all of the cases in which the oral part was involved, labeled varicosities were seen along the anteroposterior extent of the ipsilateral

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R F p and in the contralateral rRFp. No conclusive evidence of anterograde transport was found in the R F p after massive injections of the interpolar part; it should be noted that in the latter cases a relatively substantial anterograde labeling marked the medial, magnocellular division of the reticular formation. In two rats the W G A - H R P deposits were small and basically limited to the rostral part of the nucleus of the solitary tract, with only a minimal spread into the medial and inferior vestibular nuclei (Fig. 4T). In two other animals the injection sites were located in the caudal part of the nucleus of the solitary tract and also involved the hypoglossal nucleus and, very slightly, the gracile nucleus (Fig. 4U). The distribution of the anterograde labeling noticed in medullary reticular districts differed markedly according to the location of the injection site, In the rostral cases, an apparently dense terminal field was observed ipsilaterally in the R F p district subjacent to the injected territory (between levels R and U of Fig. 2), while only a modest anterograde transport was detected in the remaining ipsilateral RFp. In the caudal cases, on the other hand, labeled fibers issuing from the injection site described bilaterally an arch in the bulbar tegmentum en route to their ventrolateral medullary targets and, along their path, marked the intermediate reticular nucleus, with little if any spread over the R F p itself. Three rats received tracer deposits in the vestibular nuclei. In one animal the injection site was practically restricted to the medial vestibular nucleus (Fig. 4R). In another rat the W G A - H R P deposit was centered in the inferior vestibular nucleus with an inadvertent leakage of the marker along the pipette track in the cerebellar cortex and the posterior interpositus nucleus. The injection site in the third animal involved both the medial and the inferior vestibular nuclei (Fig. 4S). Similar observations were made in these three cases, in which few if any labeled varicosities were seen in the RFp. In fact, the anterograde labeling which was noticed bilaterally in the RFp appeared to represent, basically, fibers-of-passage en route to the contralateral vestibular nuclei (Fig. 5F). In addition a bilateral terminal field, of moderate density, was observed in the magnocellular division of the reticular formation. After injections centered in the dorsal column nuclei (Fig. 4V), no evidence of terminal labeling was found in the RFp, DISCUSSION

The present study represents the first systematic attempt to provide an overall map of the afferent connections of the R F p in a single species, the rat, using combined retrograde and anterograde tracing techniques. Our results suggest that the R F p receives its main input from several areas of the cerebral cortex, districts of the reticular formation,

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particularly the mesencephalic and medullary ones, the supratrigeminal nucleus and the deep cerebellar nuclei. Moderate to substantial sources of RFp afferents appear to come from the central amygdaloid nucleus, the parvocellular division of the red nucleus, and the orofacial and gustatory sensory cell groups (the mesencephalic, principal and spinal trigeminal nuclei and the rostral part of the nucleus of the solitary tract), whereas many other structures (including the substantia innominata, the field Hz of Forel, hypothalamic nuclei, the superior colliculus, the substantia nigra, pars reticulata, the retrorubral field and the parabrachial complex) seem to constitute more modest additional input sources. These observations, in general, confirm those of other investigators and reveal more details on the precise origin and terminal field of some of the RFp afferents. Some of the presently described projections were observed to be topographically distributed within the RFp. Thus, the medial and the "dorsolateral hump"/lateral nuclei of the cerebellum, the nucleus of Probst's bundle and the rostral part of the nucleus of the solitary tract seem to innervate the r R F p more prominently. In contrast, the parvocellular division of the red nucleus, and a constellation of structures concerned with central autonomic regulation, the granular insular cortex, the substantia innominata and the central amygdaloid nucleus appear to project more substantially to the cRFp. According to a conceptual framework originally proposed by Brodal, 15 the R F p is viewed as the sensory/associative district of the reticular core. As such, it is thought to represent an intermediate link between sensory structures and the medial magnocellular reticular fields, identified as the effector components of the reticular apparatus. The present observations in conjunction with other hodological evidence argue against this traditional concept of the role of the RFp. In fact, our findings indicate that only a restricted group of sensory nuclei related to oro-gustatory functions substantially innervate the RFp. Moreover, several structures that can conceivably channel sensory information, such as the spinal cord, the spinal trigeminal nucleus, vestibular nuclei, the cochlear nucleus, the nuclei of the lateral lemnis•cus and the superior colliculus, are known to send direct projections to the medial magnocellular reticular core (e.g. see Refs 1, 12, 15, 17, 19, 52, 56, 73, 81, 96, present anterograde observations). Prosencephalon Cerebral cortex. Cortical projections to the RFp have been described in different species essentially with anterograde tracing m e t h o d s . 6°'6j'66"75'9°'117"129'13°'131 The present combined retrograde and anterograde data, in general, confirm these reports and further underscore the impression gained from previous anterograde analyses that the R F p is largely dominated by inputs of orofacial origin.

According to the present results, the cortical projections to the RFp arise from relatively restricted territories, basically from layer V, and are chiefly contralateral. In the present material, the two cortical fields that appear to innervate more heavily the RFp are the first motor and the primary somatosensory areas. These projections issue essentially from districts that, according to electrophysiological mapping studies in the rat, 74'~24 appear to correspond to a motor representation of the jaw and tongue and a somatosensory representation of the snout. A projection from the primary jaw motor area to the R F p has recently been reported in an anterograde HRP study in the rat. 13~ Furthermore, in an anterograde investigation of the rat corticoreticular pathways, Newman et al. 7~ have pointed out that the RFp is innervated primarily by the face area of the first motor cortex, and similar findings have been reported in the opossum, 66 cat 6° and monkey. 6j In line with these anatomical data, short-latency excitatory responses, as well as burst discharges occurring in phase with rhythmical jaw movements, have been recorded in the RFp following electrical stimulation of the masticatory cortex in the rat. 72 A projection from the primary somatosensory cortex to the RFp has already been noticed in fiber degeneration material in the rat 7~'~7 and monkey. 61 In contrast to the present observations, the R F p input from the face representation of the primary somatosensory cortex was characterized as a light projection by Newman et al. 7~ This discrepancy might perhaps be ascribed to the fact that a more caudal region of the primary somatosensory cortex was explored in their study (see Figs 1 and 2 in Ref. 75). The present data indicate that additional cortical projections to the RFp originate from the secondary somatosensory area and from the granular and posterior agranular districts of the insular cortex. The existence of a projection from the granular insular area to the R F p has already been identified in the rat by Ruggiero et al. 9° with retrograde and anterograde W G A - H R P techniques. The evidence for a sparse input to the RFp from the posterior agranular insular area, suggested by the present retrograde experiments, fits well with the results of a recent Phaseolus vulgaris-leucoagglutinin (PHA-L) investigation in the ratJ 29 It is noteworthy that several of the subcortical targets of these insular districts, 2'9°'~29 including the central amygdaloid nucleus, the lateral hypothalamus, the parabrachial complex and the nucleus of the solitary tract, were seen to project to the RFp. From a functional point of view, the insular cortex appears to be involved in the regulation of a variety of visceral responses (see Refs 22, 90, 129), and its granular area has also been reported to receive thermal information of the oral cavity. 58 No other cortical area appears to send fibers to the RFp. In particular, the existence of a substantial projection from the medial division of the

Parvocellular reticular formation afferents prefrontal cortex, reported in the rat by Newman et al., v5 could not be confirmed in the present study. Other telencephalic structures. The basal forebrain continuum formed by the central amygdaloid nucleus, the dorsal division of the substantia innominata, and the lateral part of the bed nucleus of the stria terminalis is often thought as a single anatomical entity, functionally involved in multiple autonomic and behavioral mechanisms (e.g., see Refs 37, 38, 83). In the present material these three districts, but more conspicuously the medial portion of the central amygdaloid nucleus, appear to send fibers to the RFp. A projection from the medial sector of the central amygdaloid nucleus to the R F p has been reported in the rat in retrograde tracing studies 69'113 as well as with the PHA-L technique, 3° and in the cat in a combined retrograde HRP and autoradiographic investigation. 48 Recent anatomical evidence brought forth by Takeuchi et al.~°9'~~o indicates that this amygdaloreticular pathway may be relevant for the control of ingestive behavior. In combined light and electron microscopic studies, these authors demonstrated the existence of a disynaptic chain leading from the central amygdaloid nucleus to the motor trigeminal nucleus, whose intermediate link lies in the ventral part of the rRFp, ~° and that this descending amygdaloid system makes synaptic contacts with cells of the superior salivatory nucleus, located in the RFp.L~9 In line with our retrograde observations, a light projection from the bed nucleus of the stria terminalis to the R F p has been reported in an anterograde W G A HRP study in the rat. H8 Such a projection, according to autoradiographic evidence, appears to be more prominent in the cat. 46 The existence of a modest input to the RFp from the dorsal division of the substantia innominata, supported by the present retrograde and anterograde experiments, is compatible with the retrograde data reported in the rat 38 after W G A - H R P injections into the dorsomedial medulla which included the adjacent tegmental field. Interestingly enough in view of the important participation of the R F p in oral motor performances, electrophysiological studies 8v have disclosed, in the substantia innominata, units responsive to the sight and/or taste of food, and whose activity is influenced by the motivational state of the animal. Diencephalon Subthalamic region. Autoradiographic evidence 82"~2-~ and HRP retrograde experiments (see

Fig. 4 in Ref. 69) in the rat indicate a small input from the zona incerta and fields of Forel to the RFp. Besides confirming the existence of this subthalamoreticular connection, the present observations suggest that the referred pathway arises predominantly from the field H 2 of Forel. It is of note that subthalamic projections to the mesencephalic reticular formation '~7 and magnocellular bulbopontine nuclei, 96'123in contrast, originate essentially from

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the dorsal region of the zona incerta and field H~ of Forel. Hypothalamus. The present HRP retrograde data indicate that hypothalamic inputs to the RFp are relatively modest and originate essentially from the lateral hypothalamic area and the parvocellular division of the paraventricular nucleus of the hypothalamus. A small projection from the latter nucleus to the R F p has been mentioned by Mehler 69 in a retrograde HRP investigation in the rat. On the other hand, Ter Horst et al., 112 using a similar technique, described stronger and more diversified hypothalamic projections to the RFp in the rat. in their report, besides the aforementioned hypothalamic district, the dorsomedial, ventromedial and posterior hypothalamic nuclei were shown to contribute afferents to the RFp. Such discrepancies could perhaps be ascribed to different locations of the injection sites within the RFp. Thus, whereas in the present work and in Mehler's 6~ study the injections were located in the dorsal sector of the RFp, Ter Horst et al. ~2 have explored a ventral RFp district adjacent to the nucleus ambiguus. Projections from the lateral hypothalamic area and the paraventricular nucleus of the hypothalamus have also been reported in autoradiographic studies in the rat 49'5°'J~8and cat. 4~ In addition, recent PHA-L evidence in the rat 5~64 indicates that these hypothalamic districts profusely innervate the ventral part of the RFp, which also appears to receive a lighter projection from the dorsomedial hypothalamic nucleus. Using combined retrograde and anterograde tracing techniques, it has been shown that the paraventricular 5~ and the lateral hypothalamic ~' efferents give rise to a dense network around the preganglionic neurons of the superior salivatory nucleus, like those of the central amygdaloid nucleus, mentioned previously. Brainstern Reticular structures. The present retrograde findings indicate that, taken as a whole, the structures that belong to the main reticular formation constitute a very prominent bilateral source of RFp afferents. However, striking quantitative differences appear to exist among these reticular projections. The RFp seems to receive a heavy input from dorsolateral districts of the bulbar reticular core (primarily from the contralateral RFp, and also from the intermediate reticular nucleus and the nucleus of Probst's bundle), less dense but still appreciable projections from the mesencephalic reticular formation, the dorsal paragigantocellular nucleus, the ~ part of the gigantocellular reticular nucleus and the dorsal and ventral reticular nuclei of the medulla, and only a modest contingent of fibers from the oral and caudal pontine reticular nuclei, the principal and ventral parts of the gigantocellular reticular nucleus and the rostroand caudoventrolateral reticular nuclei. Similar findings, although only briefly described, have been reported by Mehler, 69 in the rat, with the

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retrograde H R P technique. In particular, his charts contain a contralateral concentration of labeled cells in districts that correspond to the RFp, the intermediate reticular nucleus and the nucleus of Probst's bundle (see Fig. 4 in Ref. 69). The existence of dense RFp commissural connections has also been pointed out on anterograde evidence with PHA-L tracer in the rat HI and autoradiography in the rat "9 and catJ 2~ Afferents to the R F p from the mesencephalic reticular formation, the oral and caudal pontine reticular nuclei, the principal and ~ parts of the gigantocellular reticular nucleus, and the dorsal and ventral reticular nuclei of the medulla have been described in anterograde tracing studies in the rat, 1~'54'H9 and are supported by the present observations which indicate the dorsal paragigantocellular reticular nucleus as a further source of R F p inputs. The presence of modest retrograde labeling noticed in our R F p cases in the ventrolateral medulla may perhaps be due to uptake of tracer by fibers-ofpassage en route to the nucleus of the solitary tract. To date, only an R F p projection from the caudoventrolateral reticular nucleus has been corroborated with anterograde tracing techniques in the rat, H9'j27 and it appears to arise from a population of nonnoradrenergic cellsJ 27 In anatomical studies 73'111'~19 RFp efferents were shown to ascend and descend in the lateral reticular core and to profusely innervate the lateral territories along their path. The medial reticular fields, on the other hand, appear to receive only a modest contingent of R F p fibers. 73'96,111'119The present findings suggest that the main sources of reticular input to the R F p arise from lateral bulbar districts. This further reinforces the view of a lateral reticular association system. While the medial bulbopontine reticular districts appear to primarily control the axial and proximal musculature, 99'~°° the lateral ones are related chiefly to orofacial musculature. 14'2°'24'34'43'45'71'72'111'114"116'119 The aforementioned profuse interconnections among the lateral districts of the reticular core would represent part of an anatomical substrate that, as stated by Siegel and Tomaszewski, 99 may allow the reticular formation to synthesize complex movement sequences from simpler elements. M i d b r a i n a n d isthmus. According to the present retrograde observations, besides the mesencephalic reticular formation, the sole substantial source of input to the R F p arising from isthmo-mesencephalic districts is the mesencephalic trigeminal nucleus. This projection has already been described with a variety of anterograde fiber tracing techniques in the rat 4'68's4'1°1 and also in retrograde investigations in the rat 4°'69'91and rabbit. 91 Ruggiero et al., 91 using HRP and W G A - H R P as retrograde tracers, reported that in the rat this projection originates exclusively from the caudal part of the mesencephalic trigeminal nucleus. The present evidence, also supported by Mehler's 69 retrograde findings in the rat (see Fig. 4

in Ref. 69), indicates that rostral mesencephalic trigeminal cells laterally bordering the periaqueductal central gray substance also contribute to the innervation of the RFp. This also appears to be the case in the rabbit. 91 The referred primary trigeminal afterents convey to the R F p information from jaw muscle spindles and periodontal receptors and appear crucially involved in orofacial reflex mechanisms (see Refs 4, 84, 91). A reciprocal projection from the R F p to the mesencephalic trigeminal nucleus has been reported in anatomical sS'91'm and electrophysiologicals5 investigations. The present data suggest that additional isthmomesencephalic projections to the RFp arise from the superior colliculus, red nucleus, substantia nigra, retrorubral field, dorsal raphe nucleus and parabrachial complex. Tectal, 69'81 nigra126'69'12° and retrorubraP 2° efferents to the R F p have been indicated in the rat with both retrograde and anterograde tracing techniques, and the nigroreticular pathway has also been confirmed by electron microscopy. 12° In line with these reports, our retrograde observations suggest that the referred RFp inputs originate from the lateral part of the intermediate layer of the superior colliculus, and from the lateral region of the substantia nigra, pars reticulata and the adjoining caudal retrorubral field. These districts were shown, in functional studies, to be involved in oral motor behaviors (see Refs 26, 31, 120). According to our retrograde observations the projection from the red nucleus to the R F p arises exclusively from the parvocellular division of that nucleus and is directed largely towards the cRFp district. A similar rubro-reticular pathway has already been documented in the cat with retrograde and anterograde tracing techniques; 47 although in that species the red nucleus cannot be subdivided into a parvocellular and a magnocellular part, the rubroreticular cells under consideration were described as much smaller than most of the rubrospinal neurons. 47 The existence of a light projection from the dorsal raphe nucleus to the RFp, inferred from our retrograde experiments, is consonant with serotoninimmunofluorescent data in the rat ~°5 and autoradiographic evidence in the cat. s° Parabrachial afferents to the R F p have already been described in the rat with retrograde tracer techniques 4° and also in autoradiographic material. 94 It should be pointed out that the parabrachial complex appears as the main recipient of ascending RFp pathways.40,73'111'l19 Our results confirm previous reports in the rat 64'~" of a projection from the central gray substance to a restricted ventral part of the bulbar tegmentum that encompasses the ambiguus nucleus and the adjacent intermediate reticular nucleus. P e r i t r i g e m i n a l district. The present retrograde resuits indicate that the R F p receives a dense projection from the supratrigeminal nucleus and only a modest one from the reticular territory that laterally and ventrally surrounds the motor trigeminal nucleus.

Parvocellular reticular formation afferents The existence of a supratrigeminal input to the R F p has been reported in the rat by Mehler 69 and substantiated in subsequent anterograde and retrograde axoplasmic transport studies. 4°'86 These studies also provided evidence of a reciprocal R F p projection to the supratrigeminal nucleus. The peritrigeminal district seems to have an important role in sensorimotor integration of oral behavior. It receives afferents from primary trigeminal fibers4'65 and the jaw area of the primary motor cortex 13°'131 and in turn innervates the trigeminal, facial and hypoglossal motor nuclei. 34'7k86'116 In electrophysiological studies peritrigeminal units were shown to be responsive to mechanical stimulation of the oral cavity and jaw movement, and the supratrigeminal nucleus was reported to exert monosynaptic inhibitory effects on masseteric-jaw-closer-motoneurons (see Refs 65, 128). Secondary sensory nuclei. The present observations indicate that, among the secondary sensory cell groups, only the sensory trigeminal complex and the nucleus of the solitary tract appear to innervate the RFp. A projection from the spinal trigeminal nucleus to the R F p has been documented in earlier fiber-degenerative experiments in the c a t 19'1°7 and on retrograde HRP evidence in the rat by Mehler. 69 The current retrograde and anterograde findings corroborate the existence of this projection and, in addition, suggest that the dorsomedial part of the main sensory trigeminal nucleus also contributes afferents to the RFp. To our knowledge the latter, rather modest, R F p input has not been identified in previous anatomical publications. In our material, the spinal cells that project to the dorsal RFp appear to be located primarily in the dorsal sector of the oral subnucleus-the sector topographically related to the mandibular branch of the trigeminal nerve. 28 No preferential distribution of the spinal trigeminal cells that give rise to RFp afferents was reported in Mehler's study. 69 The dorsal R F p also appears to receive significant direct projections from trigeminal cells whose peripheral processes join the mandibular nerve. 65 In agreement with the above-referred anatomical observations, units with a very gross somatotopic organization, whose pattern, though crude, resembles that of the adjacent trigeminal complex, have been recorded in the RFp. 77 In the context of a participation of the R F p in oral motor tasks it is interesting to note that the oral subnucleus and the dorsomedial part of the main sensory nucleus receive primary afferent projections from the oral region 98 and in turn send efferents to the motor trigeminal nucleus. 34"7HI6 The dorsomedial part of the main sensory nucleus is also reciprocally connected with the supratrigeminal nucleus. 86 Accordingly, in electrophysiological experiments a high percentage of neurons in the oral subnucleus were shown to be activated by mechanical and/or nociceptive stimuli from the oral cavity, 6 and an involvement of the dorsomedial part of the main

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sensory nucleus in the reflex control of jaw movements has been suggested (see TraversH4). In the RFp, units responsive to stimulation of intraoral mechanoreceptors have been identified.32 Anterograde transport studies in the rat, 7~'83 hamster T M and monkey, 1° and the present results suggest that the rostral and caudal parts of the nucleus of the solitary tract project to basically distinct medullary reticular districts. The rostral nucleus of the solitary tract innervates the RFp, more particularly its rostral extent (the rRFp and the rostral cRFp). Our retrograde data further indicate that this RFp input arises mainly from cells located ventrolaterally in the rostral nucleus of the solitary tract, the district reported to receive gustatory afferents and also trigeminal somatosensory fibers conveying thermal, tactile and nociceptive information (see Refs 28, 40). Similar retrograde observations have been reported in the hamster? A participation of the RFp in gustatory function is also underscored by its connections with the gustatory region of the parabrachial complex. 4°~-'5 Efferents from the caudal nucleus of the solitary tract, on the other hand, are primarily directed toward slructures located ventrally and medially to the cRFp. Reciprocal RFp connections to the above referred secondary sensory nuclei have been recently reported in a PHA-L study in the rat; ~H it is remarkable that they innervate precisely the nuclear districts that, according to the present observations, originate RFp afferents. The results of our axonal tracing experiments as well as those of others 1v'69 indicate that few if any secondary vestibular fibers project to the RFp. In consonance with these anatomical findings, only a few RFp units were found to be responsive to lateral tilt in cerebellectomized preparations, t°4 Cerebellum

From the analysis of the present material, it appears that the deep cerebellar nuclei represent an important source of input to the RFp. Our observations corroborate previous anatomical descriptions in the rat 25,33-88A26and provide information concerning the distribution of the contralateral fastigioreticular pathway in that species. In strong agreement with the retrograde findings of Rubertone et al. 88 in the rat, the present data indicate that the dorsolateral protuberance of the medial nucleus gives rise to a conspicuous projection to the contralateral RFp. Our anterograde experiments reveal that this fiber contingent emerges via the hook bundle of Russell and prominently innervates the rRFp and gradually more sparsely innervates the rostrocaudal dimension of the cRFp. This projection appears to exhibit marked interspecies variations. It has not been reported in autoradiographic investigations in the monkey 5'18 and according to fiberdegenerative evidence, it seems to be only modest in the cat. 122

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S.J. SHAMMAH-LAGNADO et al.

According to the present data, another important contingent of cerebellar fibers reaches the RFp through the ipsilateral descending limb of the brachium conjunctivum, a finding consistent with Fink-Heimer 33 and autoradiographic studies 25'~2~ in the rat. Our observations, again in strong agreement with previous retrograde data in the rat, 88'~26 indicate that the referred contingent of fibers arises primarily from the "dorsolateral hump" but also from districts of the lateral and interpositus nuclei. Cicirata et al. 27 have shown that the electrical stimulation of the "dorsolateral hump" evokes lip movements and interestingly, they have suggested that these effects could be mediated through anatomical connections with the RFp. Spinal cord

The present retrograde observations confirm the existence of a sparse spinal input to the RFp described in fiber degenerative studies 15'73and suggest that this input arises essentially from the cervical segments. A direct projection from the RFp to high spinal cord segments has been described in axoplasmic transport studies (e.g., see Refs 42, 44, 54). F u n c t i o n a l considerations

Some of the possible physiological implications of the present anatomical findings for oral motor behaviors, autonomic regulations, respiratory functions and sleep-waking mechanisms are discussed below. The R F p appears to play a crucial role in salivation, 29,5°,1°9 swallowing, ~6 chewing 72'j°8 and other complex behaviors involving the orofacial musculature. Anatomical s t u d i e s 14'24"34'43'45'71'111A16 indicate that the innervation of the motor trigeminal, facial and hypoglossal nuclei derives from local fiber connections in the lower brainstem--the propriobulbar syst e m - a n d that the R F p constitutes an important source of afferents to these motoneuronal pools. The R F p also sends projections to sensory cell groups related to oro-gustatory functions 85m'lH and thus, can potentially modulate these activities at the afferent level. The present data suggest that the R F p is dominated by inputs of orofacial origin. In fact, the R F p receives projections from the mesencephalic, main sensory (its dorsomedial part) and spinal trigeminal nuclei, the supratrigeminal nucleus and the rostral division of the nucleus of the solitary tract; these afferents convey information from jaw muscle spindles, periodontal mechanoreceptors and other intraoral tactile, thermal and nociceptive receptors, as well as gustatory signals to the RFp. Cortical projections to the R F p arise essentially from the orofacial representation of the first motor and primary somatosensory areas, and from the granular insular district, which is concerned with thermal information of the oral cavity. Physiological studies point to the

R F p as a site of convergence of peripheral and cortical information (see Ref. 14). In a series of brainstem transection studies carried out in the guinea-pig, the dorsal paragigantocellular and gigantocellular reticular nuclei were proposed to contain the central timing network for cortically induced rhythmical jaw movements (see Ref. 24). Evidence from lidocaine microinjection experiments into the pontomedullary reticular formation in the same species24 lends further support to this notion and indicates a possible participation of the intermediate reticular nucleus in the production of rhythmical jaw movements; this nucleus could either be part of the central timing network for mastication or of a circuitry that "enables" or "primes" this oscillatory network. According to the present retrograde observations, the reticular nuclei referred to above appear to project to the RFp, which could, therefore, function as an output system of the central timing masticatory network. Supporting this hypothesis Moriyama, 7: in an electrophysiological investigation in the R F p of the rat, recorded rhythmical jaw movement-related units that were antidromically activated from the motor trigeminal nucleus. Several other structures, among the presently described RFp input sources, have been shown in functional studies to evoke jaw movements. These structures include the central amygdaloid nucleus, the lateral hypothatamus, 62 the superior colliculus, the substantia nigra, pars reticulata and the mesencephalic reticular formation s9 (see Refs 26, 31, 91, 110, 120). Finally, our findings indicate that the R F p receives projections from other components of the propriobulbar system, namely the parabrachial complex, the peritrigeminal region, the intermediate reticular nucleus and the nucleus of Probst's bundle. Since this set of structures are also innervated by the R F p , 4°'73"86'1HJ~ the impression emerges of a highly interconnected system which would organize complex synergies involving orofacial musculature. The medullary lateral tegmental field seems to be importantly engaged in cardiovascular regulation. Much of the available evidence points to the intermediate reticular nucleus as a critical structure in this functional context (e.g., see Refs 7, 8, 23). It should be noted, however, that in the report of Chai et al. 23 some of the loci which evoked a large increase in arterial pressure, when stimulated electrically or chemically, are found in the dorsal part of the R F p (see Fig. 3 in Ref. 23). The anatomical picture of the dorsal RFp afferents, that emerges from the present study, indicates that this reticular district receives projections from many brain structures involved in cardiovascular regulation. These structures include territories of the insular c o r t e x , 22"9°A29 the dorsal substantia innominata, the central amygdaloid nucleus, the field H 2 of Forel, ~°~ the lateral hypothalamic nucleus, 21'6sthe parabrachial complex, the intermediate reticular nucleus 7'8'23 and the caudoventrotateral reticular nucleus (see

Parvocellular reticular formation afferents Refs 37, 38, 40, 83, 89). Many of these R F p input sources arise from limbic hypothalamic structures, a fact which would suggest an R F p role in the cardiovascular changes associated with the defense-alerting response. Several lines of evidence suggest a participation of the RFp in respiratory mechanisms. This structure coordinates special acts, such as swallowing ~6 and sneezing, 76 that are accompanied by changes of the respiratory pattern. Furthermore, in extracellular recording experiments, units with chemosensitive properties, or at least synaptically activated by pHsensitive neurons 3 were observed in the RFp. In the light of these functional findings, it is noteworthy that several of the structures identified here as sources of R F p afferents appear to be associated with respiratory control; these include the ventrolateral medulla, the parabrachial complex, the central amygdaloid nucleus, the bed nucleus of the stria terminalis and the insular cortex (see Refs 22, 30, 40, 83, 91, 102, 129). In recent reports some of the pathways through which the R F p can presumably affect the pattern of respiratory movements have been indicated. Thus, projections from the R F p to districts of the dorsomedial and ventrolateral medulla identified, respectively, with the dorsal and ventral respiratory groups have been described with anterograde PHA-L ~ and W G A - H R W °2 retrograde tracing techniques. On the other hand, both connectional and electrophysiological observations support the view that the heavy R F p input to the parabrachial complex is related to the oral/gustatory sphere, and not to respiratory phenomena (see Refs 40 and 125). In contrast to the rest of the reticular core, the RFp has been poorly explored in functional studies focused on sleep-waking mechanisms. Recently, Sakai and co-workers 93 disclosed in the RFp neurons showing tonic activation selective to paradoxical sleep; some of these units were noted to be antidromically activated from the motor trigeminal nucleus (for Refs, see Ref. 93). Anatomical data 24'34'71't16 suggest that many of the premotor neurons projecting to the motor trigeminal nucleus are distributed in the RFp. Taken together, these findings support a possible participation of the R F p in the control of the jawcloser muscular atonia occurring during paradoxical sleep. Among the sources of input to the R F p indicated in the present study, the dorsal raphe nucleus, the parabrachial complex and ventromedial bulbar reticular districts were reported to contain neurons with firing patterns phase-locked to paradoxical sleep (see Refs 36, 93, 106). A contribution of the medial bulbar reticular formation in processes that result in the motor inhibition observed during paradoxical sleep has also been suggested from neurotoxic lesion and chemical activation experiments (see Ref. 53). The mediodorsal pontine tegmentum, identified as a critical site for the generation of paradoxical sleep atonia (see Refs 53, 93, 106), according to our observations does not appear to send direct projec-

421

tions to the RFp. Recent anatomical 92 and electrophysiological93 data in conjunction with this paper indicate that commands from the mediodorsal pontine tegmentum may conceivably be transmitted to the R F p via ventromedial bulbar reticular districts. Several experimental observations, reviewed by Steriade and McCarley, ~°6 suggest that the process of cortical activation associated with electroencephalographic desynchronization appears to depend on activities widely distributed through reticular brainstem and supramesencephalic networks. Although a participation of the R F p in such phenomena awaits experimental confirmation, the hodological relationships of this reticular district support this hypothesis. The R F p sends dense projections to the parabrachial complex40.73~lll,119whose efferents join the main stream of ascending reticular pathways and distribute profusely to intralaminar thalamic nuclei and hypothalamic and subthalamic districts. 35"73'94According to the classical conceptual view of Brodal, ~5 the R F p can also act on prosencephalic targets through a synaptic link with medial magnocellular reticular territories. Finally, although with anterograde tracing techniques few R F p fibers appear to ascend beyond the rostral pons, a quite different picture emerges from fluorescent tracer investigations. 54'67 Indeed, following large diencephalic injections retrogradely labeled cells appear to be about equally distributed in both medial and lateral fields of the bulbopontine reticular core. In this context, it is significant to recall that our observations indicate that the RFp receives fairly substantial projections from districts of the reticular core that give origin to the ascending activating system (see Refs 15, 106).

CONCLUSIONS

Our results, obtained with the aid of retrograde and anterograde HRP transport techniques, provide a comprehensive account of the afferent connections of the R F p in the rat. Like other districts of the reticular core, the R F p receives projections from a host of different structures, particularly from its contralateral counterpart and other territories of the reticular formation, deep cerebellar nuclei and cortical areas. Perhaps, the hodological attribute that more specifically characterizes the R F p is its tight link with oral motor nuclei. 14'24'34"43"4531'111A16 Accordingly, the present investigation reveals that the R F p is largely dominated by inputs related to the orofacial and gustatory spheres. Taken as a whole, the present findings provide a potential neuroanatomical framework for the functionally demonstrated participation of the RFp in oral motor performances, autonomic functions, respiratory phenomena and sleep-waking mechanisms. From a conceptual point of view our data, in conjunction with other hodological evidence, call

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into question the traditional characterization o f the R F p as a relay station between sensory structures a n d the medial magnocellular reticular districts, typified as the effector c o m p o n e n t s of the reticular core.

Acknowledgements--This work was supported by grants from FAPESP (88/3978-8 and 90/2797-0), and CNPq (303264-84 and 303265-84). We thank Ana M. P. Campos, Roberto S. Nascimento and Roberto R. Valentim for expert technical assistance. M.S.M.O.C. was supported by a CAPES-PICD predoctoral fellowship.

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Afferent connections of the subthalamic nucleus: a combined retrograde and anterograde horseradish peroxidase study in the rat.

The reticular thalamic nucleus (RTN) of the rat: cytoarchitectural, Golgi, immunocytochemical, and horseradish peroxidase study.

Projections of the parvocellular reticular formation to the contralateral mesencephalic trigeminal nucleus in the rat.

A study of afferent input to the inferior olivary complex in the rat by retrograde axonal transport of horseradish peroxidase.

Horseradish peroxidase tracing of the lateral habenular-midbrain raphe nuclei connections in the rat.

Afferent connections of the nucleus raphe dorsalis in the cat as visualized by the horseradish peroxidase technique.

Afferent fibers in the hypoglossal nerve: a horseradish peroxidase study in the cat.

Afferent projections to the ventral tegmental area of Tsai and interfascicular nucleus: a horseradish peroxidase study in the rat.

The rat parvocellular reticular formation: I. Afferents from the cerebellar nuclei.

Retrograde transpost of horseradish peroxidase in afferent and efferent neurons from masseter muscle in the rat.

A combined horseradish peroxidase and Golgi study on the afferent connections of the ventrobasal complex of the thalamus in the cat.

Striatal afferent connections in the turtle (Chrysemys picta) as revealed by retrograde axonal transport of horseradish peroxidase.

The anterior ectosylvian sulcal auditory field in the cat: II. A horseradish peroxidase study of its thalamic and cortical connections.

Certain connections of the visual cortex of the monkey shown by the use of horseradish peroxidase.

A horseradish peroxidase study of the origin of central projections to the rat olfaction bulb.

Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat.

Horseradish peroxidase labeling of the efferent and afferent pathways of the avian tangential vestibular nucleus.

Demonstration of nigrothalamic connections in the cat by retrograde axonal transport of horseradish peroxidase.

Morphological heterogeneity within the cingulate cortex in rat: a horseradish peroxidase transport study.

Quantitative histological study of spinal afferent innervation on the ventral surface of the cat stomach by horseradish peroxidase (HRP) method.

Afferent projections to the cat locus coeruleus as visualized by the horseradish peroxidase technique.

Ultrastructure of the periaqueductal grey matter of the rat: an electron microscopical and horseradish peroxidase study.

Salivatory neurons innervate the submandibular and sublingual glands in the rat: horseradish peroxidase study.

Limbic projections to the cat thalamus. A horseradish peroxidase study.