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Benzodiazepine receptors in the rat hippocampal formation: Action of catecholaminergic, serotoninergic and commissural denervation
0306-4522/83/030459-06$003.00/O
.Nrum.sciwcr Vol. 8, No. 3. pp. 459 to 465, 1983 Printed in Great Britain
Pergamon Press Ltd 0 1983IBRO
BENZODIAZEPINE RECEPTORS IN THE RAT HIPPOCAMPAL FORMATION: ACTION OF CATECHOLAMINERGIC, SEROTONINERGIC AND COMMISSURAL DENERVATION M. L. NOVAS,J. H. MEDINA* and E. DE ROBERTIS Instituto de Biologia Celular and *II Cgtedra de Fisiologia, Facultad de Medicina, Universidad de Buenos Aires. Paraguay 2155 (1121) Buenos Aires, Argentina Abstract-The
problem of benzodiazepine receptor localization in the rat hippocampal formation has followed by C3H]flunitrazepam binding studies. The intraventricular injection of 6-hydroxydopamine reduced, after 14 days, the norepinephrine content of the hippocampal formation by 68.4%, and decreased the number of binding sites by 32x, without change in affinity. The intraventricular injection of 5,6 dihydroxytryptamine reduced the serotonin content by 61.5% but did not alter the C3H]flunitrazepam binding. The intraventricular bilateral injection of 0.5 pg kainic acid selectively destroyed the pyramidal neurons in area CA3 of both hippocampi and produced an increase of 28% in C3H]flunitrazepam binding, without change in affinity. These results are discussed in relation to our previous observations about benzodiazepine receptor changes after fimbria-fornix transection. The reduction in [3H]flunitrazepam binding after administration of 6-hydroxydopamine suggests the possible localization of the benzodiazepine receptors on adrenergic presynaptic terminals. The increase in binding sites after destruction of CA3 pyramidal cells, which are the site of origin of commissural fibers, is tentatively interpreted as resulting from the sprouting of mossy fibers that replace the associational-commissural projections.
been approached using several methods of selective deafferentation,
The hippocampal formation of the rat represents a large portion of the cerebral cortex with a regular neuronal organization and well known connections with the rest of the brain. A large proportion of its afferent projections enter dorsally by way of the fimbria-fornix. This route is used by cholinergic afferents coming from the medial septum,8~‘2 noradrenergic fibers from the locus coeruleus,“**’ the less developed dopaminergic input from the ventrotegmental area,23 and the serotoninergic innervation coming from raphe nuclei. I8 Also in the fimbria-fornix are commissural fibers, whose transmitter appears to be the dicarboxylic amino acid glutamate or aspartate.“l14 This connection probably also contains fibers coming from diencephalic regions. lg In a recent study from our laboratory,*l it was found that the unilateral fimbria-fornix transection resulted in considerable changes in benzodiazepine receptors in the hippocampal formation. Two days after the lesion, the maximal binding of C3H]flunitrazepam (C3H]FNZP) decreased about 38x, while after 5 days it dramatically increased by 6574, in the ipsilateral hippocampus. Because of the complexity in the inputs and outputs through the fimbria-fornix, it was
t To whom correspondence should be sent. Abbreviations: 5,6-DHT, FNZP, flunitrazepam; 5-HT, OHDA, h-hydroxydopamine.
5’6-dihydroxytryptamine; 5-hydroxytryptamine; 6459
not possible in that work to relate these receptor changes to a particular pathway. In view of these complexities, we have now approached the problem of benzodiazepine receptor localization by using more selective methods of deafferentation of the hippocampal formation. With this in mind, we have injected 6-hydroxydopamine (6-OHDA) intracerebroventricularly (i.c.v.), a neurotoxin that destroys noradrenergic and dopaminergic nerve terminals’. We also injected i.c.v. 5,6-dihydroxytrypamine (5,6-DHT) which, if associated with desmethylimipramine, preferentially affects the serotoninergic innervation.’ Finally, we have made bilateral i.c.v. injections of kainic acid which, in low doses, selectively destroys the pyramidal cells of the CA3-CA4 regions of the hippocampus.1’14 These neurons are the main source of the commissural fibers that innervate the contralateral hippocampal formation 6 (see Reference 15).
EXPERIMENTAL
PROCEDURES
Male adult Wistar rats (250-300g) were injected intracerebroventricularly on one side with 136 pg of 6-OHDA (calculated as free base), dissolved in 10 ~1 of saline containing O.ly, ascorbic acid. Sham operations, with injection of saline plus ascorbic acid, were used as control. Another group of rats received an intraperitoneal injection of desmethylimipramine HCl (25 mg/kg), 30 min before the uni-
M. L. Novas. J. II. Medina and E. De Robertis
460
Table 1. Norepinephrine
Group Control 6-OHDA 5,6-DHT
and 5-hydroxytryptamine formation
Norepinephrine @g/g) 0.573 k 0.026 (6) 0.181 & 0.04(5) 0.570 + 0.048 (3)
content in the hippocampal
5-Hydroxytryptamine % of control @g/g) 31.6* 99
0.1269 i 0.006 (8) 0.1215 k O.OOS(4) 0.0480 k 0.002 (4)
“/, of control
96 38.5**
Values are expressed as mean + SEM. Number of animals in parentheses. * P < 0.01; **P < 0.001; Student f-test.
lateral i.c.v. administration of 1OOpg (calculated as free base) of 5,6-DHT in 10~1 of saline with ascorbic acid. In this case, also, sham operations were carried out. Both groups were killed 14 days later, for determination of norepinephrine and S-HT content of the hippocampus, and [sH]FNZP binding. Kainic acid was given intracerebroventricularly as described previously.’ The rats were anesthetized with sodium pentobarbital (45 mg/kg ip.) and placed in a stereotaxic frame. Kainic acid (Sigma Co, St. Louis) in OSpg aliquots was dissolved in 2~1 of phosphate buffer saline and was slowly injected into both lateral ventricles. Controls were similarly injected with saline only. After 30 days, the rats were decapitated and both hippocampi were dissected out and homogenized in approximately 10 volumes of Tris HCI buffer, pH 7.4. A glass homogenizer fitted with a Teflon pestle was used and before the binding, the homogenate was stored at -60°C. Norepinephrine and 5-HT levels were measured fluorometrically.’ The binding with [methyl:-:3H] flunitrazepam (86.4 Ci/ mmol, New England Nuclear) was performed using the technique of Braestrup & Squire? with slight moditications.“’ Briefly, for each assay triplicate samples containing 0.2 mg protein, as determined by the Lowry method’ were suspended in 1 ml Tris HCI buffer, pH 7.4. The incubation was carried out at 4°C for 20 min, using 8 nM [sH]FNZP. To study the binding saturation, a range of I-8 nM of [sH]FNZP was used. To determine non-specific binding, parallel experiments were carried out in the presence of 3 PM flunitrazepam. The assays were terminated by filtration through Whatman GF/B glass fiber filters, with 3 washes of 5 ml each of incubation medium. Filters were dried at 100°C and counted in 5 ml Bray’s scintillation solution in a Tracer Spectrometer. Specific binding was calculated as the difference between total and non-specific binding and represented 75-80x.
in which the reduction in Bmax was accompanied by an increase in affinity.” The i.c.v. injection of 5,6-DHT together with the with desmethylimipramine, treatment previous resulted in a 61.5% reduction of 5-HT content of the hippocampal formation, while this was not affected by 6-OHDA (Table 1). In these rats, there was neither a change in the number nor in the affinity of the specific C3H]FNZP binding (Figs 1 and 2B). Confirming previous results’~‘4 the bilateral i.c.v. injection of kainic acid consistently destroyed the pyramidal cells of the region CA3 of both hippocampi. As shown in Fig. 3, this region is completely devoid of pyramidal neurons and there is marked reactive gliosis. In other regions of the hippocampus, including the dentate formation, pyramidal and granule neurons are intact. In these rats, studied 30 days after the injection of kainic acid, there was a striking and statistically significant increase in the number of [3H]FNZP binding sites compared with the sham controls (Fig. l), which
L
L
t
RESULTS
Confirming previous results (see Reference 7) the i.c.v. injection of 6-OHDA decreased the norepinephrine content of the hippocampal formation by 68.4x, while 5,6-DHT had no effect (Table 1). After catecholaminergic denervation, the binding of C3H]FNZP was reduced by about 32%, in comparison with the sham controls (Fig. 1). The saturation curves, analyzed by the Scatchard equation, showed that the reduction in binding was entirely due to a decrease in Bmax without change in affinity (Fig. 2A). This is partially at variance with what was previously observed in the cerebral cortex,
T
c
C n:s
I-
6-CWM n:,3
C
“:I0
5,6-C&IT “:I0
C “3
I_ KA ”
IO
Fig. 1. Histograms representing the percent changes in C3H]flunitrazepam binding after 6-hydroxydopamine (6-OHDA). 5,6-dihydroxytryptamine(5,6_DHT) and kainic acid (KA) in comparison with the corresponding sham controls (c); n, number of rats used. In each case at least four determinations were made. Control values for 6-OHDA = 523.4 _+27.1 fmol/mg, for 5,6-DHT = 502.9 + 29.6 fmol/mg and KA = 458.4 _+ 34.2 fmol per mg protein + SEM. 6-OHDA and 5,6-DHT are 14 days post-lesion and KA is 30 days after lesion. *P < 0.001; **P i 0.02, Student’s r-test.
“_
-
-
-__.
-
Fig. 3. Cresyl Violet-stained section of the dorsal: ~pp~carn~al formation of the rat treated with kainic acid. Observe the disappearance of pyramida) neurons in CA:, (between arrows) with gliosis and the normal aspect of the other areas. The rat was studied 30 days post-lesion. Bar indicates: 0.5 mm.
Benzodiazepine receptors in hippocampal formation amounted to about +28%. The saturation curves showed that the increase in Bmax did not involve any change in Kn (Fig 2C). In Fig. 1, the experiments on binding are expressed as percent of control +SEM, to allow a better com-
[HI
FNZP
BOUND (fmol/ma WOI.)
Scatchard plots of the saturation curves for [3H]flunitrazepam binding after (A) 6-hydroxydopamine, 5,Gdihydroxytryptamine and (C) kainic acid (open squares) and the corresponding sham controls (filled squares). Fig.
2.
463
parison of the changes in binding in the hippocampal formation with the three different lesions used here. DISCUSSION In the introduction we gave some of the main neuroanatomical aspects, related to neuronal organization and connectivity of the hippocampal formation, which justified the present study. Here, we should add that the hippocampal formation, together with some regions of the cerebral cortex and the cerebellar cortex, are the richest areas in benzodiazepine receptors in the central nervous system of mammals4 These areas can be correlated with the main pharmacological actions of benzodiazepine as anxiolytic, anticonv&ant and muscle relaxant drugs (see Reference 13). The hippocampal formation, in addition to being related to some aspects of behaviour, such as, emotional states, memory and learning, tends to generate rhythmic activity and is probably the site where most convulsive phenomena are initiated. In trying to analyze the effect of benzodiazepines on hippocampal functions, an important step would be to learn about the localization of the corresponding receptor. Benzodiazepine receptors could be localized either postsynaptically, on various neurons, or presynaptically, on some of the several nerve terminals that are afferent or intrinsic to the hippocampal formation. In a recent study, using the action of a detergent to remove preferentially the presynaptic membrane in syaaptosomes of the cerebral cortex, Sabato, Aguilar & De Robertis2* reached the conclusion that a large proportion of benzodiazepine receptors were localized presynaptically. This interpretation was strengthened by the finding that one and two days after fimbriafornix transection, there was a considerable decrease in L3H]FNZP binding in the hippocampus, suggesting that a significant ~rcentag~ of receptors could be localized in axon terminals innervating that formation.‘l In these experiments, at 5 days after the lesion, there was a great increase (+ 65oj,) in the number of receptors, which couId be attributed to a kind of postsynaptic supersensitivity. This effect appeared to be transient, since at 14 days it had decreased to only + 14%. The results obtained by the catecholaminergic denervation with GOHDA, in which there is after 14 days a significant decrease in the number of receptors (Fig. l), suggest that those missing binding sites may have been localized on presynaptic axons and nerve terminals of noradrenergic nature. A similar finding has been made in the cerebral cortex.22 It is known that the dopamine levels in the hippocampal formation are twenty times less than norepinephrine,3,23 and this suggests that the dopaminergic innervation may play a minor role in the reduction of benzodiazepine binding sites. The effect of 6-OHDA is rather selective since the serotoninergic denervation, caused by 5,6-DHT, did not change at all the number of benzodiazepine receptors. Furthermore, the eholin-
iC4
M. L. Novas. J. H. Medina and E. De RobertI\
ergic innervation also appears not to be involved, since Overstreet. Speth. Hruska. Ehlert. Dumont & Yamamura. ’ 7 after lesion of the septal region. did not find changes in c3H]FNZP binding. More interesting are the results obtained with the bilateral i.c.v. injection of kainic acid. Confirming the previous report of Nadler et ~1.‘~ we found that. at the doses used, kainic acid selectively destroyed the pyramidal cells of region CA, (Fig. 3). the main source of commissural fibers innervating the contralateral hippocampal formation’ (see Reference 15). By several criteria these fibers appear to carry aspartate or glutamate as the possible neurotransmitter (see Reference 11). The increase (+2836) in C3H]FNZP binding after kainic acid, which represents a change in receptor density withouf alteration of affinity (Fig. 2C), is difficult to interpret on the simple basis of a deafferentation of commissural fibers. Because we have studied the effect of kainic acid only 30 days after the lesion, it is not possible to compare the results with those of the fimbria-fornix transection, in which the commissural fibers and other inputs were lesioned at shorter time intervals.*’ In this previous work, we found that 14 days after the transection there is a reduction in the number of receptors with respect to 5 days. The values are only slightly higher than in the control side (+ 14”;). which suggests a difference from the lesion
by kainic acid. From the previous work of Nadler rt ctl..” we know that, after the kainic acid destruction of pyramidal neurons from which the commissural pathway originates. there is an extensive reinnervation of the remaining hippocampal pyramidal neurons and granule cells of the fascia dentata. This is done by an intense sprouting of the mossy fibers that replace the associational-commissural projections. Since we have carried out our experiments in the same way as Nadler rt L’i and obtained similar results regarding the degeneration of pyramidal neurons, we assume that in our experiments an extensive reinnervation. with increase in mossy fibers boutons, took place. It is thus tempting to postulate that the increase in (13H]FNZP binding is due to receptors localized on those sprouted mossy fibers. We are aware, however, that this is a very tentative conclusion that should be reaffirmed in the future with experiments at different time intervals and with direct observation of the sprouting with specific methods, as was done by Nadler et ~1.‘~
Acknowledyrments-This work was supported by grants from CONICET and SECYT of Argentina. We are grateful to Prof. L. M. Zieher for the determination of the amines and to A. Thorn for making the histological study of the hippocampal formation.
REFERENCES
I. Aguilar J. S.. Jerusalinsky D., Stockert M., Medina J. H. & De Robertis muscarinic receptors after kainic acid lesion of CA, and fimbria-fornix 2. Atack C. (1977) Measurement of biogenic amines using cation exchange physiol. stand. Suppl. 451, 19-60.
E. (1982) Localization of hippocampal transection. Brain Res. In press, chromatography and Buorometric assay. Acre
3. Bischoff S., Scatton B. & Korf J. (1979) Biochemical evidence for a transmitter role of dopamine in the rat hippocampus. Bruin Rrs. 165, 161-165. 4. Braestrup C. & Squires R. (1977) Specific benzodiazepine receptors in the rat brain characterized by high-affinity [‘H] diazepam binding. Proc. natn. Acud. Sci. U.S.A. 74, 3805-3809. 5. Gerson S. & Baldessarini R. J. (1975) Selective destruction of serotonin terminals in rat forebrain by high doses of 5,7_dihydroxytryptamine. Bruin Res. 85, 140-145. 6. Gottlieb D. I. & Cowan W. M. (1973) Autoradiographic studies of the commissural and ipsilateral association connections of the hippocampus and dentata area of the rat--I. The commissural connections. J. camp. Neural. 149, 3999422. 7. Kostrezawa R. M. & Jacobowitz D. M. (1974) Pharmacological actions of 6-hydroxydopamine Pharmac. Rev 26, 199-288. 8. Lewis P. R. & Schute C. C. D. (1967) The cholinergic limbic system. Brain 90, 521-540. 9. Lowry 0. H., Rosenbrough N. J., Farr A. L. & Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. 10. Loy R., Koziell D. A., Lindley J. D. & Moore R. Y. (1980) Noradrenergic innervation of the adult rat hippocampal formation. J. camp. Neural. 189, 699-710. I 1. McGeer P. L. & McGeer E. G. (198 I) Amino acid neurotransmitter. In Basic Neurochemistry (eds Siegel G. et al.) pp. 233-253. Little Brown, Boston. 12. Mosko S.. Lynch G. & Cotman C. M. (1973) The distribution of septal projection to the hippocampus of the rat. J. camp Neural. 152, 163-174. 13. Mtiller W. E. (1981) The benzodiazepine receptor. An update. Pharmacology 22, 1533161. 14. Nadler J. V., Perry B. W. & Cotman C. W. (1978) Intraventricular kainic acid preferentially destroys hippocampal pyramidal cells. Nature, Lond. 271, 676-677. 15. Nadler J. V., Perry B. W. & Cotman C. W. (1980) Selective reinnervation of hippocampal area CA, and the fascia dentata after destruction of CA3-CA4 afferents with kainic acid. Brain Res. 182, l-9.
Benzodiazepine receptors in hippocampal formation
465
16. Nadler J. V., Vaca K. W., White W. F., Lynch G. S. & Cotman C. W. (1976) Aspartate and glutamate as possible transmitter of excitatory hippocampal afferents. Nature, Lord. 260, 538-540. 17. Overstreet D. H., Speth R., Hruska R. E., Ehlert F., Dumont Y. & Yamamura H. I. (1980) Failure of septal lesions to alter muscarinic cholinergic or benzodiazepine binding sites in hippocampus of rat brain. Brain Res. 195,203-207. 18. Parent A., Descarries L. & Beaudet A. (1981) Organization of ascending serotonin systems in the adult rat brain. A radioautographic study after intraventricular administration of C3H] S-hydroxytryptamine. Neuroscience 6, 115-138. 19. Riley J. N. & Moore R. Y. (1981) Diencephalic and brain stem afferents to the hippocampal formation of the rat. Brain Res. Bull. 6, 437-444. 20. Sabato U. C., Aguilar J. S. & De Robertis E. (1981) Benzodiazepine receptors in rat brain. Action of Triton X-100 and localization in relation to the synaptic region. J. Receptor Rex 2, 119-133.
21. Sabato U. C., Aguilar J. S., Medina J. H. & De Robertis E. (1981) Changes in rat hippocampal benzodiazepine receptors and lack of changes in muscarinic receptors after fimbria-fornix lesions. Neurosci. Lett. 27, 193-198. 22. Sabato U. C., Novas M. L., Lowenstein P., Zieher L. M. & De Robertis E. (1981) Action of 6-hydroxydopamine on benzodiazepine receptors in rat cerebral cortex. Eur. J. Pharmac. 73, 381-382. 23. Scatton B., Simon H., Le Meal M. & Bischoff S. (1980) Origin of dopaminergic innervation of the rat hippocampal formation. Neurosci. Lett. 18, 125-131. 24. Storm-Mathisen S. L? Gulberg H. C. (1974) 5-Hydroxytryptamine and noradrenaline in the hippocampal region: effect of transection of afferent pathways on endogenous levels high affinity uptake and some transmitter related enzymes. J. Neurochem. 22, 793-803.
(Accepted 26 August 1982)
.Nrum.sciwcr Vol. 8, No. 3. pp. 459 to 465, 1983 Printed in Great Britain
Pergamon Press Ltd 0 1983IBRO
BENZODIAZEPINE RECEPTORS IN THE RAT HIPPOCAMPAL FORMATION: ACTION OF CATECHOLAMINERGIC, SEROTONINERGIC AND COMMISSURAL DENERVATION M. L. NOVAS,J. H. MEDINA* and E. DE ROBERTIS Instituto de Biologia Celular and *II Cgtedra de Fisiologia, Facultad de Medicina, Universidad de Buenos Aires. Paraguay 2155 (1121) Buenos Aires, Argentina Abstract-The
problem of benzodiazepine receptor localization in the rat hippocampal formation has followed by C3H]flunitrazepam binding studies. The intraventricular injection of 6-hydroxydopamine reduced, after 14 days, the norepinephrine content of the hippocampal formation by 68.4%, and decreased the number of binding sites by 32x, without change in affinity. The intraventricular injection of 5,6 dihydroxytryptamine reduced the serotonin content by 61.5% but did not alter the C3H]flunitrazepam binding. The intraventricular bilateral injection of 0.5 pg kainic acid selectively destroyed the pyramidal neurons in area CA3 of both hippocampi and produced an increase of 28% in C3H]flunitrazepam binding, without change in affinity. These results are discussed in relation to our previous observations about benzodiazepine receptor changes after fimbria-fornix transection. The reduction in [3H]flunitrazepam binding after administration of 6-hydroxydopamine suggests the possible localization of the benzodiazepine receptors on adrenergic presynaptic terminals. The increase in binding sites after destruction of CA3 pyramidal cells, which are the site of origin of commissural fibers, is tentatively interpreted as resulting from the sprouting of mossy fibers that replace the associational-commissural projections.
been approached using several methods of selective deafferentation,
The hippocampal formation of the rat represents a large portion of the cerebral cortex with a regular neuronal organization and well known connections with the rest of the brain. A large proportion of its afferent projections enter dorsally by way of the fimbria-fornix. This route is used by cholinergic afferents coming from the medial septum,8~‘2 noradrenergic fibers from the locus coeruleus,“**’ the less developed dopaminergic input from the ventrotegmental area,23 and the serotoninergic innervation coming from raphe nuclei. I8 Also in the fimbria-fornix are commissural fibers, whose transmitter appears to be the dicarboxylic amino acid glutamate or aspartate.“l14 This connection probably also contains fibers coming from diencephalic regions. lg In a recent study from our laboratory,*l it was found that the unilateral fimbria-fornix transection resulted in considerable changes in benzodiazepine receptors in the hippocampal formation. Two days after the lesion, the maximal binding of C3H]flunitrazepam (C3H]FNZP) decreased about 38x, while after 5 days it dramatically increased by 6574, in the ipsilateral hippocampus. Because of the complexity in the inputs and outputs through the fimbria-fornix, it was
t To whom correspondence should be sent. Abbreviations: 5,6-DHT, FNZP, flunitrazepam; 5-HT, OHDA, h-hydroxydopamine.
5’6-dihydroxytryptamine; 5-hydroxytryptamine; 6459
not possible in that work to relate these receptor changes to a particular pathway. In view of these complexities, we have now approached the problem of benzodiazepine receptor localization by using more selective methods of deafferentation of the hippocampal formation. With this in mind, we have injected 6-hydroxydopamine (6-OHDA) intracerebroventricularly (i.c.v.), a neurotoxin that destroys noradrenergic and dopaminergic nerve terminals’. We also injected i.c.v. 5,6-dihydroxytrypamine (5,6-DHT) which, if associated with desmethylimipramine, preferentially affects the serotoninergic innervation.’ Finally, we have made bilateral i.c.v. injections of kainic acid which, in low doses, selectively destroys the pyramidal cells of the CA3-CA4 regions of the hippocampus.1’14 These neurons are the main source of the commissural fibers that innervate the contralateral hippocampal formation 6 (see Reference 15).
EXPERIMENTAL
PROCEDURES
Male adult Wistar rats (250-300g) were injected intracerebroventricularly on one side with 136 pg of 6-OHDA (calculated as free base), dissolved in 10 ~1 of saline containing O.ly, ascorbic acid. Sham operations, with injection of saline plus ascorbic acid, were used as control. Another group of rats received an intraperitoneal injection of desmethylimipramine HCl (25 mg/kg), 30 min before the uni-
M. L. Novas. J. II. Medina and E. De Robertis
460
Table 1. Norepinephrine
Group Control 6-OHDA 5,6-DHT
and 5-hydroxytryptamine formation
Norepinephrine @g/g) 0.573 k 0.026 (6) 0.181 & 0.04(5) 0.570 + 0.048 (3)
content in the hippocampal
5-Hydroxytryptamine % of control @g/g) 31.6* 99
0.1269 i 0.006 (8) 0.1215 k O.OOS(4) 0.0480 k 0.002 (4)
“/, of control
96 38.5**
Values are expressed as mean + SEM. Number of animals in parentheses. * P < 0.01; **P < 0.001; Student f-test.
lateral i.c.v. administration of 1OOpg (calculated as free base) of 5,6-DHT in 10~1 of saline with ascorbic acid. In this case, also, sham operations were carried out. Both groups were killed 14 days later, for determination of norepinephrine and S-HT content of the hippocampus, and [sH]FNZP binding. Kainic acid was given intracerebroventricularly as described previously.’ The rats were anesthetized with sodium pentobarbital (45 mg/kg ip.) and placed in a stereotaxic frame. Kainic acid (Sigma Co, St. Louis) in OSpg aliquots was dissolved in 2~1 of phosphate buffer saline and was slowly injected into both lateral ventricles. Controls were similarly injected with saline only. After 30 days, the rats were decapitated and both hippocampi were dissected out and homogenized in approximately 10 volumes of Tris HCI buffer, pH 7.4. A glass homogenizer fitted with a Teflon pestle was used and before the binding, the homogenate was stored at -60°C. Norepinephrine and 5-HT levels were measured fluorometrically.’ The binding with [methyl:-:3H] flunitrazepam (86.4 Ci/ mmol, New England Nuclear) was performed using the technique of Braestrup & Squire? with slight moditications.“’ Briefly, for each assay triplicate samples containing 0.2 mg protein, as determined by the Lowry method’ were suspended in 1 ml Tris HCI buffer, pH 7.4. The incubation was carried out at 4°C for 20 min, using 8 nM [sH]FNZP. To study the binding saturation, a range of I-8 nM of [sH]FNZP was used. To determine non-specific binding, parallel experiments were carried out in the presence of 3 PM flunitrazepam. The assays were terminated by filtration through Whatman GF/B glass fiber filters, with 3 washes of 5 ml each of incubation medium. Filters were dried at 100°C and counted in 5 ml Bray’s scintillation solution in a Tracer Spectrometer. Specific binding was calculated as the difference between total and non-specific binding and represented 75-80x.
in which the reduction in Bmax was accompanied by an increase in affinity.” The i.c.v. injection of 5,6-DHT together with the with desmethylimipramine, treatment previous resulted in a 61.5% reduction of 5-HT content of the hippocampal formation, while this was not affected by 6-OHDA (Table 1). In these rats, there was neither a change in the number nor in the affinity of the specific C3H]FNZP binding (Figs 1 and 2B). Confirming previous results’~‘4 the bilateral i.c.v. injection of kainic acid consistently destroyed the pyramidal cells of the region CA3 of both hippocampi. As shown in Fig. 3, this region is completely devoid of pyramidal neurons and there is marked reactive gliosis. In other regions of the hippocampus, including the dentate formation, pyramidal and granule neurons are intact. In these rats, studied 30 days after the injection of kainic acid, there was a striking and statistically significant increase in the number of [3H]FNZP binding sites compared with the sham controls (Fig. l), which
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RESULTS
Confirming previous results (see Reference 7) the i.c.v. injection of 6-OHDA decreased the norepinephrine content of the hippocampal formation by 68.4x, while 5,6-DHT had no effect (Table 1). After catecholaminergic denervation, the binding of C3H]FNZP was reduced by about 32%, in comparison with the sham controls (Fig. 1). The saturation curves, analyzed by the Scatchard equation, showed that the reduction in binding was entirely due to a decrease in Bmax without change in affinity (Fig. 2A). This is partially at variance with what was previously observed in the cerebral cortex,
T
c
C n:s
I-
6-CWM n:,3
C
“:I0
5,6-C&IT “:I0
C “3
I_ KA ”
IO
Fig. 1. Histograms representing the percent changes in C3H]flunitrazepam binding after 6-hydroxydopamine (6-OHDA). 5,6-dihydroxytryptamine(5,6_DHT) and kainic acid (KA) in comparison with the corresponding sham controls (c); n, number of rats used. In each case at least four determinations were made. Control values for 6-OHDA = 523.4 _+27.1 fmol/mg, for 5,6-DHT = 502.9 + 29.6 fmol/mg and KA = 458.4 _+ 34.2 fmol per mg protein + SEM. 6-OHDA and 5,6-DHT are 14 days post-lesion and KA is 30 days after lesion. *P < 0.001; **P i 0.02, Student’s r-test.
“_
-
-
-__.
-
Fig. 3. Cresyl Violet-stained section of the dorsal: ~pp~carn~al formation of the rat treated with kainic acid. Observe the disappearance of pyramida) neurons in CA:, (between arrows) with gliosis and the normal aspect of the other areas. The rat was studied 30 days post-lesion. Bar indicates: 0.5 mm.
Benzodiazepine receptors in hippocampal formation amounted to about +28%. The saturation curves showed that the increase in Bmax did not involve any change in Kn (Fig 2C). In Fig. 1, the experiments on binding are expressed as percent of control +SEM, to allow a better com-
[HI
FNZP
BOUND (fmol/ma WOI.)
Scatchard plots of the saturation curves for [3H]flunitrazepam binding after (A) 6-hydroxydopamine, 5,Gdihydroxytryptamine and (C) kainic acid (open squares) and the corresponding sham controls (filled squares). Fig.
2.
463
parison of the changes in binding in the hippocampal formation with the three different lesions used here. DISCUSSION In the introduction we gave some of the main neuroanatomical aspects, related to neuronal organization and connectivity of the hippocampal formation, which justified the present study. Here, we should add that the hippocampal formation, together with some regions of the cerebral cortex and the cerebellar cortex, are the richest areas in benzodiazepine receptors in the central nervous system of mammals4 These areas can be correlated with the main pharmacological actions of benzodiazepine as anxiolytic, anticonv&ant and muscle relaxant drugs (see Reference 13). The hippocampal formation, in addition to being related to some aspects of behaviour, such as, emotional states, memory and learning, tends to generate rhythmic activity and is probably the site where most convulsive phenomena are initiated. In trying to analyze the effect of benzodiazepines on hippocampal functions, an important step would be to learn about the localization of the corresponding receptor. Benzodiazepine receptors could be localized either postsynaptically, on various neurons, or presynaptically, on some of the several nerve terminals that are afferent or intrinsic to the hippocampal formation. In a recent study, using the action of a detergent to remove preferentially the presynaptic membrane in syaaptosomes of the cerebral cortex, Sabato, Aguilar & De Robertis2* reached the conclusion that a large proportion of benzodiazepine receptors were localized presynaptically. This interpretation was strengthened by the finding that one and two days after fimbriafornix transection, there was a considerable decrease in L3H]FNZP binding in the hippocampus, suggesting that a significant ~rcentag~ of receptors could be localized in axon terminals innervating that formation.‘l In these experiments, at 5 days after the lesion, there was a great increase (+ 65oj,) in the number of receptors, which couId be attributed to a kind of postsynaptic supersensitivity. This effect appeared to be transient, since at 14 days it had decreased to only + 14%. The results obtained by the catecholaminergic denervation with GOHDA, in which there is after 14 days a significant decrease in the number of receptors (Fig. l), suggest that those missing binding sites may have been localized on presynaptic axons and nerve terminals of noradrenergic nature. A similar finding has been made in the cerebral cortex.22 It is known that the dopamine levels in the hippocampal formation are twenty times less than norepinephrine,3,23 and this suggests that the dopaminergic innervation may play a minor role in the reduction of benzodiazepine binding sites. The effect of 6-OHDA is rather selective since the serotoninergic denervation, caused by 5,6-DHT, did not change at all the number of benzodiazepine receptors. Furthermore, the eholin-
iC4
M. L. Novas. J. H. Medina and E. De RobertI\
ergic innervation also appears not to be involved, since Overstreet. Speth. Hruska. Ehlert. Dumont & Yamamura. ’ 7 after lesion of the septal region. did not find changes in c3H]FNZP binding. More interesting are the results obtained with the bilateral i.c.v. injection of kainic acid. Confirming the previous report of Nadler et ~1.‘~ we found that. at the doses used, kainic acid selectively destroyed the pyramidal cells of region CA, (Fig. 3). the main source of commissural fibers innervating the contralateral hippocampal formation’ (see Reference 15). By several criteria these fibers appear to carry aspartate or glutamate as the possible neurotransmitter (see Reference 11). The increase (+2836) in C3H]FNZP binding after kainic acid, which represents a change in receptor density withouf alteration of affinity (Fig. 2C), is difficult to interpret on the simple basis of a deafferentation of commissural fibers. Because we have studied the effect of kainic acid only 30 days after the lesion, it is not possible to compare the results with those of the fimbria-fornix transection, in which the commissural fibers and other inputs were lesioned at shorter time intervals.*’ In this previous work, we found that 14 days after the transection there is a reduction in the number of receptors with respect to 5 days. The values are only slightly higher than in the control side (+ 14”;). which suggests a difference from the lesion
by kainic acid. From the previous work of Nadler rt ctl..” we know that, after the kainic acid destruction of pyramidal neurons from which the commissural pathway originates. there is an extensive reinnervation of the remaining hippocampal pyramidal neurons and granule cells of the fascia dentata. This is done by an intense sprouting of the mossy fibers that replace the associational-commissural projections. Since we have carried out our experiments in the same way as Nadler rt L’i and obtained similar results regarding the degeneration of pyramidal neurons, we assume that in our experiments an extensive reinnervation. with increase in mossy fibers boutons, took place. It is thus tempting to postulate that the increase in (13H]FNZP binding is due to receptors localized on those sprouted mossy fibers. We are aware, however, that this is a very tentative conclusion that should be reaffirmed in the future with experiments at different time intervals and with direct observation of the sprouting with specific methods, as was done by Nadler et ~1.‘~
Acknowledyrments-This work was supported by grants from CONICET and SECYT of Argentina. We are grateful to Prof. L. M. Zieher for the determination of the amines and to A. Thorn for making the histological study of the hippocampal formation.
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(Accepted 26 August 1982)