Multiple benzodiazepine receptors: autoradiographic localization in normal human amygdala

Brain Research, 276 (1983) 237-245 Elsevier

237

Multiple Benzodiazepine Receptors: Autoradiographic Localization in Normal Human Amygdala DEBRA L. NIEHOFF* and PETER J. WHITEHOUSE Departments of Pharmacology and Experimental Therapeutics, Neurology, and Neuroscience, The Johns Hopkins UniversitySchool of Medicine, Baltimore, MD 21205 (U.S.A.)

(Accepted February 15th, 1983) Key words: benzodiazepines - - benzodiazepine receptors - - amygdala - - multiple receptors - - triazolopyridazines - autoradiography

Because previous studies have emphasized the importance of the amygdala in the therapeutic actions of benzodiazepines and described differences in benzodiazepine receptor distribution between human and rat, this study examined the distribution of multiple benzodiazepine receptors in normal human amygdala by light microscopic autoradiography. Benzodiazepine receptors were labeled with [3H]flunitrazepam at 5 representative rostro-caudal levels. Type 1 and Type 2 receptors were differentiated by the addition of 200 nM CL218,872 to serial sections and subsequent analysis based on the differential occupancy at Type 1 and Type 2 receptors by this drug. Results demonstrated that like the rat amygdala, human amygdala contains a higher density of benzodiazepine receptors in the basolateral nuclear complex compared to the corticomedial complex, and more Type 2 than Type 1 receptors overall. However, while rat amygdala is enriched rostrally in Type 1 receptors, this subclass in humans is elevated caudally. Pathways including the amygdala and limbic and cortical targets of its efferents may be preferential loci of benzodiazepine anxiolytic activity.

INTRODUCTION The benzodiazepines (BZs) are clinically important anxiolytic, muscle-relaxant and anticonvulsant drugsS. Localization of the sites of action of these drugs could provide important clues to defining neural pathways involved in the action of BZs. Evidence that the limbic system, particularly the amygdala, is an important locus of B Z action in a variety of mammalian species comes from several sources. In freely moving cats, a depression of the high-voltage spindling peak (40 Hz) was induced in the amygdala, but not other structures, by systemic BZ administration, while in rats, responding in a conflict paradigm increased amygdaloid neuronal discharges that were suppressed by n Z s 9,26. Behavioral studies involving electrical stimulation or lesioning of the basolateral nuclear subdivision (lateral and basal nuclei) of amygdala suggests that this region is involved in the expression of fearful behavior 21,25,28,29,

while direct application of BZs to the lateral and basolateral amygdaloid nuclei of rats was capable of eliciting antianxiety effects, as measured in the anticonflict paradigm17,23. In humans as well, direct stimulation of the amygdala produced a subjective perception of fear and anxiety, accompanied by such characteristic autonomic sequelae as increases in heart rate and blood pressure and pupillary dilation 2. The action of BZs is presumed to be mediated by BZ receptors, identified and characterized by direct binding studies. Moreover, two subtypes of B Z receptor in the CNS have now been identified, chiefly by the differential specificity of drugs such as CL218,872 (3-methyl-6[3-trifluoromethyl)phenyl]1,2,4-triazolopyridazine)12,14,15, 24. The observation that these drugs were anxiolytic and anticonvulsant but non-sedating, led to the proposition that high-affinity CL218,872 (Type 1) receptors were more intimately involved in the anxiolytic actions of BZs12,14.15,24.

* Present address for correspondence: c/o Dr. Martin Raft, MRC Neuroimmunology Research Project, Department of Zoology, University College, London WCIE 6BT, U.K. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B'.V.

238 BZ receptors can be localized by light microscopic autoradiographic techniques32. 33. Multiple BZ receptors can also be visualized by a modification which utilizes the pharmacological specificity of CL218,87234. The evidence presented above suggests that the amygdala might be expected to be a region highly enriched in Type 1 BZ receptors, particularly its basolateral subdivision. Recently, we localized multiple BZ receptors in the rat amygdala 19 (also Niehoff and Kuhar, in press). In this study, we demonstrated that BZ receptors did indeed occur in high densities in the basolateral nuclear complex; however, these receptors were primarily of the Type 2 subclass. Type 1 receptors were concentrated in the anterior aspect of the cortical and lateral nuclei. Differences in the localization of BZ receptors in rat and human cerebellum have previously been reported 32. In human cerebellum, BZ receptors were present in equivalent levels in the molecular layer and the granule cell layer, while in rats, receptors were restricted to the molecular layer. Accordingly, we have localized multiple BZ receptors in normal human amygdala and report here similarities and significant differences between these two species.

MATERIALS AND METHODS Human brain tissue was obtained at autopsy from 3 males and two females (free of neuropathology and not known to be taking any psychoactive drug immediately prior to death), following an average postmortem delay of 11 h. Subjects ranged in age from 16 to 65 years, with a mean of 40.2 years. Brains were sliced coronally in 1-2 cm slabs, and blocks of tissue containing the amygdala, uncus, surrounding temporal cortex and ventral basal forebrain were excised and mounted on brass microtome chucks with TissueTek 1I O.C.T. (Lab Tek Products, Naperville, IL) and frozen on dry ice. Eight/~m cryostat sections of these blocks were mounted on subbed microscope slides and stored at - - 2 0 °C until receptor labeling. Sixteen/~m serial sections were also thaw-mounted and employed subsequently for toluidine blue and acetylcholinesterase staining. Prior to autoradiographic experiments, the displacement of BZ receptor binding by CL218,872 was characterized in slide-mounted tissue sections 34 to

evaluate any species-specific variations in pharmacological specificity. Briefly, sections were incubated with 0.17 M Tris HCI, pH 7.4, containing 1 nM [3HI flunitrazepam ([3H]FLU. 72.4 Ci/mMol, New England Nuclear, Boston) at 4 °C for 40 rain, in the presence of increasing concentrations of CL218,872. Blanks were determined by the addition of 1 uM clonazepam. The autoradiographic labeling procedure was essentially the same, but utilized 200 nM CL218,872 to differentiate binding to Type I and Type 2 receptors. Following incubation, sections were dipped in ice-cold buffer, washed for 2 min in buffer, and dipped briefly in distilled water. Sections used for the displacement studies were wiped from the slides with a Whatman GF/B filter and counted by liquid scintillation spectrometry, while autoradiographic sections were dried under a stream of cold, dry air and apposed to emulsion-coated (NTB3, 1:1 with water) coverslips as previously described. Following an 18 day exposure, coverslips were developed and tissue sections stained with 0.8% toluidine blue. Quantification of the autoradiograms was performed by grain counting. Grain densities were sampled at 3 or 4 locations 400 ~m 2 in size randomly chosen within each nucleus and averaged to obtain a representative value for that nucleus at that rostro-caudal level. Standard errors of these measurements were less than 10%, indicating that intranuclear receptor density was relatively homogeneous. Grain counts were transformed into molar densities by comparison with a standard curve derived from brain paste standards containing known amounts of radioactivity. In the absence of CL218,872, % occupancy at both Type 1 and Type 2 receptors by [3H]FLU is equal to 100 KL/I + KL, where K = K a of [3H]FLU and L = the concentration of [3H]FLU, while % occupancy in the presence of CL218,872 is equal to 100 KL/1 + KL + K ' L ' , where K' and L' represent the K~ of CL218,872 at the particular receptor subtype and the concentration of CL218,872. Assuming a Ka for [3H]FLU of 1 nM at both Type 1 and Type 2 receptors 34, and K i values of 20 nM and 1000 nM for CL218,872 at Type 1 and Type 2 sites, respectively20,3], occupancy at both receptor subtypes by 1 nM [3H]FLU in the absence of CL218,872 is 50% while 200 nM CL218,872 reduces occupancy at Type 1 receptors by 84% and at Type 2 receptors by 10c/c

239 HUMAN AMYGDALA

Thus, BZ receptor binding can be partitioned into subtypes by the following pair of simultaneous equations: Type i + Type 2 = total specific binding 0.84 Type 1 + 0.10 Type 2 = binding displaced by 200 nM CL218,872 At each level examined, serial sections were incubated in the presence and absence of 200 nM CL218,872 to derive values for total and CL218,872displaceable binding. Sixteen /~m unlabeled sections were stained in 0.8% toluidine blue or for acetylcholinesterase by a modified Karnovsky-Roots method (Hedreen et al., submitted for publication). Such sections served as anatomical reference standards.

LEVEL 1

LEVEL 3

LEVEL 2

LEVEL 4

RESULTS The nuclear pattern of the human amygdala has been described in detaiP. Terminology and anatomical differentiation of the various nuclei are taken from this source. The use of reference standards stained for acetylcholinesterase greatly facilitated the anatomical differentiation of the various nuclei. Only the basolateral nucleus of the amygdala stains darkly for acetylcholinesterase; hence, this nucleus could be readily identified and served as an orienting landmark. In addition, certain cytoarchitectonic features were characteristic of some nuclei. For example, cells of the basolateral nucleus could be readily distinguished by their large size and abundant Nissl substance, while cells of the neighboring lateral nucleus were slightly smaller and contained a circular nucleus with a prominent nucleolus. The trilaminate structure of the cortical nucleus was also a readily identifiable feature, as were the densely packed small cells of the superficial portion of the'basomedial nucleus. Comparison of autoradiograms with acetylcholinesterase or toluidine blue-stained sections revealed that the sections used for autoradiography in this study could be classified into 5 rostro-caudal levels, separated by approximately 600-800 pm. Level 1 is the most posterior, and represents a level slightly caudal to Fig. 10 of Crosby and Humphrey 3. Level 5 is the most anterior level and corresponds to

LEVEL 5 Fig. 1. Schematic representation of the human amygdala. Level ] represents the most posterior level examined, Level 5 the most anterior. Nuclei visible at each level arc labeled; the basolateral nucleus, determined by acetylcholinestcrasc staining, is in black. Abbreviations: AA, anterior amygdaloid area; aabl, accessory basal amygdaloid nucleus, lateral portion; abl, basolateral amygdaloid nucleus; abm d, basomcdial amygdaloid nucleus, deep portion; abms basomcdial amygdaloid nucleus, superficial portion; ac, central amygdaloid nucleus; aco, cortical amygdaloid nucleus; 1, plcxiform layer; 2, cellular layer; al, lateral amygdaloid nucleus.

Fig. 8 of Crosby and Humphrey 3. Fig. 1 illustrates these 5 levels and the nuclei visible at each of them. The displacement of [3H]FLU binding by CL218,872 in sections of human amygdala was essentially the same as displacement by this compound in rat brain regions containing a heterogeneity of BZ receptors, e.g. cerebral cortex34. In the two cases examined, biphasic displacement curves with Hill coefficients less than one were obtained. The K i at the low affinity site was about 18 nM, and at the high affinity site, about 1000 nM. Thus, occupancy at Type 1 and Type 2 receptors at 200 nM CL218,872 is essentially the same in human and rat (data not shown)20,34.

240

B~

Figs. 2-4. Multiple BZ receptors in human amygdala. These 3 figures are darkfield photomicrographs, in which the tissue is invisible, and the relative brightness is associated with receptor density. The top panel (A) in each photomicrograph represents binding to a section incubated in 1 nM [3H]FLU alone, while the lower photograph (B) is of a serial section coincubated with 1 nM [3H]FLU and 200 nM CL218,872. These photomicrographs are taken from two cases, which were representative of the results in all 5 of the cases examined. Abbreviations are the same as those of Fig. 1. HP, hippocampus. Bar = 3 ram. Fig. 2 corresponds to Level 1, Fig. 3 to Level 4 and Fig. 4 to Level 5.

Figs. 2-4 are darkfield p h o t o m i c r o g r a p h s of serial sections incubated with 1 nM [3H]FLU to reveal total B Z receptor binding ( A ) or 1 nM [3H]FLU and 200 nM CL218,872 to reveal Type 2 receptors (B), at 3 representative levels: Level 1, at the caudal aspect of the amygdala, Level 4, a more anterior level, and Level 5, at the most rostral aspect, These photographs are taken from two cases, but are representative of all the cases studied. The density of B Z receptors at all levels ranged from m o d e r a t e to very high, with the lateral nucleus, accessory basal nucleus, and deep portion of the basomedial nucleus containing the highest density of receptors. The central nucleus, medial nucleus, and anterior aspect of the superficial

basomedial nucleus contained the lowest density of B Z receptors. In general, there were more Type 2 than Type 1 receptors, except at the posterior levels, which contained an appreciable portion of Type 1 receptors (Figs. 2-4). No age-, sex- or p o s t m o r t e m delay related alterations in total or subtype receptor density were observed. Within some individual nuclei, total B Z receptor density and the Type l: Type 2 receptor ratio appeared to be d e p e n d e n t on the exact locus examined along the rostro-caudal axis. For example, in the lateral nucleus, Type 1 receptors were highly enriched at posterior (Fig. 2) c o m p a r e d to anterior (Figs. 3, 4) levels, while in the cellular layer of the cortical nuclc-

241

iiii:iiii¸i!i

Fig. 3. See legendto Fig. 2. us, the reverse was true (Fig. 4 vs Fig. 2). Similarly, the deep portion of the basomedial nucleus contains more BZ receptors rostrally (Figs. 2, 3), while the plexiform layer of the cortical nucleus contains more receptors caudally (Figs. 2, 4). Other nuclei displayed little variation in receptor density throughout the amygdala, such as the basolateral nucleus (primarily Type 2 receptors), and the central nucleus

(primarily Type 1 receptors; Figs. 2, 4). Table I summarizes the average total density of BZ receptors in the entire amygdala at the given levels examined, as well as in the basolateral (lateral, basolateral, superficial and deep basomedial, and accessory basal nuclei) and corticomedial (cellular and plexiform layers of the cortical nucleus, as well as medial and central nuclei) nuclear subdivisions.

242

Fig. 4. See legend to Fig. 2.

From this table, it can be seen that BZ receptor density is relatively constant at all levels. Also, at all levels, the basolateral complex contains a higher density of BZ receptors than the corticomedial complex. Table II summarizes the Type 1 receptor proportional density in a similar fashion, and reveals that the fraction of this receptor subtype increases at caudal lev-

els. The basolateral complex in particular displays a pronounced rostro-caudal increase in the proportion of Type 1 receptors, while the corticomedial complex contains two areas of elevated Type 1 receptor density. The level 5 peak in this complex corresponds to the enrichment of Type 1 receptors in the cortical nucleus at this level (Fig. 4).

243 TABLE I

Total BZ receptors in human amygdala 'Entire amygdaloid complex' refers to all nuclei visible at that level. 'Basolateral complex' consists of lateral, basolateral, superficial and deep basomedial, and accessory basal nuclei. 'Corticomedial complex' consists of cellular and plexiform layers of the cortical nucleus and medial nucleus, plus central nucleus at levels 1 and 2 only. Total BZ receptor densities were averaged over the number of nuclei present in a given subdivision and level to obtain 'average receptor density'. Data are mean ± S.E.M., or means of two observations which varied by less than 10%.

Level No. Average receptor density of (fmol/mg tissue) cases Entire Basolateral amygdaloid complex complex only

Corticomedial complex only

1 2 3 4 5

52.2 38.1 41.2±3.6 17.7±1.4 42.1±5.6

2 2 4 4 4

54.9 50.1 47.6±2.4 38.0±7.0 54.7±3.6

57.0 59.8 50.3±2.3 42.8±6.0 56.6±3.3

TABLE II

Percentage Type I receptors in human amygdala Definitions of 'entire amygdaloid complex', 'basolateral complex' and 'corticomedial complex' are the same as in Table I. Percentage values of Type 1 receptors were averaged over the nuclei within a particular subdivision at a given level. Data are mean values of 2-4 cases + S.E.M. where appropriate.

Level

1 2 3 4 5

Average percentage Type 1 receptors Entire amygdaloid complex

Basolateral complex only

Corticomedial complex only

45.4 64.8 38.2 + 2.8 32.6 + 5.8 32.8 + 5.4

55.2 58.8 39.8 + 3.4 31.6 + 4.7 21.7 + 4.7

34.3 50.6 30.4 + 0.9 11.5 + 5.0 79.3 + 6.1

BZ receptor distribution between rat and human are notable. For example, in both species, the lateral nucleus contains the highest density of receptors overall, while the central nucleus is an area of very low receptor density 19. Both species also share a peak in Type 1 receptor density in the anterior cortical nucleus 19. Important differences between rat and human receptor distributions do exist. The most striking of these is the complementarity in Type 1 receptor density distribution along the rostro-caudal axis, with the rat amygdala containing a higher proportion of Type 1 receptors at anterior levels 19 (also Niehoff and Kuhar, in press) and human amygdala containing a higher proportion of Type I receptors at posterior levels. This is largely due to a relative enrichment in Type 1 receptors in the caudal aspects of the basolateral nuclear division in the human. In particular, the posterior lateral nucleus, which in the rat contains almost exclusively Type 2 receptors, contains 60-70% Type 1 receptors in the human. Human central nucleus is also complementary to that of the rat, being composed largely of Type 1 receptors in the former, and of Type 2 receptors in the latter species. Table III summarizes these similarities and differences. The differences in receptor distribution may reflect differences in connectivity or function in human compared to rat, or may be a phylogenetic 'rearrangement' of receptor subtypes. In any case, these data confirm previously published results identifying differences in BZ receptor distribution between rat and human in the cerebellum32 and suggest that drawing conclusions about BZ action in humans from rat receptor distributions, particularly in the case of mulTABLE III

BZ receptors in rat vs human amygdala DISCUSSION

The results of this study demonstrate that human amygdala, like the rat amygdala, contains a high density of BZ receptors, primarily of the Type 2 subclass 19. Also, like the rat, the basolateral subdivision contains a higher density of BZ receptors than the corticomedial subdivision. This suggests that in human as well as rat, the phylogenetically recent basolateral nuclei may be more important in the etiology of fearful or anxious behavior. Other similarities in

Similarities (1) Higher density of BZ receptors in basolateral nuclear complex than corticomedial complex (2) Lateral nucleus has highest density of BZ receptors, central nucleus has the lowest density (3) Overall, more Type 2 than Type 1 receptors (4) Anterior cortical nucleus enriched in Type 1 receptors

Differences (1) Type 1 receptors enriched in anterior aspects in rat but in posterior aspects in human (2) Posterior lateral and central nuclei are enriched in Type 2 receptors in rat, but contain primarily Type 1 receptors in human

244 tiple receptors may result in error. In the rat, high densities of B Z receptors are found in many of the limbic and cortical terminal fields of amygdaloid efferents, e.g., prefrontal cortex, hypothalamus and entorhinal cortex 33. Pathways which include these structures and the amygdala may be very sensitive to B Z s and may be involved in their anxiolytic properties. I n d e e d , direct injection of BZs into the mamillary body elicits potent anticonflict activity it, while the frontal cortex has been d e m o n s t r a t e d to be important in the disinhibition of punished behavior by BZs 14. These observations led K a t a o k a et al. Jl to p r o p o s e a p a t h w a y to mediate B Z anxiolytic activity c o m p o s e d of mamillary body ~ anterior thalamus 4,5, --~ frontal cortex6.7. We a d d e d the lateral amygdala ---, b a s o m e d i a l amygdala 13 ~ ventromediai h y p o t h a l a m u s 13 ~ mamillary body 22 to this pathway in the rat, and also suggested the importance of direct connections b e t w e e n the basolateral and lateral nuclei and frontal cortex 13 (Niehoff and Kuhar, in press). A m y g d a l o i d projections to tempo-

ral and frontal cortex have been d e m o n s t r a t e d in primatesJ.lo, as have most of the connections in the amygdalo-hypothalamic-cortical pathway outlined above 1,16.17.30. Therefore, the amygdalo-hypothalamic-corticai circuit and amygdalo-cortical projections may be i m p o r t a n t pathways for B Z actions in humans as well as in rats. ACKNOWLEDGEMENTS The authors gratefully acknowledge the technical assistance of Ms. Susan G o e h r i n g , Mrs. N a o m i Taylor, Mrs. R o b e r t a Proctor and the secretarial assistance of Ms. D a r l e n e W e i m e r . Dr. Juan Troncoso assisted in the p r o c u r e m e n t of h u m a n tissue, and Dr. Cheryl Kitt p r o v i d e d assistance with the amygdaloid anatomy. This work was s u p p o r t e d by MH00053, DA00266, and grants from the McKnight Foundation and L e d e r l e Laboratories. CL218,872 was the generous gift of Lederle L a b o r a t o r i e s and HoffmanLa Roche.

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