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Changes in rat hippocampal benzodiazepine receptors and lack of changes in muscarinic receptors after fimbria-fornix lesions
Neuroscience Letters, 27 (1981) 193-197 Elsevier/North-Holland Scientific Publishers Ltd.
193
CHANGES IN RAT HIPPOCAMPAL BENZODIAZEPINE RECEPTORS AND LACK OF CHANGES IN MUSCARINIC RECEPTORS AFTER FIMBRIA-FORNIX LESIONS
U.C. S,~BATO, J.S, AGUILAR, J.H. MEDINA* and E. DE ROBERTIS
Instituto de Biologla Celular y *II C6tedra de Fisiologia Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1121) Buenos Aires (Argentina) (Received June 30th, 1981; Revised version received September 10th, 1981; Accepted September 22nd, 1981)
The binding of [3H]L-quinuclidinylbenzilate to muscarinic receptors and [3H]flunitrazepam to benzodiazepine receptors were studied in the rat hippocampus after lesion of the fimbria-fornix. While the muscarinic receptors showed no change, the benzodiazepine receptors did change considerably at various time intervals. Two days after the lesion the specific [3H]flunitrazepam binding decreased 380, while at 5 days it increased 65%. After 14 days of the lesion it still was significantly above normal. These changes are due to a variation in the number of sites, and not in affinity. Possible interpretations of these results are discussed.
In recent years, the use of [3H]diazepam and [3H]flunitrazepam has demonstrated the presence of high affinity binding sites in mammalian brain. The binding is rapid, reversible, saturable and stereospecific. These, and other characteristics, have led to the interpretation of the binding as representing a true binding to benzodiazepine receptors (see refs. 3 and 20). The possible neuronal localization of this receptor has been supported by work using lesions with kainic acid, mutant mice lacking Purkinje cells [2, 4] and autoradiography [13, 17]. Furthermore, cell fractionation of the brain has demonstrated the possible localization of these receptors in synaptosomes and synaptosomal membranes [2, 14]. Recently, our laboratory has been engaged in a study of the pre- and postsynaptic localization of several central receptors using Triton X-100 to preferentially dissolve the presynaptic membrane [5]. In the case of the benzodiazepine receptor, the results obtained have suggested that in the cerebral cortex of the rat a certain proportion of these receptors are localized presynaptically at the synaptosomal membrane [15]. The hippocampus is very appropriate for carrying out localization studies; it has a well defined neuronal organization and is connected to the rest of the CNS mainly through the fimbria-fornix. In this work we have performed unilateral sections of these pathways and followed the changes in benzodiazepine receptors with [3H]flunitrazepam binding at different time intervals. Since previous studies have 0304-3940/81/0000-00013/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
194
shown that lesions of the medial septum do not change the muscarinic receptors [141, we have also carried out [3H]quinuclidinylbenzilate ([3H]QNB) binding to the hippocampus of the operated rats. Male adult Wistar rats (220-250 g) were anesthetized with sodium pentobarbital (45 mg/kg) and placed in a stereotaxic frame. A burr hole of 3 × 2 mm was drilled in the skull overlying the fimbria-fornix according to the coordinates of K0nig and Klippel [9]. A microknife was stereotaxically lowered 4.5 mm from the dura, and the section of the fimbria-fornix was performed on one side. On the contralateral side the microknife was lowered 1-2 mm above the fornix (sham operation). Control, unoperated rats of the same age and sex were also used. Localization of the lesion was done by direct microscopic visualization of frozen sections and by histological sections stained with cresyl violet. The binding studies were carried out after 1, 2, 5 and 14 days of the lesion. As indicated in Tables I and II, the binding was done at saturation. The specific binding of [3H]QNB in the partially deafferented hippocampus did not change significantly compared to the contralateral side (sham operation) and the control animals (Table I). On the other hand, the specific binding of [3H]flunitrazepam showed considerable variation with respect to the time of lesion (Table II). One day after surgery there was a small but not significant decrease. After two days, the binding decreased very significantly, i.e. about 38°7o. This decline was due to a reduction in the Brnax without an apparent change in affinity. Five days after the lesion the
TABLE I LACK OF C H A N G E S IN SPECIFIC BINDING OF [3H]L-QUINUCLIDINYLBENZILATE TO R A T H I P P O C A M P U S AFTER F I M B R I A - F O R N I X LESION Rats were sacrificed by decapitation; brains were removed and hippocampi were dissected immediately in the cold and homogenized in approximately 50 volumes of buffer using a glass homogenizer fitted with a teflon pestle. The resultant homogenate was stored at - 60°C until used. For the binding of [3H]QNB the filtration technique of Fields et al. [6] was used with slight modifications. For each assay, quadruplicate samples of hippocampus homogenates containing 0.2 mg protein, as determined by the Lowry's method [11], were resuspended in 2 ml of 50 m M PO43 , Na ÷ , K + buffer, pH 7.4. Incubations with 0.5 nM [3H]QNB (40.2 C i / m m o l , New England Nuclear) were carried out at 37 °C for 50 min. Specific binding was calculated as the difference between total binding and binding in the presence of 5 #M atropine sulfate (Mann Research) and represented over 90°7o of total binding, n = number of experiments performed. In the unoperated controls the binding was 0.94 _+ 0.1 p m o l / m g protein. Results are expressed as p m o l / m g protein +_ S.E.M. lpsilateral side
Contralateral side
Days
n
p m o l / m g protein
°7o control
p m o l / m g protein
% control
2 5 14
2 6 7
0.91 + 0.07 1.01 _+ 0.02 1.02 _+ 0.11
97 107 108
0.95 _+ 0.09 0.93 _+ 0.09 0.96 _+ 0.09
101 99 102
195
changes were of opposite sign. On the lesioned side there was an increase of about 65°70, while on the contralateral side the binding did not differ from the control. A binding curve between 1 and 8 nM [3H]flunitrazepam, submitted to Scatchard analysis, showed that the increase in specific binding did not imply a change in affinity, but in maximimal binding (contralateral, control side, K o = 4 nM, Bmax = 320 fmol/mg protein; ipsilateral lesioned side, KD = 4.5 nM Bmax = 528 fmol/mg protein). After 14 days of the lesion the [3H]flunitrazepam binding was still about + 15°70 with respect to that of the control and + 20070 above the contralateral side (Table II). Here, as well, the increase was in receptor density but not in affinity (contralateral side, K o = 3.2 nM, Bmax = 335 fmol/mg protein; ipsilateral side, K D = 3.1 nM, Bmax = 406 fmol/mg protein). The present results on the muscarinic receptor confirm previous findings [14], in which no changes in [3H]QNB binding were observed after degeneration of the cholinergic septo-hippocampal pathway [10]. These findings were interpreted as suggesting a postsynaptic localization of the muscarinic receptor in the hippocampus. However, other alternative interpretations, such as a fall in presynaptic receptors with a compensatory rise in the postsynaptic receptors, were not excluded [14]. The lesion of the fimbria-fornix is much more complex, since it involves not only the cholinergic input to the hippocampus, but also the noradrenergic [18], dopaminergic [16] and serotoninergic [18] afferents, as well as the commisural fibers. Our findings suggest that also these other afferent nerve terminals do not contain muscarinic receptors. However, from these experiments it is not possible to T A B L E II LEVELS OF B E N Z O D I A Z E P I N E R E C E P T O R S IN R A T H I P P O C A M P U S C H A N G E A T VARIOUS TIME I N T E R V A L S A F T E R F I M B R I A - F O R N I X LESION The binding of [methyl-3H]flunitrazepam (86.4 C i / m m o l , New England Nuclear) was performed using the filtration technique o f Braestrup et al. [1] with slight modifications. For each assay, triplicate samples of hippocampus homogenate containing 0.15 mg, as determined by the Lowry's method, were resuspended in 1 ml of Tris-HC1 buffer, pH 7.4. Incubation was carried out at 4 °C for 40 min with 8 nM [3H]flunitrazepam. Specific binding was calculated as the difference between total binding and a blank done in the presence of 3 /zM flunitrazepam and represented over 75o7o of total binding. Results are expressed as f m o l / m g protein _+ S.E.M. In unoperated control animals the binding was 226.4 _+ 10.9 f m o l / m g , n = number of animals operated. Three to four binding experiments were carried out for each animal. *P < 0.001 (with respect to control), Student's t-test; **P < 0.0I (with respect to control), Student's t-test; ***P < 0.01 (with respect to contralateral side), paired test. Ipsilateral side (lesion)
Contralateral side (sham)
Day
n
Bmax ( f m o l / m g prot) 070 control
Bmax ( f m o l / m g prot) 070 control
1 2 5 14
7 8 5 6
205.5 139.5 374.8 258.7
220.3 219.5 234.4 216.6
_+ 18.1 _+ 9.8 _+ 34.2 _+ 14.2
90.9 61.7" 165.5"* 114.7"**
_+ 8.6 _+ 9.9 _+ 19.2 + 9.8
97.3 96.9 105.3 95.6
196 completely discard the presence of presynaptic receptors. These could represent a small but physiological active population [8], whose changes could be difficult to detect by binding studies. These observations on the muscarinic receptor are in direct contrast with those found for [3H]flunitrazepam binding (Table II). Here, after two days of the lesion, there is a considerable decrease in binding, suggesting that a significant percentage of receptors could be localized in axon terminals innervating the hippocampus. A retrograde degeneration can be discarded, since observation of histological sections, did not reveal chromatolysis in hippocampal neurons, with no signs of gliosis. The above interpretation is in agreement with our findings using Triton X-100 for the pre- and postsynaptic localization of benzodiazepine receptors in the cerebral cortex [15]. Fuxe et al. [7] have shown that after neuronal degeneration of the dorsal hippocampus by ibotenic acid there is a reduction of only 30°70 of the [3H]flunitrazepam binding sites. They concluded that a large number of benzodiazepine receptors might be localized in hippocampal afferents. Due to the complex composition of the afferent and efferent fibers that are affected by the section of the fimbria-fornix, at the present time it is practically impossible to deduce the cause of the large increase in benzodiazepine receptors after 5 days of section (Table II). This increase could be due to the appearance of new receptors sites causing a kind of supersensitivity. In favor of this interpretation is the rise in Bmax with no change in K D. Overstreet et al. [14] found no change in the binding of [3H]flunitrazepam after lesions of the septal region. This finding suggests that the cholinergic afferents are not involved in the changes in [3H]flunitrazepam binding observed by us. Because of the close association of benzodiazepine and G A B A receptors in the CNS (see refs. 3 and 20) it would be of interest to investigate how the fimbria-fornix section affects the binding of GABA. This work is planned for the future, but present knowledge suggests that the GABAergic neurons are intrinsic in the hippocampus [19]. If this is the case, it would be another instance in which it might be possible to dissociate G A B A and benzodiazepine receptors. This work was supported by Grants from C O N I C E T and SECYT of Argentina. The excellent technical help of Mrs. Angela Veiga is acknowledged.
1 Braestrup, C. and Squires, R.F., Specific benzodiazepine receptors in rat brain characterized by highaffinity [3H]-diazepambinding, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3805-3809. 2 Braestrup, C., Nielsen, M., Biggio, G. and Squires, R.F., Neuronal localization of benzodiazepine receptors in cerebellum, Neurosci. Lett., 13 (1979) 219-224. 3 Braestrup, C. and Nielsen, M., Benzodiazepine receptors. Arzneim-Forsch. (Drug Res.), 30 (1) 5a (1980) 852-857. 4 Chang, R.S.L., Vinh, T.T. and Synder, S.H. Neurotransmitter receptor localizations: brain lesion induced alterations in benzodiazepine, GABA, c~-adrenergic and histamine Hi-receptor binding, Brain Res., 190 (1980) 95-110.
197 5 Criado, M., Aguilar, J.S., and De Robertis, E., Action of detergents and pre- and postsynaptic localization of [3Hl-naloxone in synaptosomal membranes, J. Neurobiol., 12 (1981) 259-267. 6 Fields, J.Z., Roeske, W.R., Morkin, E. and Yamamura, H.I., Cardiac muscarinic receptors, J. biol. Chem., 253 (1978) 3251-3258. 7 Fuxe, K., Kt3hler, C., Agnati, L.F., Andersson, K., Ogren, S.O., Eneroth, P. Perez de la Mora, M., Karobath, M. and Krogsgaard-Larsen, P., GABA and benzodiazepine receptor studies on their localization in the hippocampus and their interaction with central dopamine neurons in the rat brain. In E. Costa, G. Dichiara, G.L. Gessa (Eds.), GABA and Benzodiazepine Receptors, Raven Press, New York, 1981. 8 Hadhhzy, P. and Szerb, J.C., The effect of cholinergic drugs on [3H]acetylcholine release from slices of rat hippocampus, striatum and cortex, Brain Res., 123 (1977) 311-322. 9 Kt~nig, J.F.R. and Klippel, R.A., The Rat Brain. A Stereotaxic Atlas of Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 10 Lewis, P.R. and Schute, C.C.D., The cholinergic limbic system, Brain, 90 (1967) 521-540. 11 Lowry, O.H., Rosebrough, N.J. Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 12 MOhler, H., and Okada, T., Benzodiazepine receptor: demonstration in the central nervous system, Science, 198 (1977) 849-851. 13 MOhler, H., Battersby M.K. and Richards, J.G., Benzodiazepine receptor protein identified and visualized in brain tissue by photoaffinity label, Proc. nat. Acad. Sci. (Wash.), 77 (1980) 1666-1670. 14 Overstreet, D.H., Speth, R.C., Hruska, R.E., Ehlert, F. Dumont, Y. and Yamamura, H.I., Failure of septal lesions to alter muscarinic cholinergic or benzodiazepine binding sites in hippocampus of rat brain, Brain Res.r, 195 (1980) 203-207. 15 Shbato, U.C., Aguilar, J.S., and De Robertis, E., Benzodiazepine receptors in rat brain: action of Triton X-100 and localization in relation to the synaptic region, J. Receptor Res., in press. 16 Scatton, B., Sinon, H. Le Moal, M. and Bischoff, S., Origin of dopaminergic innervation of the rat hippocampal formation, Neurosci. Lett., 18 (1980) 125-131. 17 Scott Young 111, W. and Kuhar, M.J., Autoradiographic localization of benzodiazepine receptors in the brain of humans and animals, Nature (Lond.), 280 (1979) 393-394. 18 Storm-Mathisen, S. and Guldberg, H.C., 5-Hydroxytryptamine and noradrenaline in the hippocampal region: effect of transection of afferent pathways on endogenous levels, high affinity up-take and some transmitter-related enzymes, J. Neurochem., 22 (1974) 793-803. 19 Storm-Mathisen, J., GAD in the rat hippocampal region after lesions of the afferent fibre systems. Evidence that the enzyme is localized in intrinsic neurons, Brain Res., 40 (1972) 215-235. 20 Tallman, J.F., Paul, S.M., Skolnick, P. and Gallagher, D.W., Receptor for the age of anxiety: pharmacology of the benzodiazepine, Science, 207 (1980) 274-281.
193
CHANGES IN RAT HIPPOCAMPAL BENZODIAZEPINE RECEPTORS AND LACK OF CHANGES IN MUSCARINIC RECEPTORS AFTER FIMBRIA-FORNIX LESIONS
U.C. S,~BATO, J.S, AGUILAR, J.H. MEDINA* and E. DE ROBERTIS
Instituto de Biologla Celular y *II C6tedra de Fisiologia Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, (1121) Buenos Aires (Argentina) (Received June 30th, 1981; Revised version received September 10th, 1981; Accepted September 22nd, 1981)
The binding of [3H]L-quinuclidinylbenzilate to muscarinic receptors and [3H]flunitrazepam to benzodiazepine receptors were studied in the rat hippocampus after lesion of the fimbria-fornix. While the muscarinic receptors showed no change, the benzodiazepine receptors did change considerably at various time intervals. Two days after the lesion the specific [3H]flunitrazepam binding decreased 380, while at 5 days it increased 65%. After 14 days of the lesion it still was significantly above normal. These changes are due to a variation in the number of sites, and not in affinity. Possible interpretations of these results are discussed.
In recent years, the use of [3H]diazepam and [3H]flunitrazepam has demonstrated the presence of high affinity binding sites in mammalian brain. The binding is rapid, reversible, saturable and stereospecific. These, and other characteristics, have led to the interpretation of the binding as representing a true binding to benzodiazepine receptors (see refs. 3 and 20). The possible neuronal localization of this receptor has been supported by work using lesions with kainic acid, mutant mice lacking Purkinje cells [2, 4] and autoradiography [13, 17]. Furthermore, cell fractionation of the brain has demonstrated the possible localization of these receptors in synaptosomes and synaptosomal membranes [2, 14]. Recently, our laboratory has been engaged in a study of the pre- and postsynaptic localization of several central receptors using Triton X-100 to preferentially dissolve the presynaptic membrane [5]. In the case of the benzodiazepine receptor, the results obtained have suggested that in the cerebral cortex of the rat a certain proportion of these receptors are localized presynaptically at the synaptosomal membrane [15]. The hippocampus is very appropriate for carrying out localization studies; it has a well defined neuronal organization and is connected to the rest of the CNS mainly through the fimbria-fornix. In this work we have performed unilateral sections of these pathways and followed the changes in benzodiazepine receptors with [3H]flunitrazepam binding at different time intervals. Since previous studies have 0304-3940/81/0000-00013/$ 02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
194
shown that lesions of the medial septum do not change the muscarinic receptors [141, we have also carried out [3H]quinuclidinylbenzilate ([3H]QNB) binding to the hippocampus of the operated rats. Male adult Wistar rats (220-250 g) were anesthetized with sodium pentobarbital (45 mg/kg) and placed in a stereotaxic frame. A burr hole of 3 × 2 mm was drilled in the skull overlying the fimbria-fornix according to the coordinates of K0nig and Klippel [9]. A microknife was stereotaxically lowered 4.5 mm from the dura, and the section of the fimbria-fornix was performed on one side. On the contralateral side the microknife was lowered 1-2 mm above the fornix (sham operation). Control, unoperated rats of the same age and sex were also used. Localization of the lesion was done by direct microscopic visualization of frozen sections and by histological sections stained with cresyl violet. The binding studies were carried out after 1, 2, 5 and 14 days of the lesion. As indicated in Tables I and II, the binding was done at saturation. The specific binding of [3H]QNB in the partially deafferented hippocampus did not change significantly compared to the contralateral side (sham operation) and the control animals (Table I). On the other hand, the specific binding of [3H]flunitrazepam showed considerable variation with respect to the time of lesion (Table II). One day after surgery there was a small but not significant decrease. After two days, the binding decreased very significantly, i.e. about 38°7o. This decline was due to a reduction in the Brnax without an apparent change in affinity. Five days after the lesion the
TABLE I LACK OF C H A N G E S IN SPECIFIC BINDING OF [3H]L-QUINUCLIDINYLBENZILATE TO R A T H I P P O C A M P U S AFTER F I M B R I A - F O R N I X LESION Rats were sacrificed by decapitation; brains were removed and hippocampi were dissected immediately in the cold and homogenized in approximately 50 volumes of buffer using a glass homogenizer fitted with a teflon pestle. The resultant homogenate was stored at - 60°C until used. For the binding of [3H]QNB the filtration technique of Fields et al. [6] was used with slight modifications. For each assay, quadruplicate samples of hippocampus homogenates containing 0.2 mg protein, as determined by the Lowry's method [11], were resuspended in 2 ml of 50 m M PO43 , Na ÷ , K + buffer, pH 7.4. Incubations with 0.5 nM [3H]QNB (40.2 C i / m m o l , New England Nuclear) were carried out at 37 °C for 50 min. Specific binding was calculated as the difference between total binding and binding in the presence of 5 #M atropine sulfate (Mann Research) and represented over 90°7o of total binding, n = number of experiments performed. In the unoperated controls the binding was 0.94 _+ 0.1 p m o l / m g protein. Results are expressed as p m o l / m g protein +_ S.E.M. lpsilateral side
Contralateral side
Days
n
p m o l / m g protein
°7o control
p m o l / m g protein
% control
2 5 14
2 6 7
0.91 + 0.07 1.01 _+ 0.02 1.02 _+ 0.11
97 107 108
0.95 _+ 0.09 0.93 _+ 0.09 0.96 _+ 0.09
101 99 102
195
changes were of opposite sign. On the lesioned side there was an increase of about 65°70, while on the contralateral side the binding did not differ from the control. A binding curve between 1 and 8 nM [3H]flunitrazepam, submitted to Scatchard analysis, showed that the increase in specific binding did not imply a change in affinity, but in maximimal binding (contralateral, control side, K o = 4 nM, Bmax = 320 fmol/mg protein; ipsilateral lesioned side, KD = 4.5 nM Bmax = 528 fmol/mg protein). After 14 days of the lesion the [3H]flunitrazepam binding was still about + 15°70 with respect to that of the control and + 20070 above the contralateral side (Table II). Here, as well, the increase was in receptor density but not in affinity (contralateral side, K o = 3.2 nM, Bmax = 335 fmol/mg protein; ipsilateral side, K D = 3.1 nM, Bmax = 406 fmol/mg protein). The present results on the muscarinic receptor confirm previous findings [14], in which no changes in [3H]QNB binding were observed after degeneration of the cholinergic septo-hippocampal pathway [10]. These findings were interpreted as suggesting a postsynaptic localization of the muscarinic receptor in the hippocampus. However, other alternative interpretations, such as a fall in presynaptic receptors with a compensatory rise in the postsynaptic receptors, were not excluded [14]. The lesion of the fimbria-fornix is much more complex, since it involves not only the cholinergic input to the hippocampus, but also the noradrenergic [18], dopaminergic [16] and serotoninergic [18] afferents, as well as the commisural fibers. Our findings suggest that also these other afferent nerve terminals do not contain muscarinic receptors. However, from these experiments it is not possible to T A B L E II LEVELS OF B E N Z O D I A Z E P I N E R E C E P T O R S IN R A T H I P P O C A M P U S C H A N G E A T VARIOUS TIME I N T E R V A L S A F T E R F I M B R I A - F O R N I X LESION The binding of [methyl-3H]flunitrazepam (86.4 C i / m m o l , New England Nuclear) was performed using the filtration technique o f Braestrup et al. [1] with slight modifications. For each assay, triplicate samples of hippocampus homogenate containing 0.15 mg, as determined by the Lowry's method, were resuspended in 1 ml of Tris-HC1 buffer, pH 7.4. Incubation was carried out at 4 °C for 40 min with 8 nM [3H]flunitrazepam. Specific binding was calculated as the difference between total binding and a blank done in the presence of 3 /zM flunitrazepam and represented over 75o7o of total binding. Results are expressed as f m o l / m g protein _+ S.E.M. In unoperated control animals the binding was 226.4 _+ 10.9 f m o l / m g , n = number of animals operated. Three to four binding experiments were carried out for each animal. *P < 0.001 (with respect to control), Student's t-test; **P < 0.0I (with respect to control), Student's t-test; ***P < 0.01 (with respect to contralateral side), paired test. Ipsilateral side (lesion)
Contralateral side (sham)
Day
n
Bmax ( f m o l / m g prot) 070 control
Bmax ( f m o l / m g prot) 070 control
1 2 5 14
7 8 5 6
205.5 139.5 374.8 258.7
220.3 219.5 234.4 216.6
_+ 18.1 _+ 9.8 _+ 34.2 _+ 14.2
90.9 61.7" 165.5"* 114.7"**
_+ 8.6 _+ 9.9 _+ 19.2 + 9.8
97.3 96.9 105.3 95.6
196 completely discard the presence of presynaptic receptors. These could represent a small but physiological active population [8], whose changes could be difficult to detect by binding studies. These observations on the muscarinic receptor are in direct contrast with those found for [3H]flunitrazepam binding (Table II). Here, after two days of the lesion, there is a considerable decrease in binding, suggesting that a significant percentage of receptors could be localized in axon terminals innervating the hippocampus. A retrograde degeneration can be discarded, since observation of histological sections, did not reveal chromatolysis in hippocampal neurons, with no signs of gliosis. The above interpretation is in agreement with our findings using Triton X-100 for the pre- and postsynaptic localization of benzodiazepine receptors in the cerebral cortex [15]. Fuxe et al. [7] have shown that after neuronal degeneration of the dorsal hippocampus by ibotenic acid there is a reduction of only 30°70 of the [3H]flunitrazepam binding sites. They concluded that a large number of benzodiazepine receptors might be localized in hippocampal afferents. Due to the complex composition of the afferent and efferent fibers that are affected by the section of the fimbria-fornix, at the present time it is practically impossible to deduce the cause of the large increase in benzodiazepine receptors after 5 days of section (Table II). This increase could be due to the appearance of new receptors sites causing a kind of supersensitivity. In favor of this interpretation is the rise in Bmax with no change in K D. Overstreet et al. [14] found no change in the binding of [3H]flunitrazepam after lesions of the septal region. This finding suggests that the cholinergic afferents are not involved in the changes in [3H]flunitrazepam binding observed by us. Because of the close association of benzodiazepine and G A B A receptors in the CNS (see refs. 3 and 20) it would be of interest to investigate how the fimbria-fornix section affects the binding of GABA. This work is planned for the future, but present knowledge suggests that the GABAergic neurons are intrinsic in the hippocampus [19]. If this is the case, it would be another instance in which it might be possible to dissociate G A B A and benzodiazepine receptors. This work was supported by Grants from C O N I C E T and SECYT of Argentina. The excellent technical help of Mrs. Angela Veiga is acknowledged.
1 Braestrup, C. and Squires, R.F., Specific benzodiazepine receptors in rat brain characterized by highaffinity [3H]-diazepambinding, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3805-3809. 2 Braestrup, C., Nielsen, M., Biggio, G. and Squires, R.F., Neuronal localization of benzodiazepine receptors in cerebellum, Neurosci. Lett., 13 (1979) 219-224. 3 Braestrup, C. and Nielsen, M., Benzodiazepine receptors. Arzneim-Forsch. (Drug Res.), 30 (1) 5a (1980) 852-857. 4 Chang, R.S.L., Vinh, T.T. and Synder, S.H. Neurotransmitter receptor localizations: brain lesion induced alterations in benzodiazepine, GABA, c~-adrenergic and histamine Hi-receptor binding, Brain Res., 190 (1980) 95-110.
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