The pathways mediating affective defense and quiet biting attack behavior from the midbrain central gray of the cat: an autoradiographic study

Brain Research, 437 (1987) 9-25

9

Elsevier BRE 13141

The pathways mediating affective defense and quiet biting attack behavior from the midbrain central gray of the cat: an autoradiographic study Majid B. Shaikh, Jeannette A. Barrett and Allan Siegel Department of Neuroscience, UMDNJ-New Jersey Medical School, Newark, NJ 07103 (U.S.A ) (Accepted 2 June 1987)

Key words- Midbram central gray; Affective defense behavior, Qmet bmng attack behavior, 2-Deoxy-_[laC]glucose autoradmgraphy; [3H]Leucine autoradiography

The purpose of this study was to describe the pathways whicb -'aediate feline affective defense and quiet biting attack behavior elmtted from the mldbratn ~entral gray In these experiments, met,.ods of [3H]leucme and 2-deoxy-[14C]glucose (2-DG) radioautography were utilized m concert with the technique of electrical and chemical brain stimulation Affectlve defense behavmr elicited from the midbrain central gray is characterized by marked vocalization such as hissing and growling, pupdlary ddatation, unnation and piloerection In contrast, quiet biting attack elicited from the midbrain central gray lacks overt autonomic signs observed with affective defense response as well as the stalking component which is typically associated with stimulation of the lateral hypothalamus Nevertheless, central gray-elicited attack resulted in a directed bite of the neck of an anesthetized rat In a manner similar to that observed from the hypothalamus. Affective defense was elicited from the dorsal half of the midbram central gray, whde quiet biting attack was obtained following stimulation of the ventral half of the midbrmn central gray, thus indicating a functional differentiation of the central gray with respect to these two forms of aggression. In a separate series of experiments, affective defense or qmet biting attack response was identified by electrical stimulation through a cannula electrode situated in the mldbrain central gray The affective defense responses were subsequently ehcited follovcmg mlcromjections of t) L-homocysteic acid through the same cannula electrode in order to demonstrate that these responses were the result of direct stimulation of cell bodms within the central gray. Then, one of the following autora&ographtc tracing procedures was utlhzed: (1) [3Hlleuclne was injected through a cannula electrode and the animal was sacnficed after a 4- to 14-day survival period, or (2) a 2-DG solution was systemically injected and electrical stimulation was applied through the cannula electrode in order to metabolically activate the pathways associated with each of these responses. In general, the pattern of labelled target regions as indicated by 3H-ammo acid radioautography was simdar to that obtained from the 2-DG autoradiographtc analysis. The principal asccndmg pathway associated with affective defense was traced to the anteromedial hypothalamus avd medial thalamus Concerning descending projections, label was traced into the central tegmental fields of the rmdbratn and pons, locus coeruleus and motor and main sensory nuclei of the trigeminal complex With regard to quiet biting attack, a different projection pattern appears to be present Label was followed rostrally to the level of the posterior half of the perifornical hypothalamus. Caudally directed fibers associated with quiet attack appeared to be mere restricted than those linked to the affective defense response. These axons were traced primarily to the superior collicuhis, central tegmental fields and raphe complex of the rmdbrain and pons. The results suggest that: (1) the midbrain central gray serves as a criacal structure for the expression of, at least, the vocalization component of affective defense behavior smce axons associated with this response supply nuclei of the trigeminal complex; and (2) the expression of quiet biting attack from the mldbrain central gray may involve the activation of both descending fiber bundles from the central gray to the tegmentum as well as those which innervate the periforntcal hypothalamus

INTRODUCTION

gressive r e s p o n s e s c a n b e eficited b y e l e c t r i c a l o r

T w o f o r m s o f a g g r e s s i v e b e h a v i o r - - affective d e -

chemical stimulation from wide regions of the medial p r e o p t i c o - h y p o t h a l a m u s 1°,22.26, l a t e r a l h y p o t h a l a -

fense a n d q u i e t b i t i n g a t t a c k - - o c c u r u n d e r n a t u r a l

mus ~, brainstem periaqueductal gray ms and pontine

c e n d i t i o n s in t h e cat 29. I n t h e l a b o r a t o r y , t h e s e ag-

t e g m e n t u m 3,s.~. A f f e c t i v e d e f e n s e b e h a v i o r ( a n in-

Correspondence' A. Siegel, Department of Neurosoenee, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, U.S A. 0006-8993/87/$03.50 © 1987 Elsevier Science Pubfishers B V. (Biomedacal Division)

10 tra- or interspecific aggressive reaction) initially described by Ranson et al. 36 and Hess and Brugger24, involves marked affective signs such as piloerection, retraction of the ears, baring of teeth, arching of the back, marked pupillary dilatation, growling, hissing, unsheathing of claws, paw striking, urination and sympathetic cardiovascular reactions 2°'25'4s. In the laboratory, quiet biting predatory attack behavior is characterized by an initial stalking of an anesthetized rat and culminates in a bite of the back of its neck 17'46. Predatory attack behavior, in contrast to affective attack, is not characterized by marked autonomic activation with the exception of some pupillary dilatation. A number of attempts have been made to understand the anatomical substrates underlying quiet bitmg attack and affective defense behavior 4-6 IS-IS.19. 20.43,44. Concerning the pathways associated with affechve defense behavior, an initial study utilizing the Fmk-Heimer method for the staining of degenerating axons following the placement of lesions at affective attack sites was carried out by Chi and Flynn '~4. These authors were able to trace degenerating axons through the periventricular system to the antero- and posteromedial hypothalamus, midhne thalamus and midbrain central gray. Recently, studies employing horseradish peroxidase (HRP) histochemistry have suggested the presence of reciprocal connections between hypothalam~c sites associated with affective defense behavior and the mldbrain central gray5,44. In our laboratory, we have most recently described the organization of hypothalamic pathways mediating affective defense reaction utilizing SH-amino acid radloautography and 2-deoxy-[14C]glucose (2-DG) autoradlography The results of this study have indicated that the principal origin of the descending pathway from the hypothalamus to the mldbram central gray includes neurons which are situated m the anteromediai hypothalamus and medial preoptic region20,21 Concerning the anatomical substrate for quiet biting attack, Ch~ and Flynn is traced degenerating axons from attack sites situated m the lateral hypothalamus of the cat as far caudally as the ventral tegmental area and midbrain central gray. The possibility that hypothalamlc neurons linked to qmet biting attack behavior project caudally as far as the pontine tegmentum was suggested from a study which employed

HRP as a retrograde tracer 43. Our laboratory has also utilized the methods of [3H]leucine radioautography and 2-DG autoradiography to identify the functional pathways descending from the lateral hypothalamus associated with quiet biting attack 19. This study indicated that a primary projection of this behaviorally identified hypothalamic site included the midbrain central gray while fewer quantities of labeled axons appeared to innervate the region of the motor nucleus of the trigeminal nerve (N V) and adjoining pontme tegmentum. From the studies described above detailing the distribution of fibers associated with both affective defense and quiet biting behavior, it seems likely that the midbrain central gray may serve as a nodal point in the organization of the descending pathways for the expression of these behaviors. Accordingly, the present study was conducted in order to more completely identify the pathways arising from the midbrain central gray which may constitute the next link in the descending chain of neurons comprising the functional pathways for each of these behaviors. MATERIALS AND METHODS In the present study, 18 cats of both sexes that did not spontaneously exhibit aggressive behavior were used. They had free access to food and water throughout the experiment. Cats were anesthetized with pentobarbital sodium (40 mg/kg b. wt.). During aseptic surgery, stainlesssteel guide tubes (18 gauge, 10 mm in length) for electrodes were stereotaxlcally mounted (according to the atlas of Berman 7) over holes drilled through the skul! overlying the midbrain central gray. A series of bolts was anchored to the skull for purposes of securing the cat m the head holder at a later time when brain stimulation was applied and when the isotope was administered intracerebraUy. One week postoperatively, the cat was placed in a wooden observation chamber (61 x 61 x 61 cm) with a plexiglass door. Electrolyhcally sharpened, stainless-steel stylets (insulated with an oil-base paint and exposed 0.5 mm from the tip), or cannula electrode (23 gauge), also insulated with paint except at the tip, were lowered vertically through the guide tubes at 1mm steps and electrical stimulation was applied at each step to produce a behavioral response in the

ll freely moving awake cat. Stimulation was applied with biphasic, rectangular electrode pulses (0.1-0.7 mA, 62.5 Hz, 1 ms per half cycle duration). Stimuli, generated by two independent Grass S-88 stimulators, were led through stimulus isolation units to the cat. Pairs of 40,000 Q resistors in series with the cat approximated constant current conditions. The peak-to-peak current was monitored by a tektromx 502A oscilloscope. Brain activity during the interstimulus intervals was monitored with the use of a Grass 78 electroencephalograph. When an affective defense or quiet biting attack response was observed, the electrode was cemented in place with dental acrylic. The stability and threshold current was then determined by the Method of Limits. In this method, several ascending and descending series of trials were employed. Current was raised or lowered in 0.05 mA steps in a counterbalanced a-b-b-a manner (with a above threshold and b below). The response threshold for affective defewe or quiet biting attack was defined as the current strength at which the response occurred 50% of the time. The latency to hiss, growl or bite was recorded on each trial of stimulation. Monopolar stimulation of the midbrain central gray points was employed throughout the entire experiment. For each cat, a deeply anesthetized rat and food were always present in the behavioral chamber during the course of sumulation. This allowed us to determine whether predatory, or affective forms of attack or feeding responses could also be elicited from the same site. Details concerning the nature of these responses are described in the beginning of the Results section. Only those sites which produced 'pure' forms of predatory or affective attack behavior, (i.e. uncomplicated by other unrelated responses) were chosen for study.

o,L-Homocysteic acid microinjections In order to determine that the behavior obtained at a given midbrain central gray site was the result of excitation of neuronal cell bodies rather than of fibers of passage, an excitatory amino acid - - D,Lhomocysteic acid (0.1 M, pH adjusted to between 7.4 and 8.0) - - was rejected through the cannula electrode following prior ehcitation of the response in question by electrical stimulation from the same cannula electrode 2. The chemical injections (0.5 pF100 raM) were made with the aid of a 0.5-~! syringe in

conscious animals habituated to and later restrained in a head holder at which time no resistance to restraint was observed. The head holder was employed solely to facilitate the delivery of homocysteic acid in precise amounts to the desired brain region in question. The concentrations that were selected were chosen on the basis of effective values reported elsewhere in the literature 2. The volume (0.5 pl) selected was slightly higher than those reported elsewhere 2'28 but represents the minimum amount of D,L-homocysteic acid that was required to elicit an attack response from the rostral half of the periaqueductal gray. Both the hissing component of the affective defense response as well as the biting component of the quiet attack response were rehably elicited while the animal was restrained in the head holder. At sites where electrical stimulation elicited affective defense or quiet biting attack behavior, chemical stimulation resulted in a replication of each of these responses. Each injection was made over a period of 5-10 s.

[JH]Leucine radioautography For this phase of the study, 12 cats were employed. Using cannula electrodes (23-gauge hypodermic tubing) affective defense was obtained in 7 animals and quiet biting attack in 5, and these behavioral responses were reliably produced from a given cannula electrode site in the central gray over a period of 1-3 days. After an affect~ve defense or quiet biting attack response was obtained by both electncal and chemical stimulation, the cat was then anesthetized with nembutal (40 mg/kg) and a microsyringe was lowered through the cannula electrode to its tip where electncal stimulation had previously been applied to elicit the attack response. At this time, 0.25 #1 of [4,53H]leucine (spec. act. of 62 Cilmmol concentrated to 10 ~Ci/~l, Schwartz-Mann, NY) was injected over a 20- to 30-min period. After survival times of 4-14 days, the animals were given an overdose of nembutal and perfused with 0.9% saline followed by 10% formalin. The brains were removed and fixed in 10% formahn for 7-10 days. Paraffin sections of 8 ~m were mounted, deparaffinized, dipped in Eastman Kodak NTB emulsion and kept for 4 weeks at 4 °C in light-tight boxes. The slides were developed in Eastman Kodak D-19 developer and stained with Cresyl violet.

12

2-Deoxy-[14C]glucoseautoradtography In this series of experiments 6 cats were employed, Stimulation of sites in the midbrain central gray produced affective defense in 4 cats and quiet biting attack in 2 others. Following identification of a stable attack point, animals were anesthetized with nembutal (40 mg/kg) and the following protocol was utilized during 2-DG administration. Initially, 5 trials of stimulation were employed (30 s on 30 s off) at a current intensity which had previously elicited a stable affectire defense or quiet biting attack response. Then 2DG (100/zCl/kg b. wt. in 1 ml sterile saline) was inlected into the cephalic vein. Following injection of 2-DG, the same stimulation regimen was extended for an addmonal 45 trials. Rectal temperature was maintained at 37.5 °C with a heating pad. At the end of the experiment the anesthetized cat was perfused with 3% formahn. The brain was removed, blocked and frozen in freon (dichlorodifluoromethane) cooled to -70 °C with liquid nitrogen. The brain was embedded m T~ssue Tek O.C.T. compound and stored at -70 °C in a Revco freezer. Brain sections

were cut at 20/zm on a Slee cryostat at -22 °C. Sections were then placed on coverslips and exposed to Kodak SB-5 X-ray film for 2 weeks and then developed by standard procedures. Alternate sections were mounted on slides and stained with Cresyi violet to facilitate identification of the attack sites and other labelled brain regions as detected from the Xray film. RESULTS Af-fective attack behavior elicited from the midbrain central gray is characterized by marked vocalization, such as hissing and growling, pupillary dilatation, urination, arching of the back and piloerection. In contrast, quiet biting attack elicited from the central gray lacks both the overt autonomic signs observed with affective response as well as the stalking component of quiet attack which is typically associated with stimulation of the lateral hypothalamus. Nevertheless, quiet biting attack elicited from the central g.d 3 also resulted in a directed bite of the

Fig 1 Sites within the mldbramcentral gray from which affectlvedefense (squares) and quiet bmng attack (circles) behavior were ehclted, respectively Squares and circles on the right side of each section indicate the loci of lntracerebral [~H]ieuclne injections, squares and circles on the left side of each section Indicatethe sites stimulatedfolio, ing systemicadmmistratlonof 2-deoxy-[14C]glucose Note that affectlvesites are situated dorsal to those of quiet bitingattack behavior

13 neck of an anesthetized rat in a manner similar to that observed from the hypothalamus. In the present study, affective defense and quiet biting attack behavior were elicited from 18 sites w~thtn the central gray of the cat. The loci of these s~tes are indicated m maps shown in Fig. 1. Affective defense was elictted from the dorsal half of the central gray, while qmet biting attack was obtained following stimulation of the ventral half of the central gray, thus indicating a functzonal differentiation of the central gray with respect to these two forms of aggression.

In general, the pattern of labelled target regions as indicated by 3H-amino acid radioautography was simdar to that obtained from the 2-DG auto, adiographic analys~s. The relatwe distributmn of ial: .Jed regions as determined by these methods are indicated in Tables l and II and depicted in the hne drawings shown in Figs. 2, 4, 5 and 7.

Affective defense behavtor [3H]Leucme rad:oautography (cases 1-7). Tntinted leucine mjecttons were placed into affective at-

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Fig. 2 Dlstnbution of label foUowmg a [~H]leucme rejection placed into a mldbram central gra.x site from ~h~ch affect,~e defense behavior was e h o t e d The tip of the cannula electrode from x~hich sttmulat*°n x~ds applied and ~ here the mlectt°n x~as placed t~ sh°x~ n m E Note the d,stnbutton of label rostrall} into the anterior h.vpothalamus and caudall.x into the tngemmal complex and Iocu, cocraleu~ of th,' pons

14 t a c k sites l o c a t e d m t h e c e n t r a l g r a y in 7 cats. Six o f

c e n t to but d i d n o t e x t e n d i n t o the h a b e n u l a r c o m -

t h e sites w e r e l o c a t e d m the r o s t r a l h a l f o f t h e c e n t r a l g r a y at t h e l e v e l o f t h e r e d nucleus, w h i l e t h e 7th site

plex. A s e c o n d g r o u p o f l a b e l l e d a x o n s w e r e t r a c e d m a ventral direction from the midbrain-diencephalic

(case 7) was s i t u a t e d m o r e c a u d a l l y at t h e level o f t h e

juncture through posterior midline nuclei into the

r o s t r a l e n d o f t h e i n f e r i o r colliculus. A t e a c h o f t h e s e

posterior hypothalamus. This bundle of fibers passed

sites, affective d e f e n s e was e l i c i t e d b y b o t h e l e c t r i c a l

b o t h m e d i a l a n d l a t e r a l t o t h e f o r n i x in its r o s t r a l t r a -

(current threshold range: 0.2-0.35

and by

j e c t o r y , r e s u l t i n g in t h e p r e s e n c e o f l a b e l o v e r t h e

c h e m i c a l (D.L-homocysteic acid) s t i m u l a t i o n . T h e

posterior and ventromedial nuclei of the hypothala-

o v e r a l l p a t t e r n o f l a b e l l i n g was r e l a t w e l y s i m i l a r

m u s as well as t h e l a t e r a l p e r i f o r n i c a l r e g i o n i m m e -

mA)

a m o n g e a c h o f t h e cases e x a m i n e d , w i t h b u t m i n o r

diately caudal to the level of the lamina termin~lis.

v a r i a t i o n s a s s o c i a t e d with the m o r e c a u d a l i n j e c t i o n

No label could be traced rostrally beyond the ante-

in case 7 ( T a b l e I); t h e r e f o r e , o n e r e p r e s e n t a t w e

rior hypothalamus into the regions of the basal fore-

case will b e d e s c r i b e d in d e t a i l (Fig. 2). In case 1, t h e r e j e c t i o n s a c was s i t u a t e d c e n t r a l l y

brain.

w~thin t h e m i d b r a i n c e n t r a l g r a y at t h e level o f nu-

T a b l e I, d i f f e r e d s o m e w h a t f r o m t h o s e o f c a s e s 1 - 6

cleus I I I (N I I I ) a n d its d i a m e t e r was a p p r o x i m a t e l y

w h i c h w e r e j u s t d e s c r i b e d . C a s e 7, w h o s e i n j e c t i o n

0.7 m m . A s s h o w n in Fig. 2, an m t e n s e d i s t r i b u t i o n o f

site was l o c a t e d m o r e c a u d a l l y ~vithin t h e c e n t r a l g r a y

A s n o t e d a b o v e , t h e r e s u l t s o f case 7, s h o w n in

l a b e l l e d f i b e r s , p r e s u m a b l y a s s o c i a t e d d i r e c t l y with

at t h e level o f t h e a n t e r i o r a s p e c t o f t h e i n f e r i o r colli-

affective d e f e n s e b e h a v i o r , w e r e o b s e r v e d t o p a s s

culus, d i s p l a y e d o n l y s m a l l q u a n t i t i e s o f l a b e l a l o n g a

b o t h r o s t r a l l y a n d c a u d a l l y f r o m t h e i n j e c t i o n site. Ascending pathways. L a b e l l e d a x o n s w e r e t r a c e d

c e p h a l o n . In this c a s e , l a b e l was l i m i t e d t o t h e p o s -

rostrally t h r o u g h t h e c e n t r a l g r a y i n t o t h e d i e n c e p h a -

terior medial and posterior lateral hypothalamus and

Ion where at l e a s t t w o distract f i b e r b u n d l e s e m e r g e d .

to t h e p a r a f a s c i c u l a r n u c l e u s o f t h e t h a l a m u s . L a b e l

A t this level, o n e g r o u p o f l a b e l l e d a x o n s c o u l d b e

was n o t o b s e r v e d in a n y o t h e r d i e n c e p h a l i e s t r u c t u r e .

rostrally directed projection system to the dien-

f o l l o w e d f u r t h e r r o s t r a l l y into m e d i a l t h a l a m i c nuclei

Descending fibers. L a b e l l e d fibers f r o m t h e i n j e c -

which m c l u d e d t h e c e n t r u m m e d i a n u m - p a r a f a s c l c u -

t i o n site w e r e inttiaUy t r a c e d in a c a u d a l d i r e c t i o n

lar c o m p l e x ( C M - P F )

and posterior half of the me-

within the central gray. Throughout the central gray

d i o d o r s a l t h a l a m i c nucleus. L a b e l was p r e s e n t a d j a -

w h e r e label was p r e s e n t , a d d i t i o n a l g r o u p s o f a x o n s

TABLE I

[3H]Leucme-labeled regtons following inlecttons placed into mtdbram central gray sues from which affecuve defense and quwt blung attack behavior was ehctted Values in&catlve of the relatwe amount of silver grmns present over a gwen structure were assigned according to the following criteria x x x, heavy, x x, moderate; x , light; 0, not sufficiently labeled

Structures

Affecuve defense Case

Centrum medlanumparafasclcular complex Antenor hypothalamus Ventromedlal hypothalamus Lateral (penformcal) hypothalamus Posterior hypothalamus Rostralcentralgray Supenor colhculus Rostral central tegmental fields Caudal central gray Caudal central tegmental fields Raphe nucleus Locus coeruleus Motor nucleus of N V Mare sensory nucleus of N V

Quwt btung attack

1

2

3

4

5

6

7

8

9

I0

11

12

xx xx ×x xx xx ×xx 0 ×x xx xx 0 xx xx ××

x x x x x xx 0 xx xx xx 0 xx xx x

x x x x x xxx 0 xx x '< xx 0 x x x

x x x x x xxx x xx xx x x x xx xx

x x x x x xx 0 xx xx x x x xx x

x x x x xx xxx x xx xx xx 0 xx xx x

x 0 0 x x xx 0 x× xxx x 0 xx x× ×

x 0 0 x x xxx xx x x x xx 0 0 0

x 0 0 x x xxx xx xx x x x 0 0 0

0 0 x x x xxx xx xx xx xx x 0 0 0

x 0 0 x xx ×xx ×x xx xx xx xx 0 0 0

x 0 0 x xx xx× xx xx xxx xx xx 0 0 0

15 could be followed into the adjoining tegmentum. At the level of the superior coUiculus, axons were traced in a ventrolateral direction into the central tegmental fields and periruberal area. At the level of the inferior colliculus, axons were traced directly into the region of the nucleus locus coeruleus as well as into the central and lateral tegmental fields. Further caudally, groups of axons were o b s e ~ e d to innervate both motor and main sensory nuclei of N ~t and adjoining regions of the tegmentum. Small quantities of label were also noted in the rostral aspect of the spinal nucleus of N V but no label could be identified in any structure cauOal to this level (see Fig. 8 B - D ) .

2-deoxy-[t4C]glucose autoradiography (cases 13-16). In 4 animals, 2-DG was administered systemically while stimulation was applied to aff~ctive attack sites located in the dorsal half of the midb~ ain central gray (Figs. 1, 3E and 4D). Marked increase in optical densities appeared around the tip of each of the electrodes in the form of a sphere indicative of the volume-conducted activation of cell bodies, axons and axon terminals at the site of stimulation. The sphere of activation was limited to the dorsomedial central gray and did not appear to extend into other regions of the neuropil. Concerning the distribution of activated regions,

the overall patterns ot iabelhng were similar among each of these cases (Table 1I). Accordingly, the results of case 13 will be described. Increased optical densities were noted from the stimulation site along a rostrally directed pathway passing through the central gray, adjoining aspects of C M - P F complex and along the dorsomedial aspect of the hypothalamus. Increased activatton was diffusely present throughout the ventromedial hypothalamus and to a lesser extent in adjoining portions of the lateral hypothalamus. This pattern of labelling extended as far rostrally as the anterior hypothalamus Regarding the caudal pattern ot labelling following stimulation of this affective attack site, increased activation was noted throughout the central gray from the site of stimulation to the pt ",tine-midbrain border. Increased patterns of activa,ton were then followed along a pathway directed ventrolateraUy into the region of the nucleus coeruleus and adjacent motor and main sensory nuclei of N V. Diffuse increases in activation were also noted throughout the tegmental fields. Labelling here extended from the level of the injection site to the level of the motor nucleus of N V in the pons. The pattern of labelling for case 13 is shown in Figs. 3 and 4. Thus, the findings obtained with 2-DG, in general, paralleled the patterns of la-

Ftg 3 Photographs of 2-deoxy-[t~C]glucose-labeled regions following electrical stimulation of the mldbram central gra.x from ~ htch affectt~e defense behavior was elicited Arro~ m E indicates site of stlmulatton located m the medtodorsal central gra.x A r r o ~ , m all other panels depict actwated regions resulting from sttmulatton Note the presence of label ~ ~thm the dorsomedlal h~ pothalamu, I A) and the region of the nucleus locus coeruleus (F)

16

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F~g. 40uthne of the &stnbut~on of actwated regtons assocmted w~th st~mulat:on of an affectwe defense site m the m~dbram central gray following a systemic rejection of 2-deoxy-[laC]glucose Site of stimulation m this figure and m Fig 7 is m&cated with a star m D Rostral to the stte of stimulation, regions exhibiting increased aetwatlon can be seen throughout the medml hypothalamus, mldhne thalam~c nucle~as well as m the central gray Caudal ~o the s~te of stimulation, actwated regions can be observed m the central tegmental fields, locus coeruleus and motor nucleus of N V belhng observed from the analysis of cases involving injections of [3H]leucme at central gray affective attack sites. Qutet bltmg attack behavior. As noted above, the sites at which quiet biting attack behavior was elicited were somewhat ventral to those which produced affective attack. Interestingly, the pathways arising from these quiet biting attack sites also differed considerably from those associated with affectlve defense behavior. The pathways associated with quiet bitmg attack behavior from the mldbraln central gray are described below [JH]Leucme radioautography (cases 8-12). Trttiated leucine injections were placed into the mtdbrain central gray of 5 cats from which electrical and chemical sttmulatton had previously ehctted quiet biting attack behavior Again, the distribution of label followmg the placement of these mjectlons were generally similar in the cases examined (Table I). Ac-

cordlngly, the results of case 8 shown in Figs 5 and 8A, will be discussed. Ascending pathways. From the site of the injection in the ventrorostral central gray label over axons was followed to the rostral border of the central gray and then into the adjoining portions of the posterior thalamus Thalamic structures over which label was present include the C M - P F complex, lateral habenular nucleus, medial aspect of the posterior mediodorsal nucleus and posterior midhne nuclei. A group of labelled axons was also traced ventrally from the mldhne thalamus primarily into the posterior hypothalamus Smaller quantities of labelled axons extended rostrally for a short distance from the posterior hypothalamus into the adjacent periformcal hypothalamus, but could not be traced rostrally beyond this level in contrast to the ascending pathways associated with affective defense. Descending pathways. At the level of the injection

17 TABLE I! 9 14 Denss.tometrtc ratto~ of _-deory-[ C]glucose-labeled regions as determined from mmge analbsls followmg electrwal strmulatton of the m,dbram central grab' ~ltesfrom which affecttve defense and quiet biting attack ~,as ehctled

Values of 0 20 or greater were clearly visible to the naked eye and, m addmon, were shown to be statistically slgmhcant (t-test for rodependent observations comparing isotope content of the structure m question w~ih its homologue on the contralaterai side). Values were derived by determining, with the use of autoradlograph~c standards, the ratio of the difference m activation between a site ipsilateral to the stimulating electrode and the relative isotope content in the homologous structure of the contralateral side from the followmg equaUon: (A - B)/C, where A = #Cffg acnvated structure; B =/~Ci/g homotypical structure of the contralateral side, and C = l*Cl/g homotypical structure of the contralateral s~de. Structure

Affecuve defense Case

Centrum medlanum-parafascicular complex Anterior hypothalamus Ventromedial hypothalamus Lateral (perifornlcal) hypothalamus Posterior hypothalamus Rostral central gray Superior colhculus Rostral central tegmental fields Caudal central gray Caudal central tegmental fields Raphe nucleus Locus coeruleus Motor nucleus of N V Main sensory, nucleus of N V

Quret bumg attack

13

14

15

16

17

18

0 33 0 35 0.37 0 36 0.57 1 50 0 14 0 25 0 80 0 42 0 16 0 33 0 71 0 33

0 20 0 22 0 28 0 23 0 29 2 22 0 17 036 0 96 0 75 0 07 0.53 0.61 0.23

0 23 10 1 02 1 81 0 91 2 25 00 10 1 45 0 61 O.19 0.60 0 39 0 37

0 43 0 63 0 61 0 54 0 60 1 19 00 063 0 87 0 45 00 0 63 0 41 0.30

0 29 0 08 0 06 0 20 0 57 0 95 0 29 044 0 47 0 33 0 29 0 09 00 00

0 51 00 0 03 0 21 0 40 1 64 0 85 041 l.O 0 82 0.67 0.0 00 00

site in the ventral central gray, labelled axons were followed directly into the superior colliculus, where

temically while electrical stimulation was applied to quiet biting attack sites located in the ventral half of

terminals were present over all but its superficial layer. A n o t h e r group of labelled axons passed in a ventrolateral direction from the central gray where they appeared to terminate diffusely throughout the lateral and central tegmental fields at the superior colli-

the midbraln central gray (Figs. 1, 6D and 7D). Marked increases in optical densities were noted around the tips of electrodes in the form of spheres of activation similar to that described above for affec-

cular level. It was also noted that label was present over these fields at more caudal levels of the midbrain which even extended into the pons. The course of this latter pathway appeared to revolve descending axons within the central gray which passed into the tegmentum at progressively more caudal levels of the midbrain. Large quantities of label were followed directly into the raphe complex at both midbrain and pontine levels. Such a projection was not observed in our analysts of the descending pathways associated

tive defense. In these cases, however, activation at the sites of stimulation was limited to the ventral half of the midbrain central gray (Fig. 6D). Rostral to the site of stimulation, increased levels of activation could be followed along a pathway through the central gray and into the superior colhculus and C M - P F complex. Increased levels of activation could not be observed in any other thalamic nucleus. Instead, additional regions of increased activation were detected along a pathway passing ventrally from the central gray into posterior levels of the dor-

gemlnal complex or in the locus coeruleus, which d~d constitute principal target nuclei of descending fibers

solateral hypothalamus (Figs. 6 and 7). Again, no evidence of increased levels of activation were observed beyond this region within the hypothalamus m marked contrast to the more extensive 2-DG pat-

associated with central gray elicited affective de-

terns obtained following stimulation of affective at-

fense. 2-deo.~ y-[/4C]ghtcosc

tack sites within the midbrain central gray Caudal to the site of stimulation, increased levels of activation were detected along a descending path-

with affective defense behavior However, no label could be detected within any c o m p o n e n t of the tri-

atttotadtography

(ca~es 17

and 18) In 2 animals. 2-DG was administered svs-

18

A

B

C

/,

D

IF.

- .., .-'.~:.~:~ i-,

,

.

i°

-~

F

G

Fig 5 Distributionof label foilowmga [3H]leucmeinjectionplaced mto a site wlthmthe mldbramcentral grayfrom whtchqmet biting attack was ehc~ted.The tip of the cannulaelectrode fromwhichstimulationwas apphed and where the mlect~onwas placed is shown m C Note the obhque,lessof sectionsshownm A-C, and distributionof label into the raphe complex, E-I way through the central gray and ventrally from ~t to include the mldbrain raphe complex and central tegmental fields. The evidence of increased levels of activation could not be detected m any c,laer structure of the brainstem Agam, the 2-DG findings generally paralleled the observations obtained from cases revolving the placement of rejections of [3H]leucme into quiet biting attack sites within the central gray DISCUSSION In the present study the pathways arising from the midbrain central gray associated with affectwe de-

lense behavior and quiet biting attack in the cat, summarized in Fig. 9, were identified utilizing the methodologies of [3H]leucine radioautography and 2-DG autoradiography. The combination of these approaches has been employed previously in our laboratory to identify the hypothalamic pathways subserving quiet b~tmg attack, affective defense and flight behavior m the cat ~8-2x. It should be noted that for the most part, as shown in Figs. 2, 4, 5, 7 and in Tables I and II, projection patterns observed with [3H]leucine radioautography paralleled the regional 2-DG distribution within the brain for each of the attack responses considered. In

19

Fig 6 Photographs of 2-deoxy-I~4C]glucose-labelled regions following electrical stimulation of the m~dbram central gray from whtch quiet b~tmg attack was elicited Arrow m / ) depicts s~te of stimulation m the ventral central gray of the m~dbram Arrows m all other panels indicate structures with increased levels of activation following stimulation

B

\

L

C

.:..:

// >,~ 0' ":~:': /1/ ,, ".:...;../ •

"

.p

::'?.?."

1::

Fig 7 0 u t h n e of the distribution of activated regions associated wtth stimulation of a qmet bmng attack ~tc m the m~dbram central gray following a systemic injection of 2-deoxy-[l~CIglucose Note increased actwauon within posterior h.xpothalamu~ ~ -X~ and ccntral tegmental fields at the mtdbram-pontme juncture (F)-

20

F,g 8 Dark-field photomicrographs. A" [3H]leucine injection placed into a site in ventral central gray from which quiet biting attack was ehclted upon electncal and chemical stimulation, arrows indicate the region where [3H]leucme was incorporated into cells (x 30). B. labelled axons situated proximal to the motor nucleus of N V C labelled axon terminals in the central tegmentaWfields of the midbrain at the level of inferior colhculus, both following injection of [3H]leucine into the central gray site from which affectlve defense was ehclted D absence of label in the central tegmental fields of the contralaterai side of the brain whose section is shown in C (B-D. ×175) as much as both attack responses were elicited by chemical (D L-homocysteic acid) as well as by electrical stimulation, it Is reasonable to conclude that the overwhelming majority of activated fiber pathways for each behavior originate from the central gray. Our observations rephcatc those previously reported in the literature with excitatory amino a o d s 2, as well as with carbacho137, which thus lends further support

to the view that the activation of the central gray cell bodies at the level of tile superior colhculus are sufficient for elicitation of affectlve defense and quiet biting attack responses in the cat. It is also of interest to note that excitatory a m i n o acids Injected into the central gray of the sqmrrel m o n k e y can elicit vocalizat,on characteristic of a defensive reaction 28, thus suggesting that central gray may subserve similar

21

lOt lhalamus

neurons would be driven less frequently than first-order neurons and at a rate insufficient for 2-DG labelling to be visuahzed.

Affective defense behavior ,nor thalamus

nor ulus B~bng S,te

Jleus

~ry Nucleus r • Nucleus _r

Fig 9 Schematic diagram illustrating the principal efferent proJections from mldbramcentralgray s~tesassociatedwith affectlvedefense (left side) and quiet bitingattack (right side) behavior. functions in different species of mammals. Concerning the 2-DG aspect of the study, it is not at all surprising that electrical stimulation faded to actwate fibers beyond the f~.rst synapse. This issue has been considered at length in a number of previous pubhcations from our laboratory2° 49.5o. This effect is not hkely due to anesthesia since similar effects were observed in unanesthetized preparations as well. In several of our previous publications, we also demonstrated that such an effect was not frequency dependent 47-49. One possible explanation of this general finding is that the 2-DG method demonstrably labels those neurons which, upon stimulation, are driven m a one-to-one relationship. Whale first-order neurons are likely drwen at a one-to-one relationship relative to applied stimulation, it ~s likely that second-order neurons do not follow at a rate equivalent to applied stimulation because of the presence of excitatory-inhibitory sequences in those neurons. Thus, it is possible that electrical stimulation might occur dunng the inhibitory phase of the action potential in second-order neurons and, accordingly, such

The results of the present study reveal two distinctly different patterns of fiber projections from the central gray which are associated w~th affective defense and quiet biting attack behavior in spite of the relatively short distances separating the sites at which each of these behaviors was elicited. The pathways associated with affective defense are discussed immediately below while those associated with quiet biting attack will be considered later in the discussion. In a previous study2~, it was demonstrated that the principal descending affective defense pathway in the cat which arises from the hypothalamus has, as its cells of origin, the anteromedial region rather than the ventromedial nucleus. These neurons from the anteromedial region project m dense quantities to the midbrain central gray. Thus, the affective defense pathways described in the present study, represent second-order neurons from the hypothalamus which descend from the midbrain central gray to more caudal levels of the brainstem tegmentum and trigemmal complex or ascend from the central gray back to the rostrocaudal extent of the medml hypothalamus and to the medial thalamus. Concerning the descending pathways, it is possible that the projections to the pontine tegmentum constitute a downstream relay for the expression of paw striking. Such a pathway might also serve as a substrate for regulation of cardiovascular responses concomitant with affective attack. Speofically, sympathetic activation may be mamfested through a descendmg projection to the region of the locus coeruleus and surrounding noradrenerg~c neurons which have been previously shown to supply preganglionic sympathetic neurons in the intermediolateral cell column at thoracic and hlmbar levels of the spinal cord 34. Alternatively, it has also been suggested that the cardiovascular component of this response may be mediated by a relay to the solitary nucleus6. But this observation was not confirmed m the present study Perhaps, this discrepancy could be due to the possibility that, in the present study, the pathways associated w~th affective attack were traced from neu-

22 tons s~tuated somewhat more rostrally w~thin the periaqueducta! gray than those sites explored by Bandler and Tork 6 Of particular interest are the projections from the central gray to the tngeminal complex. It is likely that this pathway is essential for the regulation of the vocalization (i.e. hissing) component of the affective attack response by acting directly upon those motor neurons of N V which directly control jaw opening (i.e. the dlgastnc reflex) It may be noted that in the squirrel monkey, the pathway associated with vocahzation bears some similarity to that described above in the cat for hissing with respect to the d~stnbutlon of fibers to the midbraln and pontine tegmentum 27. These authors did point out, however, that in one case which was examined, axons from the periaqueductal gray were also traced directly into the nucleus ambiguus which presumably regulate laryngeal muscles. These observaUons suggest the possibility that sites within the central gray from which different kinds of vocalizatton can be elicited may be associated with descending pathways which differ from one another. Regarding the ascending connections from sites in the central gray from which affective defense was ehclted, it appears that the primary targets include the rostrocaudal extent of the medial hypothalamus and, to a lesser extent, medial thalam~c nuclei. This pattern of ascending projections appears to closely parallel that described previously by Hamilton 23 Since these fibers preferentially innervate the medial hypothalamus from which affectlve defense behavior can also be reliably elicited 2°,2L46, it is hkely that such ascending projections may serve as a positive feedback mechamsm for the regulation of this response. Concerning the possible putatwe transmmer actmg within the central gray and in association with aggresswe behavior, it is noteworthy to pomt out that Moss et al. 33 have demonstrated the presence of leucine-enkephalin-like lmmunoreactivlty in the cat penaqueductal gray localized to regions from which affectwe defense behavior was elicited m the present study. That the oplold peptlde system may play a significant role m regulating affective defense behavior is suggested from a recent study in our laboratory in which ~t was demonstrated that central gray inhlbmon of hypothalamlcally ehclted affective defense behavior can be blocked following administra-

tion of naloxone into central gray modulatory sites 35 Presently, experiments are under investigation to characterize the types of opiates w~thin the central gray that could effectwely alter affectwe attack behavior.

Quiet binng attack pathways Perhaps, one of the most interesting features of the present findings is that the two distinctly different kinds of attack responses can be elicited from adjoining regions of the central gray which are also associated with different patterns of projections. In this study, quiet bit:ng attack behavior was elicited from the ventral aspect of the central gray and the principal projection targets were the perifornical hypothalamus, superior colliculus, midbram and pontine tegmentum including the raphe complex. The lesults of the present study suggest two possible roles for the central gray in the regulation of quiet biting attack behavior. One likely possibility is that it constitutes a relay from the perifornical lateral hypothalamus ~9to the tegmentum of the rostral pons from which quiet attack can also be elicited by electrical stimulation ~9"42. One must then presume that neurons at this level of the pontme tegmentum could then synaptically activate both the neighboring trigemmal motor nucleus which would be essential for the occurrence of the biting component of the response as well as the main sensory nucleus whose role might include modification of the sensory fields along the lipline following the onset of this behavioral process !1"!2"31. In addition, it must also be presumed that neurons in the pontine tegmentum would make synaptic contact with other descending ret~culospinal fibers which could then modulate spinal cord motor neurons whose axons innervate the muscles involved in paw striking ~6 It is also plausible that the midbrain central gray may comprise a positive feedback route for further activation of the quiet attack mechamsm from the perifornical hypothalamus. This possibility is certainly suggested from the anatomical observations of the present experiment m which fibers from quiet attack sites in the midbrain central gray were shown to ascend to the perifornical region. It is of interest to note that the ventral central gray and lateral hypothalamus also seem functionally related with regard to the reward system as determined from selfstimulation studies 3z. Thus, it is tentatwely suggested

23 that the expression of qmet b r i n g attack behavior following stimulation of the midbrain central gray may involve concurrent activatmn of two converging parallel systems - - a descending pathway from the central gray involving the pontine tegmentum and reticulospmal fibers and an ascending pathway to the hypothalamus. From a previous study, it has been shown that the perifornical hypothalamus projects directly to the m o t o r nucleus of N V as well as to the locus coeruleus and neighboring regions of the pontine tegmentum 19. Accordingly, this hypothalamic pathway would appear to converge upon the same groups of neurons which are directly activated by ventral central gray stimulatton, which could then presumably facilitate the occurrence of the attack response. It is also of interest to note several other projections from ventral central gray sites from wllich quiet biting attack behavior was elicited. These include connections to the superior colliculus and to the median raphe of the midbrain and ports. It is possible that the projection to the superior colliculus may normally serve to aid in regulating tracking movements of the prey object within the cat's visual field. Such a mechanism would be of obvious significance in the pursuit and capture of the prey. T h e possible significance of the projection to the raphe complex can only

be suggested. Previous studies from our laboratory have indicated that stimulation of the medmn raphe of the cat can suppress quiet biting attack behavmr 39-41. In addmon, it has also been shown that pchlorophenylalanine (PCPA) admimstration facilitates the occurrence of quiet attack 3°. This finding is consistent with those obtained w~th electrical stimulation and further suggest the possibility that the serotonin (5-HT) system may serve to inhibit the quiet attack response. Presently, it would be of interest to determine the functtonal s~gnificance of the projection from the ventral central gray to the median raphe nucleus. Specific knowleclge concerning the extent to which ventral central gray stimulation could effect changes in raphe neuronal discharge patterns and in 5-HT release could be quite useful m providing further insight into the role of this transmitter in pred,~tc,ry aggression.

ABBREVIATIONS

MT P PF PT PV RE RN SC SM SN VL 5m 5S 5M 5 SP

CAE CG CI CM CP CT HA HL HP IC IP LH MD MM

locus coeruleus central gray inferior colhculus nucleus centrum medmnum cerebral peduncle central tegmental fields antenor hypothalamus lateral hypothalamus posterior hypothalamus internal capsule mterpeduncular nucleus lateral habenula medlodorsal nucleus medial mammdlary nucleus

REFERENCES 1 Bandler, R , Neural control of aggressive behavior, Trends Neurosct , 5 (1982) 390-394 2 Bandler, R., Idenaficatmn of hypothalamic and mldbrain neurones medmtmg aggresswe and delenswe behawor by lntraeerebral micromlectmns of exotatory amino acids In

ACKNOWLEDGEMENTS This research was supported by Grant NS 0794118 from the National Institutes of Neurological Dtsorders and Communicative Dtseases and Stroke. The authors also wish to thank Dr Susan Fuchs for her technical suggestions and Mrs. Rose Adler for typing the manuscript.

mammlllothalam~c tract pyramtdal tract parafasclcular nucleus praetectal area paravenmcular nucleus nucleus reumens red nucleus superior colhculus stria medullarls substantmmgra ~estibular nucleus mesencephahc nucleus of N V sensory nucleus of N V motor nucleus of N V spinal nucleus of N V

R Bandler (Ed). Modulanon of Sensonmotor Acuvey During Alterations m Behavioral States. Liss. New York, 1984. pp 369-392 3 Bandler, R J., Chl, C C and Flynn, J P., Bmng attack ehc~ted by stlmulatmn of the ventral m~dbram tegmentum of cats, Science. 177 (1972) 364-366 4 Bandler, R. and McCulloch, T , Afferents to a m~dbram

24 penaqueductal grey region in the 'defense reaction" m the cat as revealed by horseradish peroxldase II The dlencephalon, Behav Brain Res, 13 (1984) 279-285 5 Bandler. R , McCulloch, T and Dreher, B., Afferents to a mldbraln penaqueductal grey region involved m the 'defense reaction' in the cat as revealed by horseradish peroxldase. I The telencephalon, Brain Research, 330 (1985) 109-119 6 Bandler, R and Tork, I , Afferent and efferent connections of solitary tract nuclei with a mmdbralnperlaqueductal gray region mediating the defense reaction in the cat, Soc Neurosct Abstr. 12 (1986) 1155 7 Berman, A L , The Brain Stem of the Cat A Cytoarchltectonic Atlas with Stereotaxlc Coordmates, The University of W~sconsmPress, 1968 8 Berntson, G G , Blockade and release of hypothalamlcally and naturally ehclted aggresswe behaviors in cats foUowmg midbraln lesions. J Comp Physiol Psychol, 81 (1972) 541-554 9 Berntson. G G , Attack, grooming and threat ehclted by stimulation of the pontlne tegmentum in cats, Phystol Behay, 11 (1973) 81-87 i3 Berntson, G G and Beattle, M.S., Functional differentiatlor, w~thm hypothalam~c behavioral systems in the cat, Phystol Psychol, 3 (1975) 183-188. 11 Block, C H , Siegel, A and Edlnger, H . M , Effects of amygdalold stimulation upon tngemmal sensory fields of the hps that are estabhshed dunng hypothalamlcally elicited qmet biting attack m the cat, Brain Research, 197 (1980) 39-55 12 Block, C H , Siegel, A. and Edmger, H , Effects of stimulation of the substantla innomlnata upon attack behavior ehclted from the hypothalamus in the cat, Brain Research, 197 (1980) 57-74 13 Chl, C C and Flynn, J P , Neuroanatomlc projections related to bmng attack elicited from hypothalamus in cats, Brain Research, 35 (1971) 51-65. 14 Chl, C C and Flynn, J P , Neural pathways associated with hypothalam~cally ehclted attack behavior m cats, Scwnce, 171 (1971) 703-705 15 Chl, C C., Bandler, R. and Flynn, J P., Neuroanatomlc prolectlon~ related to biting attack elicited from the ventral mldbraln in cats, Brain Behav Evol, 13 (1976) 91-110. 16 Edwards, S B. and Flynn, J P , Corttcospmal control of striking in centrally ehclted attack behavior, Brain Research, 41 (1972) 51-65 17 Flynn, J P , Vanegas, H., Foote, W and Edwards, J , Neural mechanisms involved in a cat's attack on a rat In P Whalen (Ed), The Neural Control of Behavior, Academic, New York, 1970, pp 135-173. 18 Fuchs, S A G. and Siegel, A , Neural pathways medmtmg hypothalamlcally elicited flight behavior m the cat, Bram Research, 306 (1984) 263-281 19 Fuchs, S A G., Dalsass, M , Siegel, H.E and Siegel, A , The neural pathways mediating qmet biting attack behavior from the hypothalamus m the cat a functional autorad~ographic study, Aggression Behav, 7 (1981) 51-68 20 Fuchs, S A G , Edmger, H M and Siegel, A., The orgamzat~on of the hypothalamlc pathways medmtlng affectlve defense behavior in the cat, Brain Research, 330 (1985) 77-92 21 Fuchs, S A G , Edmger, H M and Siegel, A., The role of the anterior hypothalamus in affectlve defense behavior ellc~ted from the ventromedml hypothalamus of the cat,

Bram Research, 330 (1985) 93-107 22 Fuchs, S A.G., Siegel, A and Edmger, H , Neural clrcmtry subserving affectlve display elicited from the feline hypothalamus, Soc Neurosci. Abstr, 8 (1982) 973. 23 Hamilton, B L., Prolect:ons of the nucle~ of the penaqueductal gray matter in the cat, J. Comp. Neurol, 152 (1973) 45-58 24 Hess, W.R and Bragger, M , Das subkortikale Zentrum der afflektmen Abwehrreaktion, Helv. Physiol. Pharmacol Acta, 1 (1943) 33-52. 25 Hdton. S M , Hypothalamlc control of the cardiovascular responses m fear and rage. In J P Ross (Ed), The Scwnttftc Basts of Medicine Annual Revwws. Athlone, London, 1965, pp 217-238 26 Inselman, B R. and Flynn, J P., Modulatory effects of preoptic stimulation on hypothalamlcally ehcited attack m cats, Brain Research, 42 (1972) 73-87. 27 Jurgens, U and Pratt, R , Role of the penaqueductal grey m vocal expression of emotion, Brain Research, 167 (1979) 367-378. 28 Jurgens, U. and Richter, K , Glutamate-induced vocalization in the sqmrrel monkey, Blain Research, 373 (1986) 349-358 29 Leyhausen, P., Cat Behavior. The Predatory and Social Behavior of Domesuc and Wdd Cats, Garland STPM, New York, 1979 30 MacDonnell, M F , Fessok, L and Brown, S . H , Aggression and assocmted neural events m cats and effect of chlorophenylalanlne compared with alcohol, Q J Stud. AlcohoL 32 (1971) 748-763. 31 MacDonnell, M. and Flynn, J P , Control of sensory fields by stimulation of the hypothalamus, Science, 152 (1966) 1406-1408 32 Mdner, P.M and Laferrlere, A., Strength-duration characteristics of lateral hypothalamic and perlaqueductal gray reward-path neurons, Phystol Behav, 29 (1982) 857-863. 33 Moss, M S., Glazer, E.J. and Basbaum, A , The peptlderglc organization of the cat penaqueduetal gray I the distribution of ~mmunoreactwe enkephalin concentrating neurons and terminals, J Neurosct, 3 (1983) 603-613 34 Nygren, L G. and Olson, L., A new major projection from the locus coeruleus" the mare source of noradrenerglc nerve terminals m the ventral and dorsal columr~s of the spinal cord, Brain Research, 132 (1977) 85-93 35 Pott, C B , Kramer, S.Z. and Siegel, A , Central gray modulation of affective defense ~s dffferentiaUy sensitive to naloxone, Phystol Behav, m press 36 Ranson, S.W., Kabat, H. and Magoun, H W , Autonomic responses to electrical stlmulatloa of hypothalamus, preoptic region and septum, Arch Neurol Psychiatry, 33 (1935) 467-477 37 Romanmk, A., Neuroehemlcal bases of defensive behavior m animals, Acta Neurobtol Exp, 34 (1974) 205-214 38 Shalkh, M B., Brutus, M and Siegel, A , Effects of naloxone on aggressive behavior ehcited from fehne penaqueductal gray, Eastern Psychol Assoc, Abstract 57 (1986) 29 39 Shalkh, M B , Brutus, M , S~egel, H E and Siegel, A , Dlfferentml control of aggression by the mldbram, Exp Neurol, 83 (1984) 436-442. 40 Sha~kh, M B , Brutus, M., Siegel, H E. and Siegel, A , Topographically orgamzed control of aggression by the mldbrain of the cat, Brain Research, 336 (1985) 308-312 41 Sheard, M , Hypothalamically ehclted attack behavior m

25 cats. effects of raphe stamulation, J Psychaatr Res, 10 (1974) 151 Abstract. 42 Smith, D A and Fiynn, J.P., Afferent projections related to attack sates in the pontme tegmentum, Brain Research. 164 {1979) 103-119 43 Smith, D A and Flynn, J P , Afferent prolectaons to quiet attack sates m cat hypothalamus, Brain Research, 194 (1980) 29-40. 44 Smtth, D A and Flynn, J P., Afferent projections to affectire attack sites in the cat hypothalamus, Brain Research, 194 (1980) 41-51. 45 Stoddard-Apter. S L . Siegel, A and Levm. B E . Plasma catecholamlnes and cardiovascular responses following hypothalamlc stimulation m the awake cat. J Auton Nerr Syst, 8 (1983) 343-360 46 Wasman, M and Flynn, J P , Directed attack elicited from hypothalamus, Arch Neurol, 61 (1962) 220-227

47 Watson, R E , J r , Edlager, H M and Siegel, A , A [14C]2deoxyglucose analysts of the funct:onal neural pathways of the hmbac forebram m the rat. III The happocam~,al formation, Brain Res Rev . ~ (1983) 133-176 48 Watson, R E , Jr , Siegel, H E and Siegel, A , A [:~C]2deoxyglucose analysts of the functional neural pathways of the hmblc forebram in the rat V The septal area, Brain Research, 346 (1985) 89-107 49 Watson, R E , J r , Trolano, ~ , Poulakos, J , Weiner. S. and Stegel, A , A [14C]2-deoxyglucose analysis of the functional neural pathways of the hmblc forebram Jn the rat. II. The hypothalamus, Brain Res Bull, 8 (1982) 459-476 50 Watson, R E . J r , Trolano, R , Poulakos, J , Wemer, S , Block, C H and Siegel, A , A [14C]2-deoxyglucoseanalysts of the functional neural pathways of the hmbac forebram in the rat I The amygdala, Brain Res Rev. 5 (1983) 1-44