Distribution and efferent projections of corticotropin-releasing factor-like immunoreactivity in the rat amygdaloid complex

Brain Research, 382 (1986) 213-238

213

Elsevier BRE 11982

Distribution and Efferent Projections of Corticotropin-Releasing Factor-Like Immunoreactivity in the Rat Amygdaloid Complex MASAHIRO SAKANAKA1'*, TAMOTSU SHIBASAKI2 and KARL LEDERIS 1

~Department of Pharmacology and Therapeutics, Faculty of Medicine, The Universityof Calgary, Health Sciences Centre, Calgary, Alta. (Canada) and 2Departmentof Medicine, Tokyo Women's Medical College, 10 Ichigaya Kawado-cho, Shinjuku-ku, Tokyo (Japan) (Accepted February 4th, 1986)

Key words: corticotropin releasing factor-- amygdaloid complex - - cobalt-enhanced immunohistochemistry - - efferent projections

Using cobalt-enhanced immunohistochemistry, the tracing of retrograde transport of horseradish peroxidase (HRP) and experimental manipulations, a widespread localization of corticotropin-releasing factor-like immunoreactive (CRFI) structures in the rat amygdaloid complex, and CRFI-containing pathways from the amygdala to the lower brainstem, bed nucleus of the stria terminalis (bst) and ventromedial nucleus of the hypothalamus (VMH) have been demonstrated. By means of cobalt-enhanced immunohistochemistry, CRFI cells were detected in almost all the regions of the amygdala, including the central amygdaloid nucleus (Ce), basolateral amygdaloid nucleus (B1), intra-amygdaloid bed nucleus of the stria terminalis (Abst), medial amygdaloid nucleus (Me), amygdalohippocampal area (Ahi), posterior cortical amygdaloid nucleus (Aco), lateral amygdaloid nucleus (La), anterior amygdaloid area (AAA) and basomedial amygdaloid nucleus (Bm). Neural processes with CRFI were found in all of the above areas. The greatest density of CRFI fibres was observed in the Ce, the Me and Ahi. Unilateral lesions located in the Ce and adjacent areas caused an ipsilateral decrease in CRFI fibre number in the lateral hypothalamic area (LH), mesencephalic reticular formation (RF), dorsal (Dpb) and ventral (Vpb) parabrachial nuclei, mesencephalic nucleus of the trigeminal nerve (MeV) and in the lateral division of the bst (bstl). In addition, ipsilateral CRFI fibres decreased in number in the core and shell of the VMH after unilateral lesions of the corticomedial amygdala (CoM) and ventral subiculum (S). These findings suggest that (1) the CRFI cells in the Ce and adjacent areas innervate the Dpb, Vpb and MeV through the LH and RF; (2) the CRFI fibres in the bstl are supplied by the Ce and adjacent areas; and (3) the CoM and S give rise to the CRFI fibres to the VMH. The distribution of retrogradely transported HRP has confirmed these projections. Furthermore, combined HRP and immunohistochemical staining has demonstrated double labeled cells in the Ce following HRP injection into the Dpb, Vpb, MeV and bstl. This provides direct evidence for the amygdalofugal CRF-containing projections to the lower brainstem and bstl. Double-labeled cells were not seen in the CoM and S after HRP injection into the VMH.

INTRODUCTION

of efferent and afferent fibre connections in the amygdaloid complex 14,19,28,29,37,47,48,50-54,56,62,65,76,77,

The amygdaloid complex is an important component of the limbic system. Electrophysiological and behavioural studies have demonstrated that it is involved in a variety of physiological functions such as flight and defense behaviour, agonistic behaviour, respiration, salivation, chewing, heart rate, penile erection, gastrointestinal motility and ovulation 3°'31'34'43'57'61'66'72. In good agreement with this

83-85,90,91,97,103,115

apparent complexity of functions, recent studies carried out with use of degeneration, autoradiography and H R P techniques have also revealed a complexity

More recently, immunohistochemical studies have shown that various neuropeptides, including somatostatin 4"42'95'1°2, substance p15,38,58, neurotensin95,111 enkephalins26'98, vasoactive intestinal polypeptide 6°'95'1°4 and C R F 67'69'81"110, are present in the amygdaloid complex suggestive of an additional complexity of putative amygdaloid neurotransmitters. Furthermore, attempts have been made to exptore the efferent fibre connections of the peptidecontaining n e u r o n s in the amygdaloid complex by

* Present address: Department of Anatomy, Osaka Medical College, 2-7 Daigaku-Cho, Takatsuki, Osaka (569), Japan.

Correspondence: K. Lederis, Department of Pharmacology and Therapeutics, Faculty of Medicine, The University of Calgary, Health Sciences Centre, 3330 Hospital Drive N.W., Calgary, Alta., Canada, T2N 4N 1. 0006-8993/86/$03.50 (~) 1986 Elsevier Science Publishers B.V. (Biomedical Division)

214 means

of

knife

cuts

and

lesion

s t u d i e s 22"44'

93,94,96,112,113. However, little is known of the exact efferent fibre connections of the CRF neurons in the amygdaloid complex. Several suggestions have been made on this issue by Fellmann et al. 25 and Swanson et al. 11°, but experimental manipulations have not been applied to determine the precise projections of the amygdaloid CRF neurons. Using cobalt-enhanced immunohistochemistry and lesioning procedures in addition to a combined HRP tracing and immunohistochemistry, the present study describes CRF-containing pathways from the amygdaloid complex to the parabrachial nuclei, MeV, bst and VMH. MATERIALS AND METHODS Forty-nine male Sprague-Dawley rats weighing 60-90 g were used in this study. All animals were housed under constant temperature (20 °C) with a light-dark cycle of 12-h light and 12-h dark and given food and water ad libitum. The study was divided into 4 parts: (1) CRF-like immunoreactivity in the rat brain (9 normal animals, rats N 1-9), (2) lesion study (18 animals, rats L 10-27), (3) HRP study (10 animals, rats H 28-37), and (4) combined HRP and immunohistochemical study (12 animals, rats C 38-49).

CRFI in the rat brain Preparation of tissue. Four animals were prepared for analyzing the normal localization of CRFI. Each animal was anaesthetized with Nembutal (40 mg/kg) and perfused transcardially, first with 50 ml of heparinized saline, then with 30 ml of diluted Bouin's solution (pH 2.0) 2. Perfusion of the fixative was followed by a further PBS wash to clear out the Bouin's solution, then the animal was finally perfused with 200 ml of Zamboni's solution (pH 7.4) 12°. The brain was quickly removed, cut into 3 parts and sectioned at room temperature in the frontal plane at 40/~m with a Lancer vibratome. Serial sections were collected, kept in glass vials containing PBS for 2 h, then treated with ethanol (50% for 10 min, 70% for 30 min, 50% for 10 min) to facilitate the penetration of antibodies and rinsed again in PBS for 30 min. Five animals received an i.v. (25 #g/100 g b. wt.) or intra-amygdaloid (7.5 ~g/100 g b. wt.) colchicine in-

jection 24 h before sacrifice, then they were treated as described above. Antiserum against CRF. The CRF antiserum was produced in rabbits against synthetic rat CRF coupled with porcine thyroglobulin. The specificity of the antiserum was confirmed by radioimmunoassays which showed that this antiserum interacts well with rat CRF but exhibits no cross-reactivity with structurally similar peptides, ovine CRF, sauvagine and urotensin I, or with other peptides, including somatostatin, substance P, neurotensin, renin, glucagon, CCK-8, L H - R H and A C T H (T. Shibasaki, unpublished observations). The specificity of the antiserum was also checked by an absorption test, Control sections were treated with antiserum absorbed with an excess of synthetic rat CRF (15 gg/ml). The structures stained with antiserum against CRF were not seen in the sections incubated with the absorption control serum, The CRF-like immunoreactivity is defined throughout this paper as CRF immunoreactivity (CRFI). Immunohistochemical procedures. The sections were kept in a PBS solution containing 0.1% gelatin and 0.005% hydrogen peroxide for 20 min to suppress endogenous peroxidase activity of the tissue. Following a brief rinse in PBS, the sections were exposed to 10% NGS in PBS for 30 min, then rinsed in PBS containing 1% NGS and 0.25% carrageenan. Subsequently, they were stained while floating in glass vials according to the unlabeled antibody PAP method 1°~ and Co-GOD method 41's°, as follows. (1) The sections were incubated with the primary antibody diluted 1:2000 with PBS containing 1% NGS and 0.1% Triton X-100 (36-48 h at room temperature). (2) Rinsed twice in PBS containing 1% NGS, once in PBS containing 1% NGS and 0.25% carrageenan (15 min in each at 4 °C). (3) Incubated with the bridge antibody (goat anti-rabbit lgG, Sigma) diluted 1:50 with PBS containing 1% NGS and 0.1% Triton X-100 (2 h at room temperature). (4) Rinsed twice in PBS containing 1% NGS, once in PBS containing 0.25% carrageenan (15 min in each at 4 °C). (5) Incubated with PAP (DAKO) solution diluted 1:80 with PBS containing 0.1% Triton X-100 (1 h at room temperature). (6) Rinsed twice in PBS, once in 0.05 M Tris-HC1 buffer, pH 7.6 (15 min in each at 4 °C). The sections were then subjected to a slightly rood-

215 ified version of the Co-GOD method of Itoh et al. 41 and Oldfield et al. 8°, as follows. (7) The sections were washed briefly in 0.1 M Tris-HC1 buffer (pH 7.6). (8) Placed in a 0.5% solution of cobalt acetate in 0.1 M Tris-HC1 buffer (pH 7.6) for 10 min at room temperature. (9) Washed 3× in 0.1 M Tris-HCl buffer (pH 7.6), twice in 0.1 M phosphate buffer, pH 7.3 (10 min in each at room temperature). (10) Incubated for 12-16 h at 4 °C in a freshly prepared medium composed of 40 mg of DAB, 200 mg of fl-D-glucose, 40 mg of NHaC1, 0.5 U GOD (Sigma, Type V) and 100 ml 0.1 M phosphate buffer (pH 7.3). (11) Washed 3× in 0.1 M phosphate buffer, pH 7.3 (10 min in each at 4 °C). (12) The sections were mounted on gelatincoated slides, dried, dehydrated, and cover-slipped. The control sections were first incubated with antiserum absorbed with an excess of synthetic rat CRF (15 ktg/ml), after which they were processed as described above.

Lesion study Eighteen animals were used. To explore the efferent projection fields of CRFI cells in the amygdaloid complex, various CRF-containing areas such as the Ce, BI, Abst, Me, Ahi and Aco were unilaterally destroyed with the help of a stereotaxic apparatus in anaesthetized animals, by passing a DC current of 20 ~A for 30-60 min through a monopolar electrode. The animals, kept alive for 5 days after the operations, were deeply reanaesthetized, and perfused as described under 'Preparation of tissue'. Following the perfusion, the brains were quickly removed, cut into 40-ktm sections on a Lancer vibratome with close attention to the stereotaxic levels of the sections, then processed for immunohistochemistry. In this lesion study, the extent of lesion damage was carefully assessed under a bright-field microscope after Cresyi violet staining of sections containing a lesion site. HRP study Ten animals were used. In the lesion study, three CRFI-containing pathways were indicated: first, CRFI-containing pathway from the Ce, B1 and Abst to the parabrachial nuclei and mesencephalic nucleus of the trigeminal nerve, second, from the Ce, B1 and Abst to the bed nucleus of the stria terminalis, third, from the corticomedial amygdala (which consists of

the Me, Ahi and Aco) and ventral subiculum to the ventromedial nucleus of the hypothalamus. To confirm the amygdalofugal projections, HRP was iontophoretically injected into the parabrachial nuclei, MeV, bst and VMH. A 40% solution of HRP (Sigma, type VI) in a glass micropipette was connected to a high-voltage constant current, which provided a pulse-positive current (7 s on, alternating with 7 s off time). A single iontophoretic injection of HRP was given stereotaxically into the areas mentioned above in anaesthetized rats, by using a positive current of 2.5/tA for 15 min. Following a survival time of 24-48 h the animals were deeply reanesthetized and brains processed by the HRP method. Each animal was perfused transcardially with 100 ml of saline followed by 200 ml of 1.25% glutaraldehyde-0.5% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The perfusion of the fixative was frequently followed by 50 ml of 0.1 M phosphate buffer, pH 7.4, as recommended by Mesulam and Rosene 71, in order to control the suppressive effect of fixation on the HRP activity. Subsequently, the brain was removed, transferred to the same chilled buffer, 50-/~m sections cut on a Lancer vibratome and floated in a bath of the same buffer. One-half of the sections were exposed to DAB and the other sections to TMB 7°.

Combined HRP and immunohistochemical study The method developed by Wainer and Rye 116 was applied in 12 animals to obtain a direct evidence for the amygdalofugal CRFI-containing pathways shown in the lesion and HRP studies, though some modifications were made. Each animal was anaesthetized with Nembutal before the experiment. HRP was injected in a variety of brain regions such as the parabrachial nuclei, MeV, bst and VMH; a single injection of 0.04-0.5 ktl of 20% HRP dissolved in 0.2 M Tris-HCl buffer, pH 8.6, was delivered unilaterally over a 15-min period using a glass micropipette (i.d. 100/~m) mounted in a stereotaxic apparatus and connected to a 1-ktl Hamilton syringe. Each animal, kept alive for 24-48 h after the injection, was deeply reanaesthetized and then perfused transcardially with 50 ml of heparinized saline, followed by 100 ml of 0.075% glutaraldehyde-2.5% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and finally with 40

216 ml of 0.1 M p h o s p h a t e buffer ( p H 7.4). C o l c h i c i n e

port of H R P . F o l l o w i n g the p e r f u s i o n , the brain was

t r e a t m e n t was not carried out b e f o r e the p e r f u s i o n ,

quickly r e m o v e d and cut into sections of 60 ~ m thick-

since it a p p e a r e d to suppress the r e t r o g r a d e trans-

ness on a L a n c e r v i b r a t o m e . T h e sections w e r e col-

ii:~?~ ¸

c'e

13

A

BI

Ca Fig. 1. A: bright-field photomicrograph showing the CRFI structures in the central amygdaloid nucleus (Ce). Note that individual CRFI cells in the Ce are obscured by the high density of CRFI fibres. Rat N 2, frontal section, x95. B: bright-field photomicrograph showing the CRFI cells in the Ce of a colchicine-treated animal. Rat N 8, frontal section, ×95. C: bright-field photomicrograph showing some CRFI cells and a few scattered CRFI fibres in the BI of a colchicine-treated animal. Rat N 8, frontal section, x95. D: brightfield photomicrograph showing CRFI cells and fibres in the Abst of a colchicine-treated animal. Rat N 8, frontal section, x95.

217

..•

C/. •

~ , i

-..

,- -

.....

•...~I.;

,,.]

• . • o .". . .~ ~ /•- , ' " ., .--, ~i. .,":,~ "~

--~--.

, <",° 7

.. .0,

........

..-;..

:

,(,

_.:1

.r, ; / ,.,'t _ . " / !

.

.... • . : . , . . , . , o ~ .....e....'. ~--I ~;.-e:

,

.

.

.

.

.

s

•

...

'. .:

,

, , _/ i

" " ' ~

B -

~

•

t:,~,

':-':..~"

../.&~!

,

/ /'1

!-. - ,

"1

~:/<

.

.

.

i

:7

~ r ! " : : : t ; i / ' . "'.:'

"f

"

. :~..1~ie..LII. e

,

J, /(/~, ~c ~: . ; !

..'/

~ . : : ' : k,. ~; .i:; , '. r,.,. . - ; •

:. . . . . . . . .

~Oe~

• •

*~e:"',

: . ,e). " ' :

'.'/ .,'-"',

' ' :,""' : '0-%~. " * •, _ ~

....

E)

.

.

/

.

.

.

.

F

,q .,. .

,.o~J ) ' ; , n2,'_--- o ,

,. . . . . .

,

-

(i)>,el

~,',o

'~o

".

• •

,

.... "

cp

)

alj,.Fr..) . ._.A_, ~,,.*.¢ BI . . .. .j

/ I

F

, ":.;:',";

,If

,¢1r~. •

G

? . . . . . . . . .

.,-:

,¢

I

,<>1

.

C

,

D

.

:)

~;

I

,:,/

:..0,.,../:-/

::)i.~" ""'":: ':t.".': i''':°:4, o..--I 4"/"-Z'//.'.::Bm°:.:.;o2 TM /,' tee': ~ : % . : ' i ,,,

t . . . . . . .

I'. -•

,.,"1 ',/,

~;,:.,-

I ,,~

)

. . . . . . . . . . . . . .

• L,i ~.*~.~.;..~,o':,,,~

:

t~./-:L:,,;,,,:,/./ _<~,:~,~-. c ~ . < : / . - : ~ " "~ ' ' " '

,

, (. ; • l ,,t/,

,'

....

T-

~/...~°,,¢ ": .....

.,

A

'.. , .o-- ^...;B/~] o , >_ . - .. , , " q :::.:,-~t.,.'.-, 1::. ~ " " "

I .

y

:' !

/

.

/,." /j~.~+:,.,'j/ . . . ...~:~: • . . . . .

..~~.e.,~-'/oiC'.

..

-

";;'7':'

.qi-..-. % •

,, ,

,?o. •

~

, `

"/

~

::~;e " ". -:

*

:;

.r.-

H

,

:y:' o

I

/~,'.--~-/..4/

n

(

2L

Fig. 2. Schematic representation showing the CRFI structures in the rat amygdaloid complex. Large black spheres indicate CRFI cells and small dots CRFI fibres. The symbols do not represent quantitative distribution of CRFI structures. Modified frontal planes from the atlas of the 39-day-old rat brain by Sherwood and Timeras ]°°. A: A 5.9 mm. B: A 5.6 mm. C: A 5.3 mm. D: A 5.0 mm. E: A 4.7 mm. F: A 4.4 mm. G: A 4.1 mm. H: A 3.8 mm. I: A 3.5 mm. J: A 3.2 mm. K: A 2.6 mm. L: A 2.0 mm.

218

ot

LV

C

La

.Me

D

S .

~E

t

ol

B

Aco

C Fig. 3. A: bright-field photomicrograph showing CRFI cells and fine fibres in the Me of a colchicine-treated rat. Rat N 8, frontal section, × 75. B: bright-field photomicrograph showing the CRFI cells in the Ahi of a colchicine-treated rat. Rat N 9, frontal section, x 75. C: bright-field photomicrograph showing the CRFI cells in the Aco of a colchicine-treated animal. Rat N 9, frontal section, x75. D: bright-field photomicrograph showing the CRFI cells in the La of a colchicine-treated animal. (Note that a few CRFI fibres are found in A - D . ) Rat N 9, frontal section, x75. E: bright-field photomicrograph showing CRFI terminals in the caudal Ahi of a non-colchicine treated animal. Weakly stained CRFI cells are seen in the S. Rat N 3, frontal section, x75. F: bright-field photomicrograph showing CRFI terminals in the caudal Me of a non-colchicine-treated animal. Rat N 3, frontal section, x75.

219 lected serially, kept in glass vials containing 0.1 M phosphate buffer (pH 7.4) and subjected to the CoG O D method as described earlier for detection of HRP transported retrogradely 41'8°. Subsequently, the sections were processed for immunohistochemistry without cobalt intensification. The sections were then mounted, dried, dehydrated and cover-slipped.

Terminology and stereotaxic coordinates Terminology used was based upon the atlases of Krettek and Price 53, Sherwood and Timiras 1°°, Palkovits and Jacobowitz 88, K6nig and Klippe149, Paxinos and Watson 89 and Shiosaka et a1.1°]. The stereotaxic coordinates were obtained from the atlases of Sherwood and Timiras ]°° and Palkovits and Jacobowitz 88, though slight modifications were made on the basis of pilot experiments.

Lesion study An attempt was made to determine the precise efferent projection fields of the CRFI neurons observed in the amygdaloid complex. In the rats L 12, 14, 17 and 20 where the unilateral lesions were centered on the Ce, Abst and B1 (Figs. 4 and 5), marked ipsilateral changes in CRFI fibres were found in the LH, mesencephalon and lower brainstem (Fig. 6). The ipsilateral decrease in the CRFI fibres in the LH was confined to the area just medial to the amygdaloid complex (Fig. 6). In the

'

~,

_

~

.' ."

/

~

i

~'I ..: "-.

'

I

.

iC

'"'

•

,' ,'Ce

,

.,."/',

-.

.:7,~-'.

~Y..~.4"/~UJ.: .,'/ ~.~.~9."

.,".:'~ AA ,:' ,' : "-,'Y;" ,.oi " , ",~ / y ~ . :~. . . ' :."-" . . .- . ,. "7. Aco

.'"

':"I

.'." .':."

RESULTS A

Distribution of CRFI structures in the rat amygdaloid complex As shown in Fig. 1A, the Ce of normal rats contained a number of CRFI cells throughout its rostrocaudal extent, though it was sometimes difficult to identify an individual CRFI cell in the nucleus because of a very high density of CRFI fibres. Following the intra-amygdaloid colchicine injection, the number of CRFI cells in the Ce increased markedly (50-90 CRFI cells per section; Figs. 1B and 2). In addition, cells with CRFI were found in the B1 (10-15 CRFI cells per section; Figs. 1C and 2), Abst (20-25 CRFI cells per section; Figs. 1D and 2), Me (15-20 CRFI cells per section; Figs. 2 and 3A), Ahi (10-15 CRFI cells per section; Figs. 2 and 3B), Aco (15-30 CRFI cells per section; Figs. 2 and 3C), and La (10-15 CRFI cells per section; Figs. 2 and 3D). The CRFI cells in the Ahi extended medially and dorsally into the S where CRFI cells were also seen (Fig. 2). The A A and Bm contained scattered CRFI cells. Neural processes with CRFI were found in all of the above-mentioned areas. Next to the dense CRFI fibres in the Ce (Figs. 1A and 2), the Me and Ahi had a rich content of CRFI fibres (Figs. 2, 3E and 3F). Particularly in the caudal part of the Ahi, very fine terminals were observed (Figs. 2 and 3E). In contrast, B1, La and Bm contained scattered CRFI fibres (Figs. 1C, 2 and 3D).

p

B

,, . . . . . . . . . . . . . . . .

Ah,A~h,' - . : , . . -

c.

..:'."

:, . . . . . . . . .

, Ah,A~h, ~o '"" - "-

. . . .

"::'

. . . . . . . . . . . . . . . . . . . . . . . . . . . . E. . . . . . . . . .

!

A

4.

Fig. 4. Schematic representation of the lesions centred on the Ce, Abst and B1 (indicated by shaded areas). Reconstructed from the rats L 12, 14, 17, 20. Modified frontal planes from the atlas of 39-day-old rat brain by Sherwood and Timirasl°°. A: A 5.9 mm. B: A 5.6 mm. C: A 5.0 mm. D: A 4.7 ram. E: A 4.1 mm. F: A 3.8 mm. G: A 3.2 mm. H: A 2.6 mm. Note that the CoM and S are not affected by the lesions.

220 mesencephalic RF and sn, a marked reduction of ipsilateral CRFI fibres was also observed (Figs. 6 and 7A, B). Many of the CRFI fibres in the RF and sn were arranged in linear 'chains' (Fig. 7A, B), suggesting that the fibres were passing through these areas rather than terminating within them. At the level of the DSCP, changes similar to those seen in the mesencephalon were found in the RF (Figs. 6 and 7C, D). There were no or only a few changes in the periaquaductal CG (Fig. 7E, F). Caudal to the level of the DSCP, the CRFI fibres markedly decreased in number in the Dpb and Vpb and MeV (Fig. 6). Especially in the Dpb close to the SCP, the changes in the CRFI fibres were most prominent on the lesioned side (Fig. 8). Since the CRFI fibres were diffusely distributed around cell bodies in the Dpb, they appeared to terminate within the nucleus (Fig. 8A). The rostral and intermediate parts of the MeV and the intermediate part of the Vpb contained substantial numbers of amygdalofugal CRFI fibres which traversed through the areas to terminate within the most caudal parts of the MeV and Vpb (Figs. 6 and 9). No changes were observed in the Lc, laterodorsal tegmental nucleus and Dtg. More caudally, CRFI fibres were ipsilaterally decreased in the rpc (Fig. 6),

Fig. 5. Bright-field photomicrograph showing lesion affecting the Ce, Abst and B1 (indicated by arrowheads). Rat L 14, frontal section, x38.

t"

e:

A

.,'!_;,:,: ", ', "~" ,,.

; " .:

~ :[pO0"~. . . . . ", 7

:

~

"I t,

Ei"

c

. ..' '

•

".CT..rpoe." .. ",' " i

/e~

FI

j - ~ "" - " .

c '.!~: i',.,'i

~

.

"~

"-.

/ ~

G

,

~.;~.., . ~]

]

:; ( *.'-, "

. . . . .

.....--z~---~, \VV' rgi .' :

"'.'. " .' "

}

I]]

~"n" I

Pig. 6. Schematic representation showing the changes in C R F I fibres (fine dots) in the L H , RF, sn, D p b , Vph, M e V and rpc. The left side o f each plane shows the distribution o f C R F I fibres on the normal side o f the brain. M o d i f i e d frontal planes from the atlas o f the 39-day-old rat brain by Sherwood and Timiras l°° and from that o f Palkovits and Jacohowitz 88. A: A 4.7 mm. B: A 1.4 mm. C: A 0.5 mm. D: P 1.0 mm. E: P 2.0 mm. F: P 2.3 mm. G: P 2.8 mm. H: P 3.9 mm. Reconstructed from rats L 12, 14, 17, 20.

221 but this change was not as p r o m i n e n t as that seen in the Dpb, Vpb and MeV. Thus, the long descending amygdalofugal C R F I - c o n t a i n i n g fibres seemed to

pass through the L H and R F to terminate within the Dpb, Vpb and MeV. As a result of the lesions which included Ce, Abst

~!ii~i~i~!i!~i~i~ii~i~}~!!i~i!i~!!~i~i~!i!~i!!i}~i~i~!!!~!i~i~!

Fig. 7. A and B: bright-field photomicrographs showing the change in the CRFI fibres within the mesencephalic reticular formation (RF). A, control side; B, lesioned side. Many of the CRFI fibres in the RF are arranged in linear 'chains', suggesting that the fibres are passing through the area rather than terminating within it. Rat L 14, frontal section, A and B, 75×. C and D: bright-field photomicrographs showing the change in the CRFI fibres within the RF at the level of the DSCP. A, control side; B, lesioned side. Rat L 14, frontal section, C and D, ×95. E and F: bright-field photomicrographs showing the CRFI fibres in the periaqueductal CG on the control (E) and lesioned (F) sides at the level of the caudal DSCP. No or few changes in the CRFI fibres are observed within the CG. The CRFI fibres arranged in the linear 'chains' shift dorsally into the most rostral part of the Dpb. Rat L 14, frontal section, E and F, x95.

222

Fig. 8. A and B: bright-field photomicrograph showing the change in the CRFI fibres within the Dpb close to the SCP. A, control side; B, lesioned side. Note an ipsilateral decrease in the CRFI fibres in the Vpb. Rat L 20, frontal section, A and B, ×95.

and Bl (rats L 12, 14, 17 and 20; Figs. 4 and 5), CRFI fibres in the bst were also affected on the lesioned side. Although a high density of CRFI terminals was found in the bstl throughout its rostrocaudal extent (Figs. 10 and 11), only CRFI terminals located in the dorsal part of the caudal bstl were markedly decreased by the lesions (Figs. 10 and l l A , B). No changes were detected in any other areas apart from the dorsal part of the bstl (Figs. 10 and 11C, D). At the level of the AH, the ipsilateral CRFI fibres in the st arising from the amygdaloid complex decreased in number (Fig. 12). The precise projection fields in the hypothalamic area were not determined in the present study, because most of the CRFI fibres in the st became invisible just before entry to the hypothalamic area. When the destruction of the amygdaloid complex was confined to the Ce without extending into the Abst and BI (rats L 15, 16, 18), a similar but less prominent decrease in CRFI fibres was observed in the LH, RF, sn, Dpb, Vpb, MeV, bst and st. Howev-

er, the changes were not clear enough to be detected by immunohistochemistry in the rats L 10, 11, 13, 19 where the Abst or B1 were selectively destroyed. When lesions were large enough to include the CoM and S (rats L 21, 25 and 27; Fig. 13), ipsilateral CRFI fibres significantly decreased in number in the VMH (Fig. 14). In the most rostral part of the VMH, the CRFI fibres were mainly distributed to both the shell and the core of the nucleus close to the third ventricle (Figs. 14 and 15A), whereas they shifted centrally into the core of the nucleus in the caudal direction (Figs. 14 and 15B). The CRFI fibres were ipsilaterally affected at either level of the VMH by the lesions (Figs. 14 and 15B). No lesions smaller than those including the CoM and S caused noticeable alterations in the CRFI fibres within the VMH. The lesion of the La did not result in any changes. No changes in CRFI fibres were observed in any other areas apart from those mentioned above even after the total destruction of the amygdaloid complex. It was of interest to note that a significant number

223

SCP

VPb

•

"

B

A

Fig. 9. A and B: bright-field photomicrographs showing the changes in the CRFI fibres within the intermediate parts of the Vpb and MeV. Some of the CRFI fibres appear to pass through these regions to project more caudally. A, control side; B, lesioned side. Rat L 14, frontal sections, A and B, ×75. C and D: bright-field photomicrographs showing the changes in the CRFI terminals within the most caudal parts of the Vpb and MeV. A, control side; B, lesioned side. Note the ipsilateral decrease in the CRFI fibres in the Dpb close to the SCP. Rat L 14, frontal section, C and D, ×75.

:

'L

"-

A

B I

C

Fig. 10. Schematic representation showing the change in the CRF! fibres within the bst. The left side of each plane shows the distribution of CRFI fibres on the normal side of the brain. Note that the ipsilateral decrease in the CRFI fibres is confined to the dorsal part of the caudal bstl (C and D). Modified frontal planes from the atlas of the 39-day-old rat brain by Sherwood and Timiras 1°°. A: A 7.5 ram. B: A 7.2 mm. C: A 7.0 mm. D: A 6.5 mm. Reconstructed from the rats L 12, 14, 17, 20.

224

ic : ,

b~m

A

B

aC

C

11D

Fig. 11. A and B: bright-field photomicrographs showing the change in the CRFI fibres within the dorsal part of the caudal bstl. A, control side; B, lesioned side. Rat L 20, frontal section, A and B, x95. C and D: bright-field photomicrographs showing the CRFI fibres in the rostral bst on the control (C) and lesioned (D) sides. No changes are seen in either bstl or bstm. Note that individual CRFI perikarya in the bstl arc obscured by the high density of CRFI terminals within it. Rat L 20, frontal section, C and D, x95.

225 of CRFI fibres often accumulated around the lesions with occasional growth into the centre of the lesions (Fig. 16). These accumulated CRFI fibres were apparently thicker than ordinary CRFI fibres and showed sometimes growth cone-like appearance (Fig. 16, indicated by arrowheads). The accumulation of the CRF fibres was traced to the beginning of the st and to the LH close to the amygdala at the level of the A H (Fig. 17). These observations suggest the presence of amygdalopetal as well as amygdalofugal CRFI-contaning pathways; the site(s) of origin of the amygdalopetal CRFI fibres remains to be resolved. The lesion study indicated that (1) the CRFI-containing pathways originating in the Ce, Abst and BI terminate within the Dpb, Vpb, MeV and bstl, and (2) the CoM consisting of Me, Ahi and Aco gives rise to the CRFI fibres to the VMH together with the S.

HRP study Following HRP injection into the Dpb (rats H 29, 33; Fig. 18A), ipsilateral HRP-labelled cells were

found in the Ce (Figs. 18B and 19) and occasionally in the Abst (Fig. 19). Similar results were obtained when HRP was injected into either Vpb or MeV (rats H 30, 34). A heavy ipsilateral retrograde labeling was observed in the BI in addition to the Ce and Abst after HRP injection into the bstl (rats H 28, 35). In rats H 28, 37 where HRP was injected into the VMH, many retrogradely labeled neurons were present in-the Me, Ahi and S (Figs. 20, 21). However, the Aco was not significantly labelled. Thus, the HRP study demonstrated that (1) the Ce and Abst project to the Dpb, Vpb and MeV; (2) the B1 innervates the bstl together with the Ce and Abst; and (3) the Me, Ahi and S send fibres to the VMH.

Combined HRP and immunohbtochemical study. An attempt was made to obtain a direct evidence for the amygdalofugal CRFI-containing pathways shown in the lesion and HRP studies. In the rats C 39, 42, 44 where injections of HRP were mainly located in the Dpb, Vpb, MeV, 3 kinds

-

st

ic

12 B Fig. 12. A and B: bright-fieldphotomicrographsshowing the change in the CRFI fibres within the st. A, control side; B, lesioned side. Rat L 14, frontal section, A and B, ×95.

226

G/.

..-: I

" ....::

._ ~i

: .'.,:'

A

,!L°';J..~', Y

i/.:

:I', ~ ......-"-./..-I

;,/ ... ~ ,:.

- ~ ,~". "

A

, ""-

,,

.

9

:2 " " "

,:.'

~

B

,

,"

.";i

/~x~'~---,..

.

;"

.:/ i ,C.X/~;:." .....; .". ,-::

I

. . . . . . . . . . . . . . . . . .

."Ce :; .--

:,"

"" "

..., ..?'

cytoplasm (Fig. 2 2 G - I ) . These differences were easily discernible at high magnification. In control sections treated with the control serum, only retrogradely labelled cells were seen. The 3 kinds of cells were evenly distributed to the Ce throughout its rostrocaudal extent. Similar results were o b t a i n e d after H R P injection into the bstl. These findings indicated that (1) the C R F I cells in the Ce project to the D p b , Vpb, MeV and bstl and (2) the n o n - C R F I cells in the Ce also project to the same regions. Double-labelled cells were other areas apart from the Ce tion into the amygdalofugal scribed in the lesion and H R P

I. . . . . . . . . . . . . . .

',

ic

':'"

a""

,"

,

G . . . . . . . . . . . . . . . .

0,-..

~,j~

', i{/,'

~)--' I /

.---.

/ ,

!

F

A

:f ../i~.~..~::;~, ......-., ;

~~,..:;

not visualized in any following H R P injecprojection fields destudies, possibly, due

~"

c

~

"-'13~

Fig. 13. Schematic representation of the lesion including the CoM and S (indicated by shaded areas). Reconstructed from the rats L 21, 25, 27. Modified frontal plantes from the atlas of the 39-day-old rat brain by Sherwood and Timiras1°°. A: A 5.9 ram. B: A 5.6 ram. C: A 5.0 mm. D: 4.7 mm. E: A 4.1 ram, F: A 3.8 ram. G: A 3.2 mm. H: A 2.6 mm.

of cells were observed in the Ce: (1) double-labelled cells, (2) C R F I cells and (3) H R P - l a b e l l e d cells. The double-labelled cells d e m o n s t r a t e d a h o m o g e n e o u s brown reaction product throughout the cytoplasm and proximal dendrites, indicating CRF1, and a granular black reaction product over the cytoplasm, indicating the presence of the r e t r o g r a d e tracer (Fig. 2 2 A - C ) . The C R F I cells showed the diffuse brown reaction product, occasionally with dense granular brown immunostaining over the cytoplasm (Fig. 2 2 D - F ) . The H R P - l a b e l l e d cells contained only the granular black reaction product with a faintly grey

o;.:;~:~,],~ !i,ii?II;iii::: , : :,:~ vM.~J, l " B

o :i .:i~i.... ! o CHL ":": i~. ",,'::VJMHc

14c Fig. 14. Schematic representation showing the changes in the CRFI fibres within the shell and the core of the VMH. The left side of each plane shows the distribution of CRFI fibres on the normal side of the brain. Reconstructed from the rats L 21, 25, 27. Modified frontal planes from the atlas of the 39-day-old rat brain by Sherwood and Timirasl% A: A 5.0 mm. B: A 4.7 mm. C: A 4.4 ram.

227 tures in e x t r a h y p o t h a l a m i c areas including the amygdaloid complex• O t h e r intensification p r o c e d u r e s have not been used in the immunohistochemistry of C R F . The present study has shown m o r e n u m e r o u s C R F I structures in the amygdaloid complex and efferent projection fields by using the modified CoG O D m e t h o d , as c o m p a r e d with the s i l v e r - g o l d intensification of the D A B reaction product. T h e CoG O D m e t h o d was first d e v e l o p e d for detection of r e t r o g r a d e l y t r a n s p o r t e d H R P it was applied for the simultaneous ultrastructural d e m o n s t r a t i o n of anterogradely t r a n s p o r t e d H R P and immunohistochemically stained vasopressin neurons 41,s°. In the present study, several modifications of the C o - G O D procedure were introduced to improve the immunostaining of C R F and to suppress the b a c k g r o u n d staining. Briefly, cobalt acetate was used instead of cobalt chloride as r e c o m m e n d e d by W a i n e r and R y e 116.

r

VMHc

VMHs

VMHc

;

*

.

~

15B

Fig. 15. A and B: bright-field photomicrographs showing the changes in the CRFI fibres within the shell and the core of the VMH• The left side of each photomicrograph shows the distribution of CRFI fibres on the normal side of the brain. In the most rostral part of the VMH (A), the CRFI fibres are mainly distributed to both the core and the shell of the VMH, close to the third ventricle whereas they shift centrally into the core of the nucleus in the caudal direction (B). Rat L 25. frontal sections. A and B, x 70.

to the lack of colchicine t r e a t m e n t and cobalt intensification and/or due to the suppressive effect of glutaraldehyde on immunostaining (see Discussion). The amygdalofugal C R F I - c o n t a i n i n g pathways observed in the present study are r e p r e s e n t e d schematically in Fig. 23.

Q

:;:

~o

DISCUSSION Only partial immunohistochemical visualization of C R F - r e a c t i n g neuronal elements can be o b t a i n e d without intensification m e t h o d s sl't~°. M e r c h e n t h a l e r et al. 67'68 applied a s i l v e r - g o l d intensification of the D A B reaction product for the immunohistochemistry of C R F and d e m o n s t r a t e d n u m e r o u s C R F I struc-

Fig. 16. Bright-field photomicrograph showing the changes in the CRFI fibres accumulated around and into the dorsomedial edge of a lesion including the Ce. They are apparently thicker than ordinary CRFI fibres and sometimes show growth conelike appearance (indicated by arrowheads). Rat L 17, frontal section, ×75.

/,
"

: /l,.<0s

v

s

/i <-J~//

~ - " / ,

,/

A •

.v~.'''cp

B

i .~

CP

I

"7""

ic

C I

"

- ' ' "

/

""

Furthermore, low concentration of highly purified GOD (Sigma type V, 0.5 U/dl) was added to the solution of the final incubation to generate hydrogen peroxide, instead of the previously used high concentration of relatively crude GOD (Sigma type VII, 37.5-45 U/dl) 8°. The final incubation was done at 4 °C to expose the sections to small amounts of hydrogen peroxide generated at a constant rate. These modifications made it possible to obtain good immunoreaction in sections with low background staining. The modified Co-GOD method has increased the

17D •

Fig. 17. Schematic representation showing the CRFI fibres close to or in the lesions• The fibres may be traced to the LH close to the amygdala at the level of the A H (A, B), and to the beginning of the st (D) (indicated by small dots). Reconstructed from the rats L 12, 14, 15, 16, 17. Modified frontal planes from the atlas of the 39-day-old rat brain by Sherwood and Timiras I~. A: A 5.6 m m . B: A 5.3 ram. C: A 4.7 mm. D: A 3.8 mm.

;

i

"::.:-').&

'

/u.ii;d /

U

. .v',.-:~'.s

,:i)

,.: ~,-.;>:;...'.4"/ i~,/; 7E~,: "-

...>/

A

R

c,

... .,/i,,i

._.E/.',, If~.'..-.'-~o~

Y

'ii"

i

.:,/ E

•

,"

,,

.--2-.--"

"

Ce

q

,...

Ic

F

La

"- 6m'-::..."

~

'

,:'

f

.

, ,'.':~

,"

'

i

[

Fig. 18. A and B: bright-field photomicrographs showing the injection site of H R P in the D p b (A) and HRP-labelled ceils in the Ce (B). Rat H 29, frontal sections, A, x30, B, x75.

.....

:

19"

Fig. 19. Schematic representation showing the HRP-labelled cells (indicated by closed triangles) after H R P injection into the parabrachial nuclei and MeV. Reconstructed from the rats H 29, 30, 33, 34. Modified frontal planes from the atlas of the 39-day-old rat brain by Sherwood and Timirast~L A: A 5.6 m m . B: A 5.3 mm. C: A 5.0 m m . D: A 4.7 m m . E: A 4.4 m m . F: A 4.1 ram. G: A 3.8 mm. H: A 3.2 ram. Note that the C o M is not labelled.

229

~t~ .

1

Alli

.

.

.

.

.

C Fig. 20. Bright-field photomicrographs showing an injection site of HRP in the VMH (A) and HRP-labelled cells in the Me (B), Ahi (C, D) and S (D). Rat H 37, frontal sections, A, x30, B-D, x75.

sensitivity of the immunostaining to compare favourably with the silver intensification of the DAB reaction product 67,68. In the resulting Golgi-like images, the contrast has been markedly improved, neural morphology has been displayed to a good advantage and fibres have been traced for longer distances than in conventionally stained preparations. In the present study, an antiserum against rat CRF has been used instead of that against ovine CRF. The use of a homologous antiserum may have contributed to obtain more pronounced and more intense immunostaining, as compared with several previous studies carried out with use of the antisera against ovine CRF 81A1°. Skofitsch and Jacobowitz 1°5 have shown that the use of rat CRF antisera in rat brain made it possible to visualize different immunoreactive structures from those stained when using antisera against ovine CRF. Furthermore, intra-amygdaloid injection of coichicine in our studies may have enhanced

the staining of a greater number of cells in amygdaloid nuclei not previously described. Nevertheless, it should be noted that the immunostaining was still relatively weak without the application of the modified Co-GOD intensification. In good agreement with the previous studies 67's1'110, the present immunohistochemicai study has shown CRFI neurons in the Ce, Me, BI and La. In addition, CRFI cells have been demonstrated in the Abst, Ahi, Aco and S. CRFI fibres were seen throughout the rostrocaudal amygdala, particularly in the Ce, Me and Ahi. The widespread localization of the CRFI structures implies a considerable complexity of the amygdaloid CRFI-containing fibre connections. Using tritiated amino acids, autoradiographic studies have revealed that the Ce and adjacent structures (possibly Abst) send long descending fibres to terminate within the Dpb, Vpb, MeV, CG, and some

230 of these fibres project more caudally to the medullary reticular formation and to the nucleus of the solitary tract 37"52. However, the autoradiographic studies provide no information on the neurotransmitters or neuromodulators which the descending fibres contain. Fellmann et al. 25 have suggested that the CRFI cells in the Ce project to the LH via a ventral amygdalofugal pathway, but have not determined the destination of the CRFI-containing pathway. On the basis of the results obtained from our lesion study, the LH is the first region to which the amygdalofugal CRFI-containing pathway projects. Furthermore, the present study has shown that the CRFI-containing pathway arises from the Ce and possibly Abst to terminate within the Dpb, Vpb and MeV. Hopkins and Holstege 37 have demonstrated many labelled neurons in the B1 following HRP injection into the posterior hypothalamus and ventral tegmental area, but could not show any significant labelling in the same nucleus after HRP injection into the parabrachial areas. Since the present HRP study supports their results, the participation of BI in the amygdalofugal descending CRFl-containing pathway may be excluded. Significant numbers of ipsilateral CRFI fibres remained intact in the LH, RF, Dpb, Vpb and MeV even in the cases where the lesions were large enough to include the Ce and Abst (Figs. 6-9). There are several possible explanations for this: these remaining fibres might be supplied by intrinsic CRFI cells, because CRFI cells are present throughout these regions 67'81']t°. Furthermore, descending or ascending CRF-pathways from unknown origins may pass through these regions, as suggested by Swanson et al. u°. Immunohistochemical studies have demonstrated that in the parabrachial areas various neuropeptides, including SP, Enk, Nt, SRIF, CCK and CRF, are present and the localization varies from peptide to peptide 15`26`42`55`58,59'63"67`Sh98,llO'l11`lla.In contrast to the extensive knowledge on the cellular localization of neuropeptides in the parabrachial areas, few studies have been so far made to investigate the fibre connections of these neuropeptides. Kawai et al. a5 suggested by means of knife cut studies that SP fibres in the parabrachial areas originate in at least 3 unnamed sources. Thus, the long descending amygdalofugal CRFl-containing pathway determined in the present

study appears to be just the tip of the iceberg of the massive peptidergic projections within the parabrachial areas. The amygdaloid CRFI may play a role in the relay of visceral sensory information together with other neuropeptides, since the parabrachial areas receive a massive input from the nucleus of the solitary tract TM. In addition to the long descending amygdalofugal CRFI-containing pathway, the SRIF ceils in the amygdala also send descending fibres to the lower brainstem 44. Although both CRFI and SRIF cells are seen in the Ce and Abst, the projection fields of the

u:.,.) ic

/,,

:

"

,,-::i" ..'At

/'

It

/Co

I

" "

-"

.,71

./

CP

+":""+ -

',zT'::" ),m". "-

~

..'7

~,~.

,,"

A ', . . . . . . . . .

,- - - - ~ r

. . . . . .

B

I" . . . . . . . . . . . . . . . . . . . . . .

J .......

.::/ .::1

. ......

E', . . . . . . . . . . . . . . . . . . . .

.; .,':J

F I. . . . . .

y

c

i ..

T

,

Fig. 21. Schematic representation showing HRP-labelled cells (indicated by closed triangles) after H R P injection into the VMH. Reconstructed from the rats H 28, 37. Modified frontal planes from the atlas of the 39-day-old rat brain by Sherwood and Timiras I~. A: A 5.9 ram. B: A 5.3 m m . C: A 4.7 m m . D: A 3.8 m m . E: A 3.5 ram. F: A 3.2 m m . G: A 2.6 mm. H: A 2.0 mm.

231

Q A

D

2

E

¸

imp,

o,:

221

Fig. 22. A-C: bright-field photomicrographs showing double-labelled cells in the Ce. The double-labelled cells demonstrate a homogeneous brown reaction product throughout the cytoplasm and proximal dendrites, and a granular black reaction product over the cytoplasm. Rat C 42, frontal section, A-C, × 800. D-F: bright-field photomicrographs showing CRFI cells in the Ce. The CRFI cells show the diffuse brown reaction product, occasionally with dense granular brown immunostaining over the cytoplasm (E). Note that these CRFI cells are rather weakly stained in comparison with those seen in Fig. 1A, B. Rat C 42, frontal section, D-F, ×800. G-I: brightfield photomicrographs showing HRP-labelled cells in the Ce. The HRP-labelled cells contain only the granular black reaction product with a faintly grey cytoplasm. Note that a double-labelled cell is seen at the left lower corner of (G). Rat C 42, frontal section, G-I, x800.

amygdaloid S R I F cells are different from those of the amygdaloid C R F I cells. The amygdaloid S R I F cells mainly project to the pontine and m e d u l l a r y reticular formations, and to the C G without innervating the D p b , V p b and MeV. On the o t h e r hand, the amygdaloid C R F I cells mainly give rise to the massive fibre

tracts to the D p b , V p b and M e V with the scattered projection to the rpc (Fig. 6H). The two peptidergic tracts m a y affect the lower brainstem without direct interaction. F u r t h e r detailed morphological studies on the colocalization o~ C R F and S R I F within the amygdala will be required to d e t e r m i n e any interac-

232 neurons are sometimes difficult to identify because of the high density of the CRFI terminals within the bst (Fig. llC). In contrast, many CRFI neurons are visualized in the region after colchicine treatment 67't1°. Based on the autoradiographic findings and on our lesion study, the Ce and B1 appear to give rise to the CRFI fibres to the bstl. However, it is not clear whether or not the Abst projects to the bsti. In the study of amygdalofugal SP and SRIF pathways 96, the Abst was heavily labelled following H R P injection into the bstl. (The Abst is referred to as the area between the Ce and Me (see ref. 96).) Therefore, the Abst is suggested to project to the bstl together with the Ce and B1. Since the Abst is very close to the Ce, injection of isotopically labelled amino acids might involve both Ce and Abst. In such cases, the efferent projections of the Abst become indistinguishable from those of the Ce. In contrast, retrogradely labelled Abst is easily discernible in the H R P study. This may explain the discrepancy between the HRP study and autoradiographic findings. Besides the CRFI cells, the Abst has a rich content of SRIF, SP and NT cells 33'42'58"96"101, projecting to a variety of

ibstl :

• - ""

N/"

,

Abst \'-.

/ \',Ai ....

?

: DPb ! VPb MeV

23 Fig. 23. Schematic drawing indicating the amygdalofugal CRFI-containing pathways described in the present study: (1) the Ce and Abst innervate the Dpb, Vpb and MeV through the LH and RF; (2) the Ce, Abst and BI project to the bstl via the st; and (3) the CoM and S give rise to their fibres to the shell and core of the VMH.

tion. Using autoradiographic tracing of axonal connections, Kretteck and Price 52 have demonstrated that both Ce and BI project heavily to the bstl. In good agreement with this, the amygdaloid lesions have produced changes in CRFI fibres within the bstl. It may be worth noting that the decrease in the CRFI fibres was confined to the dorsal part of the caudal bstl, and not found in any other areas of the bst which also contain large numbers of CRFI fibres (Figs. 10, 11)67"11°. The CRFI fibres which are not affected by the amygdaloid lesions may be supplied by intrinsic CRFI neurons. In normal rats, the intrinsic CRFI

brain regions including the bst, hypothalamic area and limbic areas 96'1°1. From the immunohistochemical point of view, the Abst may be regarded as a different nucleus from the Ce, though its projections are very similar to those of the Ce. The present study has shown a substantial number of CRFI fibres in the st as described elsewhere 67'N°. The CRFI fibres in the st may originate in the amygdaloid CRFI neurons, because they are affected by amygdaloid lesions. Some of the CRFI fibres in the st arising from the amygdala terminate within the bstl and others appear to blend in the hypothalamic area at the level of the AH. On the basis of the previous neuroanatomical studies 52'62, the CRFI fibres in the st might project to the AH, VMH and LH. Besides CRFI, the st contains a variety of amygdaiofugal peptidergic fibres, such as SP, VIP, Enk, SRIF and NT 15'22'42'58'93'94'96'112'113. The functional significance of the peptidergic projections in the st has not been determined. In good agreement with previous neuroanatomical findings 19'52'61'65,the present study has demonstrated a CRFI-containing pathway originating in the corticomedial amygdala and S which terminates within both the core and the shell of the VMH. McBride and

233 Sutin 65 have shown heavy labelling in the Aco after injection of HRP into the VMH, but the present study and that by Luiten et al. 61 did not demonstrate any significant labelling in the Aco. Therefore, it is not clear whether or not the Aco takes part in the CRFI-containing pathway to the VMH. The present study has shown that CRFI fibres are mainly distributed to both the shell and the core of the rostral VMH, and shifted centrally to the core of the VMH in the caudal direction. In contrast to this, CCK, NT, Enk and SRIF fibres are mainly seen in the core of the VMH with scattered but significant occurrence in the VMHs 13'36'39'40'42'118'119. Furthermore, each peptide does not show similar localization to others in the VMH, suggestive of different origins and functions. Based on lesion studies, the origins of the NT, CCK and Enk fibres lie in the Me, Dpb and PVH, respectively39,4°'117. These findings may indicate other origins of the CRFI fibres which remain intact in the VMH even after the total destruction of the CoM and S (Fig. 15); it is likely that the CRFI cells located in the Dpb or PVH 67'81'11°, or those in proximity to the VMH ~°5 innervate the VMH together with the amygdaloid CRFI neurons. The functional significance of the neuropeptides within the VMH has not been determined, though several suggestions are present 17'82'1°6'1°7. The present study suggests that at least two amygdalopetal CRFI-containing pathways are present, because the accumulated fibres around the amygdaloid lesions may be traced to the beginning of the st, and to the LH at the level of the AH. In support of this, evidence has accumulated over the years that the amygdala receives inputs from a variety of brain regions 54'77'83-85'9°'91'97'1°3A15. Among these, the PVH, bst, Lc and parabrachial nuclei have a rich content of the CRFI ceils67's1,11° and thus may project to the amygdala together with the well-known aminergic and cholinergic inputs 1'3'11'21'23'24"74. Additional studies are needed to elucidate the amygdalopetal CRFI-containing projections. When HRP injection is mainly located in the Dpb, Vpb and MeV, the combined HRP and immunohistochemical study has shown 3 kinds of cells in the Ce: (1) double-labelled cells, (2) CRFI cells and (3) HRP-labelled cells. The double-labelled cells provide direct evidence for the existence of a long descending amygdalofugal CRFI-containing pathway.

However, the combined HRP and immunohistochemical method has some limitations. First, it cannot be decided whether the processes of the double-labelled cells pass through the HRP-injected area or terminate within it. Second, it does not show if the doublelabelled cells constitute major or minor connections to the HRP-injected area. Third, it does not define which parts of the HRP-injected area receive input from the double-labelled cells. The lesion study has shown that the amygdalofugal CRFI-containing pathway traverses the LH and RF to terminate within the Dpb, Vpb and MeV, and the amygdala is the main origin of the CRFI fibres in these regions. Furthermore, the lesion study has demonstrated that the amygdaloid CRFI cells mainly terminate within the Dpb close to the SCP, and within the most caudal parts of the MeV and Vpb. Lesioning, together with retrograde tracers and immunohistochemistry, seem necessary to unravel peptidergic pathways. The simple CRFI cells identified after HRP injection into the Dpb, Vpb and MeV may project to the bstl, because double-labelled cells are also seen in the Ce following HRP injection into the bstl. However, it is not clear whether or not a single CRFI cell simultaneously projects to the Dpb, Vpb, MeV, and to the bstl. To resolve this issue, the simultaneous use of two kinds of retrograde tracers and immunohistochemistry will be recommended u. The presence of the non-CRFI cells with retrogradely transported HRP raises a question of what kinds of neurotransmitters or neuromodulators these cells have. As described elsewhere, a variety of peptidergic cells is present in the Ce 117. Therefore, it is of considerable interest to examine if some of these peptidergic cells send their fibres to the lower brainstem together with the CRFI and SRIF neurons or not 44. Another problem of the combined HRP and immunohistochemical study is that a small amount of glutaraldehyde (0.075%) has to be added to the fixative to retain the activity of retrogradely transported HRP. As indicated by Eldred et al. 2°, even a small amount of glutaraldehyde may have suppressed markedly the immunostaining of CRF in the present study. Furthermore, the lack of colchicine treatment and cobalt intensification has made the visualization of CRFI cells more difficult (Figs. 1A, B and 22). As a result, the combined HRP and immunohistochemical study has failed to demonstrate double-labelled

234 cells in other amygdaloid regions apart from the Ce. The observation that only the Ce showed double-labelled cells in non-colchicine-treated rats indicates a greater density of C R F I structures in this nucleus. The comparison of H R P and fluorescent reagents may be of value in immunohistochemical studies combined with retrograde tracing 11'99. Little is known of CRFI-containing pathways which may be related to the autonomic responses and behavioural activation elicited from intracerebroventricular injection of C R F 7'8'10'27'75'1°9. O n the ba-

behavioural activation 3°,31"34"43"57,61,66,72. It is tempting to speculate that the amygdalofugal CRFI-containing pathways projecting to the Dpb, Vpb, bst and VMH may play an important role in the mediation and regulation of autonomic responses and behavioural activation. The final link between the amygdalofugal CRFI-containing pathways and occurrence of autonomic responses and behavioural activation following CRF injection into the cerebral ventricles is now open to discussion.

sis of previous behavioural, electrophysiological and neuroanatomical studies, the Dpb, Vpb, V M H and bst are all closely involved in the mediation and

ACKNOWLEDGEMENTS

integration of autonomic and behavioural responses 5'6'9"12"16'18'32'35'46'64'77-79'86'87'92. Since these

This work was supported by grants from the MRC (Canada) to K.L. (Career Investigator of the MRC) and from the Alberta Heritage Foundation for Medical Research to M.S. (AHFMR Fellow). The authors are grateful to Mrs. Marilyn Devlin for typing the manuscript.

regions are located around the cerebral ventricles, they may be affected by intracerebroventricular injection of CRF. Furthermore, the amygdaloid complex is also associated with the autonomic system and

ABBREVIATIONS A AA Abst ac

ACTH Aco AH Ahi BI Bm bst bstl bstm C Cc CCK Ce CG Co-GOD CoM CP cp CRF CRFI DAB DM Dpb DSCP Dtg Enk Ent

anterior anterior amygdaloid area intra-amygdaloid bed nucleus of the stria terminalis anterior commissure adrenocorticotropic hormone cortical amygdaloid nucleus anterior hypothalamic nucleus amygdalobippocampal area basolateral amygdaloid nucleus basomedial amygdaloid nucleus bed nucleus of the stria terminalis lateral division of the bst medial division of the bst combined HRP/immunohistochemical study corpus callosum cholecystokinin central amygdaloid nucleus central grey matter cobalt-glucose oxidase-diaminobenzidine corticomedial amygdala cerebral peduncle caudate putamen corticotropin-releasing factor CRF-like immunoreactivity 3,3'-diaminobenzidine tetrahydrochloride dorsomedial hypothalamic nucleus dorsal parabrachial nucleus decussation of superior cerebellar peduncle dorsal tegmental nucleus enkephalin(s) entorhinal cortex

f GOD H HRP I IC ic ip L La Lc LH LH-RH LV Me MeV ml mlf MoV mt N NGS NT ol ot ox P P PAP PBS PVH R rd RF

fornix glucose oxidase HRP study horseradish peroxidase intercalated nucleus of the amygdala inferior colliculus internal capsule interpeduncular nucleus lesion study lateral amygdaloid nucleus locus coeruleus lateral hypothalamic area luteinizing hormone-releasing hormone lateral ventricle medial amygdaloid nucleus mesencephalic nucleus of the trigeminal nerve medial lemniscus medial longitudinal fasciculus motor trigeminal nucleus mammilothalamic tract normal animal(s) normal goat serum neurotensin nucleus tractus olfactorii lateralis optic tract optic chiasma posterior (in legends) corticospinal tract (in figures) peroxidase- antiperoxidase phosphate-buffered saline paraventricular nucleus of the hypothalamus red nucleus dorsal raphe nucleus reticular formation

235 rgi rpc rpoc rpoo rtp S SC SCP sn

SP

nucleus reticularis gigantocellularis nucleus reticularis parvocellularis nucleus reticularis pontis caudalis nucleus reticularis pontis oralis nucleus reticularis tegmenti pontis ventral subiculum superior colliculus superior cerebellar peduncle substantia nigra substance P

REFERENCES 1 Azmitia, E.C. and Segal, M., An autoradiographic analysis of the different ascending projections of the dorsal and median raphe nuclei in the rat, J. Comp. Neurol., 179 (1978) 641-668. 2 Baker, J.R., Cytological Technique, Methuen, London, 1946. 3 Ben-Ari, Y., Zigmont, R.E., Shute, C. and Lewis, P.R., Regional distribution of choline acetyltransferase and acetylcholinesterase within the amygdaloid complex and stria terminalis system, Brain Research, 120 (1977) 435-445. 4 Bennett-Clarke, C., Romagnano, M.A. and Joseph, S.A., Distribution of somatostatin in the rat brain: telencephalon and diencephalon, Brain Research, 188 (1980) 473-486. 5 Bernardi, L.L. and Frohman, L.A., Effect of hypothalamic lesion at different loci on development of hyperinsulinemia and obesity in the weanling rat, J. Comp. Neurol., 141 (1971) 107-118. 6 Bernardi, L.L. and Skelton, F.R., Growth and obesity in male rat after placement of ventromedial hypothalamic lesion at four different ages, J. Endocrinol., 38 (1967) 351-352. 7 Britton, D.R., Koob, G.F., Rivier, J. and Vale, W., Intraventricular corticotropin releasing factor enhances behavioural effects of novelty, Life Sci., 31 (1982) 363-368. 8 Brobeck, J.R., Mechanism of the development of obesity in animals with hypothalamic lesions, Physiol. Rev., 26 (1946) 541-549. 9 Brown, M.R., Fisher, L.A., Spiess, J., Rivier, C., Rivier, J. and Vale, W.W., Comparison of the biologic actions of corticotropin releasing factor and sauvagine, Regul. Peptides, 4 (1982) 107-114. 10 Brown, M.R., Fisher, L.A., Spiess, J., Rivier, C., Rivier, J. and Vale, W., Corticotropin releasing factor: actions on the sympathetic nervous system and metabolism, Endocrinology, 111 (1982) 928-931. 11 Carlsen, J., Zaborszky, L. and Heimer, L., Cholinergic projections from the basal forebrain to the basolateral amygdaloid complex: a combined retrograde fluorescent and immunohistochemical study, J. Comp. Neurol., 234 (1985) 155-167. 12 Carrer, H.G., Asch, G. and Aron, C., New facts concerning the role played by the ventromedial nucleus in control of estrus cycle duration and sexual receptivity in the rat, Neuroendocrinology, 13 (1973/1974) 129-138. 13 Cho, H.J., Shiotani, Y., Shiosaka, S., Inagaki, S., Kubota, Y., Kiyama, H., Umegaki, K., Tateishi, K., Hashimura, E., Hamaoka, T. and Tohyama, M., Ontogeny of

SRIF st TMB vl vm VMH VMHc VMHs Vpb

somatostatin stria terminalis 3,3',5,5'°tetramethylbenzidine lateral vestibular nucleus medial vestibular nucleus ventromedial nucleus of the hypothalamus core of the VMH shell of the VMH ventral parabrachial nucleus

cholecystokinin-8-containing neuron system of the rat: an immunohistochemical analysis. I. Forebrain and upper brain stem, J. Comp. Neurol., 218 (1983) 25-41. 14 Cowan, W.M., Raisman, G. and Powell, T.P.S., The connections of the amygdala, J. Neurol. Neurosurg. Psychiat., 28 (1965) 137-151. 15 Cuello, A.C. and Kanazawa, I., The distribution of substance P immunoreactive fibres in the rat central nervous system, J. Comp. Neurol., 178 (1978) 129-156. 16 DeFrance, J.F. (Ed.), The Septal Nuclei, Plenum, New York, 1976. 17 Della-Fera, M.A. and Baile, C.A., Cholecystokinin octapeptide-containing picomole injections into the cerebral ventricles of sheep suppress feeding, Science, 206 (1979) 471-473. 18 Denavit-Saibi6, M. and Riche, D., Descending input from the pneumotaxic system to the lateral respiratory nucleus of the medulla. An anatomical study with the horseradish peroxidase technique, Neurosci. Lett., 6 (1977) 121-126. 19 de Olmos, J.S., The amygdaloid projection field in the rat as studied with the cupric-silver method. In B.E. Eleftheriou (Ed.), The Neurobiology of the Amygdala, Plenum, New York, 1972, pp. 145-204. 20 Eldred, W., Zucker, C. and Karten, H.J., Comparison of fixation and penetration enhancement technique for use in ultrastructural immunocytochemistry, J. Histochem. Cytochem., 31 (1983) 285-292. 21 Emson, P.C., Bj6rklund, A., Lindvall, O. and Paxinos, G., Contributions of different afferent pathways to the catecholamine and 5-hydroxytryptamine-innervation of the amygdala: a neurochemical and histochemical study, Neuroscience, 4 (1979) 1347-1357. 22 Emson, P.C., Jessell, T., Paxinos, G. and Cuello, A.C., Substance P in the amygdaloid complex, bed nucleus and stria terminalis of the rat brain, Brain Research, 149 (1978) 97-105. 23 Fallon, J.H., Koziell, D.A. and Moore, R.Y., Catecholamine innervation of the basal forebrain. II. Amygdala, suprarhinal cortex and entorhinal cortex, J. Comp. Neurol., 180 (1978) 509-532. 24 Fallon, J.H. and Moore, R.Y., Catecholamine innervation of the basal forebrain. IV. Topology of the dopamine projection to the basal forebrain and neostriatum, J. Comp. Neurol., 180 (1978) 545-580. 25 Fellmann, D., Bugnon, C. and Gouget, A., Immunocytochemical demonstration of corticoliberin-like immunoreactivity (CLI) in neurons of the rat amygdala central nucleus (ACN), Neurosci. Lett., 34 (1982) 253-258. 26 Finley, J.C.W., Maderdrut, J.L. and Petrusz, P., The immunocytochemical localization of enkephalin in the cen-

236 tral nervous system of the rat, J. Comp. Neurol., 198 (1981) 541-565. 27 Fisher, L.A., Rivier, J., Rivier, C., Spiess, J., Vale, W. and Brown, M.R., Corticotropin releasing factor (CRF): Central effect on mean arterial pressure and heart rate in rats, Endocrinology, 110 (1982) 2222-2224. 28 Fox, C.A., Certain basal teleneephalic centres in the cat, J. Comp. Neurol., 72 (1940) 1-62. 29 Fox, C.A., The stria terminalis, longitudinal association bundle and precommissural fornix fibres in the cat, J. Comp. Neurol., 79 (1943) 277-291. 30 Gloor, P., Amygdala. In J. Field, H.W. Magoun and V.E. Hall (Eds.), Handbook of Physiology, Vol. 2, Sect. 1, American Physiological Society, Washington, 1960, pp. 1395-1420. 31 Gloor, P., Electrophysiological studies on the connections of the amygdaloid nucleus in the cat, Electroencephalogr. Clin. Neurophysiol., 7 (1955) 243-264. 32 Grossman, S.P., Role of the hypothalamus in the regulation of food and water intake, Psychol. Rev., 82 (1975) 200-224. 33 Hara, Y., Shiosaka, S., Senba, E., Sakanaka, M., Inagaki, S., Takagi, H., Kawai, Y., Takatsuki, K., Matsuzaki, T. and Tohyama, M., Ontogeny of the neurotensin-containing neuron system of the rat: immunohistochemical analysis. I. Forebrain and diencephalon, J. Comp. Neurol., 208 (1982) 375-387. 34 Harris, V.S. and Sachs, B.D., Copulatory behaviour in male rats following amygdaloid lesions, Brain Research, 86 (1975) 514-518. 35 Hobel, B.G. and Teitebaum, P., Hypothalamic control of feeding and self-stimulation, Science, 135 (1962) 375-376. 36 H6kfelt, T., Elde, R., Fuxe, K., Johansson, O., Ljungdahl, A., Goldstein, M., Luft, R., Efendic, S., Nilsson, G., Terenius, L., Ganten, D., Jeffcoate, S.L., Rehfeld, J., Said, S., Perez de la Mora, M., Tapia, R., Teran, L. and Palacios, R., Aminergic and peptidergic pathways in the nervous system with special reference to the hypothalamus. In S. Reichlin, R.J. Rehfeld and J.B. Matin (Eds.), TheHypothalamus, Raven, New York, 1978, pp. 69-135. 37 Hopkins, D.A. and Holstege, G., Amygdaloid projections to the mesencephalon, pons and medulla oblongata in the cat, Exp. Brain Res., 32 (1978) 529-547. 38 Inagaki, S., Sakanaka, M., Shiosaka, S., Senba, E., Takatsuki, K., Takagi, H., Kawai, Y., Minagawa, H. and Tohyama, M., Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis. I. Forebrain and upper brain stem, Neuroscience, 7 (1982) 251-277. 39 Inagaki, S., Shiotani, Y., Yamano, M., Shiosaka, S., Takagi, H., Tateishi, K., Hashimura, E., Hamaoka, T. and Tohyama, M., Distribution, origin and fine structures of cholecystokinin-8-1ike immunoreactive terminals in the nucleus ventromedialis hypothalami of the rat, J. Neurosci., 4 (1984) 1289-1299. 40 Inagaki, S., Yamano, M., Shiosaka, S., Takagi, H. and Tohyama, M., Distribution and origins of neurotensincontaining fibres in the nucleus ventromedialis hypothalami of the rat: an experimental immunohistochemical study, Brain Research, 273 (1983) 299-235. 41 Itoh, K., Konishi, A., Nomura, S., Mizuno, N., Nakamura, Y. and Sugimoto, T., Application of coupled oxidation reaction to electronmicroscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method, Brain

Research, 175 (1979) 341-346. 42 Johansson, O., H6kfelt, T. and Elde, R.P., Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat, Neuroscience, 13 (1984) 265-339. 43 Kaada, B.R., Stimulation and regional ablation of the amygdaloid complex with reference to functional representations. In B.E. Eleftheriou (Ed.), The Neurology of theAmygdala, Plenum, New York, 1972, pp. 205-281. 44 Kawai, Y., Inagaki, S., Shiosaka, S., Senba, E., Hara, Y., Sakanaka, M., Takatsuki, K. and Tohyama, M., Long descending projections from amygdaloid somatostatin-containing cells to the lower brainstem, Brain Research, 239 (1982) 603 - 607. 45 Kawai, Y., Inagaki, S., Shiosaka, S., Senba, E., Takatsuki, K., Sakanaka, M., Umegaki, K. and Tohyama, M., Multiple innervation by substance P-containing fibres in the parabrachial area of the rat, Neurosci. Lett., 33 (1982) 271-274. 46 Kennedy, G.C., Hypothalamic control of energy balance and reproductive cycle in the rat, J. Physiol. (London), 166 (1963) 395-407. 47 Kevetter, G.A. and Winans, S.S., Connections of the cortico-medial amygdala in the golden hamster. I. Efferents of the 'vomeronasal amygdala', J. Comp. Neurol., 197 (1981) 81-98. 48 Kevetter, G.A. and Winans, S.S., Connections of the cortico-medial amygdala in the golden hamster. II. Efferents of the 'olfactory amygdala', J. Comp. Neurol., 197 (1981) 99-111. 49 K6nig, J.F.R. and Klippel, R.A., The rat brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, MD, 1963. 50 Krettek, J.E. and Price, J.L., Projections from the amygdaloid complex to the cerebral cortex and thalamus in the rat and cat, J. Comp. Neurol., 172 (1977) 687-722. 51 Krettek, J.E. and Price, J.L., Projections from the amygdaloid complex and adjacent olfactory structures to the entorhinal cortex and to the subiculum in the rat and cat, J. Comp. Neurol., 172 (1977) 723-752. 52 Krettek, J.E. and Price, J.L., Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat, J. Comp. Neurol., 178 (1978) 225-254. 53 Krettek, J.E. and Price, J.L., A description of the amygdaloid complex in the rat and cat, with observations on intra-amygdaloid axonal connections, J. Comp. Neurol., 178 (1978) 255-280. 54 Krieger, M.S., Conrad, L.C. and Pfaff, D.W., An autoradiographic study of the efferent connections of the ventromedial nucleus of the hypothalamus, J. Comp. NeuroL, 183 (1979) 785-816. 55 Kubota, Y., Inagaki, S., Shiosaka, S., Cho, H.J., Tateishi, K., Hashimura, E., Hamaoka, T. and Tohyama, M., The distribution of cholecystokinin octapeptide-like structures in the lower brain stem of the rat: an immunohistochemical analysis, Neuroscience, 9 (1983) 587-604. 56 Lammer, H.J., The neural connections of amygdaloid complex in mammals. In B.E. Eleftheriou (Ed.), The Neurobiology of the Amygdala, Plenum, New York, 1972, pp. 123-144. 57 Lehman, M.N., Winans, S.S. and Powers, J.B., Medial nucleus of the amygdala mediates chemosensory control of male hamster sexual behaviour, Science, 210 (1980)

237 557-559. 58 Ljungdahl, A., H6kfeld, T. and Nilsson, G., Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals, Neuroscience, 3 (1978) 861-943. 59 Lor6n, I., Alumets, R., Hakanson, R. and Sundler, F., Distribution of gastrin and CCK-like peptides, Histochemistry, 59 (1979) 249-257. 60 Lor6n, I., Emson, P.C., Fahrenkrug, J., Bj6rklund, A., Alumets, J., Hakanson, R. and Sundler, F., Distribution of vasoactive intestinal polypeptide in the rat and mouse brain, Neuroscience, 4 (1980) 1953-1976. 61 Luiten, P.G.M., Koolhaas, J.M., de Boer, S. and Koopmans, S.J., The cortico-medial amygdala in the central nervous system organization of agonistic behaviour, Brain Research, 332 (1985) 283-297. 62 Luiten, P.G.M. and Room, P., Interrelations between lateral, dorsomedial, and ventromedial hypothalamic nuclei in the rat, an HRP study, Brain Research, 190 (1980) 321-332. 63 Matsuzaki, T., Shiosaka, S., Inagaki, S., Sakanaka, M., Takatsuki, K., Takagi, H., Senba, E., Kawai, Y. and Tohyama, M., Distribution of neuropeptides in the dorsal pontine tegmental area of the rat, Cell Mol Biol., 27 (1982) 499-508. 64 Mayer, J. and Thomas, D.W., Regulation of food intake and obesity, Science, 156 (1967) 328-337. 65 McBride, R.L. and Sutin, J., Amygdaloid and pontine projections to the ventromedial nucleus of the hypothalamus. J. Comte. Neurol., 174 (1977) 377-396. 66 Meliza, L., Leung, P.M.B. and Rogers, R., Effect of anterior prepiriform and medial amygdaloid lesions on acquisition of taste-avoidance and response to dietary amino acid imbalance, Physiol. Behav., 26 (1981) 1031-1035. 67 Merchenthaler, I., Corticotropin releasing factor (CRF)like immunoreactivity in the rat central nervous system. Extrahypothalamic distribution, Peptides, 5, Suppl. 1 (1984) 53-69. 68 Merchenthaler, I., G6rcs, T. and Petrusz, P., Silver intensification of the diaminobenzidine reaction product for peroxidase immunohistochemistry, J. Histochem. Cytochem., 30 (1982) 607. 69 Merchenthaler, I., Vigh, S., Petrusz, P. and Schally, A.V., The paraventriculo-infundibular corticotropin releasing factor (CRF)-pathway as revealed by immunohistochemistry in long term hypophysectomized or adrenalectomized rats, Regul. Peptides, 5 (1983) 295-305. 70 Mesulam, M.-M., Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neuronal afferents and efferents, J. Histochem, Cytochem., 26 (1978) 106-117. 71 Mesulam, M.-M. and Rosene, D.L., Differential sensitivity between blue and brown reaction procedures for HRP neurohistochemistry, Neurosci. Lett., 5 (1977) 7-14. 72 Miczek, K.A., Brykzynski, T. and Grossman, S.P., Differential effects of lesions in the amygdala, periamygdaloid cortex and stria terminalis on aggressive behaviours in the cat, J. Comp. Physiol. Psychol., 87 (1974) 760-771. 73 Minagawa, H., Shiosaka, S., Inagaki, S., Sakanaka, M., Takatsuki, K., Ishimoto, I., Senba, E., Kawai, Y., Hara, Y., Matsuzaki, T. and Tohyama, M., Ontogeny of neurotensin-containing neuron system of the rat: immunohistochemical analysis. II. Lower brainstem, Neuroscience, 8

(1983) 467-486. 74 Moore, R.Y., Halaris, A.E. and Jones, B., Serotonin neurons of the midbrain raphe. Ascending projections, J. Comp. Neurol., 180 (1978) 417-438. 75 Morley, J.E. and Levin, A.S., Corticotropin releasing factor, grooming and ingestive behaviour, Life Sci., 31 (1982) 1459-1464. 76 Nauta, W.J.H., Fibre degeneration following lesions of the amygdaloid complex in the monkey, J. Anat., 95 (1961) 515-531. 77 Norgren, R., Taste pathways to hypothalamus and amygdala, J. Comp. Neurol., 166 (1976) 17-30. 78 Norgren, R., Projections from the nucleus of the solitary tract in the rat, Neuroscience, 3 (1978) 207-218. 79 Novin, D., Wyrwicka, W. and Bray, G., Hunger - - Basic Mechanism and Clinical Implications, Raven, New York, 1976. 80 Oldfield, B.J., Hou-Yu, A. and Silverman, A.-J., Technique for the simultaneous ultrastructural demonstration of anterogradely transported horseradish peroxidase and an immunohistochemically identified neuropeptide, J. Histochem. Cytochem., 31 (1983) 1145-1150. 81 Olschowka, J.A., O'Donohue, T.L. Mueller, G.P. and Jacobowitz, D.M., The distribution of corticotropin releasing factor-like immunoreactive neurons in rat brain, Peptides, 3 (1982) 995-1015. 82 Osumi, Y., Nagasawa, Y., Wang, Fu.L.H. and Fujiwara, M., Inhibition of gastric acid secretion and mucosal blood flow induced by intraventricularly applied neurotensin in rats, Life Sci., 23 (1978) 2275-2280. 83 Ottersen, O.P., Afferent connections to the amygdaloid complex of the rat and cat. II. Afferents from the hypothalamus and basal telencephalon, J. Comp. Neurol., 194 (1980) 267-289. 84 Ottersen, O.P., Afferent connections to the amygdaloid complex of the rat with some observation in the cat. III. Afferents from the lower brain stem, J. Comp. Neurol., 202 (1981) 335-356. 85 Ottersen, O.P. and Ben-Ari, Y., Afferent connections to the amygdaloid complex of the rat and cat. I. Projections from the thalamus, J. Comp. Neurol., 187 (1979) 401-424. 86 Palka, Y.S., Leibert, R.A. and Critchlow, V., Obesity and increased growth following partial or complete isolation of ventromedial hypothalamus, Physiol. Behav., 7 (1971) 187-194. 87 Palka, Y.S. and Sawer, C.H., Induction of estrous behavior in rabbits by hypothalamic implants of testosterone, Am. J. Physiol., 211 (1966) 225-228. 88 Palkovits, M. and Jacobowitz, D.M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain, J. Comp. Neurol., 157 (1974) 29-42. 89 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982. 90 Raisman, G., An experimental study of the projection of the amygdala to the accessory olfactory bulb and its relationship to the concept of a dual olfactory system, Exp. Brain Res., 14 (1972) 395-408. 91 Renaud, L.P. and Hopkins, D.A., Amygdala afferents from the mediodorsal hypothalamus: an electrophysiological and neuroanatomical study in the rat, Brain Research, 121 (1977) 201-213. 92 Ridley, P.T. and Brooks, F.P., Gastric secretion following hypothalamic lesions producing hyperphagia, Am. J.

238

Physiol., 209 (1965) 319-323. 93 Roberts, G.W., Woodhams, P.L., Bryant, M.G., Crow, T.J., Bloom, S.R. and Polak, J.M., VIP in the rat brain: evidence for a major pathway linking the amygdala and hypothalamus via the stria terminalis, Histochemistry, 65 (1980) 103-119. 94 Roberts, G.W., Woodhams, P.L., Crow, T.J. and Polak, J.M., Loss of immunoreactive VIP in the bed nucleus following lesions of the stria terminalis, Brain Research, 195 (1980) 471-475. 95 Roberts, G.W., Woodhams, P.L., Polak, J.M. and Crow, T.J., Distribution of neuropeptides in the limbic system of the rat: the amygdaloid complex, Neuroscience, 7 (1982) 99-131. 96 Sakanaka, M., Shiosaka, S., Takatsuki, K., Inagaki, S., Takagi, H., Senba, E., Kawai, Y., Matsuzaki, T. and Tohyama, M., Experimental immunohistochemical studies on the amygdalofugal peptidergic (substance P and somatostatin) fibres in the stria terminalis of the rat, Brain Research, 221 (1981) 231-242. 97 Saper, C.B. and Loewy, A.D., Efferent connections of the parabrachial nucleus in the rat, Brain Research, 197 (1980) 291-317. 98 Sar, M., Stumpf, W.E., Miller, R.J., Chang, K.-J. and Cuatrecasas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. Comp. NeuroL, 182 (1978) 17-38. 99 Sawchenko, P.E. and Swanson, L.W., A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques, Brain Research, 210 (1981) 31-51. 100 Sherwood, N.M. and Timiras, P.S., A Stereotaxic Atlas of the Developing Rat Brain, University of California Press, Berkeley, 1970. 101 Shiosaka, S., Sakanaka, M., Inagaki, S., Senba, E., Hara, Y., Takatsuki, K., Takagi, H., Kawai, Y. and Tohyama, M., Putative neurotransmitters in the amygdaloid complex with special reference to peptidergic pathways. In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven, New York, 1983, pp. 359-389. 102 Shiosaka, S., Takatsuki, K., Sakanaka, M., Inagaki, S., Takagi, H., Senba, E., Kawai, Y., Iida, H., Minagawa, H., Hara, Y., Matsuzaki, T. and Tohyama, M., Ontogeny of somatostatin-containing neuron system of the rat: immunohistochemical analysis. II. Forebrain and diencephaIon, J. Comp. Neurol., 204 (1982)211-224. 103 Siegel, A., Fukushima, T., Meibach, R., Burke, H., Edinger, H. and Weimer, S., The origin of afferent supply to the mediodorsal thalamic nucleus: enhancement of HRP transported by selective lesions, Brain Research, 135 (1977) 11-23. 104 Sims, K.B., Hoffman, D.L., Said, S.I. and Zimmerman, E.A., Vasoactive intestinal polypeptide (VIP) in mouse and rat brain: An immunohistochemical study, Brain Research, 186 (1980) 165-183. 105 Skofitsch, G. and .lacobowitz, D.M., Distribution of corticotropin releasing factor-like immunoreactivity in the rat brain by immunohistochemistry and radioimmunoassay:

106

107

108 109

110

111

112

113

114

115

116

117

118

119

120

comparison and characterization of ovine and rat/human CRF antisera, Peptides, 6 (1985) 319-336. Stanley, B.G., Eppel, N. and Hoebel, B.G., Neurotensin injected in the paraventricular hypothalamic area suppressed feeding in rats. In Conference on Neurotensin: a Brain and Gastrointestinal Peptide, The New York Academy of Science, New York, 1982. Stern, J.J., Cudilli, C.A. and Kruper, J., Ventromedial hypothalamus and short term feeding suppression by caerulein in male rats, J. Comp. Physiol. Psychol., 90 (1976) 484-490. Sternberger, L.A., Immunohistochemistry, 2nd edn., Wiley, New York, 1979. Sutton, R.E., Koob, G.F., LeMoal, M., Rivier, J. and Vale, W.W., Corticotropin releasing factor produces behavioral activation in rat, Nature (London), 297 (1982) 331-333. Swanson, L.W., Sawchenko, P.E., Rivier, J. and Vale, W.W., Organization of ovine corticotropin-releasing factor immunoreactive cells and fibres in the rat brain: an immunohistochemical study, Neuroendocrinology, 36 (1983) 165-186. Uhl, G.R., Kuhar, M. and Snyder, S.H., Neurotensin: immunohistochemical localization in rat central nervous system, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4059-4063. Uhl, G.R., Kuhar, M.J. and Snyder, S.H., Enkephalincontaining pathway: amygdaloid efferents in the stria terminalis, Brain Research, 149 (1978) 223-228. Uhl, G.R. and Snyder, S.H., Neurotensin: a neuronal pathway projecting from amygdala through stria terminalis, Brain Research, 161 (1979) 522-526. Vanderhaeghen, J.J., Lotstra, F., De May, J. and Gilles, G., Immunohistochemical localization of cholecystokinin and gastrin-like peptides in the brain and hypophysis of the rat, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1190-1194. Veening, J.G., Subcortical afferents of the amygdaloid complex in the rat: an HRP study, Neurosci. Lett., 8 (1978) 197-199. Wainer, B.H. and Rye, D.B., Retrograde horseradish peroxidase tracing combined with localization of choline acetyltransferase immunoreactivity, J. Histochem. Cytochem., 32 (1984) 439-443. Wray, S. and Hoffman, G.E., Organization and interrelationship of neuropeptides in the central amygdaloid nucleus of the rat, Peptides, 4 (1983) 525-541. Yamano, M., Inagaki, S., Kito, S. and Tohyama, M., An enkephalinergic projection from the hypothalamic paraventricular nucleus to the hypothalamic ventromedial nucleus of the rat: an experimental immunohistochemical study, Brain Research, 331 (1985) 25-33. Yamano, M., Inagaki, S., Tateishi, N., Hamaoka, T. and Tohyama, M., Ontogeny of neuropeptides in the nucleus ventromedialis hypothalami of the rat: an immunohistochemical analysis, Dev. Brain Res., 16 (1984) 253-262. Zamboni, L. and De Martino, C., Buffered picric-acid formaldehyde: a new rapid fixative for electronmicroscopy, J. Cell Biol., 35 (1967) 148A.

Note added in proof After submission of this manuscript, a paper was published by Moga and Gray (J. Comp. Neurol., 241 (1985) 275-284) suggesting a CRF pathway from the Ce to the parabrachial nuclei. The difference between those findings and our observations is that Moga and Gray detected double-labelled cells only in the caudal Ce, but not in the rostral Ce, and that precise projection fields of the descending amygdalofugal CRF pathway were not determined in their study.