Brain Research, 119 (1977) 21-41

21

© Elsevier/North-HollandBiomedical Press, Amsterdam - Printed in The Netherlands

AFFERENT PROJECTIONS TO THE CAT LOCUS COERULEUS VISUALIZED BY THE HORSERADISH PEROXIDASE TECHNIQUE

AS

K. SAKAI, M. TOURET, D. SALVERT, L. LEGER and M. JOUVET Ddpartement de Mddecine exp~rimentale, Facult~ de M~decine, Universitd Claude Bernard, Lyon 69008 (France)

(Accepted April 28th, 1976)

SUMMARY Using a recently developed retrograde tracer technique with horseradish peroxidase (HRP), attempts were made to identify afferent projections to the dorsolateral part of the pontomesencephalic tegmental areas, including the nucleus locus coeruleus (LC), locus subcoeruleus (LSC), parabrachialis lateralis (Pbl), K/511ikerFuse (K-F), reticularis pontis oralis (RPO), reticularis pontis caudalis (RPC), as well as an area rostral to the Pbl and dorsolateral to the brachium conjunctivum (mesencephalic reticular formation (MRF) area). It was revealed that the nucleus raphe dorsalis projects widely to all the studied pontomesencephalic tegmental nuclei. In addition, the LC was found to project to all the contralateral pontine tegmental structures which were studied. Following the injection of HRP into the dorsomedial part of the LC ('principal LC' or 'LC'), HRP labeled neurons were observed almost restricted to the nucleus raphe dorsalis. In contrast, following the injection into the ventrolateral part of the LC (LCa) or into other tegmental areas, the HRP containing neurons were observed widely distributed in the brain, extending from the diencephalon to the rhombencephalon. Especially in the case where injection was made into the LCa, numerous HRP positive cells appeared in the nucleus raphe pontis, magnus and substantia nigra, and were also identified in other brain structures, the topography of which corresponded to that of the catecholamine-containing neurons of the rat (group A1-A14). The present results confirm some previous reports on the afferent connections of the LC described in the rat, cat and rabbit, and further indicate the richness of afferent projections to the dorsolateral pontine tegmental areas. The present study also shows the heterogeneity existing between the main LC areas and the subcoeruleus areas, as well as between the dorsomedial part of the main LC (principal LC) and the ventrolateral part of this nucleus (LCa).

22 1NT RODU CTION The nucleus locus coeruleus (LC) of the cat and rat which lies in the dorsolateral part of the pontine tegmentum was shown to be rich in monoamine oxidase~7, 52, and to be composed, at least in part, of catecholamine-containing cell bodies and terminalsS,lZ,21,a6,37. Physiological studies suggest a wide range of functions of this nucleus, such as the regulation of respiration, micturition, cardio-vascular activity, motivation, as well as the regulation of sleep and wakefulness (for reviews see refs. 10, 22, 25, 48, 49). Efferent projections of the LC in both cats and rats are the object of a number of anatomical and histochemical studies, and the data indicate the widespread distribution of axons arising from cells in this nucleus 25,34-aT,45,5a,S7. However, only few afferents to this nucleus have been described in the cat4,49, 55. Recently, a retrograde tracer technique with the protein horseradish peroxidase (HRP) has been developed, and this technique has been applied in determining the cells of origin of various central fiber systems14,24, z6-~8,43. Several advantages of this technique with regard to classical retrograde degeneration techniques have been pointed out by previous investigators~4, 27,43. Therefore, in the present study, using this HRP technique, we have attempted to identify the afferent connections of the cat LC. In order to test the specificity, if it exists, of afferent projections to the LC area, afferents to the adjacent mesencephalic and pontine tegmental areas were also examined. A preliminary report has been published previously a°. MATERIALS AND METHODS The experiments were performed on 28 adult cats. Three of these animals were not injected intracerebrally, but were subjected to the same perfusion and incubation procedures as the experimental animals. A single injection of 0.1 /~1 of a 5 0 ~ H R P solution (Boehringer, Grade I) was made with a Hamilton 5 #1 syringe under stereotaxic guidance, except for the nucleus (n.) parabrachialis lateralis (Pbl) group in which double injections of 0.05 #1 was effectuated. The needle was inserted into the brain at a 45 ° angle with regard to horizontal plane in order to avoid the cerebellar tentorium. In 24 animals, the HRP solution was injected into one of the nuclei located in the mesencephalic and pontine tegmental areas as illustrated in Fig. 1. In one of these animals (case W 53), a marked outburst of H R P was observed in the cerebellar vermis, presumably at the moment of withdrawal of the needle, and thus the cat was used as a control. In the remaining one animal (case F 54), the same volume of the HRP was injected into the cerebellar vermis as a control for a possible diffusion of H R P along the needle tract. Subsequent to the injection of the H R P over period of 15-20 min, the needle was left in place for about 15 min before withdrawal from the brain. Every cat was sacrificed 13-24 h postoperatively (see details in Fig. 1), by a cardiac perfusion with a fixative containing 3~o paraformaldehyde-1 ~ glutaraldehyde in 0.1 M phosphate buffer (pH 7.2-7.4). In the present study, a relatively short survival time (usually 16-19 h)

23

~.-

P3

K F

';;

"

~

P4

P5

Fig. 1. Denomination of the pontomesencephalic tegmental areas used in this study. The localization of the HRP injections into the 24 cats are indicated with each survival time (in parenthesis). Abbreviations: 4, trochlear nucleus; 5MT, motor trigeminal tract; 5ST, spinal trigeminal tract; 6N, abducens nerve; 7N, facial nerve; BC, brachium conjunctivum; Cp, nucleus corporis pontobulbaris; CS, nucleus raphe centralis superior; Cu, cuneiform nucleus; DBC, decussation of brachium conjunctivum; DIV, trochlear decussation; GCm, griseum centrale mesencephali; GCp, griseum centrale pontis; GP, griseum pontis; IC, inferior colliculus; IP, interpeduncular nucleus; K-F, K611ikerFuse nucleus; Ldt, nucleus laterodorsalis tegmenti; Lid, dorsal nucleus of lateral lemniscus; Llv, ventral nucleus of lateral lemniscus; LC, nucleus locus coeruleus; a, locus coeruleus a; LSC, locus subcoeruleus; MLF, medial longitudinal fasciculus; mV, mesencephalic tract of trigeminus; P, pyramidal tract; PC, nucleus reticularis pontis caudalis; PO, nucleus reticularis pontis oralis; Pbl, nucleus parabrachialis lateralis; Pbm, nucleus parabrachialis medialis; RD, nucleus raphe dorsalis; RM, nucleus raphe magnus; RP, nucleus raphe pontis; Rtp, nucleus reticularis tegmenti pontis; SC, superior colliculus; SO, superior olive complex; T, nucleus of the trapezoid body; TB, trapezoid body; Td, dorsal nucleus of Gudden; Tv, ventral nucleus of Gudden; Vm, motor trigeminal nucleus; Vo, nucleus tractus spinals trigemini oralis; Vpd, Vpv, principal sensory trigeminal nucleus, dorsal and ventral divisions, respectively. Mesencephalic reticular formation (MRF) areas are shown in AI --AP0 HC planes. Nomenclature according to Taber ~6, Berman 1, Brown s and Maeda et al. 37.

was chosen, because o f r e p o r t s t h a t the r e t r o g r a d e t r a n s p o r t occurs between 72 m m a n d 120 m m / d a y 24,28, 29, a n d for certain systems a short survival time is necessary to identify the H R P c o n t a i n i n g neurons81, az, p r e s u m a b l y due to the inactivation o f the enzyme within the cell b o d y ~7. F o l l o w i n g the perfusion, the b r a i n was removed, placed in fresh fixative at 4 °C for 16-24 h, a n d then rinsed for a b o u t 24 h at 4 °C in

24 0.1 M phosphate buffer containing 5 i~o sucrose. Frontal sections 50 ktm thick were cut on a freezing microtome usually from Horsley-Clarke (HC) plane AI5 to PI5. In several cases, the sections were also taken in a plane which included the prefrontal cortex. After rewashing in Tris.HC1 buffer (0.05 M, pH 7.6) for 1-2 h, the sections were soaked for 30 min in a medium containing 0 . 0 5 ~ 3,3'-diaminobenzidine (DAB), and then incubated for 30 rain in a 0.05 ~ DAB solution containing 0.03 '~.~ hydrogene peroxide 1~,29. The sections were then washed, and mounted. Some sections were lightly counterstained with cresylecht violet. Each section was examined for the HRP containing cells under both light and darkfield illumination. Care was taken to differentiate the H R P labeled neurons from H R P labeled endothelial or vascular cells, according to the criteria of Nauta et al. 43. RESULTS

(I) Denomination of dorsolateral pontine tegmental structures In this report, we will generally use the nomenclature defined by Taber 56. Denominations of the structures used in this study are illustrated in Fig. 1. The dorsolateral tegmental areas in which the injection was made have been divided into: (1) nucleus locus coeruleus (LC); (2) locus coeruleus ct (LCa); (3) locus subcoeruleus (LSC); (4) n. parabrachialis lateralis (Pbl); (5) nucleus parabrachialis medialis (Pbm); (6) n. K~511iker-Fuse (K-F); (7) nucleus reticularis pontis oralis (RPO); and (8) nucleus reticularis pontis caudalis (RPC). In addition, the injection was also made into an area rostral to the Pbl and dorsolateral to the brachium conjunctivum (BC) at AI-AP0 HC plane (mesencephalic reticular formation (MRF) area) (as in the cats O 50, H 50, K 50, S 51). In the rabbit, Meessen and Olszewski 40 have divided the LC into two subnuclei: a more compact subventricular part and a ventrolateral part 'Pars a' with cells lying more loosely. Similarly in cats, based on cytological and fluorescence intensity criteria, Maeda et al. 37 and Leger 80 have also subdivided the LC into the two subnuclei (principal LC and LCa). Although, no precise boundary can be drawn between these two areas 1,30, we will designate, according to the previous authors a0,ST, 'principal LC' or 'LC' a region located in the dorsolateral part of the pontine periaqueductai grey (GCp) and medially to the mesencephalic trigeminal root (mV) (as in the experimental animals, R 52, W 52, J 54), and call 'LCa' a region located ventrolaterally and caudally to the principal LC, ventrally to the mV, and medially to the BC (as in the cases, B 53, E 53, E 54, H 54, O 54) (cf. Fig. 1). The results obtained in cases Q 53 and G 54, in which the injection sites extended over both LC and LCa, will be reported separately. On the other hand, in three animals (R 53, C 54, Q 54), we could not differenciate the injection into the Pbm from that into the adjoining LSC, and thus the results will be reported altogether as a result of the injection into the LSC area.

(2) Tegment o-t egmental connections As illustrated in Fig. 2, and as summarized in Table I, the LC and LCa widely

25

A

B

C

,,

o,;

~

"

(c 54) Fig. 2. Localization of HRP positive neurons obsc~ed in the brain stem following injection of HRP into the LC (A), LCa (B), and LSC (C), respectivelyi-~ts indicate HRP positive neurons. Blackened areas and surrounding shaded and stipple-outlined areas indicate the central and peripheral areas of the injection site. For explanation of abbreviations see Fig. 1.

--

area)

N. trigemini

principalis

conjunctivum

area ventral to the brachium

(MRF

MRF

--

--

--

(--)

(--)

--

--

--

(--)

(--) (--)

N. reticularis pontis caudalis (RPC) - N. reticularis pontis oralis (RPO) --

M R F area rostral to the Pbl

(+)

(--)

--

(K-F)

N. laterodorsalis tegmenti (Ldt)

N. K611iker-Fuse

(--) (--)

(+)

÷--

(--)

(--) --

--

+-- (--)

---

+

(--)

(?) (+)

-(--) +-- (÷)

N. parabrachialis medialis (Pbm) N. parabrachialis lateralis (Pbl)

+ +

++ (+) ÷ + + - - (.9)

+-+-- (--) -(--)

N. locus coeruleus a (LCa) N. locus subcoeruleus (LSC)

N. locuscoeruleusfLC)

LCa n--5

Injection site LC n:3

Studied structures

(--)

(+) (?)

(--) (--)

(.9)

--

-~-- (+)

---

+-- (+)

(.9)

+ - - (?) + - - (?)

+ -+--

L S C and Pbmn=3

(--)

(?)

--

-5

---

-(.9)

(?) (--)

(--) (--)

÷-- (÷)

+

~(?) + ÷

+ + (?) + + (?) ÷ (?)

Pbl n:2

+

--

---

--

+ +

÷ +

(.9)

(--)

(--) (--)

(+--)

(.9) (?)

+--(?) + + (.9) -(?)

K-F n:2

(--)

--

+++

--

+

(-9

(.9)

(+)

+÷+ ÷

(?)

+ --

--

(?) (+) (--)

+ + +

+

++

+

+ -++

(.9) (.9) (.9)

+ + +

RPC n= 1

+ + +

RPO n =1

-- ( + + ) --(+4) -- (--)

(.9)

(÷)

(--)

(.9)

(+)

(--)

(--)

(+++)

--(--)

_(_)

--

--

--

--

(+) --(+) (÷+) --(++~) ( + - ~ ~) - - ( - - )

(+) (+) (÷)

M R F area n:4

Ipsilateral connections are s h o w n in parenthesis. The presence o f H R P labeled neurons is indicated by ( ÷ ) , and the absence by (--). The density o f the H R P positive neurons in the various areas and nuclei is estimated subjectively following the symbols: + + + high; + + m o d e r a t e ; + low. + - - indicates that the labeled neurons are not observed in every case, while (?) indicates 'uncertainty' as to results because o f the presence o f reaction products found in structures close to the injection site.

Summary o f the tegmento-tegmental connections

TABLE I

to ox

27 project to the contralateral LC complex (LC, LCa, and LSC) and other pontine tegmental areas, namely, the nucleus parabrachialis lateralis (Pbl), K/511iker-Fuse (K-F), reticularis pontis oralis (RPO) and caudalis (RPC). As compared with the LC and LCa, only few HRP labeled neurons could be identified in the LSC area. The distribution of the HRP labeled cells was markedly similar for cats B 53, E 53, E 54, O 54, H 54, but differed from that observed in the case G 54, where the injection was made into the most rostral part of the LCa (see Fig. 1). The latter injection site corresponded to the plexus tegmentosus dorsomedialis described by RamonMoliner and Dansereau 46, which is rich in transversal and sagittal fibers. In this case, almost no HRP positive neurons were identified in the contralateral pontine tegmental areas, and few labeled neurons were observed in other parts of the brain (see below). The injection sites of the LCa group were centered either upon the pars ventromedialis of the caudal tegmentum peribrachiale or upon tegmentum entobrachiale 46, and thus located caudally and dorsally to the plexus tegmentosus dorsomedialis. In addition, the central area of these injection sites did not include the ventral aminergic bundle coming from the medulla oblongata, which runs ventral to the LCa. Taken together, the result that more cells are labeled following an LCa injection than from an LSC injection (as described above and will be described below) suggests that the labeling is a consequence of uptake by axon terminals and preterminals in the injected area rather than by axons of passage (damaged or undamaged) such as axons from the LSC past the LCa. Otherwise, it should be mentioned that following the injection into the LC complex or parabrachial nuclei, no HRP containing neurons occurred in the RPO and RPC (as well as in the nucleus gigantocellularis, see below), while such neurons appeared in the LC complex and parabrachial nuclei when HRP was injected into the RPO or RPC. In addition to the HRP labeled perikarya, we observed HRP labeled axons directed to the opposite LC complex. More frequently, these fibers crossed the midline at the dorsal and caudal part of the central superior nucleus of the raphe and beneath the ventral nucleus of Gudden. Apart from the RPC group, the HRP filled axons were also observed in the trochlear decussation, and, in few cases, in the pontine periaqueductal gray, too. Otherwise, except for the LC group, a cluster of HRP filled crossing fibers were generally observed at the decussation of the BC. Because of HRP reaction products found around the injection site, examination of possible connections within the LC complex and between the LC complex and other ipsilateral tegmental areas were not always successful. However, as summarized, in Table I, some connections could be revealed (see also Fig. 2). Interestingly enough, the MRF area (rostral to the Pbl and dorsolateral to the BC) received significant afferents from the ipsilateral LC, LCa, Pbm, Pbl, and nucleus laterodorsalis tegmenti (Ldt), while no afferent was observed from the contralateral tegmental areas, as it was the case for the Pbl.

(3) Afferentsfrom the raphe nuclei The results are summarized in Table II. The dorsal raphe nucleus widely project-

II

indicates

the

absence

Griseum

Substantia

centrale

nigra

mesencephali

--

rostralis

linearis

+

L__,

--

--

intermedius

linearis

.

centralis +

--

pontis

dorsalis

--

magnus

superior

--

LC n =~ 3

.

For

~

+

-'--

~

~

-;

-

! ~

~ ~

=

--

~ --

~ .

LCa n = 5

sections.

Infection site

corresponding

--

the

obscurus

raph6

of

pallidus

Nucleus

S t u d i e d structures

'~'

.

other

see

:

~ --

_~ _

~

-* .

.

.

.

.

LSC and Pbm n = 3

symbols

.

I.

:

--

.

-

--

--

~.

Pbl n = 2

Table

.

.

.

--

--

-

--

--

--

.

K-F n = 2

÷

--

--

~

~--

+

--

RPO n = ]

I

; w

RPC n ~ 1

,)

?

M R F area n = 4

S u m m a r y o f the afferent p r o f e c t i o n s to the p o n t o - m e s e n c e p h a l i c t e g m e n t a l areas arising f r o m the r a p h e nuclei, s u b s t a n t i a nigra, a n d m e s e n c e p h a l i c c e n t r a l gray

TABLE

tO Oo

29

D

E

F

....i

i

Fig. 3. A - C : low-power photomicrograph illustrating injection sites in cases W 52, E 54, and C 54, respectively. D: bright-field photomicrograph of H R P positive neuron in the contralateral LC following injection of H R P into the LCa (case E54) × 560. E" bright-field photomicrograph of H R P positive neurons in the area dorsolateral to the facial nucleus ipsilateral to the injection site (case E 54) x 140. F: bright-field photomicrograph of H R P labeled neuron in the contralateral nucleus tractus solitarii following injection into the RPC (case S 54) × 560. G : dark-field photomicrograph of H R P positive neurons in the ipsilateral substantia nigra following injection into the LCct (case B 53) x 120. H: higher magnification of the H R P labeled neuron seen in the substantia nigra (case B 53) x 560. I: dark-field photomicrograph of H R P positive neuron in the nucleus raphe dorsalis following injection into the LCa (case E 53) x 560.

30

1E541

Fig. 4. Localization of HRP containing neurons observed in the mesencephalon following injection of HRP into the LCa (case E 54). Dots indicate HRP positive neurons. Note the presence of HRP

labeled neurons in the substantia nigra and adjacent medial lemniscus area ipsilateral to the injection side. Abbreviations: DA, nucleus of Darkschwitsch; EW, Edinger-Westphal nucleus; FLM, medial longitudinal fasciculus; GC, griseum centrale; IP, interpeduncular nucleus; IS, interstitial nucleus of Cajal; LM, medial lemniscus; Li, nucleus raphe linearis interrnedius; Lr, nucleus linearis rostralis; NR, red nucleus; NPL, nucleus paralemniscalis; Ped, pedunculus cerebralis ; SC, superior coUiculus; SN, substantia nigra; TTC, tractus tegmentalis centralis; N III, oculomotor nucleus; l I1, oculomotor nerve. Nomenclature according to Jasper and Ajmone-Marsan 19 and Taber56. ed to the ponto-mesencephalic tegmental areas. Within the LC complex, it was the L C a which received more significant projections from this raphe nucleus (Fig. 2). In contrast, no H R P containing neurons were identified in the n. raphe centralis superior (CS). Furthermore, it should be emphasized that following the injection of H R P into the LCa, many H R P positive neurons were rather specifically observed in the raphe pontis, magnus (especially its rostral part), and linearis intermedius (see Table II).

(4) Afferents from the substantia nigra Subsequent to the injection of H R P into the LCa, we observed numerous H R P containing neurons in the ipsilateral substantia nigra, especially its caudal part. As shown in Table II, the L C a had a marked predominance for this connection. An example of the topography of the H R P positive neurons is illustrated in Fig. 4. As shown in this figure, the H R P containing cells appeared, more caudally, in the pars reticulata and adjacent medial lemniscus area. More rostrally, these labeled cells continued to appear in the pars reticulata, as well as in the pars compacta of the substantia nigra (see also Fig. 3).

(5) Afferents from other mesencephalic areas In all experimental groups except for the M R F one, H R P containing neurons

Area dorso-lateral to the superior olive complex Area surrounding the facial nucleus Area dorsal to the inferior olive complex Area surrounding the lateral reticular nucleus N. praepositus hypoglossi N. hypoglossi N. tractus solitarii N. dorsalis nervi vagi N. intercalatus

Studied structures

F o r symbols see Table I.

.

. . + + + + .

+

-. . --. . .

+ + + + +

LCa n=5

----

LC n=3

Injection site

.

. . + + +

+ +

+ + + +

L S C and Pbm n:3

+ + +

+

+ ++

Pbl n=2

- -

__

-}-__

+-+++

+ +

__

+

+

+--

+--

-F--

__

--

RPO n: 1

__

K-F n=l

Summary o f the afferent projections to the pontine tegmental areas arising from the bulbar reticular formation

TABLE III

__

+

-}-__

++ ++

++

+

+

RPC n= 1

i

\

/

~e

P6

~ 1

0

G¢

P7 °'/I/

~12

P8

i'

4

c~t

Gc RM

•

J

P9

~P1

5 (E 5z.)

Fig. 5. Localization of HRP containing neurons observed in the bulbar reticular formation and cerebellum following injection of HRP into the LCa (case E 54). Dots indicate HRP positive neurons. Abbreviations: 5 SM, 5 SP; alaminar spinal trigeminal nucleus, magnocellular and parvocellular divisions, respectively; 7G, genu of the facial nerve; 12, hypoglossal nucleus; 12N, hypoglossal nerve; AP, area postrema; CBL, lateral cerebellar nucleus; CBM, medial cerebellar nucleus; Cu, cuneate nucleus; DMV, dorsal motor nucleus of vagus; GC, nucleus gigantocellularis; IFC, infracerebellar nucleus; IFT, infratrigeminal nucleus; IN, nucleus interpositus; INT, nucleus intercalatus; IO, inferior olive complex; LR, lateral reticular nucleus; PH, nucleus praepositus hypoglossi; RB, restiform body; RFN, retrofacial nucleus; Rpa, nucleus raphe pallidus; Rob, nucleus raphe obscurus; S, nucleus of solitary tract; SA, stria acustica; Tr.s, solitary tract; VM, medial vestibular nucleus; VIN, inferior vestibular nucleus; VL, lateral vestibular nucleus; VS, superior vestibular nucleus. For other abbreviations see Fig. 1. Nomenclature according to Berman1 and Taber~.

33 were observed mainly in the ipsilateral mesencephalic periaqueductal gray, outside the dorsal raphe nucleus and laterodorsalis tegmenti (Ldt) (see Table II and Fig. 2). Furthermore, some HRP labeled cells were also identified in the Edinger-Westphal nucleus, particularly when the injection was made into the LCa, LSC, or RPC. When the injection was performed into the RPC, some HRP positive cells also occurred in other accessory oculomotor nuclei, ipsilateral to the injection side. Following the injection of HRP into the RPC or RPO, adjacent to the LC complex, some HRP positive neurons were exceptionally identified in the deeper layer of the superior colliculus. No such HRP containing neurons occurred in other experimental groups.

(6) Afferents from the bulbar reticular formation and cerebellum When the injection was centered in the principal LC, no HRP positive cells were observed in the bulbar reticular formation. In contrast, in other experimental animals, the HRP labeled cells were noticed in the structures as indicated in Table III. An example of the distribution of the HRP positive neurons observed following the injection into the LCa is illustrated in Fig. 5, Interesting enough, the topography of these labeled neurons corresponded to that of the catecholamine-containing neurons described in the rat by means of fluorescence histochemical techniqueslZ,44: area dorsolateral to the superior olive complex and ventromedial to the alaminar spinal trigeminal nucleus, area surrounding the facial and retrofacial nucleus (group A 5); area dorsal to the inferior olive complex (group A 3); area surrounding the nucleus ambiguus and lateral reticular nucleus (group A 1); nucleus tractus solitarii and dorsal motor nucleus of the vague (group A 2); and the lateral part of the roof of the fourth ventricle, just ventrolateral to the medial cerebellar nucleus (group A 4). It should be mentioned that these HRP labeled neurons generally appeared with a marked ipsilateral predominance and that the labeled neurons observed in the lateral part of the roof of the fourth ventricle occurred specifically following the injection into the LCa. Otherwise the following points should be also mentioned. (1) Following the injection into the RPC or RPO, a cluster of HRP containing cells were observed bilaterally in the nucleus praepositus hypoglossi. Further, in the former case, numerous HRP positive neurons occurred bilaterally in the infracerebellar nucleus, and in the ipsilateral retrofacial nucleus. In addition, the labeled neurons were also present in all the vestibular nuclei, but predominantly in the medial and superior vestibular nuclei; (2) the presence of the HRP positive neurons in the vestibular nuclei was also the case when the injection was made into the KSlliker-Fuse nucleus; (3) except for the case where the injection was made into the RPC, no HRP containing neurons were identified in the n.gigantocellularis or 'gigantocellular tegmental field 1'. The afferent projections from the cerebellum to the LC complex can be summarized as follows: the ventrolateral part of the medial cerebellar nucleus was labeled after HRP injection into the LCa, while the lateral cerebellar nucleus was labeled after the injection into the LSC, Pbl, or K-F. No projections from the cerebellar cortex were observed.

34 TABLE IV Summary of the afferent projections to the pontine tegmental areas arising from the hypothalamic and preoptic areas

For symbols see Table 1. Studied structures

Injection site LC n =3

LCa n:-5

L S C and Pbm n

Dorsal posterior hypothalamic area -Dorsal hypothalamic area -Lateral hypothalamic area -Perifornical area -Ventromedial hypothalamus - Subthalamic nucleus -Supraoptic nucleus -Arcuate nucleus -Suprachiasmatic nucleus -Preoptic area --

++ ++ ++ ++ + +--+. + + +. ~-

-

. +--

.

RPO n- I

RPC n-- 1

2

÷ ~÷ + -+-.

Pbl n-- 1

. -~--

-t ÷ + T ++ -+ + +-+--+--. . ++ + -. . + --

÷ -t -÷ + -----

(7) A f f e r e n t s f r o m the h y p o t h a l a m i c and preoptic areas

W h e n the injection was centered on p r i n c i p a l L C (or after cerebellar injections (case F 54); see below), no H R P l a b e l e d n e u r o n s c o u l d be identified. In c o n t r a s t , n u m e r o u s labeled n e u r o n s were o b s e r v e d in o t h e r e x p e r i m e n t a l a n i m a l s m a i n l y in the ipsilateral h y p o t h a l a m i c a r e a s such as d o r s a l a n d c a u d a l p a r t o f the p o s t e r i o r h y p o t h a l a m i c a r e a ( ' d o r s a l p o s t e r i o r h y p o t h a l a m i c a r e a ' in the T a b l e IV), d o r s a l h y p o t h a l a m i c area, lateral h y p o t h a l a m i c area, a n d areas s u r r o u n d i n g the fornix ('perifornical a r e a 9' in the T a b l e IV). T h e results are s u m m a r i z e d in the T a b l e IV, while examples o f the d i s t r i b u t i o n o f H R P l a b e l e d n e u r o n s f o l l o w i n g the injection into the L C a , Pbl o r R P C are illustrated in the Fig. 6A, B, a n d C, respectively. T h e t o p o g r a p h y o f these H R P labeled n e u r o n s was r e m a r k a b l y similar within the L C a g r o u p , within the L S C group, a n d between the R P O a n d R P C groups. I n o t h e r e x p e r i m e n t a l a n i m a l s except for the Pbl g r o u p , n o a t t e m p t was m a d e to e x a m i n e the presence o f H R P c o n t a i n i n g n e u r o n s in these areas. I t s h o u l d be n o t e d t h a t in the cases Q 53 a n d G 54 (injected into an a r e a extending over the L C a n d the m o s t rostral p a r t o f the L C a ) , the H R P l a b e l e d n e u r o n s were observed as the L C a g r o u p except t h a t no positive n e u r o n o c c u r r e d in the a r c u a t e nucleus ~1 a n d only few n e u r o n s a p p e a r e d in the d o r s a l a n d lateral h y p o t h a l a m i c areas, as well as in the perifornical area. I t s h o u l d be also m e n t i o n e d t h a t there exists similarity between the l o c a l i z a t i o n o f these l a b e l e d n e u r o n s o b s e r v e d in the h y p o t h a l a m i c areas a n d t h a t o f the catechola m i n e - c o n t a i n i n g n e u r o n s described in the r a t 3,12,1s, e.g., d o r s a l p o s t e r i o r h y p o t h a l a m i c a r e a ( g r o u p A 11), a r c u a t e nucleus ( g r o u p A 12). N o p r o j e c t i o n s f r o m the f r o n t a l cortex were observed.

35

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/

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-I

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(s 5 4 ) Fig. 6. Localization of HRP containing neurons observed in the bypotbalamic and preoptic areas following injection of HRP into the LCa (A), Pbl (B) and nucleus reticularis pontis caudalis (C), respectively. Dots indicate HRP positive neurons• Abbreviations; Ar, arcuate nucleus; arid, area hypothalamus dorsalis; CA, commissura anterior; Ch, chiasma opticum; CI, capsula interna; En, nucleus entopeduncularis; Fil, nucleus filiformis; FT, fasciculus thalamicus; Fx, fornix; GP, globus pallidus; Ha, hypothalamus anterior; HL, hypothalamus lateralis; Hp, hypothalamus posterior; Hvm, hypotbalamus ventromedialis; MFB, medial forebrain bundle; MI, nucleus mamillaris lateralis; Mm, corpus mamillare; NHvm, nucleus bypothalami ventromedialis; Ped, Pedunculus cerebralis; PVH, nucleus periventrieularis hypothalami; RPO, regio preoptica; Sch, nucleus suprachiasmaticus; SMx, commissura supramamillaris; SO, nucleus supraopticus; STh, nucleus subthalamicus; TMT, tractus mamillothalamicus; ZI, zona incerta. Nomenclature according to Jasper and AjmoneMarsan TM and Christ 9.

36

(8) Results obtainedfrom control animals In 3 uninjected but incubated control animals, no HRP labeled-like neurons were detected in any part of the brain. This negative finding rules out any possible confusion between retrograde HRP granules and granules endogeneously present in the brain such as lipofuscine, neuromelanin, etc.41, 43. When the injection was made into the cerebellar vermis, as in case F54, a number of H R P labeled neurons were detected: (1) bilaterally in the pontine grey, but with a marked contralateral predominance; (2) bilaterally in Taber's n. corporis pontobulbaris (Cp)56; (3) in the contralateral dorsal accessory nucleus of the inferior olive, and (4) bilaterally in the lateral reticular nucleus. Some HRP positive cells were also observed in the cuneate nucleus and in the inferior vestibular nucleus as well. Otherwise, no H R P labeled neurons were detected in any part of the brain. A cluster of H R P containing neurons were also identified in the same structures where the injection was made into an area extending over the LCa and LSC with a marked spread of HRP into the cerebellum (case W 53). In such a case, numerous HRP positive cells also occurred: (1) bilaterally in the superior, medial and inferior vestibular nuclei; (2) in the contralateral fastigial or medial cerebellar nucleus; (3) bilaterally in the nucleus praepositus hypoglossi; (4) bilaterally in the alaminar spinal trigeminal nucleus; (5) in the contralateral inferior olive complex, and (6) in the nucleus reticularis tegmenti pontis with contralateral predominance. The results are thus in general agreement with those reported by previous workersS, 16,47,54. The distribution of these labeled neurons was strictly different from that observed in our experimental animals, indicating that some slight diffusion of HRP solution observed along the needle tract was not effective in eliciting the retrograde transport of HRP. DISCUSSION (l) In the present study, a small volume (0.1 #1) of highly concentrated H R P solution (50 ~ ) was injected in all animals by means of a syringe system. The extent of H R P stained regions around the injection site, however, varied from animal to animal, and extended over several adjacent tegmental nuclei (cf. Figs. 2 and 3). This diffusion may be explained, at least in part, by the fact that some part of the LC complex can be considered as an 'open' nucleus ~s,46. Such a diffusion might be a limitation for the mapping of the specific projection to the subdivisions of the LC complex. However, several authors have pointed out that the effective zone of uptake tends to be limited to the tissue immediately surrounding the needle tip z°,zT,al,43. Our data confirm this interpretation, since the topography of the H R P labeled neurons was usually different when H R P was injected into the adjacent nuclei, and some specificity or predominance was usually apparent in the afferent connections of the given structure, e.g., presence of labeled neurons in the raphe pontis, magnus and substantia nigra with the injection into the LCa (see Table II). However, in the raphe pontis or substantia nigra, some labeled neurons were also identified following the injection into the adjacent LSC area. In this case, it is unclear whether the labeled neurons are really due to the uptake by axon terminals or preterminals located within the LSC area or due to the diffusion of

37 HRP into the LCa area. Therefore, the possibility of uptake in the adjacent nucleus by diffusion and subsequent retrograde transport cannot be excluded. Similarly, the possibility of uptake and subsequent transport by axons of passage that may be undamaged or damaged by the needle cannot be excluded27. Thus, as has been pointed out by LaVai127, any final conclusion cannot be drawn until further complementary fiber degeneration or autoradiographic experiments and further physiological experiments can be accomplished. Therefore, these limitations should be taken into consideration when the results of HRP studies are evaluated. (2) The present findings give further evidence of projections from the nucleus raphe dorsalis (NRD) to the LC area, as reported in the rat and cat with autoradiographic techniques4,11. In this regard, our data further suggest that the projections mainly arise from the rostral part of the NRD and that the LCa receives more significant projections from this raphe nucleus. The present study further supplies evidence of projections from the n. raphe pontis and magnus to the LC complex4, but supplies no evidence that could confirm the existence of LC afferents originating in the n. raphe centralis superior, the latter connection being reported in the cat by an autoradiographic study4 and suggested by a neurochemical study in the rat3L Further study is needed to resolve this problem. Recently, Saper et al. 51 have reported with autoradiographic techniques in the rat projections from the ventromedial nucleus of the hypothalamus to the LC and peribrachial areas. Our findings support in part their findings, but, in the cat, this connection seems to be less important than the projections arising from neighboring hypothalamic areas such as arcuate nucleus and perifornical area. On the other hand, Mizuno and Nakamura 4z have reported in rabbits, with degeneration study by electron microscope, a projection to the LC area originating in the ipsilateral hypothalamic areas. According to them, the effective lesions which lead to degeneration within the LC area are: (1) lateral and dorsal posterior hypothalamus; (2)anterior, lateral and medial hypothalamus, or (3) posterior hypothalamus with some supramammillary area involvement. Thus our findings are in general agreement with those reported by them. Our data clearly indicate, however, the presence of some contralateral projections to the LC area and further specify the cells of origin of the LC afterents arising from the hypothalamic areas. The same authors 42 have also reported that lesions placed in the mesencephalic central gray or mesencephalic dorsal tegmentum resulted in electrondense degenerated synaptic bags in the ipsilateral LC, while no degeneration occurred with lesions of the cerebral cortex, thalamus, or inferior colliculus. Our data in the cat thus accord with their findings in the rabbit. Afferents to the LC originating from the mesencephalic central gray have been also reported by Russe149. With regard to afferent projections from the cerebellum to the LC area, Snider 55 has recently reported with degeneration staining techniques that electrolytic lesions of the nucleus fastigii (NF) or cerebellar cortex resulted in degenerating terminals on cells in the LC, especially its ventrolateral part, corresponding to our LCa. In our study, following the injection into the LCa, some HRP neurons were specifically observed in the medial cerebellar nucleus or NF, especially its ventrolateral part

38 (see Fig. 6). Therefore, our data disclose further evidence of projections to the LC area from the deep cerebellar nucleus, but do not give any evidence of projections from the cerebellar cortex. Whether this discrepancy comes from limitations of our HRP technique remains uncertain. Buda et al. 7 have recently demonstrated in the rat that unilateral lesions of the LC resulted in a marked increase in tyrosine hydroxylase activity in the opposite LC, indicating a possible coerulo-coerular connection. Our results obtained in the cat might give an anatomical basis for this neurochemical study. Furthermore, the present results disclose the abundance of projections to the LC complex arising from whole brain structures rich in catecholamine-containingneurons described in the rat (group A1 to A14)3,t2,18,44. In this respect, the presence of projections from the substantia nigra to the LC should be emphasized, especially as this finding could open new frontiers for new investigations. In addition, our present results strongly suggest an interaction between the LC area (particularly LCa and LSC) and other catecholaminergic cell groups located from the diencephalon to the rhombencephalon, although the aminergic nature of these HRP labeled neurons remains to be determined. This anatomical data in the cat might be also applied, at least in part, to the rat, since H~Skfelt et al. 17 have recently reported that the rat LC, particularly its ventral part, receives a moderately dense adrenergic input. The presence of HRP containing neurons in the raphe nuclei (dorsalis, pontis and magnus) with the injection into the LCa also suggest an interaction existing between the catecholamine-containing cell groups and the serotonin-containing cell groups. Furthermore, in the light of results on LC efferents reported by Loizou37 and other authors 36,37,45,53 and on LC afferents as reported in this study, it seems also plausible that there exists a reciprocal innervation between the LC area and the brain structures in which HRP positive neurons were identified in this study. In conclusion, the present study indicate, on the one hand, the richness of afferent connections of the LC complex and other pontine tegmental nuclei and strongly suggest, as suggested by previous workers z0,37, the heterogeneity existing between the main LC area and the LSC area, as well as between the dorsomedial part of the main LC (principal LC) and the ventroJateral part of this nucleus (LCa). On the whole, the present results are in favor of the presence of anatomical connections existing within different central monoaminergic cell groups 7,17,22,23. Finally, the wealth and heterogeneity of the afferents to the dorsolateral pontine tegmental areas arising from the hypothalamus, raphe nuclei, and bulbar reticular formation might provide some anatomical basis concerning the functions of the LC complex and adjacent pontine tegmental areas, such as respiration s, cardiovascular activityTM, and sleep TM 22,23,29. ACKNOWLEDGEMENT

This work was supported by INSERM (U 52), CNRS (LA 162) and DRME (Grant No. 75-024).

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41

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