Neuronal localisation of benzodiazepine receptors in cerebellum

Neuro~ienee LetterJ, 13 (1979) 219--224 © ]~sevier/North-Holland Scientific Publishers Ltd.

NEURONAL LOC_ALIgATION OF BENZODIAZ~INE IN C E R E B E L L U M

C. B ~ U P ,

219

RECEPTORS

M. NIELSEN, G. BIGGIO and R~F. SQUIRES

(C.B.) Reseqreb Laboratories, A/$ Ferrosan 5, Sydmarken, DK-2860 Soeborg (Denmark), (M.N.) Psyehoplmrm4~ological Research Laboratory Dept. E, Sct. Hans Mental Hospital, DK-4000 Ro~ilde, (Denmark), (G.B.) University o f Cagliari, Sardinia (Italy) and (R.F.S.) CNS Biology, ly,ederle Laboratories, Pearl River, N Y 1096~ (U.S.A.)

(Received February 21st, 1979) (Revised vermionreceived A ~ 23rd, 1979) (Accepted May 4th, 1979)

SUMMARY The ~ellularlocallsation of benzodiazepine receptors was s~udied. Kainic acid induced neuronal lesions (2 X 0.25--2 × 2/Jg; 2--26 days in rat cerebellum decreased specific binding of [3H]flunitrazepam dow~ to 35% of controls. Specific binding of [3H]flunitrazepam was also decreased (to 8 0 % of controls) in the cerebellum of mutant nervous mouse (nr/nr) where Purkinje cellsare degenelated but not in the mutant weaver mouse where granule cellsare degenerated. These results show that benzodiazepine receptors are located mainly on neurons; both on Purkinje cellsand other neurons~ but no~ to a great extent on ~anule cells.

There is good evidence that the high affinity binding sites for [3H]diazepam and [3H]flunitrazepam in the mammalian brain are pharmacologically and clinically relevant receptors for benzodiazepines in vivo [ 2--4, 14,15,20,21]. Our u n d ~ d i n g of the celhdaz localisation of the benzodiazepine receptors, however, is still limited [ 10]. We present here evidence ~',~ benzodiazepine receptors in rat cerebellum almost completely disappear after injection of the neurot0xic agent kainic acid, thus indicating an aimost exclusive neuronal locailsation. Results with mouse mutants of the weaver and nervous type indicate that cembellaz granule cells lack benzodiazepine receptors while Purkinje cells are one of several cell types in cerebellum canting benzodiazepine receptors. Benzodiazepine receptor levels were measured with [3H]flunitrazepam as ligand (1-[methyl-3H], 87 Ci/mmol NEN, Boston, MA) since flunitrazepam labels the same receptors as [SH]diazepam [4] and flunitrazepam bindir. ~,is

220

technically super or to diazepam binding with respect to sensitivity and accuracy. The flit ration technique [ 1] with small modifications was used. Cerebellar tissue ,14--62 mg) was usually homogenized in 2 ml, Tris--HCl, 50 raM, pH 7.1 at 0°C. Since cerebeUar tissue of treated Rnimnl~ often differed appreciable in weight from controls, higher homogenisation volumes were used in some experiments and aliquots of equal volume and tissue concentration were then applied t o minimize artifacts. The homogenate was centrifuged at ,/.8,000 × g for 10 rain and the pellet was r e s u s ~ e d in approx. 1000 vols. (original tissue weight) of Tris--HCl 50 mM (pH 7.1) and used directly for binding assays. We used "total membrane fractions' instead of 'P2' f r ~ i o n s since large amounts of specific [3H]flunitrazepam binding sites are ; in the cerebellar PI fraction and since appreciable amounts of b i ~ g sites are also found in the 'microsomal fraction' [6] which is included in our 'total membrane fraction' (protein in this fraction approximates 70 mg/g original tissue). The membrane suspension was incubated for 40 rain at 0°C with 1 nM [3H]flunitrazepam in duplicate, fil:;ered and counted for tritium [1]. All values presented are specific binding, which is total binding minus binding in the presence of 3 * 10 -6 M diazepam. Cer~bellurn was selected for the present study since the neuronal organisation in this structure is well defined and possesses a limited number of neuron types and since it has intermediate levels of benzodiazepine receptors [1,14]. Cerebellum contains five types of neurons, the granule cells, Purkinje cells, basket cells, stellate cells and Golgi cells. All these ceU types, except the granule cells, are destroyed by kainic acid, probably because all four types receive glutamate input from the granule cells [ 9]. We observed a profound, dose dependent, decrease in cerebeUar benzodiazepine receptors after kainic acid (Table I). Most results were obtained 9 - 1 0 days after kainic acid but large decreases could be measured already after 2 days. The strong decreases observed are apparently at variance with our own preliminary results showing variable non-significant reduction in benzodiazepin receptors in cerebellum 9 - 1 0 days after a single injection of 2 #g kainic acid. Two injections of kainic acid 2 rain and 1 mm apart (see legend to Table I), however, result in pronounced and reproducible effects. The results after kainic acid strongly indicate that benzodiazepine receptors are located on neurons. A partial, kainic acid-induced, decrease in benzodiazepine receptor concentration in rat substantia n'm~ra [ 4] and basal ganglia of patients dying with Huntington Chorea [16,22] a disease associated with known neuronal degeneration, also points to a neuronal localisation. The present results do not support the contention that benzodiazepine receptors are localised mainly on ~ cells in rat brain [8], at least not in the cerebellum. The small (30--35% of controls) levels of benzodiazepine receptors l e f t a f t e r neuronal degeneration does not allow for much glial localisation; since the absolute amount of receptors r e ~ g are even lower due to reduced tissue weight. The appat'evlt lack Of glial localisation is also

221

TABLE I REDUCED LEVELS OF BENZODIAZEPINE RECEPTORS AND GLUTAMIC ACID DECARBOXYLASE (GAD) IN RAT CEREBELLUM AFTER LOCAL KAINIC ACID INJECTION Male WMar rats (180--210 g) were anesthetized with mebumal (50 mg/kg i.p.). Kainic acid (in 2 × 1 ~I iBoton~c saline, pH 6.9--7.1) was injected through a Hamilton syringe type 701 (outer diameter 0.5 mm) at a Sl~ccd of 0.5 ~ I / m ~ . The needle remained in position for 2 rain after each injection, lnjectiows were made only in left cerebellar hemisphere, 2 m m lateral to the midline, 0.5 nun anterior to the edge between os interparietsle and os 8upraoccipitale and 1.4 and 2.4 mm (two injecti(~ns) below the skull ~mrfaee. Blocks o f brain ti~ue, including the injection site, were dissected and symmetric blocks from the right hemisphere served as controls. Specific [3H]flunitrazepam binding was determined u described in the text. GAD was amayed by adding 100 #1 of crude brain homogenate (approx. 2 mg original t ~ u e ) to 100 , I buffer (phosphate 9 raM, pH 7.0) containing 1-[14C]glutamate (1.7 raM, 20--50 #Ci/mmol) and pyridoxal phosphate (0.07 raM) all final concentrations, and incubating the suspension for 30 min at 37 ° C. ['4C]CO~ was liberated with 200 #1 2-N sulphuric acid, trapped in Protosol, and counted by liquid scintillation counting. Control levels of GAD averaged 257 ± 9 cpm/mg tissue (mean ± S.E.M of 42 values), blanks obtained with boiled tissue evolved approx. 10--15% of total [14C]CO2. Scatchard analyses in an experiment similar to experiment 5 showed that the Bran was decreased to 33% of the control (5 values), while the affinity constant was not increased by kainic acid. Protein determinations by the Lowry method showed that the protein level (mg/g tMue) was decreased to 70 ± 3% of control (~nean ± S.E.M. of 5 vjlues) in a schedule like Expt. 6. The experiments in this table were all done at Set. HJms Mental Hospital, Denmark. Similar series of experiments using [ 3H ]diazepam ins~ad of [aH]flunitrazepam but otherwise almost identical parameter, was done at University of Cagliari, Sardinia, w~th very similar results (not shown).

Expt No.

1 2

3 4 5

6

Kainic acid (~g)

Weight of sample (rag)

Days

N

Specific [3H]flunitrazepam binding (I nM, 0°C)

2 x 0.25 Control 2 x 0.5

47±2 56 ± 1 28±2

Control

35 ± 2

2 X 0.75 Control 2 X 2.0 Control 2 X 2.0 Control

20 ± 1 35 ± 2 43±4 44 ± 5 19± 1 30± 1

10

11

2

9

9

8

2 X 2.0 Control

ZO ± 1 34 ± 2

26

8

9

12

9

7

GAD %

pmol/g tissue

%

16.6±0.7 19.1 ± 0.6 1 2 . 1 ± 1.3

88*

9 6 ± 11

58***

3 6 ± 13 **~

50***

23 -+ 2 **~"

20.7 ± 1.0

*P < 0.05; * * P < 0.01; * * * P < 0.001.

9.1 ± 0.5 18.6 ± 0.8 12.8±0.5 20.2 -* 2.0 7.7 23.3 9.5 30.4

± 1.0 ± 0.7 +- 1.8 ± 0.4

63**

8±

5"*"

35***

16 +_ 2***

31"**

25 +- ~***

222

indicated by the !ack of brain specific [1] benzodiazepine receptors on primary cultures of mouse cerebral astroglial cells [5] and C~-giioma cells (Solomon Snyder, personal communication) (both these p l ~ t r a t i o n s contain binding of the periphenfl kind, which is di~-etmm di~pl,lu~able but no clonazepam displaceable [3H]diazepam binding [1]). Granule cells in cerebellum survive kainic acid treatment [9]. The small amounts of benzodiazepine receptors remaining after kainic acid (30--35%) might therefore be located on granule cells. This is apparently not the case in mice. Benzodiazepine receptom are not decrmm~ in the weaver mutant (wv/wv) (Table H) which is almost completely devoid of cerebeBar granule cells at the age of 20 days [ 13,17]. The increased receptor density may be caused by decreased total cerebellar weight. The kainic acid experiment does not differentiate between basket cells, Purkinje cells, stellate cells and Golgi cells. Purkinje ceils selectively degenerate in the nervous mutant mouse (nr/nr) from the age of about 23 days [ 11,18]. The density of benzodiazepine receptors are slightly (20%) decreased in the nervous mouse (Table H and ref. 19) as well as in the Purkinje cell deficient mouse (pcd/pcd) at the age of 30 days (Braestrup unpublished). Thi~ partial depletion of benzodiazepine receptors, in contrast to a much larger depletion reported in the P2 fraction of nervous mice [12], indicates

TABLE II BENZODIAZEPINE RECEPTOR IN CEREBELLUM OF THE WEAVER AND NERVOUS MUTANT MOUSE Weaver mice (Be CBA/51) were wv/wv and controls ~ere phenotypic normal ]itternmtes (wv?/+). Nervous mice (C~HeB/J) were nr/nr and normal B, CBA/51 (+/+ mice or phenotypic normal littermates (nr/+) at the same age were used as controls. Values are the mean ± S.E~M. of (N) values. *P < 0.05; **P < 0.01; ***P < 0.001. Genotype

wv/wv +/w'v or +/+ nr/nr a +/+ nr/nr nrl+

Age days

20 20 53 53 53 53

Cerebellar weight (mg)

14.0 44.0 38.1 49.0 32.0 54.0

± 1"** ±3 ± 2* ±3 ± 1"** ±1

Specific [3H ]flunitrazepam binding (1 riM, 0"C) pmol/g tissue

%

13.6 ± 0.4*** 10.1 ± 0.7 29.0 ± 1.7"* 37.3 ± 1.4 29.61 ± 1.5" 37.0 ± 2.0

134 78 80

N

5 7 5 5 7 9

aProtein concentration (mg/g tissue) was not different (35%, P > 0.05) in these two groups. The experiment was repeated using Ps membranes and we found similar difference between the two groups.

223 t h a t benzodiazepine receptors are located on several kinds o f neurons in the cerebellum. T h e lesions described here might also be useful for investigation o f th e possible relationships [7] between b e n z o d i a z e p i n e receptors ,~nd GABAreceptors. A?KNOWLEDGEMENT This s t u d y was aided by a grant f r o m the Danish Medical Council Jr. No. 5 1 2 - 8 4 0 5 a nd 512- 8404. J. G u e n e t , Institut Pasteur, kindly supplied breeding, pairs of weaver and nervous mice. REFERENCES 1 Brae~zp, C. and Squires, R.F., Specific benzo ~azepine receptors in rat brain characby high-aff'mity [~I]diazepam binding, i'coc. Natl. Acad. Sci. (USA), 74 (1977) 3805--3809. 2 Brae~lzp, C., Albrechtsen, R. and Squires: R.F., High densities of benzodiazepine receptors in human cortical areas, Nature (Lond.), 269 (1977) 702--704. 3 Bra~trup, C. and Squires, R~F., Pharmacological characterisation of benzodiazepir.e receptors in the brain, Europ. J. Pharmacol., 48 (1978) 263--270. 4 ~ p , C. and Squires, R.F., Brain specific benzodiazepine receptors, Brit. J. Prychiat., 133 (1978) 249--260. 5 Brae~Tup, C., Ni~en, C., Squires, R.F. and Schousboe, A., Lack of brain specific benzodiazepine receptors on mouse primary astroglial cultures, Neurosci. Lett., 9 (1978) 45--59. 6 Braestrup, C., Squires, R.F., Bock, E., Torp Pedersen, C. and Nielsen, M., Benzodiazepine receptors: cellular and subcellular localkation in brain. In J.~. Tillement (Ed.), Advances in Pharmacology and Therapy, Vol. 7, Pergamon Pren, 1978, pp. 173--185. 7 Guidotti, A., Toffano, G. and Costa, E., An endogenous protein moGulates the affinity of GABA and benzodiazepine receptors in rat brain, Nature (Lond.), 275 (1978) 553-'555. 8 Htmn, F.A. and Henke, D~J., Ccrlular localkation of [3H ]diazepam receptors, Ne~lropharmacology, 17 (1978) 985--988. 9 Herndon, R~M. and Coyle, J.T., Selective destruction of neurons by a transmitter agonig, Science, 198 (1977) 71--72. 10 I v a n , L.L., GABA and benzodiazepine receptors, Nature (Lond.), 275 (1978) 477. 11 Landis, 8.C., U i t ~ c t u r a l changes in the mitochondria of cerebellar Purkmje cells of m~rvous mutant mice, J. Cell Biol., 57 ( 1 9 3) 782--797. 1~ Liplm, A.8., Simo, M.C., Coupet, J. and B~r, B., Evidence that benzodiazepine receptors reside on cerebellar Purkinje cell~: studies with 'nervous' mutant mice, Life M . , 23 (1978) 2213. 13 Mallet, J., Huch~, M., Pougeois, R. and Changeux, J.-P., Anatomical physiological and biochenfleal studies on the cerebellum from mutant mice. HI. Protein differences associated with the weaver, ~ e r e r and nervotts mutations, Brain Res., 103 (1976) 291--312. 14 M6hl~r, H. and Oksda, T., Banzodiazepine receptor: demonstration in the central

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