Effects of treatment with imipramine and clonazepam on an animal model of panic disorder

Varied Manifestations of Acute Intermittent Porphyfia

atOLPSVOV.A~t't

745

1994",36:744-747

given twice daily for 3 days. Autonomic hyperarousal is managed with propranolol and excitement with low-dose antipsychotics. Intravenous dextrose and hematin have a role only when instituted early on in the attack.

Case Report This is the report of a case of AlP who presented with episodic, variable psychiatric symptoms. DS, the 14-yearold patient, is the eldest of seven siblings from a ruraljoint family. Febrile illness had precipitated respiratory distress and death in one sister and three brothers of DS, when they were 2 1/2 years, 4 months, 3 months, and 21/2 years of age, respectively.

First Episode DS was brought to the Medical Emergency services of the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, with a 7-day history of headache, retardation, and muteness. The symptoms had started with mild fever, severe cramp-like epigastric pain, and headache. Following this he developed fearfulness, insomnia, and progressive psychomotor retardation. This progressed into muteness, refusal of food, and near-total cessation of movement. In the Emergency room, he was investigated to find the cause of his fever and stupor. He had no leukocytosis and a culture of urine was sterile. Cerebrospinal fluid (CSF) microscopy and biochemistry revealed no abnormality. CSF and blood cultures were sterile. His chest x-ray showed no abnormality. Blood smear for malarial parasite was negative and blood Widal was not suggestive for enteric fever. His electroencephalogram, computed tomography (CT) scan of brain and urine porphobilinogen (done thrice) revealed no abnormality. He was treated with haloperidol 5 rag/day and trihexyphenidyl 4 rag/day. Despite the anticholinergic cover, he developed six episodes of severe nu-'--' . .". . . U l l i i l dy S torda W ithi n - mAo . u . !-_ ,r,.:...._~ • r a n w , ~ m a n age d w ith ,,,u,~,,.: nous diazepam and by stopping haloperidol. He was then switched to the less-potent agent, chlorpromazine, 200 mg/day. The patient recovered completely within 20 days and was discharged after 25 days of hospitalization.

Second Episode At home, the patient remained well for 8 days and then developed fever, abdominal pain, and nausea, followed by severe headache. He became intensely apprehensive, fearful, and fell into a stupor within 48 hr. He was readmitted and the diagnosis of acute intermittent porphyria was entertained as a strong possibility. The urine was found to be positive for porphobilinogen then and on three subsequent occasions. The patient was treated with chlorpromazine 200 nag/day after which he recovered completely in 3 weeks.

Third Episode Three days after recovery, he d e v e l ~ abdominal pain, vomiting, and headache, followed by elation, distracu~ility, and social disinhibition. This hypomanic phase gradually disappeared in 8 days without a change in ~ r e p y .

Fourth and Fifth Episodes Two days after the third episode, the patient once again developed abdominal pain, headache, and he became fearful. He became confused and disoriented to time, place, ar.H person and the Babinski's sign was positive on the left side. He was immediately given rapid infusion of one pint of 10% dextrose. He recovered from this delirium in 3 hr. A similar attack was seen 2 days after recovery. In view of a very high frequency of attacks, 4 pints of 10% dextrose was infused over a 24-hr period to try and prevent further episodes.

Sixth Episode Two days after the last episode, the patient again developed abdominal pain, vomiting, headache, and became confused and disoriented. He developed echolalia, posturing, ambitendency, and became stuporous. His blood pressure was found to be high and fluctuating, along with tachycardia (pulse 130/rain). He was given 200 g of glucose orally, followed by intravenous glucose, but it did not abort this episode. The patient was continued on 300 mg of chlorpromazine daily and was given a carbohydrate-rich diet. Propranolol 40 rag/day was added to control the peripheral autonomic fluctuations. Despite the fact that the cause of fever could not be ascertained, the patient was treated empirically with a course of cloxacillin for 7 days, for possible underlying infection. The patient gradually recovered in a week's time. No further episodes occurred and he was discharged 10 days after recovery from the last episode. He was advised to continue chlorpromazine 300 rag/day for the period of 1 month, which was then gradually tapered off over the next 15 days. In addition, the patient was advised to take a high~ carbohydrate diet and was given a list of drugs that he should not take (Appendix I). Because the patient lived in a remote rural area, the local doctor was contacted ~ d advised regarding management of acute episodes, whenever the need arose. The patient was symptom-free when followed-up 20 months after the last discharge. Screening of the other family members did not show any urine prophobilinogen positivity. Regular follow-up of the siblings is planned, however.

Discussion Although it is reported in the literature that psychiatric manifestations of AIP vary considerably, it is not clearly

746

PJ. Santoshand S. Malhotra

nlOLPSYCHtA'rRY 1994:36:744,-747

APPENDIX 1

Unsafe PorphyHnogenic Drugs (Drugs that can precipitate an attack): Alpha methyldopa Alcohol Amphetamines Aminopyrine Amitriptyline Barbiturates Busulfan Carbamazepine Chlorambucil Chloroform Chloramphenicol Chlorpropamide Chlordiazepoxide Cimetidine Cionidine Cyclophosphamide Cycloserine Dimenhydrinate Dichloralphenzone Oral contraceptives Oxazolidinediones Pargyline Paraldehyde Progresterone Probenacid Pyrimethamine Phenylbutazone Primidone Pentazocine Pyrazinamide

Ergot preparations Erythromycin Ethylchlorvynol Estrogens Eucalyptol Fentanyl Furosemide Glutethimide Gold preparations Griseofulvin Hydantoins Halothane Hydralazine Imipramine Ketamine Lidocaine Meprobamate Methoxyflurane Metochlopramide Rifampicin Spironolactone Steroids Succinimides Sulfonamides Sulfonylureas Theophylline Tranylcypromine Tetracyclines Valproate

documented that they vary in the same patient during different episodes. This case illustrates the varied psychiatric manifestations of Alp in different episodes. Within a span of 2 months, the patient had presented with a panorama of reversible psychiattic syndromes ranging from catatonic stupor and hypomanic excitement, to delirium. All the episc~deshad a typical acute onset, with abdominal pain and severe headache, leading onto psychiatric symptoms of fear and anxiety. These were teen followed by either withdrawal, catatonia, hypomania, or delirium with labile hypertension. Each episode was managed symptomatically with intravenous dextrose and chlorpromazine, resulting in complete recovery. As high-potency neuroleptics like haloperidol induced severe dystonic reaction, with worsening of

his clinical condition, he was given chlorpromazine, which has often been recommended as the initial line of management of AlP (Goldberg et ai 1983; Lishman 1987). In psychiatric practice, a diagnosis of AlP is to be suspected in all patients with brief episodes of psychiatric manifestations accompanied by epigastric pain. The symptoms are of acute onset and recovery is usually complete, in the present case, although the diagnosis of AlP was suspected early, the negative porphobilinogen in urine was misleading. The false-negative result was probably caused by the time lag between the sample collection and testing, if porphobilinogen is estimated in urine after 10 min of voiding, a false-negative result could occur due to oxidation of porphobilinogen (Reio and Wetterberg 1969). Subclinical infection probably precipitated the repeated attacks. This stopped once the infection was treated empirically with antibiotics. In the hyperacute phase, the attacks were aborted by administration of intravenous hypertonic dextrose. Glucose prevents porphyrogenesis by blocking the induction of delta amino levulenic acid (A ALA) synthetase in the liver (Khanderia and Bhattacharya 1984). Intravenous hematin also has similar therapeutic effects (Pietach 1982). A high-carbohydrate diet has a prophylactic role in AIP, but is insufficient to treat acute attacks. Propranolol can be used to control the labile hypertension and tachycaralia in AIP (Hacks 1970). Low doses of antipsychotics are safe in AlP and hence psychotic features warrant their use. Chlorpromazine and trifluoperazine have been recommended in AIP (Goldberg et al 1983; Lishman 1987), however. Management of AlP includes strategies for prevention of attacks by avoiding a large number of drugs considered to be porphyrogenic. A high-carbohydrate diet is recommended prophylactically to prevent further episodes (Tshudy 1964). A detailed list of drugs contraindicated in the patient (Appendix I) was prepared and given to the patient, to be handed over to doctors before accepting any treatment. A very important cautior, in psychiatric practice is to refrain from using intravenous pentothal for either exploration, diagnostic clarification, or therapy in patients with probable AIP. Pentothal sodium is a very potent porphyrogenic drug that could be lethal if used in patients with AlP. The nature of the disorder imposes severe restrictions on the management of ordinary physical illness, as a large number of commonly used drugs are porphyrogenic. Seizures can be controlled in AIP with IV diazepam (Reynolds and Miska 1981). Abdominal pain should be managed with either paracetamol, morphine, or dihydrocodiene. Penicillins are safe in treating sepsis. Patients ought to be shifted to Intensive Care Units whenever neurological signs appear, as bulbar palsies are common in such patients. Four of the patient's siblings had died during infancy and early childhood, the cause of which is uncertain. Although

Varied Manifestations of Acute Intermittent Porphyria

nlot. PSVCtttA't~V

747

t 994",36:744-747

clinical manifestations of AlP axe rare before puberty, it remains a speculation whether these deaths were related to

the disease. Detailed pedigree studies of sach cases migt~ throw light on the vicissitudes ofthe disorder.

References

Ackner B, Cooper JE, Gray CH, Kelly M (I 962): Acute Porphyria. A neuropsychiatric and biochemical study. J Psvchosom Res 6: !-24. Barclay N (I 974): Acute intermittent porphyria in childhood. Arch Dis Child 49:404-406. Beauvais P, Klein ML, Denave L, Martel C (1976): Intermittent porphyria at four months of age. Arch Fr Pediatr 33:987-992. Bonkowsky HL, Schady W (l,e,2): Manifestations of acute porphyria. Semin Liver Dis 2:198- i 23. Boon FFL, Ellis C (1989): Acute intermittent porphyria in a children's psychiatric hospital. JAm Acad Child Adolesc Psychiatry 28(4):606--(309. Chisolm JJ (1987): Defects in heine pigment metabolism. In Betman RE, Vaughan VC (eds.), Nelson Textbook of Pediatrics i 3th ed. Philadelphia: Saunders, pp 35 !-357. Hacks LM (1970): Propranolol in acute porphyria. Lancet i: 363. Goldberg A (1959): Acute intermittent porphyria: A study of 50 cases. Q J Med 28,183-209. Goldberg A (1968): Diagnosis and treatment of the porphyria. Proc Roy Soc Med61:193-196. Goldberg A, Moore MR, McColl KEL, Brodie MJ (1983): Prophyrin metabolism and the porphyria. In Weatherall DJ, Ledingham JG, Warreil DA (eds). Oxford Textbook of Medichw, Vol. 1. Oxford: Oxford University Press, pp 98 i-989.

Khanderia U, Bhattacharya A (1984): Acute in,~ermittent potphyria: Pathophysio!ogy and treatrnem. Pha~cotherapy 4{3):144-150. Kreimir-Birnbaum M. Bannerman R {1975): Acute intermittent porphyria in childhood: a neglected diagnosis. Arch Dis Child 50:49~ -*.95. Lishman WA {1987): Acute porphyria, in Organic Psychiato': The Ps).'chological Consequences of Cerebral Disorder. 2nd ed. London: Blackwell Scientific. pp 482-485. Pietach CA {1982): Hematin therapy for the porphyric attack. Semin Liver Dis 2:12.$--131. Reio L, Wetterberg L (1969): False porphobilinogen reactions in the urine of mental patients. JAMA 207:148-150. Reynolds AC, Miska RM (1981): Safety of anticonvulsants in hepatic porphyria. Neurolog).' 31:480-483. Stein JA. Tschudy DP (1970): Acute intermittent porphyria. A clinical and biochemical study of 46 patients. Medicine 49: i-16. Tishler PV, Woodward B, O'Conner Jet al ( 1985): High prevalence of intermittent acute porphyria in a psychiatric patient population. Am J P~,chiatr)" 142:1430-1436. Tshudy DP, Wetland FH, Collins A, et al (1964): The effect of carbohydrate feeding on the inductors of aminolevulinic acid synthetase. Metabolism ! 3:396-406.

Effects of Treatment with Imipramine and Clonazepam on an Animal Model of Panic Disorder Anantha Shekhar

Tonic GABAergic inhibition in the dorsomedial hypothalamus (DMH) has been shown to regulate a constellation of behavioral and physiological responses that resemble a human panic attack. The present stud), was aimed at testing if the panic-like response elicited by injecting gamma-aminobutyric acid (GABA) antagonists into the DMH can be blocked by pretreating the animals with the antipanic drugs, imipramine and clonazepam. Rats were fitted with arterial catheters and bilateral chronic microinjection cannulae in the DMH. Their baseline heart rate, respiratory rate, blood pressure rout "'anxiety" (measured by the social interaction test) responses to injection of the GABAa antagonist bicuculline methiodide (BMI) into the DMH were recorded. After treatment in a double-blind manner with vehicle, imipramb~e (5 and 15 mg/kg, 7 days), and clonazepam (5 mg/kg, 3 days), the response to BMI microinjection into the DMH was once again recorded. Both imipramine and clonazepam, but not vehicle treatments blocked the BAIl response. Key Words: Gamma-aminobutyfic acid, dorsomedial hypothalamus, heart rate, blood pressure, social interaction, anxiety

Introduction Blocking gamma-aminobutyfic acid (GABA^) neurotransmission in the dorsomedial hypothalamus (DMH) has been shown to elicit sudden, dramatic increases in heart rate (HR) and respiratory rate (RR) and smaller increases in blood pressure (BP) in anesthetized (DiMicco and Abshire 1987) and conscious (Wible et al 1989) rats. Similar physiological responses have also been obtained with injections of excitatory amino acids into the DMH of rats (Soltis and DiMicco 1990). We have shown in behavioral studies with conscious

From the Departments of Psychiatry, Pharmacology and Toxicology, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, IN. Address reprint requests to Anantha Shekhar, MD, PhD, Department of Psychiatry, 791 Union Drive, liaR Indiana University Medical Center, Indianapolis, IN 46202. Received August 30, 1993; revised March 15, 1994.

© 1994 Society of Biological Psychiatry

animals that GABA blockade in the cardiostimulatory region of the DMH elicits not only increases in HR, BP, and RR but also "escape"-oriented locomotion (Shekhar and DiMicco 1987), a selective enhancement of "fear" or "avoidance" responses without affecting nonaversive "approach" responding (Shekhar et al 1987) and an increase in experimental anxiety as measured in the "conflict" (punishment-induced suppression of lever presses for sweetened milk; Shekhar et al 1990), elevated plus-maze (Shekhar 1993) and social interaction (SI) tests (Shekhar and Katner 1994). In addition to the respiratory, and cardiovascular activation as well as anxiogenic-like effects, GABA^ blockade in the DMH also elicits increases in plasma catecholamines (Wible et al 1989), plasma adrenocorticotropic hormone (ACTH) (DiMicco et al 1992) and plasma corticosterone (Keim and Shekhar, unpublished data). All of these data suggest that the GABA neurons in the dorsome0006-3223/94/$07.00

Animal Model of Panic

dial hypothalamus regulate a constellation of physiological and behavioral responses associated with intense anxiety states similar to human panic attacks. Based on this phenomenologicai similarity and the fact that medial hypothalamic structures have long been implicated in the regulation of the "fight-or-flight" response (Hilton 1982), we have proposed that GABA^ blockade in the DMH of rats could be a potential model of human panic disorder. More recently, we have shown that chronic subthreshold GABA dysfunction in the DMH of rats makes them susceptible to physiological arousal with intravenous sodium lactate infusions (Shekhar et al 1993). Demonstrating that the physiological arousal and anxiogenic effects of GABA^ blockade in the DMH can be blocked by clinically effective antipanic drugs, would further strengthen the similarities to panic attacks. Therefore, the aim of the present study was to test if the response elicited by hypothalamic GABA^ receptor blockade would show successful attenuation with clinically effective antipanic medications, imipramine (Kline 1964; Lydiard 1988) and cionazepam (Tesar et al 1987). The effects of chronic treatments with vehicle, imipramine, and clonazepam on the increases in HR, BP, RR, and the decreases in SI elicited by injecting the GABA^ antagonist bicuculline methiodide (BMI) into the DMH of rats were studied.

t i m PSVOUATRY

749

polyethylene (PE 50) tubing. The whole system was filled with BMI solution (50 pmole/250 nl) ~ the syringes were placed on a Sage pump set to deliver 250 nl in 39 sec. T ~ internal cannula along with the guide cannula was lowered into the DMH at an angle of 106 from the vertical plane the skull coordinates with respect to bregma being 1.2 mm posterior, 1.7 nun lateral, and 8.5-9.0 mm ventral. At this site, 50 pmoles of BM! in 250 nl volume was injected over 30 sec and the HR response was noted. An increase in HR of at least 50 beaffmin was the criterion used to define a reactive site. If microinjection at these coordinates did not y;~eld a reactive site, the same process was repeated after repositioning the cannula 0.2 mm medial, lateral, anterior or posterior to the above coordinates until a reactive site wus found. After the HR had returned to baseline, the opposi~ cannula was placed at a reactive site following the same procedure. After placing both cannulae at the desired sites, the guide cannulae were fixed at these positions with steel screws anchored to the skull and cranioplastic cement (all supplies obtained from Plastic Products, Roanoke, VA). The injection cannulae were then removed ~ the guide cannulae were sealed with steel wire dummy cannulae. The rats were allowed to recover in individual cages with ad lib water and food.

Experimental Protocol Methods and Materials

Implantation of Microinjection Cannulae b~the Dorsomedial Hypothalamus Experiments were conducted with male Sprague-Dawley (275-325 g) rats that were individually housed and allowed free access to food and water. Cannulae were implanted bilaterally in the cardiostimulatory region of the DMH using a technique routinely employed in our laboratory (Shekhar et al 1990). Rats were anesthetized with pentobarbital (50 mg/kg, IP) and arterial catheters were inserted into the femoral artery.. The catheters were made of 5 cm of 0.01 in tygoa : ~ c fused with cyciohexanone inside 30 cm of 0.02 in tygon tubing (Sanders and Shekhar 1991). The catheters were filled with heparinized saline (25 units/ml) and routed subcutaneously to the dorsal aspect of the neck where they were stabilized using a leather jacket. The animal was then fixed into a stereotaxic instrument (David Kopf Instruments, Tujunga, CA) with the incisor bar at a +5 ° angle from the horizontal plane. The arterial catheter was connected via a pressure transducer to a Dynograph (Sensormedics R511) and BP and HR were measured throughout the surgery. Two stainless steel injection cannulae (33 gauge, 12 mm length) were placed within guide cannulae (26 gauge, 10 mm length) mounted on to the stereotaxic apparatus on either side and connected to l0 ul Hamilton syringes by

After at least 3 days of recovery, the femoral arterial catheter of the conscious rat was connected to the Dynograph in order to measure HR and BP. After a 5-rain baseline heart rate and blood pressure recording, microinjection cannulae were inserted into the guide cannulae. Rats received a bilateral 250 nl in intracranial (IC) microinjection of BMI (20 pmoles). One minute after completion of injection, the injection cannulae were withdrawn. Heart rate and BP were monitored for a lO-min period from the time of injection in their home cages and any changes in relation to baseline were noted. At the end of the 10-min period, the animals in the clonazepam and the imipramine 5 mg/kg series were placed in the social interaction test (File 1980), an ethologically based test of experimental anxiety in rats (Lister 1990). The apparatus itself consisted of a solid wooden box with an open roof 36" long by 36" wide with walls 12" high. A video camera was fixed above the box and all behavioral tests were videotaped. The "experimental" rat and an unfamiliar "partner" rat were both placed in the center of the box and allowed to freely interact for a period of 5 min. The number of seconds of nonaggressive physical contact (grooming, sniffing, crawling over and under, etc.) initiated by the "experimental" rat was then counted. The actual time in seconds that the "experimenta W''rat spends, ;,nteracting with the "partner" rat over the 5-min period was measured as the SI time. The SI time has been shown to be a reliable measure of experimental anxiety in rats and anxiolytic drugs such as

"/50

BIOL PSYCHIATRY 1~;36:748-758

diazepam increase SI time, whereas anxiogenic drugs decrease SI time. Sessions were scored at a later time by two raters of whom at least one was blind to any drug treatment. Reliability for the time of social interaction was found to be excellent for the two scorers (r = 0.97). In another series of rats, the BMI injections were given tu~er light pentobarbital anesthesia (20 mg/kg) and the increases in RR elicited by BMI were recorded. These animals were not tested in SI and were treated with imipramine (15 mg/kg ~ l y ) and re~estedwith BMI 7 days later for RR changes. Once the baseline responses to BMI were recorded, the rats were randomly divided into either vehicle- or drug-treatment groups. Imipramine, a tricyclic antidepressant (Kline 1964; Lydiard 1988) and clonazepam, a benzodiazepine (Tesar et al 1987), two drugs that are well documented as effective antipanic drugs but belong to two completely different classes of compounds were selected for this study. In the imipramine series, there were three sets of treatment groups: (l) vehicle (n = 4) and imipramine 15 mg/kg (n = 6) for HR and BP measurements; (2) vehicle (n = 4) and imipramine 15 mg/kg (n = 4) for RR measurements under light anesthesia; (3) vehicle (n = 5) and imipramine 5 mg/kg (n = 5) for HR, BP, and SI measurements. All vehicle and imipramine treatments were given as single, daily IP injections in the evening for 7 days and the experimenters were blinded to the type of drug treatment given to each rat. The cionazepam series had vehicle (n = 5) and clonazepam 5 mg/kg (n = 5) treatments given as single, daily evening IP injections for 3 days. After completion of the treatment periods (7 days for the imipramine and 3 days for the clonazepam groups), the HR, BP responses to microinjection of BMI 20 pmolesJ250 nl into the DMH of rats were recorded the next day. The SI responses were recorded only for the clonazepam and imipramine 5 mg/kg series. Three days after stopping the drug treatments, the HR, BP, and SI responses to injection of BMI into the DMH of rats was once again recorded to check for the return of the reaction. After completion of the experiments, rats were injected IP with a lethal dose of pentobarbital. Microinjections sites were marked by injection of 250 nl of 50% India ink. The brains were then removed, fixed in 10% neutral buffered formalin and later sectioned into 50 um slices and stained with neutral red. The exact site of injection was then determined by comparing the sections with the atlas of Paxinos and Watson (1986). All data were expressed as mean and SEM. Statistical analyses were done by using either analysis of variance (ANOVA) coupled with Student NewmanKeul's test, repeated measure ANOVA coupled with the least mean-square test or paired t-test. Results Microinjection of 50 pmoles of BMI into the DMH of rats elicits a significant increase in HR under pentobarbital an-

A. Shekhar

esthesia (78 +_ 5 and 82 _ 6 beats/rain on the right and left sides, respectively). These changes begin immediately after starting the microinjection and the peak effects are seen in ! 0 min or less on both sides. The results of the histological confirmation of the injection sites are represented in Figure I. All the reactive sites that elicited increases in HR with BM! injection are within the region of the DMH as defined by the atlas of Paxinos and Watson (1986).

Effects of lmipramine Treatment Figure 2(A) summarizes the effects of chronic treatment with imipramine on the HR response elicited by BMI injection into the DMH of rats. During baseline measurements, BMI injections into the DMH elicit a significant increase in HR as ~-.~hpared to vehicle (saline) injections in all three chronic IP drug-treatment groups. After 7 days of treatment, both imipramine groups but not the vehicle group showed significant suppression of the BMI-induced increases in HR [Figure 2(A), day 7 BMI]. Three days after stopping the IP drug treatments [Figure 2(A), day 10 BMI], the placebo group continued to show increases in HR, whereas the imipramine 5 mg/kg group was beginning to show recovery from the suppression of the HR response to BMI injection. The effects of imipramine treatment on the BP responses elicited by BMI injection into the DMH are shown in Figure 2(B). At baseline, BMI injections into the DMH once again elicit a significant increase in BP in all three groups as compared to saline injections. After 7 days of IP treatments, only the 15 mg/kg dose of imipramine significantly suppresses the BP response elicited by BMI injection, and this suppression of BP response is lost after stopping the treatments for 3 days [Figure 2(B), day 10 BMI]. In contrast to HR effects, only the higher dose of imipramine appears to suppress the BP response elicited by BMI injections into the II~KAL.I LP J,Vll 1.

Figure 3 presents a summary of the effects of imipramine treatments on the anxiogenic-like effects of BMI injection into the DMH of the rats placed in the social interaction test. Only the group of rats that received 5 mg/kg of imipramine and their vehicle controls were tested in the social interaction test as this behavioral measurement was begun after studies with the imipramine 15 mg/kg series were completed. At baseline, BMI injections into the DMH significantly decrease SI time in both groups suggesting an anxiogenic-like effect. After 7 days of treatment, imipramine, unlike vehicle, completely reverses this BMI induced suppression (Figure 3, day 7), indicating a blockade of the anxiogenic-like effects of the BMI injections. At day 10, after stopping the imipramine treatments for 3 days, BMIinduced suppression of SI is once again seen in both the treatment groups. The vehicle-treated rats show a continued

Animal M o d e l

of

Panic

SmL P S Y Q U A ~ y 1994;36:74~75~

I

.o °

1:

0

I0 Interaural

6.70 mm

Bregrna - 2.30 mm

o

,o [nteraura|

6.20 mm

Bregma - 2.80 mm

1

9

0

10 Interaural

5.86 mm

B r e g m a - 3 . 1 4 mrn

Interaural

5.70 mm

B r e g r n a - 3 . 3 0 rnrn

I

,

/

/ ( l k

°,ov.o

-: .

LH

1

9

0 --

'10 Interaural

5.40 rnm

Bregma - 3.60 mm

Interaural 4.84 mm

Bregma - 4.16 mm

0

••••••l•'••••••••••••••••••••••••••••••••••••••••••••••••••••l•••••••••••I••'•fI•

7

6

5

4

3

2

1

0

1

2

3

4

5

6

7

Figure I. Schematic representation of the results of histology showing the bilateral injection sites in the hypothalamus in 28 of the 30 rats used in this study. The sections from two rats could not be pr~essed for technical reasons. The sites that were reactive to injection of BMI (closed circles) by cliciting increases in HR under anesthesia were mostly in the region of the DMH. Brain sections are represented according to the atlas of Paxinos and Watson (1986). The distances from the interaural line and bregma are given under each section. The scales on the fight and left indicate the distances in millimeters form the ventral and dorsal surfaces. The scale at the bottom indicates the distance in rmllimeters lateral to the midline. DMH, dorsomedial hypothalamus; F, fornix; LH, lateral hypothalamus; mt, mamillothalamic tract; PH, posterior hypothalamus; V, 3rd ventricle; VMH, ventromedial hypothalamus.

?51

752

A. Shekhar

BIOLPSYCHIATRY 1994;36:748-758

All

1410" ,~

.

I"1 Vehicle (n=9) ~ Imipramine 5 mg/Kg (n=S) BE Imipramine 15 mg/Kg (n=6)

$

.E

= Yl .

BaselineSalineBaselineBMI Day 7 BMI Day 10 Bluff

I,.P. B=

~'

30-

~J o

E E

* "

=o ~

.

Q,

,,-/ 1/

0

/f 1J

0

/i

10

,-///'1

~//

//

U

"~'

[m

.,'/

BaselineSalineBaselineBldl Day 7 BMI Day 10 BMI I I.P. Treatment J * Significaatly different from baseline saline by re!)eated measure AhDVA, p
Figure 2. Effects of chronic treatment with vehicle and two doses of imipramine on the increases in (A) HR and (B) BP elicited by injecting BMI into the DMH of rats. Afte.r baseline HR measurement following saline and BMI injections, rats were treated with daily IP iajections of vehicle or im~pramine for 7 days. Day 10 measurements were taken after stoppwg drug treatments for 3 days. Data are presented as means + SEM.

anxiogenic-like effects of BMI injections throughout the experiment. Table l summarizes the effects of chronic imipramine treatments on the respiratory stimulation elicited by BMI injections into the DMH. At baseline, both groups show significant increases in RR after BMI injections into the DMH. After 7 days of imipramine and not vehicle treatments, these increases in RR are completely blocked.

Effects of Clonazepam Treatment In the clonazepam series, microinjection of BMI into the DMH once again elicits significant increases in HR as compared to saline injections at baseline [Figure 4(A)]. After 3 days of IP drug treatments, however, only the clonazepamtreated animals show a significant suppression of the HR response. Three days after stopping the drug treatments, the clonazepam induced blockade of the HR response elicited

Animal Model of Panic

emL ~'tCmA~ V 1994;,.~7'4g-7~

"0 140 c

O u G m

753

I"1 Vehicle ( n = 5 )

120

T

---

I~1 Imipramine 5 mg/Kg (n=5)

•=- loo

E

* /

80

T

e-

.o

~,

*

u m L_

40'

•~

20,

, m

*

.

~

]

U 0

BaselineSalineBaselineBMI Day 7 BMI Day 10 BMI [I.P. Treatments] • Significantly different from baseline saline by repeated measure ANOVA, p
by BMI injections into the DMH is no longer seen [Figure 4(A), day 6 BMI]. The vehicle treatments have no significant effect on the BMI response throughout the experiment. The effects of clonazepam treatment on the BP response elicited by BMI injection into the DMH are shown in Figure 4(B). The BP response to B M I at baseline is significantly different from saline. After 3 days of clonazepam treatment, the BMI response is not significantly different from saline injections at baseline [Figure 4(B)]. Figure 5 summarizes the results of clonazepam treatment on the anxiogenic-like effects of BMI injection into the DMH as measured by the SI test. At baseline-, all animals show a significant decrease in SI after BMI injec-

tions compared to saline injections into the DMH (Figure 5, baseline), suggesting increases in experimental anxiety with BMI injections. After 3 days of treatment, rats show a significant reversal of the SI suppression by BMI injections with only clonazepam and not vehicle (Figure 5, day 3). This reversal of BMI induced SI suppression by clonazepam is lost 3 days later after stopping the drug treatments (Figure 5, day 6). ll'~g. . . . . vg,~aoaa

g_,.,

The data presented above clearly suggests that chronic treatment with two effective but chemically unrelated anti-

Table 1. Effects of Chronic Imipramine Treatments on the Increases in the Respiratory Rate (RR) Elicited by BMI Injections (20 pmoles/side) into the DMH of Lightly Anesthetized Rats Pretreatment baseline Daily IP

D a y 7 o f treatment

Baseline R R (breaths/

Increases in R R (breaths/

Baseline R R (breaths/

Increases in .r,tR (brear~hs/

treatment

minute)

minute)

minute)

minute)

Vehicle (n = 4) I m i p r a m i n e 15 m g / k g (n = 4)

76 _ 9 81 ± ! 0

33 _ 7 ~ 29 _ 8 ~

84 ± 11 72 ± 8

30 __- 8 ~ 5+_3 ~

All data are presented as mean + SEM. ° Significantly different from baseline by paired t-test, p < 0.05. bSignificantly different from vehicle treatment by ANOVA coupled with Student Newman Keul's test. p < 0.05.

7~

A. Slltekhar

BIOL PSYCHIATRY 1~;36:748~758

All A

['1 Vehicle (n-S) J Clonazepam 5 mg/Kg (n=5)

t=

E m

100 er

#0 I

m

G .S

T

t

"

W

40

G ~t

20 U

BaselineSalineBasetinei~ll Day 3Bldl Day

ll:t,. T , ~ , ~ t Bill 2::

61~U

l

a0" Vr

o er

E E er

20

Ul I/) e~ o o

10

.S ItJ O m t~

¢J

o

_= * Significantly different from baseline saline by repeated measure ANOVA, p<0.05 # Significantly different from baseline BMI by repeated measure ANOVA, p<0.05

Figure 4. Effects of chronic treatment with vehicle and clonazepam on the increases in (A) HR and (B) BP elicited by injecting BMI into the DMH of rats. After baseline HR measurement following saline and BMI injections, rats were treated with daily IP injections of vehicle or clonazepam for 3 days. Day 6 measurements were taken after stopping drug treatments for 3 days. Data are presented as mean __.SEM. panic drugs, imipreanine and clonazepam, blocks the physiological and behavioral responses to GABAA blockade in the DMH of rats. The fact that the BMl-induced panic-like reactions were blocked effectively after 3 days of clonazepam treatment resembles the clinical situations with humans. Benzodiazepines like clonazepam (Tesar et al 1987) and alprazolam (Ballenger et al 1988) become rapidly effective in blocking panic attacks. Although response to

imipramine in patients with panic disorder usually takes at least 2 weeks or longer, the response to BMI injection into the DMH was blocked by I week of imipramine treatment. This may not be too far off from the situation in human panic attack. Much of the initial treatment period with imipramine in patients with panic disorder is spent in gradually building up to a therapeutic dose while the rats received maximal dosage from day I of treatment. The blocking of the panic-

Animal Model of Panic

emt. PsvooA~n~v 1994;36:74g-'/~

in qD c O (.t q) I/)

755

100 (n=S) zepam 5 mg/Kg (n=5) 80

C

. !

¢D

E

. m

60

4~ O 4~ CJ m Im

. s

0) 4J C

. m

40

20

I

t~ .,m, (J O (n

_8_8_8_8_8_8_8_8_8Saline ~___nie Baseline BMI Day 3 BMI

Day 6BM!

I.P. treatment[ * Significantly different from baseline saline by repeated measure ANOVA, p<0.05 # Significantly different from baseline BMI by repeated measure ANOVA, p
like response to BMI injections with both imipramine and clonazepam were beginning to be lost 3 days after stopping the treatments (Figures 2-5), suggesting that the DMH was still intact and the loss of response was not due to any mechanical damage. So far, we have only tested two effective anti-panic drug treatments to validate this response. To fully validate this model, however, we need to test other effective treatments such as monoamine oxidase inhibitors (Kelley et al 1971) and selective serotonin reuptake inhibitors like fluoxetine (Gorman et al 1987) as well as known ineffective treatments o.... I, (Sh,,~han ~,H h as U U.l .~ a.V.l . ~ a.v a;n,, a aa~.,.a • v~, J , ~t 1983). Another important aspect of the above results is that in the present experimental model treatment with imipramine has effects that are similar and comparable to clonazepam on the responses elicited by injecting BMI, a GABAA antagonist. This suggests a possible common mode of action for tricyclic antidepressants and benzodiazepines in this model. Some available data does support the interaction of tricyclic antidepressants with GABA neurotransmission. Chronic treatment with antidepressants is known to elicit significant increases in GABAB binding, a change that is proposed as a possible mechanism of action of diverse antidepressants (Lloyd 1985). Tricycfics are reported to block GABA uptake in vitro (Gottesfeld and Elliot 1971; Lloyd and Morselli 1987) and enhance release of GABA (Korf and Venema 1983; Petty 1986). In the learned helplessness model of

depression, bicuculline reverses the effects of imipramine suggesting a GABAergic mechanism for the effects of imipramine (Bartholoni et al 1986). All these data strongly implicate a GABAergic mechanism for the antidepressant effects of tricyclic antidepressants. The present findings are in agreement with these data and provide further evidence of an interaction between the GABA/benzodiazepine system and tdcyclic antidepressants. Although the reaction obtained by GABAAblockade in the DMH of rats is clearly a "fight-or-flight" response, p~mc attack~ in their phenomenolo~ ~ p e a r to be just such atavistic "fight-or-flight" responses that occur in humans. Therefore, the response elicited by stimulating the DMH of rats may be an essential survival reflex that is preserved across all mammalian species. In fact, some available human neurosurgical data does support such a hypothesis. Human hypothalamotomy involving the posterior hypothaiamic area caused a decrease in anxiety and was termed "sedative surgery" (Kuhar 1986). Electrical stimulation prior to lesioning of the posterior region of the hypothalamus under local anesthesia in humans as reported by Schvarcz (1977) and Sano et al (1975) consistently elicited an increase in heart rate, respiration, and fear or anxiety. In one of the studies (Schvarcz 1977), the increase in heart rate was actually used to localize the area for subsequent lesioning, much like we have used the heart rate response to localize our reactive sites.

756

A. Si,ekhar

BIOL PSYCHIATRY 1~',36:748-758

In order to fully support a role for DMH in panic responses, one would also have to show that (1) intense fearstates (such as presenting the rat with a cat) would activate this area, (2) blockade of DMH activity blocks the paniclike response, (3) DMH stimulation produces a terrorizing state that fully abolishes social interaction, and (4) DMH stimulation is so aversive as to elicit conditioned avoidance that is not easily extinguished. Although many such experiments have not yet been conducted, currently available data does provide some answers. We have shown that by subjecting rats to fear-potentiated startle, a model of acute anxiety, there is a dramatic three-to fourfold increase in the norepinephrine content in the DMH (Shekhar et al 1994), suggesting involvement of this region in fear responses. Blocking the DMH activity with muscimol injections in rats that are confined to the open arm of an elevated plus-maze, a situation that is extremely stressful to rats, not only ameliorates the behavioral effects but blocks the increases in HR, BP and plasma norepinephrine levels (Shekhar et al 1993a). Lesioning of the perifomical hypothalamus including the DMH blocks the physiological concomitants of emotional behavior in primates (Smith and DeVito 1984). If the dose of BMI injected into the DMH is increased to 50 pmoles or greater, we can elicit intense fear states and a flight response where the social interaction will be reduced to 0 sec. We have intentionally lowered the dose so that we can measure the social interaction effects of drug treatments. Finally, GABA^ receptor blockade in the DMH does enhance previously acquired avoidance responses (Shekhar et al 1987) although whether it will elicit conditioned avoidance by itself has not been tested so far. Two of the currently prevailing theories about the etiology of panic disorder are the respiratory center abnormality leading to suffocation false-alarms (Kline 1993) and the hyperactivity of the locus ceruleus (Redmond and Huang 1979). As data in Table l suggests, the DMH is intimately involved in the central regulation of respiration and could very well be associated with changes resulting in abnormal sensitivity of the medullary respiratory centers. Further, chronic GABA dysfunction in the DMH makes rats susceptible to physiologic arousal with intravenous lactate infusions, much like patients with panic disorder (Shekhar et al 1993b). The mechanism of lactate-induced panic in patients is presumed to be a respiratory center response (see Kline 1993) and GABA dysfunction in the DMH appears to induce a similar physiological hyperresponsivity in rats. The DMH also has close interaction with the locus ceruleus. Lesioning of the hypothalamus is known to decrease the physiological arousal elicited by stimulating the locus ceruleus (Przuntek and Philippu 1973). Fear responses dramatically increase the norepinep,hrine content of the DMH (Shekhar et al 1994) and lesioning the DMH with 6-OH dopamine reduces the physiological responses elicited by

stimulating the DMH (Shekhar and Katner, unpublished data, 1994). All these data indicate a close interaction between the DMH and the locus ceruleus. Thus the role of DMH in panic-like responses appears consistent with the current understanding of the pathophysiology of panic and may add another anatomical site toward completing an as yet poorly understood panic circuitry. The known neuroanatomicai connections of the DMH also supports its proposed key integrative role in anxiety and panic-like responses. The DMH has ascending afferents from spinal cord (Smith and DeVito 1984), locus ceruleus, raphe nuclei, nucleus of the solitary tract, periaqueductal grey (Swanson 1987), septum and bed nucleus of the stria terminalis (Conrad and Pfaff 1976). Within the hypothalamus, it receives large projections from paraventricular nucleus (Conrad and Pfaff 1976) and the lateral hypothalamus (Saper et al 1979). The amygdalar pathway responsible for autonomic arousal appears to project to the lateral hypothalamus (LeDoux et ai 1988), which has dense connections with the DMH. The DMH also has connections with the hippocampus and the subiculum (Swanson 1987). The DMH has efferent projections to a number of areas that regulate emotional and physiological responses. Within the hypothalamus, its major target is the paraventricular nucleus (Ter Horst and Luiten 1986), which is thought to be responsible for stress-related release of ACTH and sympathetic arousal (Saper et al 1976; Swanson 1987). The DMH also projects to the periaqueductal grey, nucleus tractus solitarius, subretrofacial nucleus, the nucleus ambiguus, and the intermediolateral column of the spinal cord. All these connections are essential to integrating a constellation of autonomic and behavioral responses associated with stress and anxiety. Therefore, the afferent connections of the DMH are such that it has a variety of sensory, limbic, and cognitive input although its efferent connections enable it to be a coordinated output center of stress responses. Indeed, the DMH has been described as a maior inteL~q'atorn f emntional and autonomic responses (Bernardis and Bellinger 1987; DiMicco et al 1992). In summary, GABAergic inhibition in the DMH appears to regulate a constellation of behavioral and physiological responses that resemble a human panic attack. In the present study we tested whether the increased HR, BP, and RR as well as the anxiogenic-like SI responses elicited by injecting the GABA^ antagonist BMI into the DMH can be blocked by pretreating the animals with antipanic drugs imipramine and clonazepam. Both imipramine and clonazepam, but not vehicle treatments, blocked the physiological and behavioral effects of inhibiting GABA function in the DMH of rats. . . . . . .

;.¢

.

.

.

.

.

.

.

o r -

.

.

.

.

.

.

.

.

.

.

.

This study was supported by PHS grant MH 45362. The author thanks Jason Katner, Lora Sims, Stan Keim, and William Rusche for technical assistance.

Animal Model of Panic

a~

PSYCHIATRY

757

|~;36:748~T58

References Ballenger JC, Burrows GD, DuPont R, et al ( 1988): Alprazolam in panic disorder and agoraphobia: Results from a multicenter trial, !: Efficacy in short-term treatment. Arch Gen P~chiato" 45:413-422. Bartholini G, Scatton B, Zivkovic B, Lloyd KG (1986): On the mode of antidepressant action of GABA receptor agonists and monoamine uptake inhibitors. In Bartholini G, Lloyd KG, Morselli PL (eds), GABA and Mood Disorders. New York: Raven Press, pp 105-11 I. Bernardis LL, Beilinger LL (1987): The dorsomedial hypothalamic nucleus revisited: An update. Brain ICesRev 12:321-38 I. Conrad LC, Pfaff D W ( 1976): Efferents from the medial forebrain and hypothalamus of the rat: An autoradiography of the anterior hypothalamus. J Comp Neurol ! 69:22 !-262. DiMicco JA, Abshire VM ( 1987): Evidence for GABAergic inhibition of a hypothalamic sympathoexcitatory mechanism in anesthetized rats. Brain Res 402: I-! O. DiMicco JA, Soltis RP. Anderson JJ. Wible JH Jr (1992): Hypothalamic mechanisms and the cardiovascular response to stress. In Kunos G, Ciriello J (eds), Central Neural Mechanisms in Cardiovascular Regulation, voi. 2, Boston: Birkhauser, pp 52-79. File SE (1980): The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide-like drugs. J Neurosci Meth 2:219-238. Gorman JM, Liebowitz MR, Fyer AJ et al (1987): An open trial oI fluoxetine in the treatment of panic attacks. J Clin P~,chopharmacol 7:329-332. Gottesfeld Z, Elliot KAC ( 1971 ): Factors that affect the binding and uptake of GABA by brain tissue. J Neurochem 18:683690. Hilton SM (1982): The defense arousal system and its relevance for circulatory and respiratory control. J Exp Biol 100:159174. Kelley D, Mitchell-Heggs J, Shean D (1971): Anxiety and the effects of sodium lactate assessed clinically and physiologically. BrJPsychiatry ! 19:129-141. Kline DF (1964): Delineation of two drug responsive anxiety syndromes. P@,chopharmacology 5:397-408. Kline DF (1993): False suffocation alarms, spontaneous panics, and related conditions: an integrative hypothesis. Arch Gen Psychiatry 50:306-317. Korf J, Venema K (1983): Desmethylimipramine enhances the release of endogenous GABA and other neurotransmitter amino acids from the rat thalamus. JNeurochem 40:946-950. Kuhar MJ (! 986): Neuroanatomical substrates of anxiety. Trends Neurosci 9:307-31 !. LeDoux JE, Iwata J, Cichetti P, Reis DJ ( 1988): Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear. J Neurosci 8:25172529. Lister RP (1990): Ethologically based animal models of anxiety disorders. Pharmacol Ther 46:32 i-340. Lloyd KG, Morselli PL (1987): Psychopharmacology of GABAergic drugs, In Meltzer HY (ed) Psychopharmacology, the

third generation of progress. New York: Raven Press, pp 183195. Lloyd KG, Thuret F:, Pilc A ( 1985): Upregulation of gamma-aminobutyric acid (GABA) B b i ~ i n g sites in rat frontal cortex: A common action of repeated administration of ~fferent classes of antidepressants and electroshock. J Pharmacol Er~p Ther 235:191-199. Lydiard BR ( 1988): P h ~ a c o l o g i c a l treatrnem of ~ i c disorder. Ps).'ch Annals 18:468--472. Paxinos G, Watson C ( 1986): The Rat Brain in Stereotaxic Coordinates. 2nd ed. New York: Academic Press. Petty W (1986): GABA mechanisms in learned helplessness, In Bartholini G, Lloyd KG, Morselli PL (eds). GABA and Mood Disorders. New York: Raven Press, pp 61--66. Przuntek H, Philippu A (1973): Reduced pressor responses to stimulation of the locus coeruteus after lesion of the posterior hypothalamus. Naunyn-Schmiedeberg's Arch Pharmacol 276(2):119-122. Redmond Jr DE, Huang ~ (1979): Current concepts. 11. New evidence for a locus coeruleus-norepinephfine connection with anxiety. Life Sci 25(26):2149-2162. Sanders SK, Shekhar A (1991): Blockade of GABA^ receptors in the re,on of the anterior basolateral anaygdalaof rats elicits increases in heart rate and blood pressure. Brain Res 576:I01-110. Sano K, Mayanagi Y, Hiroaki S, Motohide O, Ishijima B ( 1970): Results of stimulation and destruction of the posterior hypothalamus in man. J Neurosurg 33:689-707. Saper CB, Loewy AD, Swanson LW, Cowan WM (1976): Direct hypothalamo-autonomic connections. Brain Res I 17:305-312. Saper C B, Swanson L W, Cowan W M (1979): An autoradiographic study of the efferent connections of the lateral hypothalamic area of the rat. J Comp Neurol 183:707-720. Schvarcz JR ( i 977): Results of stimulation and destruction of the posterior hypothalamus: a long term evaluation. In Sweet WH, Obrador S, Martin-Rodriguez JG (eds), Neurosurgical Treatments in Psychiatry, Pain and Epilepsy. Baltimore: University Park Press, pp 429-438. Sheehan DV, Bao BCH, Davidson JD, et al (1983): Lack of efficacy of a new antidepressant (bupropion) in the treatment of panic disorder with phobias. J Clin Psychopharmacol 3:28-31. Shekhar A ( 1993): GABA receptors in the dorsomedial hypothalamus of rats regulate experimental anxiety in the elevated plusmaze test: I. Behavioral measures. Brain Res 627:9-16. Shekhar A, DiMicco JA ( 1987): Defense reaction elicited by injection of GABA antagonists and synthesis inhibitors into the posterior hypothalamus m r~ts. Neuropharmacology 26:407417. Shekhar A, Katner JS (1994): Dorsomedial hypothalamic GABA regulates anxiety in the social interaction test. Pharmacol Biochem Behav (in press). Shekhar A, Hingtgen JN, DiMicco JA ( 1987): Selective enhancement of shock avoidance responding elicited by GABA blockade in the posterior hypothalamus of rats. Brain Res 420:118128. Shekhar A, Hingtgen JN, DiMicco JA (1990): GABA receptors in

758

BIOLPSYCHIATRY 1994;~:748-758

Shekhar A, Keim SR, Simon JR ( !993b): Sodium lactate infusions provoke physiological arousal in an animal model of panic disorder. Soc Neurosci Abstr 19:7. Shekhar A, Kamer JS, Rusche WP, Sajdyk TS, Simon JR (1994): Fear-potentiat~:i startle elevates catecholamine levels in the dorsomedial hypothalamus of rats. Pharmacol Biochem Behav 48:525-529. Smith OA, DeVito JL (1984): Central neural integration for the control of autonomic responses associated with emotion, Annu Rev Neurosci 7:43--66. Soltis RP, DiMicco JA (1991): GABAAand excitatory amino acid receptors in dorsomedial hypothalamus and heart rate in rats. Am J Physio1260:R 13-R20. Swanson LW (1987): The hypothalamus, in Bjorklund A, Hokfelt T, Swanson LW (eds), Handbook of Chemical Neuroanatomy, vol 5. Amsterdam: Elsevier, pp 1-104.

A. Shekhar

Ter Horst GJ, Luiten PGM (1986): Projections of the dorsomedial hypothalamic nucleus of the rat. Brain Res Bull 16:231-248. Tesar GE, Rosenbaum JF, Pollack MH, et al (1987): Clonazepam versus alprazolam in the treatment of panic disorder:. Interim analysis of data from prospective, double-blind, placebo-controlled trial. J Clin Psychiatry 48(suppl): 16-19. Wible JH Jr, Luft FC, DiMicco JA (1988): Hypothalamic GABA suppresses sympathetic outflow to the cardiovascular system. Am J Physio1254:R680-R687. Wible JH Jr, Luft FC, DiMicco JA (1989): Hypothalamic GABA and sympathetic regulation in the spontaneously hypertensive rats. Hypertension 14:623--628.