Functional Relationship between the Hypothalamic Vigilance Area and PAG Vigilance Area

Physiology & Behavior, Vol. 62, No. 3, pp. 675–679, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/97 $17.00 / .00

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Functional Relationship between the Hypothalamic Vigilance Area and PAG Vigilance Area YU-FEI DUAN, 2 RAY WINTERS, 1 PHILIP M. MCCABE, EDWARD J. GREEN, YING HUANG AND NEIL SCHNEIDERMAN Department of Psychology, University of Miami, Coral Gables, FL 33124 USA Received 8 February 1996; Accepted 6 December 1996 DUAN, Y.-F., R. W. WINTERS, P. M. MCCABE, E. J. GREEN, Y. HUANG AND N. SCHNEIDERMAN. Functional relationship between the hypothalamic vigilance area and PAG vigilance area. PHYSIOL BEHAV 62(3) 675–679, 1997.—The vigilance reaction is characterized by a large bradycardia, a pressor response, and inspiratory apnea in anesthetized rabbits and the inhibition of movement in conscious rabbits. This affective response pattern can be elicited by electrical stimulation of the dorsolateral hypothalamus (the hypothalamic vigilance area) or the ventrolateral periaqueductal gray (the periaqueductal gray vigilance area). The present study sought to advance our understanding of the functional relationship between the hypothalamic vigilance area (HVA) and the periaqueductal gray vigilance area (PVA) by measuring the effects of transverse transections of the caudal portion of the ventrolateral PAG (vlPAG) upon the cardiovascular responses elicited from the dorsolateral hypothalamus and the rostral vlPAG. Selective transverse transections of the caudal vlPAG significantly reduced the magnitudes of the bradycardia and pressor response elicited by stimulation of the PVA rostral to the transection site, but had minimal impact on the cardiovascular responses evoked by stimulation of the HVA. These findings suggest that the cardiovascular responses elicited by stimulation of the vlPAG are mediated by a neural pathway that is parallel, at least in part, to the one that subserves the response elicited from the HVA. The results also provide support for the view that the PAG is not an essential structure in the mediation of the autonomic components of affective behaviors involving behavioral inhibition. q 1997 Elsevier Science Inc. Hypothalamus

Cardiovascular

Affective behavior

Periaqueductal gray

AFFECTIVE behaviors are integrated somatic and visceral response patterns that are evoked when environmental circumstances pose a threat to an organism. The vigilance reaction is a type of affective behavior that has been the focus of research efforts in our laboratory in recent years (3,4,6). It is characterized by a large bradycardia, a pressor response, and inspiratory apnea (or shallow tachypnoea) in anesthetized rabbits (6) and the inhibition of motor activity in conscious rabbits (4). It can be elicited by electrical stimulation of the dorsolateral portion of the posterior hypothalamus (the hypothalamic vigilance area, HVA) or the ventrolateral PAG (the PAG vigilance area, PVA). When identical or nearly identical autonomic/behavioral response patterns can be elicited by stimulating different central nervous system (CNS) sites, it is natural to query about the morphological and functional discreteness of the neuronal circuitry that sub-

serves the responses evoked. Indeed, there is good reason to believe that the CNS pathways that subserve the autonomic and behavioral components of affective behaviors are neuroanatomically partitioned. As a case in point, we know from human psychophysical studies that the autonomic components of an affective behavior such as the defense reaction can be evoked in stressful situations involving active coping, such as preparation for public speaking (7). Similarly, the autonomic components of the vigilance reaction can be elicited in stressful situations involving inhibitory coping such as the cold pressor task (7). The results of studies by Smith and colleagues (8) are also consistent with the view that the autonomic and behavioral components of affective behaviors are neuroanatomically fractionated. Baboons were trained by operant conditioning techniques to respond to colored lights by either pressing a lever, feeding,

1

To whom requests for reprints should be addressed. Current address: National Institutes of Health, National Institute of Neurological Disorders and Stroke, 10 Center Drive, 10/5N214, MSC 1424, Bethesda, MD 20892-1424. 2

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DUAN ET AL. of the perifornical hypothalamus totally abolished or severely attenuated the cardiovascular response to the tonal stimulus but presentation of the tonal stimulus still led to lever suppression, thereby suggesting fractionation of the autonomic and behavioral components of this affective behavior involving behavioral inhibition. Studies of classically conditioned affective behavior in the rat also suggest that the hypothalamus is an essential structure in the neuronal circuitry that mediates the autonomic components of affective behaviors involving behavioral inhibition. Lesions in the amygdala and the medial portion of the medial geniculate nucleus were found to abolish both the autonomic (pressor response) and behavioral (inhibition of somatic motor activity) components of the affective response to a conditioned acoustic stimulus, whereas lesions in the lateral hypothalamus were found to abolish the cardiovascular component of the response, but not the somatic motor component of the response (5). Lesions in the PAG were found to disrupt the somatic motor component of the affective behavior but not the conditioned changes in blood pressure. In addition to providing evidence for neuroanatomical fractionation of the autonomic and somatic components of affective behavior, the studies of Smith and colleagues (8) and LeDoux and coworkers (5) provide evidence that the hypothalamus is an essential component of the neuronal circuitry that mediates the autonomic components of affective responses in which there is suppression of ongoing behavioral activity. Moreover, the work of LeDoux and associates suggests the working hypothesis that the PAG is not a component of the neuronal pathway that mediates the autonomic component of this type of affective behavior (behavioral inhibition), though integrated somatic/autonomic affective responses such as the defense reaction and vigilance reaction can be elicited by electrical stimulation of the PAG. The present investigation tests this idea by assessing the effects of PAG lesions upon the autonomic components of another type of affective behavior involving the suppression of ongoing behavior, the vigilance reaction. More specifically we examined the effects of selective surgical transections within the PAG vigilance area (PVA) upon the cardiovascular responses elicited by electrical stimulation of the hypothalamic vigilance area (HVA). METHODS

Subjects

FIG. 1. Schematic drawings of the periaqueductal gray vigilance area. Outer edges of the closed circles indicate the outer boundaries of the sites that yielded all of the cardiorespiratory components of the vigilance reaction. The posterior (P) level in mm is referenced to bregma. Abbreviations: DN, nucleus Darkschewitsch; EW, nucleus Edinger–Westphal; IN, nucleus interpeduncularis; PAG, periaqueductal gray; RN, nucleus ruber; SC, colliculus superior; III, nucleus n. oculomotorii.

or executing a mild leg exercise response, depending on the color of the light. Classical conditioning techniques were used to develop a conditioned emotional response (CER) to a tonal stimulus that had been paired with an electric shock to the abdominal skin. The CER included a neurally mediated tachycardia, renal vasoconstriction, and pressor response, followed by secondary increases in catecholamine secretions by the adrenal glands. The tonal stimulus for the CER was only presented during the lever pressing and led to a decrease in response rate. Bilateral lesions

Subjects were twenty adult New Zealand albino rabbits, of both sexes, weighing between 2.5 and 3.5 kg. Fourteen of the subjects were used to determine the neuroanatomical site in the PAG that yielded the cardiovascular and respiratory responses that were identical to those that were elicited by electrical stimulation of the hypothalamic vigilance area. Six different subjects were used, in a within-subjects design, to determine the effects of transverse transection of the caudal portion of the ventrolateral PAG upon the cardiovascular responses elicited from the hypothalamic and PAG sites that yield the autonomic components of the vigilance reaction. Surgery The animals were anesthetized with isoflurane (2%–5%) in pure oxygen. The trachea was cannulated with an endotracheal tube and connected to an anesthetic machine and respiration signal transducer. A catheter was inserted into the femoral artery to record blood pressure. Bipolar hypodermic electrodes were placed under the skin of the chest and hindlimb to record EKG. Body temperature was maintained at 377C by a heating pad.

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FIG. 2. Schematic illustration of the locations and extents of caudal PAG transections (P 10.0). The transections are 2 mm apart in the dorsal/ventral plane. Darkened area indicates the transection that was found to be effective in reducing the cardiovascular response elicited by stimulation of the vlPAG rostral to the transection site. Abbreviations: HI: hippocampus; PAG, periaqueductal gray; PV, nucleus pontis ventralis; RA, nucleus raphis; RAP, nucleus raphis pontis; TN, nucleus n. trochlearis.

A dorsal craniotomy was performed after the animal’s head was placed in a stereotaxic instrument. The dorsal surface of the skull was exposed by a midline incision of the skin and subcutaneous tissue; the tissue was then retracted bilaterally. A dental drill was used to open a bilaterally symmetrical rectangular area in the skull (A 2.0 to P 12.0, L 4.0). The saggital sinus was ligated on both sides and one ligated end was cut so that it could be folded over. This procedure served to remove major blood vessels from the midline of the brain before subsequent insertions of the blade used for transection. Bone wax was used to stop minor bleeding. In general, there was minimal blood loss during this procedure. Bipolar stainless steel stimulating electrodes were placed in the hypothalamus and in the PAG. The details of the electrical stimulation protocol that we use in this type of study are described in earlier papers (3,4,6). The mean stimulating current (100 Hz, 0.5 ms pulse duration, 10 s train) intensities were between 150 and 200 mA, and between 300 and 350 mA for the hypothalamic stimuli. In general, the current intensity required to evoke the largest cardiorespiratory response was found to be much lower for the vlPAG (about 150 mA) than for the hypothalamic vigilance area stimulus (about 300 mA). The stereotaxic coordinates were P 6.0–9.0, L 0.5–1.5, V 8.0–13.0 for the PAG and P 0.0–2.5, L 1.0–2.0, V 10.0–12.0 for the hypothalamus. The HVA and PVA were also identified by cardiorespiratory responses to electrical stimulation. The cardiorespiratory components of the vigilance reaction are a pressor response, bradycardia and inspiratory apnea. Repeated stimulation of the HVA and PVA led to the same responses. After the HVA and PVA sites were located, the two electrodes were secured to the skull with dental cement. The portion of the dental cement located at the posterior end of the PAG stimulating electrode was shaped by hand so as to form a vertical cliff along the shaft of the electrode,

thereby leaving room for subsequent insertion of the transection blade posterior to the PAG electrode. The distance between the PAG stimulating electrode and the transection blade was approximately 3 mm. Procedure After placing stimulating electrodes in the PVA and HVA, a vertical blade was mounted on the stereotaxic instrument in order to transect the caudal portion of the PAG (P10.0). Four transverse transections were made 2 mm apart in the dorsal/ventral plane (V 7.0, 9.0, 11.0, 13.0). Cardiovascular responses to HVA and rostral PVA stimulation were measured at each of the four blade positions. The blood pressure data from one animal were lost due to technical difficulties with the arterial catheter. At the end of the experiment, electrolytic lesions were placed at the stimulation sites in the hypothalamus and PAG. The animal was then sacrificed by an overdose of pentobarbital and perfused transcardially with physiological saline, followed by 4% formaline. Three days later, the brain was sectioned to assess the depths of the surgical transections in the midbrain. Brain sections were also processed for the Prussian blue reaction to identify the locations of the stimulating electrodes. The cardiovascular data were statistically analyzed using Student t-tests with the significance level set at 0.05 for two tailed tests. RESULTS

The schematic diagram of Fig. 1 shows the PAG sites where the cardiorespiratory components of the vigilance reaction were elicited by electrical stimulation. Hypothalamic sites that yield the vigilance reaction are provided in earlier publications from this laboratory (3,4,6). The outer edges of the closed circles in Fig. 1 indicate the outer boundaries of the sites that yielded all

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DUAN ET AL. changes in the magnitudes of the cardiovascular responses to PVA or HVA stimulation. The effects of the caudal PAG transections (V13.0) were quantified and the results are illustrated in the bar graphs shown in Fig. 3. The magnitudes of the bradycardia and pressor response elicited by vlPAG stimulation rostral to the transection site (V13.0) were greatly attenuated, though they were not completely abolished, by caudal vlPAG transection (Fig. 3A,B). Comparisons of the changes in blood pressure and heart rate elicited by PAG stimulation before and after the vlPAG transection revealed significant reductions (t (5) Å 3.2, p õ 0.05 for heart rate; t (4) Å 3.4, p õ 0.05, for blood pressure). As can be seen in Fig. 3, heart rate and blood pressure responses elicited by HVA stimulation were not significantly affected by transverse transection of the caudal PAG (V13.0). DISCUSSION

FIG. 3. Changes of the magnitudes of heart rate and blood pressure responses to HVA and PVA stimulation before and after the caudal PAG transection at P 10.0, V 13.0. *p õ 0.05, n Å 5–6, paired t-tests for comparisons between the changes before and after the PAG transection.

of the cardiorespiratory components of the vigilance reaction. Most of the ventrolateral PAG (vlPAG) stimulation sites were located in the intermediate and caudal parts of the PAG where the vlPAG is well developed. In an attempt to assess the functional relationship between the PVA and HVA, the caudal PAG was transected in 2 mm increments in the dorsal/ventral plane. A schematic diagram of the four blade positions is shown in Fig. 2. The tip of the surgical blade was designed so that the lower part of the blade was restricted primarily to the ventral and ventrolateral PAG. The shaded area on the tip of the blade at the V 13.0 position shows the level of transection which reduced the magnitudes of the cardiovascular responses elicited by stimulation of the vlPAG rostral to the transection site (P 10.0); none of the blade positions significantly affected the magnitude of the cardiovascular responses elicited by HVA stimulation. Transections dorsal to the ventral and ventrolateral PAG (V13.0) did not produce noticeable

Human psychophysical studies demonstrate that the autonomic components of affective behaviors such as the defense reaction can be elicited in stressful tasks that require active coping such as preparation for public speaking, and the autonomic components of the vigilance reaction can be elicited in stressful situations involving inhibitory coping such as the cold pressor task (7). These observations indicate partitioning of the CNS pathways that mediate the autonomic and behavioral components of these responses to emotional stressors. Studies of conditioned emotional responses in baboons (8) and classically conditioned affective behavior in the rat (5) suggests that, when emotional stressors elicit behavioral inhibition, the hypothalamus is an essential structure in the neuronal circuitry that mediates the autonomic components of the affective response. Moreover, the conditioning studies prompt the hypothesis that the integrity of the PAG is not essential to the mediation of the autonomic components of emotional responses involving behavioral inhibition, even though integrated somatic/autonomic affective responses such as the defense reaction and vigilance reaction can be elicited from this structure. The present study served as a test of this hypothesis by determining the effect of transverse transections of the caudal vlPAG upon the cardiovascular components of the vigilance reaction elicited from the hypothalamic vigilance area and from the PAG vigilance area. Electrical stimulation of the dorsolateral hypothalamus (hypothalamic vigilance area; HVA) in rabbits evokes a response pattern that is nearly identical to the one elicited by stimulation of the vlPAG (PAG vigilance area; PVA). Blood pressure increases are coupled with a profound bradycardia and inspiratory apnea in anesthetized animals (6), and motor activity is inhibited in conscious animals (4). Because identical or nearly identical response patterns are elicited by stimulation of the HVA and the PVA, it seems reasonable to suggest that the two regions are components of an integrated neural system that is responsible for the expression of this type of affective behavior. Indeed, there is neuroanatomical evidence for widespread connections between the hypothalamus and the PAG (1). Despite the similarities in the responses elicited by the HVA and vlPAG, the HVA-elicited cardiovascular response pattern was not significantly altered by transverse transection of the caudal vlPAG. In contrast, the pressor response and bradycardia elicited by vlPAG stimulation at sites rostral to the transection were significantly reduced in magnitude by transection of this portion of the vlPAG. These observations provide evidence that the cardiovascular responses elicited by electrical stimulation of the vlPAG are mediated by a neural pathway that is parallel, at least in part, to the one that subserves the response elicited from the HVA. It should be noted,

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however, that we did not stimulate in the most caudal regions of the PAG and we can not exclude the possibility that there are critical collaterals at these sites. Also, we did not make lesions lateral to the PAG to block the effects of the stimulation of the HVA and there is a possibility that this route to the vlPAG is important. The results of the present study provide additional support for the view that the PAG is not an essential structure in the mediation of the autonomic components of affective behaviors involving behavioral inhibition. The findings are subject to several interpretations. One possibility is that the two responses patterns, hypothalamic vigilance response and PAG vigilance response, are essentially the same but elicited by different stimuli. Carrive (2) has hypothesized that the primary function of the PAG is somatosensory integration and that the vlPAG is involved in the processing of subcutaneous and visceral noxious stimuli that would be associated with inescapable visceral pain and with mus-

cle fatigue. The prevailing wisdom is that rostral structures such as the hypothalamus are involved in the processing of higher order stimuli rather than the direct processing of incoming stimuli. For example, the HVA may respond to conditioned stimuli in which the animal has learned to respond to a threatening situation by inhibiting movement and remaining alert. Another possibility is that the behavioral response associated with the hypothalamic site and PAG site are not identical. Carrive (2) reports that vlPAG stimulation leads to hyporeactive immobility that can range from a simple decrease in spontaneous activity to a ‘‘waxy’’ immobility with signs of atonia. We have found that the characteristics of the immobility elicited by HVA stimulation are dependent upon stimulus intensity and though we have observed most of the behavioral responses reported by Carrive, stimulation of the HVA can increase extensor muscle tone (4). ACKNOWLEDGEMENTS

The present study was supported by NIH HL 36588 and HL 07426.

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