Positron emission tomographic studies of cerebral benzodiazepine-receptor binding in chronic alcoholics

l'ositron Emssron 1 omograpmc - - P n 1 1 n 1. a n

of terebral benzo&azepme-Keceptor

Sid Gilman, MD,* Robert A. Koeppe, PhD,? Kenneth Adams, PhD,fS Douglas Johnson-Greene, PhD$ Larry Junck, MD,* Karen J. Kluin, MS,*S James Brunberg, MD," Susan Martorello, MS,*

and Mary Lohman, BA*

Positron emission tomography was used with ["C]flumazenil (FMZ) and ['8F]fluorodeoxyglucose to study GABA type Aibenzodiazepine (GABA-NBDZ) receptors and cerebral metabolic rates for glucose (1CMRglc) in 17 male patients with severe chronic alcoholism (ALC), 8 with (ACD) and 9 without alcoholic cerebellar degeneration (non-ACD). In comparison with male normal controls of similar ages, the ALC group had significantly reduced FMZ ligand influx ( K ) , FMZ distribution volume (DV), and ICMRglc bilaterally in the medial frontal lobes, including superior frontal gyrus and rostral cingulate gyrus; the ACD group had significant reductions of K,, DV, and lCMRglc bilaterally in the same distribution, and also in the superior cerebellar vermis; and the non-ACD group had significant reductions of K,, DV, and ICMRgIc bilaterally in the same regions of the frontal lobes but not in the superior cerebellar vermis. When compared with the non-ACD group, the ACD group had significant reductions of and DVbilaterally in the superior cerebellar vermis. The results suggest that severe chronic alcoholism damages neurons containing GABA-NBDZ receptors in the superior medial aspects of the frontal lobes, and in patients with clinical signs of ACD, neurons containing GABA-NBDZ receptors in the superior cerebellar vermis.

Gilman S, Koeppe RA, Adams K, Johnson-Greene D, Junck L, Kluin KJ, Brunberg J, Martorello S, Lohman M. Positron emission tomographic studies of cerebral benzodiazepine-receptor

binding in chronic alcoholics. Ann Neurol 1996;40: 163- 171

Excessive and prolonged alcohol intake can result in extensive neuronal cell loss in the human cerebral cortex [ 1-31. Generalized cerebral atrophy, particularly of the frontal lobes, has been recognized in chronic alcoholics for many years [I] , and recent studies reveal reduced numbers of neurons in the superior frontal region of the cerebral cortex in chronically alcohol- dependent subjects compared with control subjects of similar age and sex [ 3 ] . The neuronal loss appears to be site-specific since no significant decrease is found in the motor cortex [3], anterior cingulate or middle temporal gyri [4], although neurons in the superior frontal, cingulate, and motor areas are shrunken [4]. The dendritic arbors of layer I11 pyramidal neurons in the superior frontal and motor areas are reduced in chronically alcohol-dependent patients compared with controls [ 5 ] . The number of large neurons in the superior frontal cortex is reduced, and the number of large neurons in the motor cortex is unchanged [6]. The

number of small neurons in the frontal cortex is also unchanged, probably because the cellular loss affects only large neurons [6, 71. These findings support the notion of greater vulnerability of neurons in the frontal cortex in alcohol-related cerebral injury and are consistent with demonstrated focal cerebral atrophy on anatomical imaging [S] , decreased local cerebral metabolic rates for glucose (1CMRglc) in the medial portions of the frontal lobes in positron emission tomography (PET) studies with ['8F]fluorodeoxyglucose (FDG) [9], and abnormalities in neuropsychological tests that are widely thought to reflect frontal lobe dysfunction D O ] .

y-Aminobutyric acid (GABA) is an abundant and important inhibitory neurotransmitter in the central nervous system [11, 121. Two types of receptors medi- ate GABAergic neurotransmission, GABA-A and GABA-B. The GABA-A receptor is an oligomeric, inte- gral membrane glycoprotein complex belonging to the

From the *Department of Neurology; TDivision of Nuclear Medi- cine, Department of Internal Medicine; $Division ofNeuropsycho1- ogy, Department of Psychiatry; SDivision of Speech Pathology, De- partment of Physical Medicine and Rehabilitation; \\Division of Neuroradiology, Department of Radiology, University of Michigan; and 'Psychology Service, Ann Arbor Veterans Affairs Medical Cen- ter, Ann Arbor, MI.

Received Jan 4, 1996, and in revised form Feb 20. Accepted for publication Feb 21, 1996. Address correspondence to Dr Gilman, Department of Neurology, University of Michican Medical Center, 1500 E. Medical Center Dr, Ann Arbor, MI 48109-0316.

-

Copyright 0 1996 by the American Neurological Association 163

family of receptor-operated ion channels [l 1, 131. This receptor contains several functionally coupled binding sites for pharmacologically specific agents that modulate GABA receptor/chloride channel function [ 141, including binding sites for benzodiazepines (BDZs), bar- biturates, and picrotoxin-like convulsants. These three components are functionally coupled and reside on the oligomeric protein complex that regulates ion perme- ability [ 121. The BDZ, barbiturate, and convulsant receptors appear to be allosterically linked to low-affinity GABA agonist sites [13]. The latter are concentrated mostly in layer V of the cerebral cortex, cingulate cortex, hippocampal region, amygdala, anterior and medial hypothalamus, superior colliculus, substantia nigra pars reticulara, periaqueductal gray, and molecular layer of the cerebellum [15]. Among other functions, BDZ receptors are thought to be important in influ- encing anxiety in humans [ 161.

Ethanol activates a number of specific receptors, including N-methyh-aspartate (NMDA) [ 171, dopa- minergic [ 181, P-adtenergic [ 191, serotonergic [20], and GABAergic [21]. Ethanol strongly affects GABA- A receptors, stimulating chloride ion flux through channels activated by GABA [21], and at least some of the actions of ethanol may be mediated through enhancement of function of the BDZ-sensitive GABA- A/chloride channel complex [22]. GABA agonists en- hance the actions of ethanol, and GABA antagonists reduce ethanol intoxication [22]. Also, compounds with inverse agonist actions at the GABA-A/BDZ receptor reduce ethanol intoxication [23]. Chronic exposure to ethanol in rats significantly decreases GABA-A receptor-mediated chloride uptake in cerebral cortical synaptoneurosomes [24]. Muscimol and pentobarbital stimulation, as well as ethanol augmentation of muscimol-stimulated chloride flux, are decreased with chronic ethanol exposure. These findings suggest that chronic exposure to ethanol may decrease the level of mRNAs coding for the cx subunit of the GABA-A receptor. This decrease may reflect altered processing of mRNA encoding GABA-A-receptor proteins.

The GABA-A/BDZ receptor may be important in the pathogenesis of chronic alcoholism. One hypothesis is that central GABAergic hypofunctioning, due to genetic inheritance and/or the result of chronic ethanol intoxication, may facilitate further alcohol consumption [25]. According to this hypothesis, an individual consumes ethanol to restore GABAergic neurotransmission and to prevent anxiety. If this hypothesis is correct, a reduced number of GABA neurons or of GABA-A/BDZ receptors could decrease the capacity to modulate anxiety in chronically alcohol-dependent persons. This is in keeping with clinical appraisals of severe chronic alcoholics as having decreased effective control as well as defective anxiety management [lo].

Investigations with FDG and PET of chronically al-

Table l. Ages o f the Subjects

Normal Controls ALC Subjects ~

FDG FMZ ACD non-ACD Studies Studies

Number 8 9 15 14 Ages (yr) 54 2 6 49 2 10 50 2 1 1 46 % 13

( 2 SD)

All subjects were male.

ALC = all chronic alcoholic subjects; ACD = chronic alcoholic subjects with clinical evidence of alcoholic cerebellar degeneration; non-ACD = chronic alcoholic subjects withour evidence of alcoholic cerebellar degeneration; FDG = [ l R F ] f l ~ ~ r ~ d e ~ ~ y g l u ~ ~ ~ e ; FMZ = ["C]flumazenil; SD = standard deviation.

coholic patients demonstrated hypometabolism in the rostral frontal regions of the brain as well as decreased performance on select neuropsychological tests thought to measure frontal lobe functioning [3, 26, 271. These findings could result from neuronal loss or impaired function in the rostral frontal lobe or in remote sites from transneuronal effects (diaschisis). The recent development of ["C]flurnazenil (FMZ) as a ligand for PET makes it possible to study GABA-A/BDZ-receptor density quantitatively in the brain of living patients with chronic alcoholism and to determine whether these sites or the neurons containing these sites are damaged. The present studies were undertaken to ex- amine lCMRglc and changes in the regional binding of FMZ to GABA-A/BDZ receptors in patients with severe chronic alcoholism.

Subjects and Methods Patient Groups and Normal Subjects The studies were approved by the Institutional Review Boards of the University of Michigan Hospitals and the Ann Arbor Veterans Affairs Medical Center (AAVAMC), and in- formed consent was obtained from all patients and normal control subjects. We studied 17 male chronically alcohol- dependent patients recruited predominantly from the Alco- hol Treatment Unit of the AAVAMC (Table 1). All patients met the Diagnostic and Statistical Manual o f Mental Disorders, fourth edition (DSM-IV) [ZS], diagnostic criteria for alcohol dependence and all except 1 exceeded a weekly intake of 560 gm of ethanol over 2 of the 3 preceding years. The 1 excep- tion was a severely alcoholic patient who had been abstinent for 60 months at the time of study. Most patients had at least one hospitalization for alcoholism, and most previously achieved sobriety for no more than 1 month since beginning the chronic use of alcohol until the current period of abstinence. These patients are characterized as "heavy" drinkers by existing epidemiological data, both in duration and inten- sity of alcohol intake [29]. Patients were excluded if they had any history of polydrug abuse as defined by the quantitative criteria of the National Institute on Drug Abuse in its National Collaborative Study of Polydrug Abuse [30]. Other exclusion criteria were neurological disorders apart from

164 Annals of Neurology Vol 40 No 2 August 1996

those due to alcohol, including stroke, birth complications, learning disorders, or other acquired or developmental disorders carrying neurological or neuropsychological risk. Pa- tients with closed head injury with loss of consciousness ex- ceeding 30 minutes were also excluded. This criterion was used because head injury with loss of consciousness for less than 30 minutes is associated with a favorable neurological outcome, including full recovery. Blood alcohol levels were measured on the day of the scan in patients whose behavior suggested alcohol ingestion or frank intoxication. O n this basis, 1 subject was excluded from study before the scan was initiated, and measurements in 3 ocher subjects revealed blood alcohol levels of zero.

Eight of the chronic alcoholic subjects studied had a diagnosis of alcoholic cerebellar degeneration (ACD) (see Table 1). The diagnosis of ACD was made on the basis of the criteria described above for alcohol dependence along with convincing and converging evidence of ACD from the patients’ history, physical examination, and neurological examination, supported by laboratory tests, including anatomical imaging, to exclude other diseases. The diagnosis required the history of a gait disorder occurring in the course of severe, chronic alcohol dependence, usually with associated malnutrition, in conjunction with evidence on neurological examination of an ataxia of gait of the cerebellar type but without severe ataxia of upper extremity movements or speech. The gait disorder could have developed suddenly or slowly. Disorders of sensory function sufficient to cause gait ataxia could not be present.

The findings in the patients were compared with data obtained from male normal control subjects of comparable age distribution consisting of 15 for PET studies with FDG and 14 for studies with FMZ (see Table 1). Normal control subjects were excluded if they had a history of alcohol abuse or dependence or if they met any of the exclusion criteria of the alcoholic group.

All patients and control subjects received a complete niedi- cal and neurological history and neurological examination. The subjects were examined in the absence of medications that could influence cognition, the motor system, or the PET studies. Alcoholic patients and control subjects taking BDZs at the time of study were excluded. Five of the alcoholic patients had received chlordiazepoxide 50 mg four times daily for a maximum of 4 days during the alcohol detoxifica- tion process following admission to hospital. This medication was discontinued at least 6 weeks prior to PET study in all cases except 1 in whom the medication was discontinued 13 days prior to study.

The alcoholic subjects were studied with PET after they had achieved at least 30 days of sobriety except for 1 in whom the PET study was conducted following 17 days of abstinence. At the time of study, the patients were consum- ing a normally nutritious diet and had no evidence of ketosis at the time of study, as determined by urinalysis.

Positron Emission Tomography These studies were performed similarly for all subjects. The subjects fasted for 4 hours before the scan and were studied lying supine and awake in a quiet room, alert but not speak- ing, with eyes open from 5 minutes before injection until

completion of the scan. A catheter was placed in a radial artery for blood sampling. PET scans were performed following intravenous injection of 22 ? 2 mCi of FMZ and, 90 minutes later, 10 mCi of FDG.

The subjects were imaged with either a SiemendCTI 9311 08-12 or a Siemens ECAT EXACT-47 scanner. Images from both scanners were reconstructed to a resolution of 8 to 9 mm full-width at half-maximum (FWHM) in-plane. Since the axial sampling of the EXACT-47 scanner is twice as fine as the 931, two adjacent levels from the EXACT were aver- aged, providing images with the same axial spacing and nearly the same resolution as the 931 scanner. Attenuation correction was calculated by the standard ellipse method. The PET studies were analyzed with a compartmental model and parameter estimation technique that provides pixel-by- pixel determinations of each measurement, thus creating “functional” images [3 11.

We labeled flumazenil with carbon- 11 at high specific ra- dioactivity using a methylation process [32]. Blood samples were taken as rapidly as possible during the first 2 minutes after tracer injection and then at progressively longer intervals throughout the remainder of the study. We took a total of 25 to 30 samples per scan. The samples were centrifuged, and the plasma radioisotope concentrations were measured in an NaI well counter. Plasma levels of radiolabeled metabo- lites of FMZ were determined by a rapid Sep-Pak CI8 car- tridge chromatographic technique [33] in the samples taken at I minute, 2 minutes, and every sample from 3 minutes until the end of the scan. Dynamic PET scans were performed for 60 minutes beginning immediately after injection. Radioactive fiducials with 5 pCi of ”C were placed on each subject’s scalp. Computer routines automatically determined the locations of these fiducials and used this informa- tion to correct for patient motion. The studies with FMZ provided quantitative measurement of ligand influx ( K J , which is highly correlated with flow because the single-pass extraction fraction for flumazenil is greater than 50% [31]. The studies also provided measurement of FMZ distribution volume ( D V ) , which is linearly related to the density of available receptor sites divided by the ligand dissociation con- stant (Bmax‘/KD). The methods for BDZ receptor-binding measurement, including the assumptions and limitations and the performance of FMZ, are published [31, 33, 341. In the FDG studies, data were acquired 30 to 90 minutes after injection and quantified utilizing the static scan method of Hutchins and colleagues [35]. Data acquired with the Sie- mens/CTI 931/08-12 scanner were obtained as two inter- leaved image sets.

PET Data Analysis Data from the PET studies were mapped into a standard stereotactic orientation utilizing an automated method for rotational and translational transformation [36] followed by linear scaling and nonlinear warping of individual brains [37]. After anatomic standardization, pixel-by-pixel t-statistic images, corrected for multiple comparisons, were produced with methods derived from the work of Friston and associ- ates [38] and Worsley and co-workers 1391. The PET studies were displayed as three matched image sets (one each for FDG, FMZ 4, and FMZ DV) . PET data acquired from

Gilman et al: FMZ and FDG Binding in Alcoholics 165

the normdl controls were compared with data obtained from the patient groups. The patients with severe chronic alcoholism (ALC) were compared with the normal controls and also, after the patients were divided into two groups, with those with alcoholic cerebellar degeneration (ACD) and those without ACD (non-ACD). The data from each group were combined and comparisons were made between groups by subtracting data pixel by pixel. Significant differences between groups portrayed in color were projected onto a surface-rendered magnetic resonance (MR) image volume. The maximum activation level normal to and within 15 mm of the cortical surface was projected onto the surface display [ d o ] .

Magnetic Resonance Imaging M R imaging was performed in 5 patients at 1.5 T and in 8 patients at 0.5 T utilizing coronal T1 (repetition time [TR] 500-600 msec/echo time [TE] 18-25 msec) and axial T 2 (TR 2,500-3,000 mseclTE 80-90 msec) images. Slice thick- ness was 5 mm with 2- to 2.5-mm intervals between slices.

ma1 control) and PET measure (FDG ICMRglc, FMZ K , and FMZ D V ) as factors was performed on these mean nor- malized data for each of the 10 stereotactically defined VOIs. Kendall rank-order correlations were performed to assess the relationship between the extent of tissue atrophy determined by MR imaging and the degree of FDG or FMZ decline measured by PET.

Results The Figure illustrates regional differences in PET measures between groups mapped onto surface representations of the brain generated from a standardized MR image set (shown in gray scale). The upper left quadrant shows comparisons between normal control and all alcoholic subjects. The upper right quadrant- shows differences between normal control and the ACD sub- group of alcoholics, while the bottom left shows differences between normal control and the non-ACD sub-

MR Data Analysis MR images were rated by a neuroradiologist (J.B.) who was blinded to the other data in the study. He used perceptual analysis for evidence of cortical volume loss at the medial surfaces of the cerebral hemispheres, based on the width of the interhemispheric fissure and on the size of the subarach- noid spaces bordering the gyri. Cerebral cortical volume loss was rated on a 0 to 4 scale and characterized as absent, mini- mal, mild, moderate, or severe in each of five regions that were localized relative to the position of the corpus callosum. The five regions were the medial surfaces of the cerebral hemispheres anterior to the genu of the corpus callosum (region l), the medial surfaces of the cerebral hemispheres above the anterior (region 2) , middle (region 3), and posterior (region 4) thirds of the corpus callosum, and the medial surfaces of the cerebral hemispheres posterior to the splenium of the corpus callosum (region 5). With the same rating scale, atrophy was assessed in five other areas, the frontal and parietal convexities, superior cerebellar vermis, inferior cerebellar vermis, and cerebellar hemispheres. The central sulcus, evi- dent in images of all subjects, was used to differentiate the frontal and parietal convexities.

Data obtained from both FMZ and FDG PET studies were analyzed further with quantitative volume-of-interest (VOI) data derived from regions defined stereotactically to match the areas of the MR image sets that were used to assess tissue atrophy. To compare the degree of abnormality across the three PET measures, individual VOI values for every subject were expressed as a fraction of the normal control group mean for each parameter in each particular region. Following this step, the mean value for the control group in every region became 1.0 for each of three PET measures, while the correct standard deviations and coefficients of vari- ation were maintained since each individual (both control and patient) was adjusted by the appropriate normal control group mean. Thus, a value of 0.9 represents a 10% decrease from the normal mean, independent of what parameter or which region is being examined. A two-factor analysis of variance (ANOVA), with group (ACD, non-ACD, and nor-

b (Upper left) Su face representations o f data obtained fiom positron emission tomography studies mapped onto standard magnetic resonance images illustrating differences between normal control subjects (Norm) and all alcoholic subjects (ALC) for local cerebral metabolic rates for glucose (LCMRglc) (top row), inj9u.x o f ["C$umazenil (FMZ K,) (middle row), and distribution volume of [I' CIj9umazenil (FMZ DV) (bottom row). The images show, fiom left to right, right medial and lefi medial views of the cerebrum. Statistical comparisons are made pixel by pixel, with t values indicated in the rainbow scale. Signijicant differences at the p < 0.05 level are portrayed in red or yellow. Signi5cant differences between groups are found in the medial fiontal lobe, including the superior frontal gyrus and rostral portion of the cingulate gyms. (Upper right) &$ace representations o f data obtained fiom positron emission tomography studies mapped onto standard magnetic resonance images as in the upper le$ illustration, showing differences between normal control subjects (Norm) and subjects with clinical evidence of alcoholic cerebellar degeneration (ACD). Si'iJcant diferences are seen in the medial fiontal lobe, including the superior frontal gyrus and rostral portion of the cingulate grus, and in the superior aspect of the cerebellar vermis. (Lower left) Suvface representations of data obtained from positron emission tomography studies mapped onto standard magnetic resonance images as in the upper left illustration, demonstrating differences between normal control subjects (Norm) and chronic alcoholic subjects without clinical evidence o f alcoholic cerebellar degeneration (non-ACD). Signijicant diferences are seen in the medial frontal lobe, including the superior frontal gyrzw and rostral portion of the cingulate gyrus. (Lower right) Su face representations of data obtained from positron emission tomography studies mapped onto standard magnetic resonance images as in the upper left illustration, showing differences between chronic alcoholic subjects without clinical evidence of alcoholic cerebellar degeneration (non-ACD) and chronic alcoholic subjects with clinical evidence of alcoholic cerebellar degeneration (ACD). Signijicant diferences are seen in the superior aspect of the cerebellar vermis.


Fig.

Table 2. Regional Group Means for Patients With Alcoholic Cerebellar Degeneration (ACD) and Without Alcoholic Cerebellar Degeneration (non-ACD) Expressed as a Fraction o f Normal Control Group Means for the Three PET Measures

VOI

FDG ICMRglc FMZ K FMZ DV

non-ACD ACD non-ACD ACD non-ACD ACD

Medial cereb ctx area 1,' 0.966 0.95 1' 0.954 0.949 0.965 0.961 Medial cereb ctx area 2L 0.960 0.948' 0.926' 0.918' 0.938' 0.925' Medial cereb ctx area 3,',h 1.007 0.969 0.972 0.92Td 0.937' 0.931' Medial cereb ctx area 4 0.978 1.010 0.984 1.01 1 0.992 1.003 Medial cereb ctx area 5 0.978 1.053 0.991 1.029 1.029 1.033 Frontal convexity 0.974 1.027 0.999 1.040 1.013 1.042 Parietal convexity 0.981 1.017 0.997 1.046 1.021 1.053 Sup cerebellar vermis",b 1.125' 0.84Fd 1.074 0.788',d 0.950 0. 772',d Inf cerebellar vermis" 1.116' 0.925d 1.071 0.890d 0.993 0.8 5 5',d Cerebellar cortex 1.072' 1.047 1.038 0.976 0.932 1.027

'Two-factor analysis of variance (ANOVA) significant for differences across groups at p < 0.05. "Two-factor ANOVA significant for differences across posirron emission tomographic (PET) measures ar p < 0.05. 'Two-tailed t tesr significantly different from normal control ar p < 0.05. dTwo-tailed t tesr significantly different between non-ACD and ACD groups at p < 0.05.

FDG = ['8F]fl~orode~~yg~u~o~e; ICMRglc = local cerebral metabolic rates for glucose; FMZ = [llC]flumazenil; k; = ligand inflow; DV = disrribution volume; cereb = cerebral; ctx = cortex; Sup = superior; Inf = inferior.

group. The lower right compares directly the ACD and non-ACD subgroups. In each quadrant, the top row of images is generated with lCMRglc data from FDG studies. The middle and bottom rows show the ligand transport ( K ; ) and distribution volume (DV) from FMZ studies. Images in each row show right and left medial views of the cerebrum. Pixel-by-pixel comparisons are made and t values at statistical significance levels of p < 0.05, uncorrected for multiple comparisons, are shown in a rainbow color scale. Pixels reach- ing statistical significance after correction for multiple comparisons appear in the yellow to red range.

significant differences between the entire alcoholic group and controls (upper left) for ICMRglc, FMZ K [ , and FMZ DVare found in the medial frontal lobe, including the superior frontal gyrus and rostral portion of the cingulate gyrus. Significant differences for comparisons of the non-ACD (lower left) and ACD (upper right) subgroups separately to controls were detected in these same frontal regions; however, the ACD group shows an additional area of significant decrease cen- tered in the superior aspect of the cerebellar vermis. Direct comparison of the two alcoholic subgroups (lower right) demonstrated significantly lower FMZ transport (6) and binding density ( D V ) in ACD than non-ACD patients.

Table 2 reports the group means for the non-ACD and ACD patient groups, expressed as a fraction of the normal control group mean for all three PET measures in each of the 10 stereotactically defined VOIs. Results of the two-factor ANOVA revealed statistically significant main effects across groups in medial cortical regions 1 ( p = O.O011), 2 ( p < O.OOOl) , and 3 ( p <

O.OOOl) , as well as both superior ( p < 0.0001) and inferior cerebellar ( p < O.0001) vermis. Significant main effects were also found across the three PET measures for medial cortical region 3 ( p = 0.022), where greater decreases were observed in patient groups with FMZ and FDG studies, and superior cerebellar vermis ( p = 0.0061), where, again, FMZ studies showed greater decreases. A significant interaction effect for group and PET measure was detected only in the superior cerebellar vermis ( p = 0.046).

Subsequent direct comparisons between groups (see Table 2) yielded similar results to those from the pixel- by-pixel analysis presented in the Figure. Areas of significant decrease were found in medial frontal lobe, including portions of the superior frontal and rostral cingulate gyri. Significant decreases in lCMRglc were found in medial cortical VOIs 1 and 2 for the ACD group compared with normal control. Significant decreases in FMZ 4 were found in medial cortical VOI 2 for both ACD and non-ACD subgroups, while in the ACD group, medial cortical VOI 3 also achieved significance. Medial cortical VOIs 2 and 3 were significantly decreased in FMZ DVfor both ACD and non-ACD subgroups. Significant decreases in all three PET measures were observed in the superior cerebellar vermis for ACD patients compared with normal controls. In addition, the decrease in FMZ DV reached significance in the inferior cerebellar vermis. No significant decreases in either superior or inferior cerebellar vermis were observed for non-ACD patients. An unexpected increase in ICMRG was observed, however, in both superior and inferior cerebellar vermis for non- ACDs compared with controls.


Table 3. Kendall Rank-Order Correlations Between MRI absent neurons cannot be determined from these Atrophy Index and PET Measure studies.

VOI FDG lCMRglc FMZK, FMZDV

Medial cereb CM area 1 Medial cereb ctx area 2 Medial cereb ctx area 3 Medial cereb ctx area 4 Medial cereb ctx area 5 Frontal convexity Parietal convexity Sub cerebellar vermis Inf cerebellar vermis Cerebellar cortex

-0.380 -0.061 -0.457 -0.015

0.036 0.188

-0.272 -0.594" -0.699b -0.115

-0.322 -0.484 -0.339 -0.255 -0.215

0.314 -0.272 -0.651" -0.642b -0.230

-0.409 -0.272

0.280 -0.075 -0.036

0.188 -0.242 -0.623a -0.499

0.345 _____

'Kendall rank-order correlation significant at p < 0.05. bKendall rank-order correlation significant at p < 0.01.

MRI = magnetic resonance imaging; PET = positron emission tomography; FDG = [I"] fluorodeoxyglucose; lCMRglc = local cerebral metaboliL rates for glucose; FMZ = ["C]flumazenil; k; = ligand inflow; DV = distribution volume; cereb = cerebral; ctx = cortex; Sup = superior; Inf = inferior.

Comparisons of VOI data in ACD and non-ACD patients demonstrated a primary difference between groups in the cerebellar vermis. Statistically significant decreases in ACD compared with non-ACD patients were detected in both superior and inferior cerebellar vermis for ICMRglc, K , and D V (see Table 2). One additional significant decrease was observed for ACD compared with non-ACD subjects in FMZ k; in medial cortical VOI 3 .

Table 3 gives correlations between the MRI-based measure of tissue atrophy and each of the PET measures for the 10 VOIs. Significant correlations were found for all three PET measures in the superior cerebellar vermis, and for ICMRG and k; but not DVin the inferior cerebellar vermis. No significant correlations between degree of atrophy and any of the PET measures were detected in the medial frontal regions.

Discussion This investigation demonstrated in the ALC group, compared with normal controls, significantly reduced ICMRglc bilaterally in the medial aspects of the frontal lobes, including the rostral cingulate and superior frontal gyri. These findings are consistent with previous investigations [9] and provide more complete maps of the regions affected. Studies with FMZ revealed a similar distribution of decreased and DK suggesting that both blood flow and GABA-NBDZ binding are diminished in this region. Some of the changes in ICMRglc, 4, and DVmay reflect tissue atrophy. Fur- thermore, the relative contribution to the changes in DV of reduced receptors on remaining neurons versus

Eight chronically alcoholic patients studied had clinical evidence of ACD, a disorder usually associated with both excessive alcohol intake and severe rnalnutri- tion [41, 421. Comparison of this group with the normal controls revealed significantly decreased lCMRglc, k;, and DVin both medial aspect of the frontal lobes and the superior portions of the cerebellar vermis, re- flecting decreased synapses and neuronal cell bodies in these sites. Comparison of the ACD and non-ACD groups of alcoholic subjects revealed significant differences in ICMRglc, K , and DVin the cerebellar vermis and not in the frontal region, indicating that the degree of injury to the frontal lobes is approximately equal in these groups.

The studies suggest that severe chronic alcoholism damages neurons directly in the rostra1 medial portions of the frontal lobes, and that additional injury occurs in the superior cerebellar vermis in the ACD sub- group. The studies with FDG show approximately the same distributions of abnormalities as the studies with FMZ, indicating that the changes in lCMRglc result from direct cerebral injury. and not from remote effects on other sites through diaschisis. The data acquired in this study could result from tissue atrophy resulting in partial volume averaging rather than reduction of GABA-A/BDZ receptors on preserved neurons or loss of neurons containing these receptors. Comparison of assessments of the degree of atrophy with the PET data, however, revealed significant correlations only in the superior and inferior aspects of the cerebellar vermis for all measurements, including k;, D K and 1CMRglc. These findings suggest that atrophy of tissue in the cerebral cortex cannot account for the marked decrease of ICMRglc and receptor binding found there.

The GABA-A/BDZ receptor may be important in the pathogenesis of chronic alcoholism. One hypothesis is that in chronic alcoholic persons, decreased central GABAergic function, due to genetic inheritance and/ or chronic ethanol intake, leads to further alcohol consumption [25]. Thus, the alcoholic subject may con- sume ethanol to restore GABAergic neurotransmission and to prevent the development of anxiety. In the present study, we found no evidence for a generalized loss of GABA-A/BDZ receptors manifested as diffusely decreased OK instead, we found evidence for focal loss involving the superior frontal gyrus and rostral cingulate gyrus. These regions of the cerebral cortex are thought to be important in modulation of affect and motivation, inhibition of certain types of behavior, selective attention, and in higher cortical functions such as abstraction, rule, and concept formation. Consider- ing these important functions of this region, it is possible that reduction of neurons in this structure con-


tributes to the excessive drinking behavior through decreased inhibition, inability to engage in efficient and flexible problem solving, and inability to identify high- risk situations.

Several studies have been reported of the density of GABA-A/BDZ receptors in the brains of human alcoholics at postmortem, and the results are inconsistent. One biochemical study revealed increased GABA binding but no change in BDZ binding in the superior frontal gyrus of alcoholic human subjects compared with normal controls 1431. In another study, no difference was found in the density of BDZ-binding sites in the frontal lobes between alcoholics and controls, but a decrease was found in the cerebellum of alcoholics [44]. In a third set of studies, a 20 to 25% decrease in BDZ binding was found in the frontal cortex [45] and a 30% decrease in the hippocampus [46] of chronic alcoholics compared with age-matched nonal- coholic controls. The decrease was site specific; no change in BDZ-receptor density was found in the temporal cortex, putamen, or caudate nucleus [46]. In a fourth study, the affinity of BDZ-binding sites for ['HI diazepam was decreased in the frontal lobe of alcoholic subjects compared with controls, but the affinity for ['Hlflunitrazepam was increased [47].

Studies of the effects of chronic ethanol administration on GABA-A/BDZ-receptor binding in experimental animals have produced conflicting results. Two studies demonstrated decreased BDZ receptors in the brains of experimental animals exposed chronically to alcohol [48, 491, but several others reported no change [50-521.

The results of this investigation indicate that severe chronic alcoholism, with or without alcoholic cerebellar degeneration, leads to sustained injury to the superior medial aspects of the frontal lobes as manifested by diminished metabolic rates, reduced blood flow, and diminished distribution volume of GABA-AIBDZ receptors. Although the present study indicates that the injury affects neurons containing GABA-A/BDZ receptors, we consider it likely that other neuronal elements are affected as well. The reason that this region should be vulnerable to injury to alcohol is not clear, nor is it clear why the cerebellar vermis should be selectively vulnerable in alcoholic patients. We expect that contin- uing investigations with other ligands in chronic severe alcoholics will disclose evidence of much more wide- spread involvement of the central nervous system, pos- sibly including subcortical as well as cerebral cortical and cerebellar sites.

These investigations were supported in part by NIH grants AA 07378, NS 15655, and AG 08671 and by a sharing agreement for PET studies between the Ann Arbor Veterans Affiairs Medical Cen- ter and the University of Michigan.

W e thank the personnel of the PET Center of the Division of Nuclear Medicine for production of the PET isotopes and acquisi- tion of the scans, and Dr David Kuhl for assisrance. We also thank Drs Judith Marks, Phillip Kroll, and Stanley Berent and Ms Kitty Heiss for their help in this study.

References 1.

2.

3.

4.

5 .

6.

7.

8.

9.

10,

11

Courville CB. Effects of alcohol in the nervous system of man. Los Angeles: San Lucas Press, 1955 Ferrer I, Fabregues I, Rairiz J, Galofre E. Decreased numbers of dendritic spines on cortical pyraniidal neurons in human chronic alcoholism. Neurosci Lett 1986;63:115-119 Harper CG, Kril J, Daly J. Are we drinking our neurones away? Br Med J 1987;294:534-536 Kril J, Harper CG. Neuronal counts from four cortical regions of alcoholic brains. Acta Neuropathol 1983;79:200-204 Harper C, Corbett D. Changes in basal dendrites of cortical pyramidal cells from alcoholic patients-a quantitative Golgi study. J Neurol Neurosurg Psychiatry 1990;53:856-861 Harper C , Kril J . Patterns of neuronal loss in the cerebral cortex in chronic alcoholic patients. J Neurol Sci 1989;92:81-89 Harper C, Kril J. If you drink your brain will shrink. Neuro- pathological considerations. Alcohol Alcohol 1991 (suppl 1):

Carlen PL, Penn RK, Fornazzari L, et al. Computerized tomographic scan assessment of alcoholic brain damage and its po- tential reversibihy. Alcohol C h Exp Res 1986; 10:226-232 Gilman S, A d a m K, Koeppe RA, er al. Cerebellar and frontal hypometabolism in alcoholic cerebellar degeneration studied with positron emission tomography. Ann Neurol 1990;28:

Grant I , Adams KM, Reed R. Intermediate-duration (subacute) organic mental disorder of alcoholism. In: Grant I, ed. Neuro- psychiatric correlates of alcoholism. Washington, DC: Ameri- can Psychiatric Press, 1986 Haefely WE. The GABA-benzodiazepine interacrion fifteen years later. Neurochem Res 1990;15:169-174

375-380

775-785

12. Matsumoto RR. GABA receptors: are cellular differences re- flected in function? Brain Res Rev 1989;14:203-225

13. Olsen RW, McCabe RT, Wamsley JK. GABA, receptor sub- types: autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system. J Chem Neuroanat 1990;3:59-76

14. Olsen RW. GABA-benzodiazepine-barbiturate receptor inter- actions. J Neurochem 1981;37:1-13

15. McCabe RT, Wamsley JK. Autoradiographic localization of subcomponents of the macromolecular GABA receptor complex. Life Sci 1986;39:1937-1945

16. Smith DE, Wesson DR, eds. The benzodiazepines: current standards for medical practice. Lancaster, England: MI]' Press, Ltd, 1985

17. Lovinger DM, White G, Weight FF. Ethanol inhibits NMDA- activated ion currents in hippocampal neurons. Science 1989; 243: 1721-1724

18. Brodie MS, Schefner SA, Dunwiddie RV. Ethanol increases the firing rate of dopamine neurons of the rat ventral tegmental area in vitro. Brain RKS 1990;508:65-69

19. Valvcrius P, Hoffman I'L, Tabakoff B. Hippocampal and cerebellar P-adrenergic receptors and adenylate cyclase are differen- tially altered by chronic ethanol ingestion. J Neurochem 1989; 52:492-497

20. McBride WJ, Murphy JM, Gatto GJ, et al. Serotonin and dopamine systems regulating alcohol intake. Alcohol Alcohol 199 1 (suppl 1):411-416

21. Suzdak I'D, Schwartz RD, Skolnick P, Paul SM. Ethanol stim- ulates y-aminobutyric acid receptor-mediated chloride trans-


port in rat brain synaptoneurosomes. Proc Narl Acad Sci USA 1986;83:407 1-4075

22. Harris RA, Allan AM. Involvement of neuronal chloride channels in ethanol intoxication, tolerance and dependence. In: Ga- lanter M, ed. Recent development in alcoholism, vol 5. New York: Plenum, 1987:313-325

23. Suzdak PD, Glowa JR, Crawley JN, et al. A selective imidazo- benzodiazepine antagonist of ethanol in the rat. Science 1986; 234: 1243-1247

24. Morrow AL, Montpied P, Lingford-Hughes A, Paul SM. Chronic ethanol and pentobarbitol administration in the rat: effects on GABA,, receptor function and expression in brain. Alcohol 1990;7:237-244

25. Ollat H, Parvez H , Parvez S. Review: alcohol and central neurotransmission. Neurochem Int 1988;13:275-300

26. Adams KM, Gilman S, Koeppe RA, et al. Neuropsychological deficits are correlated with frontal hypometabolism in positron emission tomography studies of older alcoholic patients. Alco- hol Clin Exp Res 1993;17:205-210

27. Adams KM, Gilman S, Koeppe R, et al. Correlation of neuropsychological function with cerebral metabolic rate in subdivi- sions of the frontal lobes of older alcoholic patients measured with [I"] fluorodeoxyglucose and positron emission tomography. Neuropsychology 1995;9:275-280

28. American Psychiatric Association. Diagnostic and statistical manual of mental disorders. 4th ed. Washington, DC: Ameri- can Psychiatric Association, 1994

29. Adams KM, Grant I, Carlin A, Reed R. Self-reported alcohol consumption in four clinical groups. Am J Psychiatry 1979; 138:445-449

30. Wesson D, Carlin A, Adams KM, Beschner G. Polydrug abuse. New York: Academic Press, 1978

31. Koeppe RA, Holrhoff VA, Frey KA, et al. Compartmental analysis of ["Clflumazenil kinetics for the estimation of ligand transport rate and receptor distribution using positron emission tomography. J Cereb Blood Flow Metab 1991;1:735-744

32. Maziere M, Hantraye P, Prenant C, et al. Synthesis of RO 1788 with "C: a specific radioligand for the in vivo study of central benzodiazepine receptors by positron emission tomography. Int J Appl Radiat Isotope 1984;35:973-976

33. Frey KA, Holthoff VA, Koeppe RA, et al. Parametric in vivo imaging of benzodiazepine receptor distribution in human brain. Ann Neurol 1991;30:663-672

34. Holthoff VA, Koeppe RA, Frey KA, et al. Differentiation of radioligand delivery and binding in the brain: validation of a two-compartment model for ["C] flumazenil. J Cereb Blood Flow Metabol 1991;11:745-752

35. Hutchins GD, Holden JE, Koeppe RA, et al. Alternative approach to single-scan estimation of cerebral glucose metabolic rate using glucose analogs, with particular application to isch- emia. J Cereb Blood Flow Metab 1984;4:35-40

36. Minoshima S, Koeppe RA, Mintun MA, et al. Automated de-

tection of the intercommissural line for stereotactic localization of functional brain images. J Nucl Med 1193;34:322-329

37. Minoshima S, Koeppe RA, Frey KA, Kuhl DE. Anatomic standardization: linear scaling and nonlinear warping of functional brain images. J Nucl Med 1994;35:1528-1537

38. Friston KJ, Frith CD, Liddle PF, Frackowiak RSJ. Comparing functional (PET) images: the assessment of significant change. J Cereb Blood Flow Metab 1991;11:690-699

39. Worsley KJ, Evans AC, Marrett S, Neelin P. A three-dinien- sional statistical analysis for CBF activation srudies in human brain. J Cereb Blood Flow Metab 1992;12:900-918

40. Minoshima S, Frey KA, Koeppe RA, et al. A diagnostic approach in Alzheimer's disease using three-dimensional stereotactic surface projections of fluorine-18 FDG PET. J Nucl Med 1995;36: 1238- 1248

41. Victor M, Adams RD, Collins GH. The Wernicke-Korsakoff syndrome and related neurologic disorders due to alcoholism and malnutrition. 2nd ed. Philadelphia: FA Davis, 1989

42. Phillips SC, Harper CG, Kril J. A quantitative histological study of the cerebellar vermis in alcoholic patients. Brain 1987; 110:301-314

43. Tran VT, Snyder SH, Major LF, Hawley RJ. GABA receptors are increased in brains of alcoholics. Ann Neurol 1981;9:289-292

44. Korpi ER, Uusi-Oukari M, Wegelius K, et al. Cerebellar and frontal cortical benzodiazepine receptors in human alcoholics and chronically alcohol-drinking rats. Biol Psychiatry 1992;3 1:

45. Freund G, Ballinger WE. Decrease of benzodiazepine receptors in frontal cortex of alcoholics. Alcohol 1988;5:275-282

46. Freund G, Ballinger WE. Loss of synaptic receptors can pre- cede morphologic changes induced by alcoholism. Alcohol A- cohol 1991 (supp1 1):385-391

47. Dodd PR. Benzodiazepine binding sites in alcoholic cirrhotics: evidence for gender differences. Metab Brain Dis 1995;10:93- 103

48. Freund G. Benzodiazepine receptor loss in brains of mice after chronic alcohol consumption. Life Sci 1980;27:987-992

49. Volicer L, Biagioni TM. Effect of ethanol administration and withdrawal on benzodiazepine receptor binding in the rat brain. Neuropharmacology 1982;21:283-286

50. Bosio A, Lucchi L, Spano PF, Trabucchi M. Central toxic effects of chronic ethanol treatment: actions on GABA and benzodiazepine recogition sites. Toxicol Lett 1982; 13:99- 103

51. Burov W, Yukhanonov RT, Maisky AI, et al. The role of benzodiazepine and opiate receptors in the development of ethanol dependence: experimental approach. In: Parvez S, ed. Progress in alcohol research, vol 1. Utrecht, Netherlands: VNU Science Press, 1985: 163-175

52. DeVries DJ, Johnston GAR, Ward LC, et al. Effects of chronic ethanol inhalation on the enhancement of benzodiazepine binding to mouse brain membranes by GABA. Neurochem Int 1987;10:231-235

774-786


Documents

Positron emission tomographic studies of cerebral benzodiazepine-receptor binding in chronic alcoholics