Hypothalamic, midbrain and bulbar areas involved in the defense reaction in rabbits

Physiology & Behavior, Vol. 49, pp. 493-500. ©PergamonPress plc, 1991. Prin~din the U.S.A.

0031-9384/91 $3.00 + .00

Hypothalamic, Midbrain and Bulbar Areas Involved in the Defense Reaction in Rabbits 1 CARRIE G. M A R K G R A F , RAY W. WINTERS, 2 DAVID R. L I S K O W S K Y , PHILIP M. McCABE, E D W A R D J. GREEN AND NEIL SCHNEIDERMAN

Department o f Psychology, University o f Miami, Coral Gables, FL 33124 Received 15 August 1990

MARKGRAF, C. G., R. W. WINTERS, D. R. LISKOWSKY,P. M. McCABE, E. J. GREEN AND N. SCHNEIDERMAN.Hypothalaraic, midbrain and bulbar areas involved in the defense reaction in rabbits. PHYSIOLBEHAV 49(3) 493-500, 1991.--The present study mapped neuroanatomicalsites in the hypothalamusand periaqueductal gray (PAG) of the rabbit which, when stimulated electrically, evoked the cardiorespiratory componentsof the defense reaction (CRDR). This included increases in heart rate, blood pressure, hindlimbblood flow and respirationrate. All of the componentsof the CRDR were elicited by electricalstimulation of the posterior hypothalamus, at sites dorsal and medial to the fornix. Althoughthere were regions throughout the PAG in which electrical stimulationelicited concomitantincreasesin blood pressure, hindiimbblood flow and respirationrate, only stimulationof the dorsal PAG evoked tachyeardia. Injectionof horseradish peroxidase into the rostral ventrolateralmedulla (RVLM) led to heavy retrograde and antemgrade labeling in the region of the hypothalamusthat yielded the CRDR when stimulatedelectrically. Heavy labeling was also observed in the dorsal and ventral PAG. The results of this study provide evidence that the posterior hypothalamus and the dorsal PAG are nodal structures in the mediationof the CRDR and that cells in posterior hypothalamus, dorsal PAG and ventral PAG make monosynapticconnectionswith the RVLM. Defense reaction

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tion of the same region in both species (1, 36, 64). Attempts by investigators to elicit the defense reaction by injecting excitatory amino acids into the hypothalamus have not been successful (29,34). In contrast, microinjections of GABA antagonists into the posterior hypothalamus of the rat (3,18) and the cat (8, 61, 62) elicit the cardiovascular/respiratory changes characteristic of the defense reaction; escape behaviors are elicited when GABA antagonists are injected at the same site (19, 50, 57). Electrical stimulation or chemical stimulation of the periaqueductal gray (PAG) with excitatory amino acids has been found to elicit the CRDR in the cat (6, 13, 14) and the rat (7, 34, 40, 64); electrical or chemical stimulation of the PAG also evokes the behavioral components of the defense reaction (6, 13, 64). Research from several laboratories has focused upon identifying the bulbar structures that mediate the CRDR elicited by stimulation of the hypothalamus or PAG. Several converging lines of evidence implicate the neurons of the rosttal ventrolateral medulla (RVLM) in the mediation of the cardiovascular components of the response. Neuroanatomical tracing experiments provide evidence for monosynaptic connections between the PAG defense area and the RVLM in the cat (44). Electrolytic lesions of the RVLM block the cardiovascular responses elicited by electrical stimulation of the PAG defense area (45). Kainic acid lesions of

THE present investigation sought to determine the neuroanatomical sites in the hypothalamus and periaqueductal gray (PAG) of the rabbit which, when stimulated electrically, evoke the cardiorespiratory components of the defense reaction (CRDR). Horseradish peroxidase (HRP) histochemical techniques were used to test the idea that the rostral ventrolateral medulla (RVLM) is an essential component of the efferent pathway that mediates the CRDR. The response of an animal to threatening or stressful stimuli, referred to as the defense reaction, is characterized by behavioral, cardiovascular, and respiratory changes that serve to prepare the animal for "flight or fight" (1,2). The cardiorespiratory components of the defense reaction include increases in cardiac output, heart rate, blood pressure, and hindlimb blood flow, accompanied by hyperventilation (1, 2, 10, 58). The behavioral components appear to be species specific with the cat showing aggressive behaviors or flight (2, 6, 12, 32, 41) and the rat and rabbit showing escape behaviors (4, 19, 25, 41, 57, 64). The neural organization subserving the defense reaction has been studied extensively in the cat and rat. The behavioral components of the defense reaction can be evoked by electrical stimulation of the ventral pedfornical hypothalamus in the eat (12,32) and the rat (64). The CRDR can be elicited by electrical stimula-

1This research was supported by grants HL36588, HL07426, and NS24874. ZRequests for reprints should be addressed to Ray W. Winters, Ph.D., Department of Psychology, University of Miami, P.O. Box 248185, Coral Gables, FL 33124. 493

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ABBREVIATIONS

A ac APT cp Fx 1C LH MG ml MT NK NL ON OT PAG PH PVT RN SC SN VMH

amygdaloid region anterior commissure anterior pretectal thalamic nucleus cerebral penduncles fornix inferior colliculus lateral hypothalarnus medial geniculate medial lemniscus mammillothalamictract nucleus Darkschewitsch nucleus lateralis oculomotor nucleus optic tract periaqueductal gray posterior hypothalamus paraventricularhypothalamus red nucleus superior colliculus substantia nigra ventromedialhypothalamus

the RVLM suppress the tachycardia and renal nerve activity produced by stimulation of the hypothalamic defense area (46) and topical application of glycine in the RVLM attenuates the pressor component of CRDR elicited from the hypothalamus (9, 30, 33). In addition, the discharge rate of single neurons in the RVLM is altered by stimulation of the hypothalamic defense area (11, 36, 42) and the PAG defense area (36, 42, 54). Attempts to delineate the PAG and hypothalamic defense areas in the rabbit have yielded inconsistent findings. Evans (22,23) reported bradycardia and facilitation of the baroreceptor reflex from electrical stimulation of the ventral perifomical region of the hypothalamus. Similarly, he did not find an increase in hindlimb blood flow by electrically stimulating this site (24). Azevedo, Hilton and Timms (5) report that electrical stimulation of the perifornical area evoked hindlimb vasodilation and increases in blood pressure, but with variable changes in heart rate and ventilation. Tan and Dampney (59) found that microinjections of glutamate into the ventromedial nucleus of the hypothalamus or the PAG led to increases in hindlimb blood flow and suggested that these two regions were the integrative sites for the defense reaction of the rabbit. In view of the conflicting results from studies of the rabbit's defense area(s), a systematic exploration of the hypothalamus and PAG in this species, with the aim of mapping the sites that produce the defense reaction, seems particularly worthwhile. Similar investigations in other species have demonstrated that the neuroanatomical sites associated with the CRDR elicited in anesthetized animals are excellent predictors of the sites that will evoke both the behavioral and cardiorespiratory components of the defense reaction in conscious animals (1, 10, 57, 64). Accordingly, one goal of the present study was to provide a map of the neuroanatomical sites in the PAG and hypothalamus from which the CRDR can be elicited in the rabbit. A second goat of the present study was to assess the neuroanatomical connections between regions in the hypothalamus and in the PAG that yield the CRDR when stimulated electrically and the RVLM. There is very little known about the neuroanatomical relationships among the cardiovascular regions of the hypothalamus, PAG, and the RVLM of the rabbit, but in view of the results of studies with the

cat and rat it is reasonable to hypothesize that the RVLM L~ a neuroanatomical structure in the pathway that mediates the CRDR. In the present study we used anterograde and retrograde neuroanatomical tracing techniques to determine if the RVLM receives monosynaptic projections from neuroanatomical sites that. when stimulated electrically, yield the CRDR. METHOD

Electrical Stimulation Experiments The experiments were performed on 27 New Zealand albino rabbits (2.2-2.5 kg body weight). Anesthesia was initially induced by intravenous (IV) injections of ethyl carbonate (urethane; 1.5 g/kg) into the marginal ear vein, and maintained by IV injections into the right femoral vein. After the animal was anesthetized the left and fight femoral arteries and the left femoral vein were cannulated. A flow probe was placed around the right femoral artery and connected to an electromagnetic square wave flowmeter (Zepeda Instruments, Model SWF-4). Subcutaneous stainless steel electrocardiograph leads were placed above the fight shoulder and left haunch. The animal's head was placed in a Kopf stereotaxic instrument and secured with zygomatic arch clamps. The skull was subsequently trephined above the stimulation site. Blood pressure was monitored with a Statham model 23 DB pressure transducer that was connected to a catheter in the left femoral artery. Respiration rate was measured by inserting a tube from a model PT5 Grass volumetric pressure transducer into one limb of a "y"-shaped tracheal cannula and amplifying the transduced signal with a Grass model 7P1 DC amplifier. Body temperature was monitored continuously with a rectal thermistor probe (Electro-Temp) and regulated by an electric heating pad. A Grass model 7 polygraph and Macintosh Plus computer (with Maclab software) were used for on-line monitoring and storage of heart rate, blood pressure, and respiration rate data. Bipolar epoxylite-insulated stainless steel electrodes, with a DC resistance between 35 and 75 kfL were placed in either the hypothatamus (11 animals) or the periaqueductal gray (n= 16). Electrical stimuli were 10-s trains with a pulse duration of 0.5 ms, at 100 Hz, and an intensity between 80 and 200 p,A. The stimuli were delivered by a Grass model S-88 stimulator coupled to a Grass model PSIU6 photoelectric isolation unit with a constant current unit. The hypothalamus and PAG were systematically explored by varying the dorsal/ventrai position of the electrode in 150-txm steps and observing cardiorespiratory responses to electrical stimulation at each site. This procedure was repeated for a series of electrode tracks separated by approximately 500 Ixm in the mediolateral plane and 500 ~m in the rostral/caudal plane. Cardiovascular and respiratory measures were taken continuously during the 10-s interval immediately prior to the onset of stimulation (baseline measure) and during the 10-s stimulation period. The response measure used for quantitative analyses was the maximum value of the heart rate, blood pressure and respiration rate observed during the 10-s baseline interval and during the 10-s stimulation period. Henceforth, this value is referred to as the peak value. Stimulation sites were marked with electrolytic lesions by passing a 200-1xA anodal current, for 30 s, through the stimulating electrodes. At the conclusion of each experiment the rabbit was sacrificed with an IV injection of sodium pentoharbital and perfused transcardially with 2,0 liters of saline followed by 2.0 liters of 10% formalin. The brain was removed and 40-micron frozen coronal sections taken using a Reichert-Jung 2800 frigocut microtome. The sections through the hypothalamus or the PAG were stained

CNS CONTROL OF RABBIT DEFENSE REACTION

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FIG. 1. Hypothalamicsites that yielded the CRDR when stimulatedelectrically. (A) Line drawing composite of 3 serial sections(AP - 1.0); (B) composite of 3 serial sections (AP - 1.5). Atlas from Shek,*~Cen, and Wisneiwski (57).

with neutral red and viewed with a Nikon Optiphot microscope.

Neuroanatomical Tracing Experiments Twenty-two New Zealand albino rabbits (2.0-2.5 kg) were studied in these experiments. After inducing anesthesia by injecting sodium pentobarbital (Nembutal, 60 mg/kg) into the marginal ear vein, the femoral artery and vein were cannulated, the animal's head was placed in the stereotaxic instrument and the dorsal surface of the rostral medulla was exposed. The femoral vein was used for subsequent injections of Nembutal to maintain anesthesia. Blood pressure was monitored from the femoral artery in the manner described above. Fifteen to 30 nl of wheat germ agglutinin-horseradish peroxidase (1.5% WG-HRP) were injected into the RVLM via pressure injections through glass micropipettes (tip outside diameter of 15-25 microns) attached to a Medical Systems pico-injector. The target of the injections was the subretrofacial nucleus. RVLM pressor sites were first determined by passing an electric current through the micropipette. The current intensity of the 10-s stimulus train was between 30 and 50 IxA, with a pulse duration and frequency of 0.25 ms and 100 Hz, respectively. This electrical stimulus elicited an increase in blood pressure of approximately 30 mm Hg. After locating the most responsive pressor area in the RVLM, HRP injections were made with 2-3 pulses of compressed gas;

injections were given 15 min apart. The pipette was left in place for 30 min before the wound was closed and the animal returned to its home cage. After 48 h the animals were perfused transcardially with 0.9% saline, followed by 1.25% glutaraldehyde and 1% paraformaldehyde in 0.1 m phosphate buffer. The brain was removed, stored over night in 30% sucrose/phosphate buffer, and frozen sections (40 microns) were taken using a freezing microtome. Every fourth section was saved for WG-HRP reaction and adjacent sections were counterstained with cresyl violet. All tissues were processed for HRP histochemistry according to the Mesulam (48) tetramethylbenzedinetechnique, but with the following variations: 1) A 1% gelatin solution was used for the incubation and reaction procedure, rather than distilled water; 2) at the end of the incubation period the solution was discarded and replaced with a fresh solution containing hydrogen peroxide. The tissue was examined for HRP reaction product using light- and darkfield microscopy. The resultant retrograde and anterograde labeling was mapped using a drawing tube attached to the microscope. Photomicrographs were made with a Nikon Optiphot microscope and UFX-II photomicrographic system. Injection sites were examined to determine the amount of spread of HRP. Although there were differences among the animals with respect to the magnitude and the pattern of the spread of HRP from the injection site, there were no substantial differences in the density of labeling in the PAG and hypothalamus, when the focus of the injection site was the subretrofacial nucleus. RESULTS

Hypothalamic Stimulation Experiments A systematic exploration of the hypothalarnus with discrete electrical stimuli revealed that all of the cardiovascular and respiratory components of the CRDR could be elicited by stimulation of the posterior hypothalamus at sites that were dorsal and medial to the fornix (Fig. 1). Although it was possible to elicit one or more components of the CRDR in other regions of the hypothalamus, the complete response pattern was only evoked at the sites in the posterior hypothalamus shown in Fig. 1. The data from the active sites in the hypothalarnus were pooled and the median (peak) values for blood pressure, heart rate, hind-

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respiration rate, p < 0.0, t(10) = 6.0, were found to he significantly larger than baseline values.

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FIG. 3. Sites in PAG that yielded blood pressure, hindlimb blood flow, and respirationrate increasesin response to electricalstimulation.These

sites were associated with tachycardia in the dorsal PAG and bradycardia in the ventral PAG. (A) Camera lucida drawing of rostral PAG (AP - 8.0) section depicting dorsal stimulation sites (closed circles) and ventral stimulation sites (open circles). (B) Camera lucida drawing of caudal PAG section (AP - 9.0). Symbols represent dorsal and ventral sites as in (A).

limb blood flow, and respiration rate during the 10-s stimulation period were calculated. A site was considered to he active if the change in the cardiorespiratory response was visually apparent in the polygraph record. A comparison of these values with the peak baseline (10-s period prior to stimulation) values are shown in Fig. 2. Wilcoxon signed-rank tests were used to determine if median peak systolic blood pressure, hindlimb blood flow, respiration rate and heart rate were statistically different from the baseline values. Heart rate, p < 0 . 0 1 , t(10)=6.0, systolic blood pressure, p < 0 , 0 1 , t(10)=6.0, blood flow, p < 0 . 0 1 , t(10)=6.0, and

There is some evidence from studies of the PAG in the rat (25, 34, 54, 55, 64) that the defense reaction is elicited by stimulation of the dorsal region but not the ventral portion of the PAG. Indeed, this appears to be the case for the rabbit also. Although there were a number of regions in both the ventral and dorsal PAG that yielded a pressor response, an increase in hindlimb blood flow, and an increase in respiration rate, these components of the CRDR were always accompanied by bradycardia in the ventral PAG. Figure 3 shows a schematic drawing of the neuroanatomical sites in the rostral PAG (A} and caudal PAG (B) at which electrical stimulation elicited an increase m blood pressure, hindlimb blood flow, and respiration rate. This response pattern was coupled with tachycardia at the dorsal sites (closed circles) and bradycardia at the ventral sites (open circles). The data from the PAG sites were pooled and median peak heart rate, blood pressure, hindlimb blood flow. and respiration rate during the 10-s baseline period and during the 10-s stimulation period were calculated. A comparison of these values for the dorsal PAG and ventral PAG are shown in Fig. 4. A statistical analysis, using Wilcoxon signed-rank tests, revealed that heart rate, p < 0 . 0 1 , t(8)= 4.5, systolic blood pressure, p<0.01, t(8)= 4.5, hindlimb blood flow, p<0.01, t(8) = 5.0, and respiration rate. p<0.01, t(8)= 4.5, were significantly larger than baseline values for stimulation sites in the dorsal PAG. The pattern was different at the ventral sites in the PAG. Systolic blood pressure during stimulation was significantly higher. p < 0 . 0 1 , t(10)= 5.5, than baseline but heart rate during stimulation was significantly lower, p < 0 . 0 1 , t(10) = 5.5, than baseline.

FACING PAGE HG. 5. H R P labelingin posteriorhypothalamus from injectionsin the rostralventrolateralmedulla. (A) Line drawing of composite of 4 serialsections (AP ~ 1.5); (13)darkfieldphotomicrograph of posteriorhypothalamic section showing dense cell body labeling(36 x ); (C) darldieldphotomicrograph of enclosed region in (13)(180 x ).

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The present study delineated the hypothalamic and PAG regions in which electrical stimulation evoked the CRDR in the rabbit. All of the components of this response could be elicited in the posterior hypothalamus, at sites dorsal and medial to fornix. and in the dorsal PAG. There were regions in the ventral PAG in which electrical stimulation elicited increases in blood pressure, respiration rate, and hindlimb blood flow but this response pattern was always accompanied by bradycardia, rather than the tachycardia that is characteristic of the CRDR. Both the dorsal PAG and the ventral PAG showed heavy anterograde and retrograde labeling in neuroanatomical tracing experiments in which HRP was injected into the RVLM; dense labeling was also observed in the portion of the posterior hypothalamus in which electrical stimulation evoked the CRDR. B

FIG. 6. HRP labeling in PAG from injectionsin the rostral ventrolateral medulla. A: Line drawing composite of 3 serial sections in rostral PAG (AP -8.5); B: Line drawing composite of 6 serial sections (AP - 10.5) from caudal PAG.

Hindlimb blood flow, p<0.01, t(10)=6.0, and respiration rate, p<0.Ol, t(10)=6.0, during stimulation were significantly larger than baseline values.

Neuroanatomical Tracing Experiments Microinjections of WG-HRP into the RVLM led to dense anterograde and retrograde labeling in the dorsomedial region of the posterior hypothalamus. Labeling was bilateral but with much heavier deposits observed on the side ipsilateral to the injection site. Figure 5A shows a schematic drawing of data from 4 serial sections through the posterior hypothalamus; Fig. 5B and C show darkfield photomicrographs of labeling in the dorsomedial region. A comparison of these findings with those of the hypothalamic stimulation experiments reveals that the region in the hypothalamus which yielded the CRDR from electrical stimulation was similar to the one in which dense labeling resulted from microinjections of HRP into the RVLM. HRP injections into the RVLM also produced dense anterograde and retrograde labeling in the PAG. The schematic drawings in Fig. 6A is a composite of 3 serial sections through the rostral mesencephalon. Figure 6B shows a schematic drawing from 6 serial sections in the caudal mesencephalon. In general, the HRP studies did not reveal any substantial differences between the dorsal PAG and the ventral PAG.

Hypothalamic Stimulation Experiments The results of our studies in the hypothalamus are, for the most part, consistent with findings from other investigations of the CRDR of the rabbit. Evans (23,24) was not able to elicit the cardiovascular components of the response by stimulating the ventral perifomical area; likewise we could not elicit all of the components of the CRDR from this region. Tan and Dampney (59} elicited an increase in hindlimb blood flow from the ventromedial hypothalamus of the rabbit. We also observed an increase in hindlimb blood flow from stimulation of the ventromedial hypothalamus but all of the components of the CRDR could not be evoked by stimulation this region. It is difficult to determine if our active hypothalamic sites overlap those reported by Azevedo, Hilton and Timms (5) because their findings were presented in abstract form and a detailed description of their stimulation sites was not provided. Studies of the defense reaction in the cat and rat have identified two hypothalamic sites in which the cardiorespiratory and behavioral components of the response can be elicited. Electrical stimulation of ventral perifornical region evokes both components of the response in the cat (1,2) and the rat (64). Microinjections of GABA antagonists into the posterior hypothalamus of the rat. in a region that is dorsal and medial to the fornix, elicit the cardiorespiratory components of the defense reaction (3,18) and escape behaviors (19, 25, 50, 56). Electrical stimulation or chemical stimulation (with GABA antagonists) of this region in the posterior hypothalamus in the cat elicits the CRDR (8, 20, 61-63) and locomotion (20,62). The region in the hypothalamus that yielded the CRDR in the present study appears to correspond to the defense area in the posterior hypothalamus of the cat and rat thal has been identified by injections of GABA antagonists. The observation that we could not elicit the CRDR in the ventral perifomical area indicates that the neuronal mechanisms that integrate and regulate the defense reaction in the rabbit are different from those that subserve the defense reaction in the cat and rat. Studies from other laboratories also demonstrate species differences in the neuronal circuitry that underlie these animal's responses to stressful stimuli. Electrical stimulation of the central nucleus of the amygdala (ACE) of the rabbit elicits bradycardia and inhibition of movement (4), accompanied by facilitation of the baroreceptor reflex (51), whereas stimulation of the ACE (or the axons of neurons from this region) elicits the defense reacuon in cats (37,49) and inhibition of the baroreceptor reflex (49.60). These species differences are not unexpected. As pointed out elsewhere (49), the rabbit's typical response to stressful or threatening stimuli is quite different than those of the cat or rat. The cat and rat usually show escape behaviors or defensive aggression. Even though the rabbit often shows escape behavior. "freez-

CNS CONTROL OF RABBIT DEFENSE REACTION

499

ing" or "playing dead" (4,22) are more common in this species than in the cat or rat.

neurons which mediate the response are localized preferentially in the caudal region.

Periaqueductal Gray Stimulation Experiments

HRP Neuroanatomical Tracing Experiments

There is ample evidence that neurons in the PAG of the cat and rat are a part of the neuronal circuitry that subserves the autonomic and behavioral components of the defense reaction. All of the components of the response can be elicited by electrical stimulation (1,55, 64), chemical stimulation with excitatory amino acids (6, 13, 34, 40), or chemical stimulation with GABA antagonists (17,54). Moreover, the defense reaction can be elicited by electrical stimulation of the PAG or with natural stimuli when the PAG has been isolated from rostral structures by complete section of the brainstem between the midbrain and diencephalon, large lesions of the hypothalamus, or surgical isolation of the hypothalamus (21, 26, 28, 38, 39). The present investigation of the cardiorespiratory components of the defense reaction in the rabbit revealed that it was possible to elicit the complete CRDR by electrical stimulation of the dorsal PAG. Electrical stimulation of the ventral PAG produced increases in blood pressure, hindlimb blood flow, and respiration rate but these responses were always accompanied by bradycardia. Several investigators present evidence for differences in the responses elicited from the dorsal and ventral PAG of the rat. Electrical stimulation (25,64) or chemical stimulation with DLhomocysteic acid (34) or GABA antagonists (54) elicited the defense reaction in the rat when the stimulation site was the dorsal PAG but not when it was the ventral PAG. Similarly, the stimulation-produced analgesia coupled with the defense reaction can only be elicited from the dorsal PAG of the rat (45). Studies in the cat provide evidence that the PAG defense area forms a cylindrical column lateral to the cerebral aqueduct with the rostral end in a position that is dorsal to the caudal end (6) and that the

It is now well established that neurons in the rostral ventrolateral medulla play an essential role in the mediation of phasic changes in blood pressure and heart rate, and in the setting of the resting level of vasomotor tone (11, 15, 16, 27, 52, 53). In the present study, injections of HRP into the RVLM led to anterograde and retrograde labeling in the dorsomedial region of the posterior hypothalamus, the dorsal PAG and the ventral PAG. The hypothalamic site corresponds to the one that yielded the CRDR from electrical stimulation. Electrical stimulation of the PAG sites that showed heavy labeling elicited increases in blood pressure, hindlimb blood flow and respiration rate, coupled with tachycardia at the dorsal sites and bradycardia at the ventral sites. These findings are consistent with the view that the RVLM mediates, via monosynaptic connections, one or more of the cardiovascular components of the responses elicited from the PAG and hypothalamus. When this portion of the RVLM in the rabbit was stimulated electrically or chemically (with L-glutamate) in our laboratory (31) we observed pressor responses coupled with bradycardia. The pattern of labeling that we observed is similar to the one reported for the cat (44) after injections of HRP into the nucleus paragigantocellularis lateralis (PGL), an area that corresponds to the injection site in the present study. Lovick (44) observed dense labeling in the dorsomedial hypothalamus and the caudal ventrolateral PAG. The connections from PAG were found to be less extensive than those observed in the present study and Lovick did not observe dense labeling in the dorsal PAG. Whether these discrepancies are due to species differences or differences in injection techniques remains to be determined.

REFERENCES 1. Abrahams, V. C.; Hilton, S. M.; Zbrozyna, A. W. Active muscle vasodilatation produced by stimulation of the brain stem: Its significance in the defcnce reaction. J. Physiol. (Lond.) 154:491-513; 1960. 2. Abrahams, V. C.; Hilton, S. M.; Zbrozyna, A. W. The role of active muscle vasodilatation in the alerting stage of the defence reaction. J. Physiol. (Lond.) 171:189-202; 1964. 3. Abshire, V. M.; Hankins, K. D.; Roehr, K. E.; DiMicco, J. A. Injections of 1-Allyglycine into the posterior hypothalamus in rats causes decreases in local GABA which correlate with increases in heart rate. Neuropharmacology 27:1171-1177; 1988. 4. Applegate, C. D.; Kapp, B. S.; Underwood, M. D.; McNall, C. C. Autonomic and somatomotor affect of amygdala central nucleus stimulation in awake rabbits. Physiol. Behav. 31:353-360; 1983. 5. Azevedo, A. D.; Hilton, S. M.; Timms, R. J. The defence reaction elicited by midbmin and hypothaiarnic stimulation in the rabbit. J. Physiol. 301:56P-57P; 1980. 6. Bandler, R.; Carrive, P. Integrated defense reaction elicited by excitatory amino acid microinjection in the midbrain periaqueductal grey region of the unrestrained cat. Brain Res. 439:95-106; 1988. 7. Bander, R.; Depaulis, A.; Vergnes, M. Identification of midbrain neurones mediating defensive behaviour in the rat by microinjections of excitatory amino acids. Behav. Brain Res. 15:107-119; 1985. 8. Bauer, R. M.; Vela, M. B.; Simon, T.; Waldrop, T. G. A GABAergic mechanism in the posterior hypothalamus modulates baroreflex bradycardia. Brain Res. Bull. 20:633--642; 1988. 9. Bloch, R. J.; Feldrnan, P.; Bousquet, P.; Schwartz, J. Relationship between ventromedullary clonidine-sensitive area and the posterior hypothalamus. Eur. J. Pharmacol. 45:55-60; 1977. 10. Bolme, P.; Ngai, S. H.; Uvntls, B.; Wallenberg, L. R. Circulatory and behavioural effects on electrical stimulation of the sympathetic vasodilator areas in the hypothalamus and the mesencephalon in unanaesthetized dogs. Acta Physiol. Scand. 70:334-346; 1967.

11. Brown, D. L.; Guyenet, P. E. Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats. Circ. Res. 56:359-369; 1985. 12. Brown, J. L.; Hunsperger, R. W.; Rusuold, H. E. Defence attack and flight elicited by electrical stimulation of the hypothaiamus of the cat. Exp. Brain Res. 8:113-124; 1969. 13. Carrive, P.; Bandler, R.; Dampney, A. L. Somatic and autonomic integration in the midbrain of the unanesthetized decebrate cat: A distinctive pattern evoked by excitation of neurones in the subtentorial portion of the midbrain periaqueductal grey. Brain Res. 483:251258; 1989. 14. Carrive, P.; Dampney, R. A. L.; Bandler, R. Excitation of neurones in a restricted portion of the midbrain periaqueductal grey elicits both the behavioral and cardiovascular components of the defence reaction in the unanaesthetised decerebrate cat. Neurosci. Lett, 81:273-278; 1987. 15. Caverson, M. M.; Ciriello, J.; Calaresu, F. R. D. Direct pathway from cardiovascular neurons in the ventrolateral medulla to the region of the intermediolateral nucleus of the upper thoracic cord: An anatomical and electrophysiological investigation in the cat. J. Auton. Nerv. Syst. 9:451-475; 1983. 16. Dampney, R. A. L.; Goodchild, A. K.; Robertson, L. G.; Montgomery, W. Role of ventrolateral medulla in vasomotor regulation: A correlative anatomical and physiological study. Brain Res. 249:223235; 1982. 17. Depaulis, A.; Vergnes, M. Elicitation of intraspeciflc defensive behaviors in the rat by microinjection of picrotoxin, a GABA antagonist, into the periaqueductal gray matter. Brain Res. 367:87-95; 1986. 18. DiMicco, J. A.; Abshire, V. M. Evidence for GABAergic inhibition of a hypothalamic sympathoexcitatory mechanism in anesthetized rats. Brain Res. 402:1-10; 1987. 19. DiScala, G.; Schmitt, P.; Karli, P. Flight induced by infusion of

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