ADRENERGIC RECEPTOR REGULATION IN POSTTRAUMATIC STRESS DISORDER

Bruce D. Perry, M.D., Ph.D.
Steven M. Southwick, M.D.
Earl L. Giller, Jr., M.D., Ph.D

The ChildTrauma Academy
www.ChildTrauma.org

This is an Academy version of a chapter originally appearing in "Biological Assessment and Treatment of Posttraumatic Stress Disorder" E.L. Giller, Jr. Ed. , Progress in Psychiatry, D. Spiegel, M.D., Series Editor. American Psychiatric Press, Inc.

Official citation:  Perry, BD, Southwick, SW, Yehuda, R and Giller, EL Adrenergic receptor regulation in post-traumatic stress disorder. In: Advances in Psychiatry: Biological Assessment and Treatment of Post Traumatic Stress Disorder (EL Giller , Ed.). American Psychiatric Press, Washington, DC, 87-115, 1990.

INTRODUCTION

Post-Traumatic Stress Disorder (DSM-III-R, PTSD) is a clinical syndrome characterized by prominent affective symptoms (dysphoria, irritability, anxiety) and by a 'hyperactive' sympathetic nervous system (see DaCosta 1871, Bury 1918, Frazer and Wilson 1918, Crile 1940, Dobbs and Wilson 1960, Horowitz et al. 1980, Brende 1982 ). A high percentage of combat veterans, rape victims , sexual abuse victims and survivors of catastrophic events experience symptoms of PTSD (see Terr 1983, Blanchard et al. 1983, Boehlein et al. 1985, Birkhimer et al. 1985, Bleich et al. 1986, McLeer et al. 1988). These long term psychological and physical effects of exposure to traumatic stressors have been described for centuries . Despite the prevalence of this syndrome in our generation, and the descriptive validation of many generations before, PTSD remains a controversial diagnostic syndrome. Few studies have attempted to study directly the pathophysiology of PTSD. Among the reasons for this are difficulties in recruiting a population of individuals with PTSD willing to participate in a research protocol, and the ever-present complexities of co-morbidity. Substance abuse, personality disorders and other Axis I diagnoses, primarily Major Depressive Disorder (MDD), are seen with high frequency in most PTSD cohorts. In the present chapter a series of studies examining platelet alpha2-adrenergic receptors in PTSD will be presented. These studies, demonstrating altered peripheral adrenergic receptor measures in PTSD, will be discussed in context of the physiological responses to 'stress', both acute and chronic. We will suggest that the 'typical' central (CNS) and peripheral nervous system responses to acute stress are mediated, in part, by adrenergic receptors which, when 'hyper-stimulated' during severe or chronic stress, can become permanently altered in their capacity to respond to future stressors. Further, we will suggest that this adrenergic receptor 'dysregulation' may be related to the symptoms associated with PTSD.

ADRENERGIC RECEPTOR REGULATION AND STRESS

The neurophysiology of stress has been studied extensively in man and in animal models (see Selye 1936, Mason 1968 and 1975, Stone 1975 and 1988). Acute 'stress' is associated with a variety of physiological responses including the activation of the HPA - axis with a concomitant peripheral release of ACTH, epinephrine and cortisol , a significant increase in centrally- controlled peripheral sympathetic nervous system tone, and the 'activation' of a variety of neurochemical systems in the CNS. One of the most critical of these systems is the noradrenergic nucleus in the locus coeruleus (Korf 1976, Redmond and Huang 1979). This region controls noradrenergic tone and activity throughout the midbrain and in important forebrain areas including the cortex (Foote et al. 1983). The LC has been shown to be critical in many regulatory functions including the regulation of affect, 'irritability', locomotion , arousal , attention and startle (Korf 1976, Foote et al. 1983, Andrade and Aghajanian 1984, Bhasharan and Freed 1988 ). Another key neural system in the brain, also an adrenergic/noradrenergic system is the ventral tegmental nucleus (V.T.N.) which is involved in regulation of the sympathetic nuclei in the pons/medulla (Moore and Bloom 1975).

Both the L.C. and the VTN nuclei have adrenergic receptors which are involved in modulation of the adrenergic or noradrenergic afferentation and efferent outflow (U'Prichard et al. 1984, Vantini et al. 1984). Alpha2-adrenergic receptors, both pre- and postsynaptic, play important roles in meditating the effects of these key systems (Perry et al. 1983, Vantini et al. 1984). Manipulation of alpha2 adrenergic receptors in these areas by specific alpha2-adrenergic drugs results in a variety of behavioral effects ( see Krystal et al., this volume). More direct evidence of the primary importance of noradrenergic and adrenergic systems and their receptors in the stress response comes from 'stress' inducing paradigms in animals (for review see Stone 1988 ). Chronic stress (e.g., footshock, 'handling') results in altered beta and alpha2-adrenergic receptor functioning (e.g., decreased alpha2 receptors and the less efficient coupling of beta and alpha2 receptors to adenylate cyclase) in many brain regions (U'Prichard and Kvetnansky 1980, Stone et al. 1986, Stone 1988). These changes are felt to reflect homeostatic changes resulting from the increased activity of the adrenergic and noradrenergic systems mediating the CNS stress response (Stolk et al. 1984, Stolk et al. 1985 ). Further evidence for the critical role of CNS alpha2-adrenergic receptors is seen in two inbred strains of rat, F344 and Buffalo, which have very different physiological responses to 'stress'. A major CNS neurochemical difference between these animals is in the number and regulation of alpha2-adrenergic receptors in the LC and VTN regions (Perry et al. 1983, Vantini et al. 1984, Stolk et al. 1984 )

One of the most useful paradigms for the study of stress is the 'learned helplessness' (LH) or inescapable shock (IS) model (Anisman et al 1979, Krystal et al. in press, and Krystal this volume). In this well known paradigm a variety of 'behavioral' deficits and concomitant neurochemical alterations are observed following exposure to IS (see Murberg et al. this volume). The similarities between the signs and symptoms seen in rats exposed to inescapable shock (stress) and those seen in PTSD suggests that the IS paradigm may be a good animal model of PTSD (van der Kolk et al. 1985). Adrenergic and noradrenergic systems and their receptors are involved in the mediation and recovery from the observed behavioral changes following IS (Anisman et al. 1979, Cassens et al. 1980, Anderson et al. 1984). It is of interest to note that under certain conditions tricyclic antidepressant medications (somewhat efficacious in PTSD, see Frank et al., this edition) attenuate the effects of IS in animals (Petty and Sherman 1979 and 1980, Sherman and Petty 1980, Kitada et al. 1981).

THE PLATELET ALPHA-2 ADRENERGIC RECEPTOR AS A MODEL

Brain noradrenergic and adrenergic systems and their receptors, then, play key roles in a variety of important affective and behavioral changes associated with the stress response. In turn, these initially-adaptive changes are pathologically altered in PTSD (and perhaps, other psychiatric disorders). Unfortunately, direct investigation of the role of these systems and their receptors in human psychiatric disorders has been difficult. A variety of indirect methods have been developed, including challenge paradigms, measurement of peripheral catecholamines and their metabolites, and measurement of adrenergic receptors and their functioning in peripheral tissues (e.g., platelets and lymphocytes).

Beginning in the late 1970s, when the platelet alpha2- adrenergic receptor was first radiolabeled, investigators have utilized this readily available peripheral receptor as a marker for the central alpha2 receptor. There are a number of technical (e.g., Karliner et al. 1982 ,Perry and U'Prichard 1984) and conceptual difficulties (Kafka and Paul 1987) with these studies. Despite these problems, many investigators have used radioligand binding techniques to measure directly platelet alpha2-adrenergic receptors in a variety of psychiatric populations. The most promising findings are in major depressive disorder. The results of studies in MDD have been inconsistent; some studies have suggested an increased total alpha2 receptor number (Garcia-Sevilla et al. 1981); others, an increase in one of the affinity states of the alpha2 receptor (Daiguchi et al. 1981, Doyle et al. 1983, Garcia-Sevilla et al. 1987), or changes in the ratio of affinity states, affinity of ligand, or direct demonstration of an altered receptor-effector efficiency (Pimoule et al. 1983, Seiver et al. 1984, Garcia- Sevilla et al. 1986, Kafka et al. 1986, Wolfe et al. 1987 ). Taken as a whole these studies do suggest some altered peripheral alpha2 receptor/effector functioning in affective disorders.

Since 1977 many of the complexities of the alpha2- adrenergic receptor/effector systems and their regulation have been worked out (see Harden 1983, Lefkowitz et al. 1984), making appropriately designed binding studies easier to interpret. As with alpha2-receptors in other tissues (including brain), the platelet alpha2 receptor is actually comprised of multiple membrane components (Fig. 1): the receptor/recognition site (R) with the agonist (and antagonist) binding sites; the N component: a protein heterotrimer which links the R to the third component , the second messenger (e.g., Ni links to the catalytic moiety of adenylate cyclase: AC). In the intact system, the multiple components of this receptor/effector system are in a dynamic steady-state which is influenced by a variety of factors, including the amount of agonist present, the pH, temperature, and the prevalence of certain nucleotides (e.g., GTP) and metal ions (e.g., Na +).

In the process of performing standard radioligand binding assays, this steady-state is 'frozen'. The binding measures (KD, Bmax, see below) reflect a summation of a set of dynamic events which in turn are related to the 'regulation' of the receptor/effector complex. The equilibrium dissociation constant or KD reflects the tightness of binding of the radioligand to the receptor (affinity). The Bmax is the 'maximal' number of binding sites which are labeled by the radioligand. In these radioligand binding studies, the free R is labeled with low affinity by agonists; this binding site is also called the alpha2-(L) or low affinity state. The R-Ni complex is labeled with higher affinity by agonists, thus it is called the alpha2-(H) or high affinity state.

In the present studies we utilized the antagonist radioligand, 3H-rauwolscine (RAUW: Perry and U'Prichard 1981). This radioligand labels the 'affinity' states of the alpha2 receptor differently than an agonist radioligand (Perry and U'Prichard, 1984). The high affinity component of rauwolscine binding is the free R (or alpha2-(L) affinity state) while the low affinity component of rauwolscine binding is the R-Ni complex (or alpha2-(H) affinity state). The advantage of this radioligand is that under appropriate assay conditions, unlike an agonist ligand such as 3H-clonidine, it can measure the total number of receptor binding sites, both R and R-Ni in the membrane.

The studies presented here also utilized another measure obtained from radioligand binding studies, the ratio of affinity states (i.e., the number of free R relative to the 'coupled' R-Ni sites or alpha2 (L)/alpha2(H)). Again, the equilibria between the multiple affinity states of the alpha2-receptor are complex but in general the ratio of affinity states derived from membrane binding studies is thought to reflect the efficiency of the receptor-effector coupling and, therefore is a more physiologically-significant measure of receptor-effector functioning than receptor number or radioligand affinity alone (Kent et al. 1980, Delean et al. 1981, Supiano et al. 1987). A higher 'ratio' of R/R-Ni would suggest that the alpha2 receptor recognition protein, R, is less efficiently coupled to the second messenger.

PLATELET ALPHA2-ADRENERGIC RECEPTORS IN PTSD

The strong suggestion of altered peripheral (and likely CNS) alpha2 adrenergic receptor regulation in other affective disorders and the clear relationships between adrenergic and noradrenergic systems in the stress response prompted us to assay of platelet alpha2 adrenergic receptor binding measures in PTSD (Perry et al. 1987, Perry 1988). Well aware of the limitations of the peripheral model utilized, we nonetheless felt that careful application of radioligand binding techniques would provide useful information regarding the possibility of altered peripheral alpha2 receptor regulation in PTSD relative to controls and other psychiatric groups (e.g., major depression and borderline personality disorder).

In our initial studies of the platelet alpha2-adrenergic receptor in PTSD, we performed two basic experiments: (1) an extended saturation study (12 concentrations of the radioligand) which would allow us to derive the KD, Bmax and relative proportion of affinity states (ratio); and (2) a competition study with 14 different concentrations of (-)-epinephrine 'competing' for RAUW-labeled sites; this allows determination of the Ki (or affinity) of (-)-epinephrine at the platelet alpha2 affinity states and provides another measure of the relative ratio of affinity states. The basic radioligand binding methods utilized have been described in detail elsewhere (Perry and U'Prichard 1984 ).

Our population of PTSD subjects was largely inpatient (75 % ), male ( 95 % , average age 38 ). Research Diagnostic Criteria were used for diagnoses other than PTSD and DSM-III criteria for PTSD. Exclusion criteria included active substance abuse, major medical illness, or use of medication known to directly interfere with adrenergic receptors. The total population of PTSD subjects was 21 Of this number, 13 met RDC for Major Depressive Disorder. The control population was comparable with regard to age, sex, and medical health.

Saturation studies (Fig. 2, Table I) demonstrated fewer total platelet alpha2-adrenergic receptor binding sites in the PTSD subjects compared with controls. Furthermore, when using a computerized (LIGAND; Munson and Rodbard 1980) curve fitting program to analyze the multiple components of the RAUW saturation isotherm, two sites of interaction for both control and PTSD subjects were demonstrated (i.e., alpha2-(H) and alpha2-(L), see above). No population differences were seen in the affinity (KD) of RAUW for either site (Table 1). The observed decrease in total number of RAUW sites was due to fewer of alpha2-(H) site (the R-Ni complex) in the PTSD subjects. The ratio of the affinity states ( alpha2-(L)/alpha2-(H) x 100) was much higher in the PTSD subjects (Table 1).

(-)-Epinephrine competition studies (Figure 3, Table 2) were consistent with these findings. Epinephrine appeared to be a less potent inhibitor of RAUW specific binding in the PTSD membranes; the competition curve was somewhat steeper and shifted to the right (Fig 3 ). LIGAND analysis of the (-)-epinephrine competition curves revealed two sites of interaction. No population differences in the affinity of (-)-epinephrine for the two sites were seen. More of the total alpha2-adrenergic receptors were in the low (alpha2-(L)) and fewer in the high affinity state (alpha2-(H)) in the PTSD membranes, relative to controls. This accounted for the observed apparent decrease in (-)-epinephrine potency. Again the observed difference in the relative prevalence of the alpha2 affinity states was reflected by the ratio of R/R-Ni (as above); in PTSD the ratio was two times the control value (see Table 2).

The presence of fewer membrane binding sites and an altered ratio of affinity states is seen in other adrenergic receptor/effector systems exposed to chronically high levels of endogenous agonist in the CNS (e.g., Perry et al., 1983). In a similar manner, down-regulation and 'uncoupling' of platelet alpha2-receptors has been demonstrated in clinical states associated with increased levels of circulating catecholamine: congestive heart failure (Weiss et al. 1983), aging (Supiano et al. 1987), hypertension (Hollister et al. 1981 and 1986).

The results in PTSD, then, suggest that these platelet alpha2 receptors have been exposed chronically to high levels of endogenous agonist. Indeed, this appears to be the case in PTSD. Mason and co-workers (this volume; and Kosten et al. 1986) have demonstrated elevated and sustained excretion of urinary catecholamines, likely reflective of circulating catecholamines. In addition, a recent report of an attenuated lymphocyte beta- adrenergic receptor-mediated cyclic AMP production seen in PTSD is consistent with desensitization secondary to excess circulating catecholamine (Lerer et al. 1987). High levels of circulating catecholamines and a secondary down-regulation of platelet adrenergic receptors are consistent with an overactive sympathetic nervous system as seen in PTSD. Chronic exposure to agonist (as is presumed in the PTSD subjects) decreased the total number of receptors and also changed the steady-state between the affinity states (R and R-Ni) which is reflected as an increase of the ratio of alpha2-(L) to alpha2-(H) (Figure 3).

These findings are different than those found by us and others in major depressive disorder, where there are also increases in circulating catecholamines (Roy et al. 1985). In our MDD population (subjects with no cormorbid personality disorder or PTSD), we found more total alpha2 receptor binding sites relative to controls but we still observed the altered ratio of affinity states (i.e., an increase in R/R-Ni) similar to that seen in PTSD (see Table 2 ). When we took our total population of PTSD subjects and separated them on the basis of co-morbidity, we were unable to demonstrate any differences in any of the binding parameters (KD, Bmax, ratio) between the whole PTSD population and PTSD with no co-morbid diagnoses, PTSD with co-morbid substance abuse history (data not shown), PTSD with co- morbid borderline personality disorder, or PTSD (data not shown) and co-morbid major depressive disorder (see Table 2 and Figure 2). This, in concert with our findings in the 'pure' MDD population, strongly suggested that some aspects of the pathophysiology of PTSD are distinct from MDD.

THE DYSREGULATION HYPOTHESIS AND PTSD

The single time point studies described above provided limited information regarding the actual dynamic functioning and regulation of the alpha2 receptor in PTSD. The values obtained were reflective only of the alpha2 receptors in the membrane. The number of receptors present in the membrane and 'accessible' to agonist (or radioligand) depends upon a variety of complex intracellular processes (see Harden 1983, Sibley et al. 1986, Perry 1988). Critical in regulating the number of receptors in the membrane, and receptor-mediated signal transduction, is the complex process of receptor 'internalization'(see Fig. 3 and Harden, 1983). In brief, it appears that following agonist binding, the receptor recognition protein is physically taken inside the cell where it may then be recyled. Therefore, at any given moment, a certain percentage of the cell's total receptor population is inside the cell or 'internalized'. The rate of internalization, in turn, is dependent upon a variety of factors; among these, agonist concentration, duration of exposure, rate of degradation, rate of phosphorylation, and other as yet unidentified processes. Despite our relative ignorance of the specific molecular mechanisms involved in these processes, it is clear that they play a critical regulatory role in alpha2- receptor/effector functioning.

In order to study these processes, 'dysregulation' experiments were performed (Perry 1986; Perry 1988). These studies attempted to examine some of the dynamic processes involved in receptor regulation by using an 'in vitro' incubation of intact platelets with agonist. Briefly, an intact platelet preparation isolated from a single subject was washed extensively in isotonic saline (25 C) and then suspended in buffered (Tris HCl, 50 mM) Dulbecco's Modified Eagles Medium. The platelets were incubated at 37 C (see Figures) with or without 100 uM (- )epinephrine. At specific times, platelets were removed and membranes were prepared for radioligand binding studies (Perry and U'Prichard 1984; centrifugation techniques were used under which 'light' vesicles containing 'internalized' receptor would not be sedimented). The platelet membranes were washed to remove the excess agonist (this to assure that any observed 'downregulation' was not due to retained agonist, Karliner et al., 1982). Radioligand binding assays were then performed in the membranes using standard techniques (Perry and U'Prichard, 1984 ).

Intact, isolated platelets from controls and PTSD subjects were incubated with (-)-epinephrine for 1, 2.5, and 5 hours. Binding studies were performed in the membranes prepared following these incubations. Saturation studies demonstrated loss of RAUW specific binding sites (Table 3 and Figure 4 ) in the membranes prepared from both control and PTSD subjects. Again, biphasic saturation isotherms were obtained, and no changes in the affinity of RAUW for the two sites of interaction were observed in either the PTSD or control membranes following incubation (data not shown). The progressive loss of RAUW binding sites observed in both controls and PTSD subjects during epinephrine incubation was due primarily to the loss of the alpha2-(H) affinity state (Table 3). The alpha2-(L) state decreased but less dramatically. The ratio of affinity states (free R/R-Ni) increased dramatically with incubation . Competition studies in these same membranes also demonstrated a significant change in the relative prevalence of alpha2-affinity states with agonist incubation with an increase alpha2-(L) (R) and a decrease in alpha2-(H) (R-Ni).

Acute agonist incubation in controls resulted in a decrease in receptor number, shift in equilibria between R and R-Ni (reflected as an increase in the ratio) and an apparent 'selective' loss of R-Ni, such that the control binding parameters following acute agonist exposure look very much like the PTSD subjects at baseline. Again, these findings are similar to that seen following acute agonist exposure in other receptor linked-adenylate cyclase systems (e.g., Chioffi and El Fakahany, 1986).

These studies also suggested, among other things, that the rate of loss of RAUW binding in the PTSD membranes was increased relative to controls. In order to directly study this, the specific binding of a single concentration of RAUW (3.0 - 4.0 nM) in platelet membranes prepared from the intact platelets incubated for various times was examined (see Fig. 4). In controls, the rate and extent of loss in RAUW specific binding was quite reproducible, with a 40-50 % loss at 5 hours with a t1/2 of approximately 2 hours. In PTSD subjects, the extent of loss is greater, with approximately a 60 - 80 % decrease from original values at 5 h. The rate of loss is also increased. Using KINETIC, a computerized program for determining rates, the rate of loss of RAUW specific binding was increased in the PTSD subjects.

RECEPTOR-EFFECTOR 'FATIGUE' AND PTSD

These preliminary dysregulation studies, in concert with the the single time point assays presented earlier, suggest that the platelet alpha2-adrenergic receptor in subjects with PTSD are desensitized to agonist. Thus, with any given exposure to agonist less alpha2-mediated second messenger effect would be expected. On the other hand, the same 'signal' (i.e., chronic agonist) which 'uncouples' and the receptor and second messenger, appears to have resulted in an accelerated mechanism for 'down- regulation' , in other words, a higher percentage of membrane receptors will be taken out of the membrane and internalized following any given agonist exposure. The PTSD subjects had a more rapid and extensive apparent internalization following agonist incubation which was accompanied by a very large increase in the 'ratio' (more rapidly 'uncoupled' from cyclase; more free R in a membrane preparation and, 'in vivo', likely less capable of interacting with Ni). The molecular adaptations involved in these processes (i.e., uncoupling of receptor and second messenger, 'down regulation', and altered internalization functioning ) are pervasive and important regulatory processes by which many membrane bound receptors can be controlled to maintain intracellular homeostasis (Harden 1983, Lefkowitz et al. 1984). In many intact cell systems (Harden, 1984), 'in vivo' in the brain (Perry, et al., 1983), and in animal models of stress, 'higher than control' levels of agonist result in down- regulation of adrenergic membrane receptors.

Chronic exposure to 'higher than control' levels of agonist (in PTSD) results in a receptor-effector system less capable of responding to agonist increases associated with subsequent stressors. The platelet alpha2-adrenergic receptor-effector system in the PTSD subjects was 'overtaxed' and easily 'fatigued'. Does this chronic exposure to agonist alter in a more permanent fashion the ability of the cell to express receptor ? In PTSD are similar processes taking place which result in a sensitized alpha2-adrenergic receptor/effectors in key systems in the CNS ? A number of studies suggest this is likely (see Krystal et al., in press). Furthermore, peripheral sympathetic activity appears to be involved in regulation of the LC (Elam et al. 1984, Svensson 1987, Baskaran and Freed 1988). An overactive sympathetic nervous system, then, may directly alter the adrenergic receptors in the locus coeruleus by feedback mechanisms, and therefore these peripheral findings may have more significance than merely being a reflection of circulating catecholamine levels.

It is clear that permanent intraneuronal changes which lead to alterations in expression of receptor proteins do take place with agonism in mature neuronal systems. This, in turn, is related to the molecular bases for 'memory' and learning (see Kandel and Schwartz 1982, Goelet and Kandel 1986). In this regard, the dissociative and 'flashback' symptoms of PTSD could be conceptualized as 'state or physiological memories' not typically associated with the more conventional concepts of cognitive memory. Indeed the descriptions of flashbacks by many individuals suffering from PTSD suggest activation of a set of multimodal sensorial memories.

IMPLICATIONS: THEORETICAL AND CLINICAL

Are the pathophysiological mechanisms at play in PTSD similar to those in other psychiatric disorders ? The diathesis-stress model of psychiatric illness suggests that a predisposition (genetic or developmental) for a specific psychiatric disorder exists which can be differentially expressed in an individual depending upon the degree of 'biopsychosocial' stressors. Combat exposure, rape and other severe trauma would qualify as 'biopsychosocial' stressors. Yet not all individuals with these experiences develop signs and symptoms of PTSD. Indeed, the biological and psychological makeup which an individual has prior to these traumatic experiences must play an important role in the adaptive or maladaptive (both neurochemical and psychological) coping mechanisms. Some individuals may be 'sensitized' to subsequent stressors, other may be extremely resilient.

Where do these predispositions come from ? Are they genetic or are they developmentally determined ? Again animal studies have provided some interesting parallels which may provide important insight to these clinical phenomenon. In inbred strains of rat, genetic differences in the catecholamine synthetic enzyme PNMT (which converts norepinephrine to epinephrine) have been linked to a cascade of neurochemical alterations including alpha adrenergic receptor regulation in the locus coeruleus which, in turn, result in an altered capacity of the animals to respond to a variety of stressors (Perry et al. 1983, Vantini et al. 1984, Stolk et al. 1984). Is the diathesis in PTSD some genetically-coded 'altered' step in a critical neurochemical cascade involved in mediating the stress response (see Weiland et al. 1986) ? If so, are the observed platelet alpha2 receptor changes related to this ? An equally plausible explanation for the 'diathesis' in PTSD may be developmental (see Suomi in press). The development of behavioral and neurochemical changes in the CNS following inescapable shock has been suggested as an excellent animal model for PTSD. What happens to the neurochemical systems responsible for mediating normal stress respones in animals exposed to severe stressors during development ? A number of fascinating studies in animals demonstrate the exquisite sensitivity of the developing CNS to stress. It appears that the neurophysiological capabilities of the adult to respond to stress is determined by early life experiences. In rats exposed to perinatal handling stress major alterations in the ability of the rat to 'learn' and to respond appropriately to stressors are seen later in life (Weinstock et al. 1988). The most interesting aspect of these studies is that exposure to unpredictable stress resulted in deficits while exposure to consistent, daily stress resulted in 'improved' or superior behavior; these animals were 'resilient'. In a similar fashion, in humans, increased psychiatric symptoms and disorders are observed in adults who have severe early life stressors (Brown and Harris 1977, Lloyd 1980, Rutter 1984). Indeed, in a recent report by Breier and co-workers (1988), an altered neuroendocrine axis and a higher incidence of 'psychiatric' symptomatology was observed in children suffering a major losses and continued family 'distress' during development. One can speculate on equivalent 'controlled' or daily stress and uncontrollable, non- scheduled stressors in the development of a human. An infant who is allowed to have an 'optimal' degree of frustration, one who can control, during rapproachment, his own optimal degree of 'tension, anxiety' (i.e., stress) and return to mother for comfort , is one who's developing CNS is establishing an appropriate neurochemical milieu for the development of a flexible, maximally-adaptive physiological apparatus for responding to future stressors. A child who is reared in an inconsistent, unpredictable environment (see Spitz and Wolfe 1946) will likely have a evoked in his developing CNS a milieu which will result in a poorly homeostatic, 'dysregulated' stress neurochemistry . One would hypothesize that such a child would be susceptible to the development of more severe signs and symptoms when exposed to psychosocial stressors.

FUTURE DIRECTIONS

These studies suggest a variety of follow up projects. The findings of down-regulated and uncoupled alpha2-adrenergic receptor binding sites needs to be replicated and correlated with peripheral measures of circulating catecholamine (i.e., plasma and 24 hour urine norepinephrine and epinephrine). In addition, other peripheral receptors, such as the lymphocyte beta adrenergic receptor should be examined for similar down- regulation. At present our laboratories are proceeding with these studies.

Similar studies would be useful in other, non-combat PTSD populations and should include prospective, longitudinal and treatment outcome studies. Longitudinal studies in children, examining a variety of psychological, social and neurochemical parameters are planned at the Harris Center for Developmental Studies. Understanding what significance these peripheral measures have will require ultimately looking directly at the CNS. In order to do this, challenge paradigms, post-mortem studies, and in vivo visualization techniques (e.g., SPECT and PET) will be useful. In concert with these studies, further work on the neurochemistry associated with these promising animal models of PTSD are needed. Of particular interest in this area is examination of pre and perinatal 'environmental' factors (including 'stress') involved in determining neurochemical phenotypes of the adult animal (Perry 1988). Finally, further examination of the role of 'stressors' and the neurochemistry of the acute and chronic stress response in the pathophysiology of other psychiatric disorders is needed.

In summary, these preliminary studies have provided evidence of the sustained distress (reflected by persisting overactive sympathetic nervous system) in PTSD. It is not apparent to us that the observed changes in the PTSD subjects are related necessarily to any genetic difference in alpha2 receptors or their regulation which may be preserved in the CNS. Neither does this downregulated, desensitized system appear to be specific to PTSD. We do see similar findings in Borderline Personality Disorder, but not in Major Depressive Disorder. Despite this, the adrenergic receptor changes observed in PTSD platelets may be worth further investigation for two major reasons. First, to track 'distress' in an individual as a function of some intervention. Indeed, we have found downregulated platelet alpha2-adrenergic binding sites associated with anxiety may be restored toward normal following benzodiazepine treatment which decreases symptoms (in preparation). Secondly, more important, studying the altered mechanisms of peripheral receptor regulation in PTSD may provide useful models for the molecular mechanisms involved in altered receptor functioning in the CNS noradrenergic systems in PTSD subjects.

ACKNOWLEDGEMENTS

The authors would like to thank the many courageous veterans and their families who elected to participate in our studies. Technical assistance was provided by Janet Giunti, Alex Ackles and Helen Spencer. These studies were supported by VA funds. BDP is supported by the Harris Center for Developmental Studies and the Brain Research Foundation.

Portions of the data presented in this chapter were originally presented at The 1986 Meeting for the Society of Neurosciences, New Research at the American Psychatric Association 1987 Meetings and at the 1987 Meeting of the Society of Biological Psychiatry .

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FIGURES

Figure 1. Schematic representation of the multiple membrane components of the alpha2-adrenergic receptor-effector system. This cartoon illustrates the major components of the alpha2- adrenergic receptor-effector complex as well as other second messenger systems likely involved in alpha2-receptor functioning. From left to right, the receptor/recognition site (R) with the agonist (and antagonist) and sodium binding sites; the Ni component: a protein heterotrimer which links the R and the catalytic moeity of adenylate cyclase (AC). The three protein components of Ni ( i, , ) have interactions with other membrane bound receptors (e.g., in the platelet the 5-HT2-serotonergic) as well. In addition, other N-heterotrimers may link the alpha adrenergic receptor/recognition site with other second messenger systems (e.g., k, , may link to a voltage sensitive Ca channel). A Na-H antiporter (Isom et al., 1987) likely links the alpha2-R (in a nucleotide sensitive fashion,i.e., via another N heterotrimer) with a membrane bound phosphodiesterase which cleaves phosphatidylinositol (PI) to result in inositol triphosphate (IP3) and diacylglycerol (DG), two more key intracellular 'second messengers'.

In radioligand binding studies, the free R is labeled with low affinity by agonists; this binding site is also called the alpha2-(L) affinity state. The R-Ni complex is labeled with higher affinity by agonists, thus it is called the alpha2-(H) affinity state. The antagonist rauwolscine labels these sites with inverse affinities (see Perry and U'Prichard, 1983): the high affinity component of rauwolscine binding is the free R (or alpha2-(L) affinity state) while the low affinity component of rauwolscine binding is the R-Ni complex (or alpha2-(H) affinity state).

Figure 2. Platelet Alpha2-Adrenergic Receptor Binding Sites in PTSD: Increased 'Ratio' of Alpha22-(L) to Alpha2-(H) in PTSD.

Extended saturation studies were performed on membranes prepared from platelets as described in text. The ratio was determined by taking (the number of alpha2-(L)/the number of alpha2-(H) sites) x 100. Values represent the mean + S.E.M. See Table 2 for other values.

Figure 3. (-)-Epinephrine displacement of RAUW-specific binding to platelet alpha2-adrenergic receptor binding sites: PTSD vs. controls. Platelet membranes were prepared and, extensively washed and competition studies were performed using 3.0 nM RAUW. 14 concentrations of unlabeled epinephrine were used and specific binding was determined by 100 uM (-)-norepinephrine. Competition curves were obtained using LIGAND (Rodbard and Munson, 1980). This figure is from a representative experiment performed in triplicate; refer to Table 2 for specific values.

Figure 4. Platelet Alpha2-Adrenergic Receptor Binding Sites Following 'In Vitro' Exposure to Epinephrine: Increased Rate of'Downregulation' in PTSD. Intact platelets were incubated for various times with 10 -4 M (-)-epinephrine and membranes were prepared as described in text. The specific binding of 4.0 nM RAUW was then determined. Values represent mean + S.E.M. N = 8 for controls, N = 5 for PTSD.