CSF diazepam-binding inhibitor concentrations in panic disorder

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CSF Diazepam-Binding Inhibitor Concentrations in Panic Disorder Richard Payeur, R. Bruce Lydiard, James C. Ballenger, Michele T. Laraia, Mark D. Fossey, and Joseph Zealberg

Diazepam-binding inhibitor (DBI) is a neuropeptide that has been detected in the brain and cerebrospinal fluid (CSF). Previous studies have suggested the possible role of DBi as a potential endogenous anxiogenic ligand modulating GABAergic transmission at the benzodiazepine.GABA receptor complex. The measurement of DB! immunoreactivity (DBIIR) in CSF of panic-disorder patients and normal controls was undertaken to assess whether there were differences in the CgF concentration of this peptide to assess possible relationships with other monoamines and peptides, Lumbar CSF was obtained from 18 panic patients (4 men, 14 women) and 9 controls (5 men, 4 women). As a group, no significant differences were found between panic patients' CSF concentration of DBi-IR (1.12 +. 0.27 pmol/mL) and normal volunteers (1.23 +_ 0.27 pmol/mL). No gender differences were demonstrated. However, we did find a positive correlation between CgF levels of DB! and CSF corticotro~,in releasing hormone (CRH) in our panic p,,,ients.

Introduction Panic disorder is a potentially disabling anxiety disorder characterized by sudden onset of intense anxiety or fear associated with symptoms such as palpitations, shortness of breath, chest pain, and other autonomic symptoms (American Psychiatric Association 1987). Panic attacks can be blocked by a variety of pharmacological agents, including benzodiazepines (BZD) (Ballenger et al 1988). The hypothesized role of the GABAergic system in the pathophysiology of panic attacks is suggested by the antipanic effects of BZDs but is not yet clear. Receptors for gamma-aminobutyric-acid (GABA) are found throughout the central nervous system (CNS) and generally exert an inhibitory influence on neuronal firing via their action on chloride channels (Hamill et al 1983). GABA receptors also contain a benzodi~epine-binding site that enhances the potency of neuronal inhibition when a benzodiazepine agonist binds to these receptors (Study and Barker 1982). It is believed that inhibition of the noradrenergic neurons, particularly in the locus cocruleus, may conucibut¢ to the antipanic effects of BZDs (Charley et ~1 1986; Grant et al 1980). Other ligands can also bind to GABA receptors such as the inverse-agonist methyl-B-carboline-3-carboxylate (B-CCE), which elicits an attenuation of social inter-

Froln the Medical University of South Carolina: P,cpartment of Psychiatry. Charleston, South Carolina 29425. A&lress reprint requests to Dr, R, Bruce Lydiard, Department of Psychiatry. Medical University of South Carolina, 171 Ashley Ave,, Charleston. SC 29425. Received November 25, 1991; ~evi~.d A~ri! ]9, I~2. © 1992 Society of Biological Psyc,hiatry

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actions. This effect can be reversed by the BZD receptor antagonist flumazenil (RO151788), in rats (File 1982). All of these compounds are man-made and researchers have been looking for an endogenous ligand corresponding to the BZD-binding site. In 1983 Guidotti and colleagues described diazepam-binding inhibitor (DBI), a ligand that displaced diazepam at the BZD-beta carboline receptor site and caused an anxiogenic effect in rats (Guidotti et al 1983). DBI was subsequently found in humans (Ferrero et al 1986). In rats, DBI inhibits the binding of flunitrazepam and B-CCE to the BZD-GABA receptors. This action is prevented by the benzodiazepine-antagonist flumazenil RO15-1788 (File 1990). DBI is a peptide consisting of 104 amino acids in humans, and exhibits a high affinity to BZD-beta carboline binding sites. This anxiogenic ligand appears to cause downregulation of the GABA^ receptor complex in animals and man (Barbaccia et al 1986; Ferrero et al 1986). In studies utilizing antibodies against DBI, DBl-like immunoreactivity (DBI-IR) was found in high concentrations in cortical and limbic areas, cerebellum, and brainstem of rats and humans (Ferrarese et al 1989). Many of these areas are believed to be involved in mediating fear, arousal, and panic anxiety (Ferrero et al 1986). Only two previous studies examined the potential relationship between DBI and anxiety in humans. Roy et al measured DBI-IR in the cerebrospinal fluid (CSF) of alcoholics and found no significant correlation between CSF DBI and the anxiety rating subscore of the Hamilton Rating Scale for Depression (HRSD) (Roy et al 198%). George and colleagues found no significant difference in CSF concentration of DBI in patients with panic disorder and alcohol dependence compared to alcoholics free of panic attacks or controls (George et al 1990). The possible role of HPA axis dysregulation in anxiety has been reviewed recently (Gold et al 1990). Roy and co!leagues reported a positive correlation between CSF concentrations of DBI and CRF in depressed p.~.tients, advancing the hypothesis that DBFs action on GABA^ receptors might play a role in the release of CRH and the activation of the HPA axis response to stress (Roy et al 19891)). In vitro administration of GABA or BZD on hypothalamic cells inhibits the secretion of CRH (Calogero et al 1988). To date, no studies have examined DBI function in patients with primary anxiety disorders. In a sample of panic patients and normal volunteers, we measured DBI-IR in CSF to further assess the possible role of DBI in pathological anxiety.

Methods Eighteen patients (4 men, 14 women), were diagnosed via Structured Clinical Interview for DSM-III (SCID) (Spitzer and Williams 1982) and met DSM-III criteria for panic disorder or agoraphobia with panic attacks. Patients with major depression were included only if panic disorder symptoms preceded the onset of major depression and depression was not the principal diagnosis. Five patients admitted to occasional (but not daily) use of a benzodiazepine between 2 and 7 days prior to the lumbar puncture; the remaining s~bjects were drug free for at le~.~l 2 w~.e~s. Nine normal controls (5 men, 4 women) were found to be free of any Axis I psychiatric diagnosis via administration of the SCID. Additionally, no first-degree relative of normal controls h~:d a psychiatric diagnosis per patient history. All subjects were admitted to the Clinical Research Center of the Medical University of South Carolina overnight prior to the lumbar puncture procedure. Subjects had beer: maintained on a low-monoamine diet for the 4 previous days, had at least 9 hr of bed rest ano were fasting overnight prior to the lumbar puncture. Lumbar CSF was obtained between 8 AM and 10 AM in the lateral de~.ubitus position. Samples from an

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2.0 1.8 1.6 1.4 CSFOBI-IR 1.2 pMoles/ml 1.0 0.8 0.6 0.4 0.2 0

e e Q

B

3r

Q @

Psnl¢

Controls

n. 18

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Figure 1, CSF DBI levels in panic disorder patients and controls.

aliquot of the ! 5th to 26th cc of CSF were immediately frozen on dry ice and stored at -70°C until assay. ,'I~.e radioimmunoassay procedure was parformed in the laboratory of A. Guidotti, PhD, Fidia Georgetown Institute for the Neurosciences. The assay used human DBI (H-DBI) labeled with iodine 125 bound to antiserum directed toward H-DBI (Barbaccia et al 1986). Corticotropin-releasing hormone was also measured in these CSF samples (Fossey et al 1990). Several rating scales were used in this study. For the week preceding the procedure, patients were asked to record the frequency and type of panic attacks and to complete the global phobia scale. In the evening before the lumbar puncture the following ratings were aaministercd: Beck depression scale, HRSD (21 items) (Hamilton 1960), Hamilton Rating Scale for Anxiety (HRSA) (14 items) (Hamilton 1969), Sheehan Clinician Rated Anxiety, SCL-90 (Derogatis et al 1973), and the Multiple Affect Adjective Check List (MAACL). On the morning of the lumbar ~uncture the HRSA and MAACL we:e repe,~:ed prior to the procedure. Groups were compared by analysis of variance (ANOVA). Correlation analysis were performed via Pearson's correlation.

Results The mean age of panic patients (n = 18) was 36.5 __. I0.0 years and of normal controls (n -- 9) was 28.6 *- 5.'/years. The distribution of H-DBI concentrations in the CSF of the 18 patients and 9 controls is shown in Figure I, The values were normally distributed in both the control and patient groups, No significant differences were observed between males and females within or between groups, No significant differences were observed in the CSF concentrations of the pamc disorder patients (I. 12 + 0,2'/pmol/mL) and controls (1.23 - 0.27 pmol/mL) according to an ANOVA. We found a positive correlation between CSF levels of DBI and CSF CRH in our panic disorder patients (r = 0.54, p < 0.03, n --- 17), but not in normal controls (r = 0.16, p < 0.6/(NS), n -- 9). Four patients in the panic group had a Hamilton depression score ~> 18. These four patients do not differ in DBI-IR levels from those with a Hamilton depression score < 18 (p = 0.40). Also, the five patients who used a benzodiazepi.-.e in the week before the lumbar puncture are not significantly different in DBI levels than those drug-bee for more than 2 weeks (p = 0.64). No correlation between DBI and

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relevant clinical variables (Total Hamilton Anxiety, Total Hamilton Depression, Global Phebia, and panic attack frequency) in the prior week was discerned. Discussion Because of its potential role as an endogenous anxiogenic peptide, DBI, in the panic group, might have been expected to differ from normal controls. However, there were no detectable differences found. We did find a correlation between DBl-like immunoreactivity and CSF concentrations of CRH in panic patients but not in contrvis. CSF levels of CRH have been reported to be increased in depressed patients but not in panic patients (George et el 1990; Roy et al 1989). Our findings in panic patients agree with the findings of Roy et al that CSF DBI is positivel~•correlated with CSF CRH (Roy et al 1989b). There are several p~ "sible reasons we failed to find any group differences in DBI-like immunoreactivity. It may be that there were no significant differences or that we failed to observe differences because of the small sample size. Also it may be that the pattern of DBI secretion in panic-disordered patients is different than normals, and our method did not detect this. For example, CSF DBI was reported to be elevated in depressed patients in whom a more chronic activation of CRH neurons is speculated to occur (George et al 1990); abnormal secretion patterns of CRH have been postulated to be more brief and intermittent in panic disorder (Gold et al 1990). If DBI does modulate CRH secretion we might expect more episodic increases in CSF DBI in panic disorder as well (Calogero et al 1988). Also, the CNS origin of CSF DBI measured is unknown. It is unclear whether there is a peripheral source of DBI that contributes to CSF levels as well. It might be that a small but critical pool of DBl-containing neurons in the CNS functions abnormally but total CSF DBI measures are not significantly affected. Our DBI measure was a single determination taken between panic episodes. If DBI secretion is abnormal in panic, it might also be episodic and therefore not reflected by our method. Alternatively, biologically active subunit.s of DBI exist (Costa and Guidotti 1990); it may be that differences in CNS activity of one of these subunits exists, but was not detected. Finally, there may not be abnormally high levels of DBI in patients with panic disorder. The recent report by Nutt et al which indicated that flumazenil, a BZD antagonist, was anxiogenic in panic.disorder patients argues against overproduction of an endogenous inverse-agonist in panic-disorder patients (Nutt et al 1990). Yet, indirect effects of DBI on steroid synthesis pathways, a pathway not antagonized by flumazenil, may in turn modulate GABA function (Costa and Guidotti 1991), were not assessed in this study. In summary, this is the first report of CSF DBI in panic disorder patients without other Axis 1 diagnoses. Although there was no difference in CSF DBI levels between these patients and controls, a positive correlation between CSF DBI and CSF CRH suggests a possible influence of DBI in the regulation of the HPA axis. The different biological actions of this interesting molecule remain to be explored. This research was supportedin l~artby NIH GrantM01RR001070.

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