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Cholinergic and GABAergic forebrain projections to the habenula and nucleus interpeduncularis: Surgical and kainic acid lesions
287
Brain Research, 275(1983) 287-297 Elsevier
Cholinergic and GABAergic Forebrain Projections to the Habenula and Nucleus Interpeduncularis: Surgical and Kainic Acid Lesions A. CONTESTABILE* and F. FONNUM**
Norwegian Defence Research Establishment, Divisionfor Toxicology, P.O. Box 25, 2007 KjeUer(Norway) (Accepted February 2nd, 1983)
Key words: acetylchofine - - choline acetyltransferase-- G A B A - - glutamate decarboxylase - - habenula-- nucleus interpeduncularis - - septum - - nucleus triangnlaris septi - - nucleus septo-fimbrialis
The forebrain cholinergic and GABAergic projections to the habenula and nucleus interpeduncularis have been investigated by means of surgical and kainic acid lesions. Bilateral transection of the stria medullaris caused a 50% decrease of choline acetyltransferase in both the habenula and nucleus interpeduncularis, and a 65% decrease of glutamate decarboxylase in the habenula. Electrolytic lesions of the posterior septum (nucleus triangnlaris septi and nucleus septo-fimbrialis) accounted for at least 30-40% decrease of the cholinergic parameter in the habenula and nucleus interpeduncularis. Moreover, the choline acetyltransferase decrease in the habenula appeared restricted to the medial part of the nucleus. Kainic acid injections causing very large neuronal destruction in the nucleus of the diagonal band of Broca, and more than 70% decrease of choline acetyltransferase in the dorsal hippocampus, did not affect the cholinergic parameter in either the medial or lateral habenula or nucleus interpeduncularis. Kainic acid injections in the nucleus entopeduncularis resulted in a 40% decrease of glutamate decarboxylase in the habenula. Kainic acid injections in the nucleus of the diagonal band were accompanied by a 40% decrease of glutamate decarboxylase in the medial subdivision only. The present study points at the nuclei of the posterior septum as the source of a major cholinergic projection to the habenula and nudens interpeduncularis, and reveals a previously unsuspected GABAergic input from the nucleus of the diagonal band to the medial habenula. INTRODUCTION
The neural pathway constituted of stria medullaris-habenula-fasciculus retroflexus-nucleus interpeduncularis, is one of the most important connections between forebrain centers and midbrain structures. These anatomical connections have been extensively studied by lesion methods~,30,32 and, more recently, by techniques based on anterograde and retrograde transport of tracer moleculesS,1S,tg,2s,36. The orion of the cholinergic input to the habenula and nucleus interpeduncularis has been a controversial issue. It was first suggested that the cholinergic input to the nucleus interpeduncularis originated in the habenulaex; both the medial17 and the lateral part 7,33 have been suggested. More recently, an additional cholinergic input originating in the nucleus of the diagonal band of Broca and reaching the habenu-
la and nucleus interpeduncularis through the stria medullaris-fasciculus retroflexus system, has been proposedX3,14. GABAergic projections to the lateral habenula have been supposed to originate in the nucleus entopeduncularis and the nucleus of the diagonal band of Brocal6, 29. Furthermore, a direct habenula-interpeduncular substance P projection has been proposed on the basis of experimental studiesT,20. In addition to the discrepancies in the neurochemical literature, there are several important controversial points concerning the anatomical relationships between the different nuclei4,1sA9,28,36. The present paper is a reappraisal of the neurotransmitters involved in the main projections from forebrain areas to the habenula and nucleus interpeduncularis by using not only the conventional lesion methods but, whenever possible, the selective neuron cell body lesions caused by stereotaxic injec-
* Permanent address: Institute of Comparative Anatomy, University of Bologna, 40126 Bologna, Italy. ** To whom correspondence should be addressed.
288 tions of kainic acid. In particular, neurochemical parameters related to cholinergic and GABAergic projections have been studied. Particular attention has been paid to some important projections recently revealed by the use of neuroanatomical tracer methods from two small nuclei of the posterior septum, the nucleus triangularis septi and the nucleus septo-fimbria1is18.36. EXPERIMENTAL PROCEDURES
Experimental Male Wistar rats weighing 200-250 g were anesthetized with Valium (Roche) (0.4 rag/100 g body weight, i.p.) and Hypnorm vet. (Mekos) (0.7 ml/100 g body weight, s.c.). The animals were placed in a David Kopf stereotaxic frame for the subsequent experimental manipulation. The stereotaxic coordinates of the Koenig and Klippe123 atlas were used. (1) Bilateral transection of the stria medullaris was performed by lowering small razor blade splits (2 mm wide) to a depth of 6.5 mm below the skull at the A/P coordinate +5800. The razor blade splits were secured to a holder connected to the vertical pillar of the stereotaxic frame. In order to avoid damage of the sinus sagittalis, the medial edge of the blade was placed 0.5 mm lateral to the midline and inserted in the brain at an angle of 10° . Five days after the operation the rats were decapitated, the brains were immediately removed and sliced in a cold room with a Sorvail tissue chopper. The part of the brain rostral to the habenula was transferred to a cryostatic chamber and sectioned for histological confirmation of the lesion. The habenula and nucleus interpeduncularis were dissected from the tissue chopper slices under the stereomicroscope and homogenized in 150 #l (left and right habenula) or 90/zl (nucleus interpeduncularis) of 0.32 M sucrose for enzyme and protein determination. Unoperated animals of the same lots were used as the control. (2) The area of the posterior septum (nucleus triangularis septi and nucleus septo-fimbrialis) was lesioned using a tungsten electrode insulated except at the tip, delivering a current of 5 m A for 20 s at the following coordinates: A/P + 6600, M/L +_ 0.1, D/V + 0.8. Five or 6 days after the operation the animals were killed, areas of interest were dissected for neurochemical analysis and homogenized as specified
above. The rostral part of the brain was sectioned in the cryostat in order to verify the lesion extension. The posterior septum of an additional group of animals was electrolytically lesioned for enzyme determination. The brains of these animals were immediately frozen under a COe stream, transferred to the cryostat, cut at 40/.tm thickness and freeze-dried 11. The habenular region was dissected from the freezedried sections under the streomicroscope, using very small razor blade splits fixed to a holder. The stria medullaris layer overlying the habenula proper was cut and discarded. The medial and lateral habenula were separated, weighed with a fish-pole balance and assayed as previously described 11. For each animal, 18-20 samples of each habenular subdivision were dissected in the area comprised approximately between the coordinates 23 + 3350 and + 4150. The nucleus interpeduncularis was similarly dissected and 12-14 samples were taken from approximately coordinates23 + 1250 and + 1950. The usual dry weight range of individual samples was 0.5 pg for the medial habenula, 2-4~g for the lateral habenula and 3.5-6.5 pg for the nucleus interpeduncularis. As control for both experimental groups, sham-operated animals were used in which the electrode was positioned 0.5 mm above the lesion point and no current was delivered. (3) Kainic acid was injected bilaterally in different brain areas as follows. Posterior septum: 0.5 or 0.75 pg in 0.5/~1 saline at A/P + 6600, M/L + 0.5, D/V + 0.8. Nucleus entopeduncularis: 0.4pg in 0.5 pl saline at A/P + 5200, M/L + 2.4, D / V - - 1.8. Nucleus of the diagonal band of Broca: 1/~g in 0.5 gl saline at A/P + 7600, M/L + 0.5, D / V - - 2.2. For each experimental group controls were animals in which 0.5 pl saline had been injected in the different nuclei. The injections of kainic acid and saline were performed with a stainless steel cannula connected to a 20 gl Hamilton syringe mounted o n a slow-injection apparatus working a t a rate of 0.25 gl/min, over a period of 2 min. The cannula was left in situ for 5 min at the end of each injection. Kainic acid was dissolved in 0.9% NaCi and adjusted to pH 7.2-7.3 with NaOH. Both kainic acid and salinetreated animals were killed and processed for biochemical assays as detailed above. The survival time was 5-6 days.
289 An additional experimental group of rats with kainic acid injections in the nucleus of the diagonal band was used for freeze-dried sections. In this experimental group the medial and lateral habenula were separately dissected and 24 samples were obtained for each habenular subdivision in the area comprised between the coordinates mentioned above. Twelve alternate samples were assayed for choline acetyltransferase (CHAT) and 12 for glutamate decarboxylase (GAD). E n z y m e assays
Homogenates were activated with Triton X-100 (0.3% final concentration) before assays for enzyme activities. ChAT 10, GAD1,11 and acetylcholinesterase (ACHE) 35 were assayed by previously published methods. The activity was expressed either as protein weight26 or as dry weight 11. Histology
The part of the brain in which lesions or kainic acid injections had been performed, was cut in the cryostat at 40/~m thickness. The sections, mounted on slides, were stained with May-Grunwald and Giemsa in order to verify the extension of the lesion or to evaluate the neuronal damage caused by kainic acid. Other rats, in which kainic acid or saline had been injected in the various nuclei, were perfusion fixed with 10% formalin and used for histological purposes. The brains of these rats were cut with the freezing microtome and stained with cresyl violet. RESULTS Lesion experiments
Bilateral transection of the stria medullaris caused
a 50% drop of ChAT in the habenula and nucleus interpeduncularis, and a 65% decrease of G A D in the habenula (Table I). No significant decrease of A C h E was observed in either nuclei. Lesions performed in the area of the posterior septum caused a signicant decrease of ChAT in both the habenula and nucleus interpeduncularis (35% and 40% respectively, Table II). Further experiments carded out with freeze dried sections to allow better dissection (Table II) revealed more than 30% decrease of ChAT in the medial habenula and nucleus interpeduncularis, while the decrease found in the lateral habenula was not statistically significant. No decrease of G A D and AChE was noticed in habenula or nucleus interpeduncularis (Table II). Histological controls showed that differential destruction of the nucleus triangularis septi and nucleus septo-fimbrialis was obtained, while no encroachment of the stria medullaris occurred (Figs. 1 and 2). The necessity to avoid damage of the stria medullaris reduced the extension of the lesions so that in no case was complete destruction of both nuclei of the posterior septum achieved. The nucleus triangularis septi was usually almost completely destroyed, with the exception of its rostralmost and sometimes its caudalmost part (Fig. 1). The medial parts of the nuclei septo-fimbrialis were usually completely destroyed, while the lateral parts were in most cases partially spared (Fig. 1). Kainic acid experiments
Kainic acid was injected in the posterior septum, the nucleus entopeduncularis and the nucleus of the diagonal band as specified in Experimental Procedures No remarkable neurotoxic effect of kainic acid
TABLE I Effect of bilateral transection of the stria medullaris on enzymes in the habenula and nucleus interpeduncularis
Values are expressed as the mean _S.E.; the number of animals is in brackets. Enzyme activities are expressed as/~mol/gprot./h for ChAT and GAD, and mmoi/g prot./h for ACHE. Habenula
ChAT GAD AChE
Nucleus interpeduncularis
Control (8)
Lesioned (8)
Control (8)
Lesioned (8)
201 + 12.7 431 + 23.7 6.28 + 0.25
102 + 4.5** 151 + 3.7** 5.60 + 0.22
1200 + 74.5 451 + 10.3 13.37 + 0.33
646 + 39.1'* 409 + 22.4 13.45 + 0.62
** P < 0.001 (Student's t-test).
290 TABLE II Effect of electrolytic lesions of the posterior septum on enzymes in the habenula and nucleus interpeduncularis Values are expressed as the mean +S.E.; the number of animals is in brackets. Enzyme activities are expressed as/tmol/g prot./h for ChAT and GAD, and mmol/g prot./h for ACHE. For freeze-dried sections ChAT activity is expressed as/*mol/g dry weight/h (see the text for details). (a) Fresh tissue homogenates Nucleus interpeduncularis
Habenula
ChAT GAD AChE
Sham (9)
Lesioned (9)
Sham (9)
Lesioned (9)
184 + 8.7 437 + 15.6 5.69 + 0.39
130 + 5.3** 443 + 17.8 5.75 + 0.38
1098 + 50.9 475 + 27.7 11.28 + 0.64
676 + 35.2** 488 + 20 11.97 _ 0.98
(b) Freeze-dried sections Medial habenula
ChAT
Lateral habenula
Nucleus interpeduncularis
Sham (7)
Lesioned (6)
Sham (7)
Lesioned (6)
Sham (7)
Lesioned (7)
721 + 41.9
500 + 25.9*
105.1 + 8.88
85.3 + 6.86
961 + 44.8
666 + 38.6**
* P < 0.002; ** P < 0.001 (Student's t-test). could be noticed in the nuclei of the posterior septurn. In histological controls the neural damage appeared restricted to a small area a r o u n d the center of the injection site. The majority of the n e u r o n a l population was therefore unaffected by kainic acid in both nuclei of the posterior septum. I n addition, preliminary experiments showed no modification of enzyme activities in the h a b e n u l a and nucleus interpeduncularis after such injections.
Bilateral injections of kainic acid in the nucleus of the diagonal b a n d caused extensive neural damage to the nucleus itself and partially to the adjacent areas (Figs. 3 a n d 4). The vertical limb of the nucleus appeared more sensitive to the neurotoxic effect of kainic acid, since complete n e u r o n a l disappearance was usually observed in this area for all the rostrocaudal extension of the nucleus. The n e u r o n s of the horizontal limb of the nucleus were less sensitive to
TABLE III Effect of bilateral injections of kainic acid (1/~g) in the nucleus of the diagonal band on enzymes in the habenula and nucleus interpeduncularis Values are expressed as the mean +S.E.; the number of animals is in brackets. Enzyme activities are expressed as/~moi/g prot./h for ChAT and GAD and ramol/g prot./h for ACHE. For freeze-dried sections enzyme activities are expressed as/~mol/g dry weight/h. (a) Fresh tissue homogenates Nucleus interpeduncularis
Habenula
ChAT GAD AChE
Saline (11)
Kainic (9)
Saline (11)
Kainic (9)
173 + 5.6 375 + 22.9 5.36 + 0.14
171 + 12 326 + 19.1 5.55 + 0.22
1112 + 44.9 415 _+14.4 10.12 __.0.32
1005 +__36.9 465 + 13.5 10.88 + 0.53
(b) Freeze-dried sections Medial habenula
ChAT GAD
Lateral habenula
Saline (5)
Kainic (5)
Saline (5)
Kainic (5)
750 + 25.8 153.4 + 10.7
749 + 54.7 91.2 + 2.1"*
98.6 + 12.2 640 _+48.3
107.7 + 3.8 617 _+61.3
** P < 0.001 (Student's t-test).
291
O
Fig. 1. Schematic drawing showing the extension of the electrolyric lesions in the area of the posterior septum from the approximate coordinates 6300 (A) to 6900 (D) of the Koenig and Klippel (1963) atlas. Fig. 2. Example of a lesion in the area of the posterior septum at the level of the nucleus triangularis septi. Note the undamaged striae medullaris (arrows). x32. the neurotoxic action. Some of the large neurons of the horizontal limb were usually spared, with some differences among the various cases examined, also depending on the distance from the injection site. After examination of histological controls, only those cases were selected in which at least 70-80% destruction of the horizontal limb could be assessed by light microscopic analysis. To confirm the actual damage of the nucleus, the dorsal hippocampus overlying the habenular nuclei was dissected and assayed for ChAT activity. Kainic acid injections in the diagonal
band nucleus caused a dramatic decrease of ChAT in the dorsal hippocampus (saline-injected animals (11) 66.4 + 1.7/~mol/g prot./h; kainic add-injected animals (9) 18.5 + 2.3/~mol/g prot./h). Kainic acid lesions of the nucleus of the diagonal band which led to a greater than 70% drop of ChAT in the dorsal hippocampus, did not affect ChAT levels in either the habenula or nucleus interpeduncularis (Table III). G A D activity showed a small decrease in the habenula which was, however, not statistically significant (P ~ 0.1). AChE was unaffected in both the habenula and nucleus interpeduncularis. When the analyses were performed on freezedried sections, which enabled us to separate the medial and lateral habenula, a significant decrease of G A D (40%) was observed in the medial habenula, while ChAT remained unaffected (Table III). Bilateral injections of kalnic acid in the nucleus entopeduncularis led to a virtually complete disappearance of the neuronal population (Figs. 5 and 6). The injections also partially affected the adjacent region of the lateral hypothalamus, which showed a less dramatic decrease of neuronal population. Kainic acid injections in the nucleus entopeduncularis caused marked decrease of G A D in the habenula (40%), leaving ChAT unaffected (Table IV). A C h E activity was not affected. Histological controls did not show any evidence of distant neuronal damage in the nucleus entopeduncularis after kainic acid injections in the nucleus of the diagonal band or, conversely, in the nucleus of the diagonal band after injections in the nucleus entopeduncularis. There was no evidence of damage to neurons of the habenula and nucleus interpeduncularis. TABLE IV Effect of bilateral kainic acid (0.4 l~g) injections in the nucleus entopeduncularis on enzymes in the habenula
Values are expressed as the mean +S.E.; the number of animals is in brackets. Enzyme activities are expressed as/~mol/g prot./h for ChAT and GAD and mmol/g prot./h for ACHE.
ChAT GAD AChE
Saline (8)
Kainic (9)
164 + 5.7 384 + 14 4.67 + 0.13
188 + 9.4 236 + 5.8** 4.59 + 0.15
** P < 0.001 (Student's t-test).
292
Figs. 3 and 4. Effect of kainic acid on the nucleus of the diagonal band. In the kainic acid-injected nucleus (Fig. 3) an almost complete disappearance of nerve cell bodies is apparent in comparison with the saline-injected nucleus (Fig. 4). x60. Figs. 5 and 6. Effect of kainic acid on the nucleus entopeduncularis. In the kainic acid- injected nucleus (Fig. 5) a complete loss of nerve cell bodies and a remarkable glial proliferation can be noticed in comparison with the saline-injected nucleus (Fig. 6). x 100.
293 DISCUSSION
The septal cholinergic system to the habenula and nucleus interpeduncularis
Fig. 7. Diagram summarizing the present knowledge on the identity of neurotransmitters involved in the forebrain projections to the habenula and nucleus interpeduncularis and in the connections between the two nuclei. Solid lines indicate projections for which clear evidence of the neurotransmitter involved has been reached. A question mark indicates that the identity of the putative neurotransmitter still raises some doubt. Dashed lines indicate that the neurotransmitter involved in the projection is unknown or that the available information is too scarce. Note that anatomical studies36indicate that the nucleus septo-funbrialis (NSF) only projects to the medial habennla, whereas the nucleus tdangulads septi (NTS) projects to both the medial habenula (MH) and the nucleus interpeduncularis (NIP). Since both nuclei are affected by a decrease of ChAT after lesions of the posterior septum, we assume that the projection from the nucleus triangularis septi is cholinergic; it is likely, but not certain, that also the projection from the nucleus septo-fimbdalis is cholinergic. The projection from the nucleus entopeduncularis (NE) to the lateral habenula (LH) is GABAergic whereas no conclusive data exist for the area of the lateral hypothalamus (LHy). The projection from the nucleus of the diagonal band (NDB) to the medial habenula is GABAergic whereas we could not confirm that the same is true for the lateral habenula. No information is available concerning the connection between the lateral habenula and the nucleus interpeduncularis. The majority of the available anatomical and neurochemical data seems to point at the medial habenula as the source of the cholinergic and a substance P projection to the nucleus interpeduncularis. However, the question cannot be considered completely settled, since selective lesions of the neurons of the medial habenula are at present very difficult to obtain.
The results of the p r e s e n t p a p e r only partially supp o r t the conclusions o f previous studies. The overall picture of the cholinergic input from the septal areas to the h a b e n u l a and nucleus interpeduncularis appears, in fact, rather different as far as the nuclei of origin are concerned. The present results confirm that about 50% of the cholinergic activity in the h a b e n u l a and nucleus interpeduncularis originates from a forebrain innervation conveyed through the stria medullaris13,14. H o w e v e r , the main source of this cholinergic innervation does not a p p e a r to be the nucleus of the diagonal band13,t4 but the nuclei of the posterior septal division (Table II). The lesion o f these nuclei in fact accounts for a conspicuous portion of C h A T decrease in the habenula and nucleus interpeduncularis after stria medullads transection (Table I). M o r e precisely, since the nucleus trangularis septi is known from anatomical study6, 36 to p r o j e c t to both the h a b e n u l a and nucleus interpeduncularis, we m a y assume from o u r results that both projections are cholinergic. T h e nucleus septo-fimbrialis only projects to the medial h a b e n u 1a18,36. O u r results suggest that also this p r o j e c t i o n is cholinergic, but, since no selective lesions of the nucleus septo-fimbrialis were achieved in the present study, this question cannot be considered finally settled. It was unfortunate that in the present study kainic acid did not show appreciable neurotoxic effect towards the neurons of the two nuclei of the posterior septum, since kainic acid lesions allow selective degeneration of neuronal cell bodies sparing fibers of passage. In our cases, however, careful examination of histological controls could exclude lesions of the stria medullaris and, of course, of the nucleus diagonal band which lies very far from the lesioned area. M o r e o v e r , no decrease of G A D was noticed in the habenula, while lesions of the stria medu|laris would have affected G A D levels to a larger extent than C h A T levels (Table I). It should also be stated that the quantitative d a t a of the cholinergic input in the present study are m i n i m u m values, since the lesions e m p l o y e d always s p a r e d part of the septal nuclei involved.
294 The results obtained with kainic acid injections in the nucleus of the diagonal band of Broca gave indirect support to our conclusions. Our results in fact strongly suggest that a major cholinergic innervation to the habenula and nucleus interpeduncularis is unlikely to derive from the nucleus of the diagonal band. Kainic acid lesions caused a very large, even if not complete, neuronal loss in the nucleus of the diagonal band and a substantial drop of ChAT in the hippocampus, which is known to be a major target of cholinergic input from the nucleus of the diagonal band25,27,31. The same animals, however, did not show any decrease of ChAT in the habenula and nucleus interpeduncularis. Moreover the experiments carded out on freeze-dried sections revealed that ChAT levels were unaffected in both the medial and lateral habenula (Table III). Since complete neuronal destruction could not be achieved in the nucleus of the diagonal band by using kainic acid, the neurons projecting to the habenula and nucleus interpeduncular may have been selectively spared. As far as the habenula is concerned, however, the decrease of GAD activity recorded in the medial habenula after kainic acid injection in the nucleus of the diagonal band, indicates that at least some of the neurons of the diagonal band projecting to the habenula do degenerate. Furthermore, electron microscopic observations showed the presence of degenerating axon terminals in the habenula and nucleus interpeduncularis after kainic acid injections in the nucleus of the diagonal band5, 6. On the basis of all the previous considerations, a cholinergic projection from the nucleus of the diagonal band to the habenula and nucleus interpeduncularis must be regarded, if present, as subsidiary to the major cholinergic innervation from the nuclei of the posterior septum demonstrated by the present paper. The previous identification of the nucleus of the diagonal hand as the primary source of the septal cholinergic input to the habenula and nucleus interpeduncularis based on electrolytic lesions13,14 could be the result of encroachment of fibers coming from the posterior septum. The question, however, requires anatomical confirmation since the exact course of fibers coming from the nucleus triangularis septi and nucleus septofimbrialis and joining the stria medullaris is not known. The possibility that our lesions in the posterior sep-
tum could have encroached cholinergic fiber systems from the nucleus of the diagonal band to the habenula and nucleus interpeduncularis, is not supported by the current anatomical literature. There is in fact general agreement that the frimbria and fornix fibers coming from the nucleus of the diagonal band and crossing the area of the posterior septum, only distribute to the hippocampal formation, the entorhinal area and the cingulate gyrusS,37. Fibers coming from interpeduncularis take an entirely different route, entering at first the medial forebrain bundle and subsequently leaving it for stria medullaris at the level of the anterior hypothalamusS. Neither transection of the stria medullaris nor lesions of the posterior septum can account for more than half of the cholinergic activity in the habenula and nucleus interpeduncularis. It is well known that transection of the fasciculus retroflexus or electrolytic destruction of the habenula, leads to almost complete loss of ChAT in the nucleus interpeduncularis7,21. We therefore suggest, in agreement with preVious hypotheses13,14, that the remaining activity in these two nuclei is due to intrinsic cholinergic neurons of the habenula, also projecting to the nucleus interpeduncularis. Habenular injections of kainic acid which selectively and massively destroy neurons of the lateral habenulaS,9,38, do not result in ChAT decrease in the interpeduncular nucleus 38. This negative finding points at the medial habenula, which massively projects to the nucleus interpeduncularis 4,19, as being the source of the habenulo-interpeduncular cholinergic projection. This conclusion is partially supported by immunohistochemical results which showed ChAT-positive neurons in the medial habenula in the guinea pig 17, bur not in the cat 22. As far as AChE is concerned, there is no significant decrease of this enzyme as a consequence of the various lesions performed. This situation suggests that the AChE conveyed by afferent inputs to the habenula and nucleus interpeduncularis is a minor fraction of the enzyme produced in situ, possibly by cholinoceptive neurons. This suggestion is in agreement with the fact that large populations of AChE-positive neurons are demonstrated in the habenula and in the nucleus interpeduncularis by electron microscopic histochemistry (Contestabile, personal observation) and with the observation that complete bilateral habenular lesions result in only minor decrease of
295 AChE stain in the rostralmost tip of the nucleus interpeduncularis, while the histochemical reaction has completely disappeared from the fasciculus retroflexus9. It is interesting to compare the present results with the picture of the nervous connections derived from anatomical observations. As far as habenula is concerned, the posterior septum looks like a better candidate than the nucleus of the diagonal band for the source of the major cholinergic input. The innervation from the nucleus triangularis septi and nucleus septo-fimbrialis is in fact massive and largely concentrated in the medial habenula18, 36, which is the area with the highest ChAT level and is, from the present study, the only habenular subdivision affected by a reduction of ChAT after lesion of the posterior septurn (Table II). On the contrary, the input from the nucleus of the diagonal band of Broca is quantitatively much smaller and is distributed to both the medial and the lateral habenulaS, TM. In addition, the present study helps in solving a major discrepancy from the anatomical literature regarding the nucleus interpeduncularis. The decrease of ChAT in the nucleus interpeduncularis after lesion of the posterior septum, confirms the projection described from anterograde protein transport36 and an electron microscopic studyS,6. The failure of methods based on the retrograde transport of horseradish peroxidase to demonstrate this pathway4,2s, shows that negative results derived from peroxidase studies should be considered with caution~8.
The forebrain GABAergic system to the habenula The use of combined lesion techniques and kainic acid injections partly confirms previous studies based on electrolytic lesions15,16A9 and give additional information on the GABAergic input to the habenula. Bilateral transection of the stria medullaris results in a 65% drop of G A D in the habenula. The destruction by kainic acid of the nucleus entopeduncularis, which is known to project massively to the lateral habenula3,1s,37, accounts for at least a 40% decrease of G A D in the habenula. Since kainic acid virtually destroyed all the neuronal population of the nucleus entopeduncularis, it seems likely that additional GABAergic input(s) does reach the habenula through the stria medullaris. The use of freeze-dried sections and the
separate assay of the medial and lateral habenula, allow us to identify the nucleus of the diagonal band of Broca as the source of a previously unsuspected GABAergic input to the medial habenula, while a possible GABAergic projection from the nucleus of the diagonal band to the lateral habenula 16 was not apparent in the present study. The presence of the GABAergic projection to the medial habenula was not evident using homogenates of the whole habenula, because it was most likely masked by the disproportion of GAD levels between the lateral and the medial habenula. The use of high doses of kainic acid in the nucleus of the diagonal band, necessary to obtain relevant neuronal destruction in this nucleus, may raise the question if some neuronal degeneration could have affected the nucleus entopeduncularis, or even the habenula and nucleus interpeduncularis, by way of the distant damage mechanism recently described in another neural system 4. As reported above, no signs of such neuronal damage were noticed by examination of histological controls. In addition, degeneration of neurons in the nucleus entopeduncularis would have affected GAD levels in the lateral habenula and this was not the case in our experiments. Finally, recent electron microscopic observations5,6 showed terminal degeneration, but not degeneration of intrinsic cell bodies, in the habenula and nucleus interpeduncularis after kainic injections in the nucleus of the diagonal band. A further possible source of GABAergic input to the habenula might be identified in the area of the lateral hypothalamus which lies very close to the ventral tip of the nucleus entopeduncularis and which has been shown to project to the lateral habenula TM. However, due to the close vicinity of the two regions and to the extreme sensitivity of the nucleus entopeduncularis to small doses of kainic acid, it seems very difficult to study possible differential projections from the two areas. Finally, as far as the nucleus interpeduncularis is concerned, the present paper demonstrates that no GABAergic input reaches this nucleus through the stria medullaris system. The diagram of Fig. 7 is an attempt to summarize the most important information of the neurotransmitters involved in the system constituted of the stria medullaris-habenula-nucleus interpeduncularis, in the light of the present results and those of other investigators.
296 ACKNOWLEDGEMENTS
bile was s u p p o r t e d by a t r a i n e e s h i p of the T r a i n i n g P r o g r a m m e in B r a i n a n d B e h a v i o u r R e s e a r c h of the
T h e e x c e l l e n t technical assistance of Mrs. E v y
European Science Foundation.
I v e r s e n is gratefully a c k n o w l e d g e d . Dr. A . C o n t e s t a REFERENCES 1 Albers, W. and Brady, R., The distribution of glutamic decarboxylase in the nervous system of the rhesus monkey, J. biol. Chem., 234 (1959) 926-928. 2 Akagi, K. and Powell, E. W., Differential projections of the habenular nuclei, J. comp. Neurol., 132 (1968) 263-274. 3 Carter, D. A. and Fibiger, H. C., The projections of the entopeduncular nucleus and globus pallidus in rat as demonstrated by autoradiography and horseradish peroxidase histochemistry, J. comp. Neurol., 177 (1978) 113-124. 4 Contestabile, A. and Flumerfelt, B. A., Afferent connections of the interpeduncular nucleus and the topographic organization of the habenulo-interpeduncular pathway: an HRP study in the rat, J. comp. Neurol., 196 (1981) 253-270. 5 Contestabile, A. and Villani, L., The use of kainic acid as an auxiliary tool in the study of controversial anatomical connections, Neuroscience, 7, Suppl.. (1982) $43 (abstract). 6 Contestabile, A. and Villani, L., The use of neuron-destroying agents for tracing neuroanatomical connections: experimental study in the septo-habenulo-interpeduncular system of the rat, J. cornp. Neurol., submitted. 7 Cuello, A. C., Emson, P. C., Paxinos, G. and Jessel, T., Substance P containing and cholinergic projections from the habenula, Brain Research, 149 (1978) 413--429. 8 Domesick, V. B., Projections of the nucleus of the diagonal band, Anat. Rec., 184 (1976) 391-392. 9 Flumerfelt, B. A. and Contestabile. A., Acetylcholinesterase histochemistry of the habenulo-interpeduncular pathway in the rat and the effects of electrolytic and kainic acid lesions, Anat. Embryol., 163 (1982) 435-446. 10 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 75 (1975) 407-409. 11 Fonnum, F., Storm-Mathisen, J. and Walberg, F., Glutamate decarboxylase in the inhibitory neurons. A study of the enzyme in Purkinje cell axons and boutons in the cat, Brain Research, 20 (1970) 259-275. 12 Fonnum, F., Grofova, I., Rinvik, E., Storm-Mathisen, J. and Walberg, F., Origin and distribution of glutamate decarboxylase in substantia nigra of the cat, Brain Research, 71 (1974) 77-92. 13 Gottesfeld, Z. and Jacobowitz, D. M., Cholinergic projection of the diagonal band to the interpeduncular nucleus of the rat brain, Brain Research, 156 (1978) 329-332. 14 Gottesfeld, Z. and Jacobowitz, D. M., Cholinergic projection from the septal-diagonal band area to the habenular nuclei, Brain Research, 176 (1979) 391-394. 15 Gottesfeld, Z.. Massari, J., Muth, E. A. and Jacobowitz, D. M., Stria medullaris: a possible pathway containing GABAergic afferents to the lateral habenula, Brain Research, 130 (1977) 184-189. 16 Gottesfeld, Z., Brandon, C., Jacobowitz, D. M. and Wu, J. Y., The GABA system in the mammalian Habenula, Brain
Res. Bull., 5, Suppl. 2 (1980) 1-6. 17 Hattori, T., McGeer, E. G., Singh, V. K. and McGeer, P. L., Cholinergic synapses of the interpeduncular nucleus, Exp. Neurol., 55 (1977) 666-679. 18 Herkenham, M. and Nauta, W. J. H., Afferent connections of the habenular nuclei in the rat. A horseradish peroxidase study, with a note on the bouton-of-passage problem, J. comp. Neurol., 173 (1977) 123-146. 19 Herkenham, M. and Nauta, W. J. H., Efferent connections of the habenular nuclei in the rat, J. comp. Neurol., 187 (1979) 19--48. 20 Hong, J. S., Costa, E. and Yang, H.-Y. T., Effects of habenular lesions on the substance P content of various brain regions, Brain Research, 118 (1976) 523-525. 21 Kataoka, K., Nakamura, Y. and Hassler, R., Habenulo-interpeduncular tract: a possible cholinergic neuron in the rat brain, Brain Research, 62 (1973) 264-267. 22 Kimura, H., McGeer, P. L., Peng, G. H. and McGeer, E. G., The central cholinergic system studied by choline acetyltransferase immunohistochemistry in the cat, ]. comp. Neurol., 200 (1981) 151-201. 23 Koenig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 24 Lenn, N. J., Synapses in the interpeduncular nucleus: electron microscopy of normal and habenula-lesioned rats, J. comp. Neurol., 166 (1976) 73-100. 25 Lewis, P. R., Shute, C. C. D. and Silver, A., Confirmation from choline acetyltransferase analyses of a massive cholinergic innervation of the rat hippocampus, J. Physiol. (Lond.), 91 (1967) 215-224. 26 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurements with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 27 Malthe-Sorenssen, D., Odden, E. and Walaas, I., Selective destruction by kainic acid of neurons innervated by putative glutamergic afferents in septum and nucleus of the diagonal band, Brain Research, 182 (1980) 461--465. 28 Marchand, E. R., Riley, J. N. and Moore, R. Y., Interpeduncular nucleus afferents in the rat, Brain Research, 193 (1980) 339-352. 29 Nagy, J. I., Carter, D. A., Lehmann, J. and Fibiger, H. C., Evidence for a GABA-containing projection from the entopeduncular nucleus to the lateral habenula in the rat, Brain Research, 145 (1978) 360-364. 30 Nauta, W. J. K., Hippocampal projections and related neural pathways to the midbrain of the cat, Brain, 81 (1958) 319-341. 31 Oderfeld-Novak, B.. Narkiewicz, O., Bialowas, J., Dabrowska, J., Wieraszko, A. and Gradowska, M., The influence of septal nuclei lesions on the activity of acetylcholinesterase and choline acetyltransferase in the hippocampus of the rat, Acta neurobiol., 34 (1974) 583-601. 32 Raisman, G., The connections of the septum, Brain, 89 (1966) 317-348. 33 Sastry, B. R., Zialkowski, S. E., Hansen, L. M., Kavanagh, J. P. and Envoy, E. M., Acetylcholine release in in-
297 terpeduncular nucleus following stimulation of habenula, Brain Research, 164 (1979) 334--337. 34 Schwob, J. E., Fuller, T., Price, J. L. and Olney, J. W., Widespread pattern of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study, Neuroscience, 5 (1980) 991-1014. 35 Sterri, S. H. and Fonnum, F., Isolation of organic anions by extraction with liquid anion exchangers and its application to micromethods for acetylcholinesterase and 4-aminobutyrate aminotransferase, Europ. J. Biochern., 91 (1978) 215-222.
36 Swanson, L. W. and Cowan, W. M., The connections of the septal region in the rat, J. comp. Neurol., 186 (1979) 621-656. 37 Van der Kooy, D. and Carter, D. A., The organization of the efferent projections and striatal afferents of the entopeduncular nucleus and adjacent areas in the rat, Brain Research, 211 (1981) 15-36. 38 Vincent, S. R., Staines, W. A., McGeer, E. G. and Fibiger, H. C., Transmitters contained in the efferents of the habenula, Brain Res., 195 (1980) 479-484.
Brain Research, 275(1983) 287-297 Elsevier
Cholinergic and GABAergic Forebrain Projections to the Habenula and Nucleus Interpeduncularis: Surgical and Kainic Acid Lesions A. CONTESTABILE* and F. FONNUM**
Norwegian Defence Research Establishment, Divisionfor Toxicology, P.O. Box 25, 2007 KjeUer(Norway) (Accepted February 2nd, 1983)
Key words: acetylchofine - - choline acetyltransferase-- G A B A - - glutamate decarboxylase - - habenula-- nucleus interpeduncularis - - septum - - nucleus triangnlaris septi - - nucleus septo-fimbrialis
The forebrain cholinergic and GABAergic projections to the habenula and nucleus interpeduncularis have been investigated by means of surgical and kainic acid lesions. Bilateral transection of the stria medullaris caused a 50% decrease of choline acetyltransferase in both the habenula and nucleus interpeduncularis, and a 65% decrease of glutamate decarboxylase in the habenula. Electrolytic lesions of the posterior septum (nucleus triangnlaris septi and nucleus septo-fimbrialis) accounted for at least 30-40% decrease of the cholinergic parameter in the habenula and nucleus interpeduncularis. Moreover, the choline acetyltransferase decrease in the habenula appeared restricted to the medial part of the nucleus. Kainic acid injections causing very large neuronal destruction in the nucleus of the diagonal band of Broca, and more than 70% decrease of choline acetyltransferase in the dorsal hippocampus, did not affect the cholinergic parameter in either the medial or lateral habenula or nucleus interpeduncularis. Kainic acid injections in the nucleus entopeduncularis resulted in a 40% decrease of glutamate decarboxylase in the habenula. Kainic acid injections in the nucleus of the diagonal band were accompanied by a 40% decrease of glutamate decarboxylase in the medial subdivision only. The present study points at the nuclei of the posterior septum as the source of a major cholinergic projection to the habenula and nudens interpeduncularis, and reveals a previously unsuspected GABAergic input from the nucleus of the diagonal band to the medial habenula. INTRODUCTION
The neural pathway constituted of stria medullaris-habenula-fasciculus retroflexus-nucleus interpeduncularis, is one of the most important connections between forebrain centers and midbrain structures. These anatomical connections have been extensively studied by lesion methods~,30,32 and, more recently, by techniques based on anterograde and retrograde transport of tracer moleculesS,1S,tg,2s,36. The orion of the cholinergic input to the habenula and nucleus interpeduncularis has been a controversial issue. It was first suggested that the cholinergic input to the nucleus interpeduncularis originated in the habenulaex; both the medial17 and the lateral part 7,33 have been suggested. More recently, an additional cholinergic input originating in the nucleus of the diagonal band of Broca and reaching the habenu-
la and nucleus interpeduncularis through the stria medullaris-fasciculus retroflexus system, has been proposedX3,14. GABAergic projections to the lateral habenula have been supposed to originate in the nucleus entopeduncularis and the nucleus of the diagonal band of Brocal6, 29. Furthermore, a direct habenula-interpeduncular substance P projection has been proposed on the basis of experimental studiesT,20. In addition to the discrepancies in the neurochemical literature, there are several important controversial points concerning the anatomical relationships between the different nuclei4,1sA9,28,36. The present paper is a reappraisal of the neurotransmitters involved in the main projections from forebrain areas to the habenula and nucleus interpeduncularis by using not only the conventional lesion methods but, whenever possible, the selective neuron cell body lesions caused by stereotaxic injec-
* Permanent address: Institute of Comparative Anatomy, University of Bologna, 40126 Bologna, Italy. ** To whom correspondence should be addressed.
288 tions of kainic acid. In particular, neurochemical parameters related to cholinergic and GABAergic projections have been studied. Particular attention has been paid to some important projections recently revealed by the use of neuroanatomical tracer methods from two small nuclei of the posterior septum, the nucleus triangularis septi and the nucleus septo-fimbria1is18.36. EXPERIMENTAL PROCEDURES
Experimental Male Wistar rats weighing 200-250 g were anesthetized with Valium (Roche) (0.4 rag/100 g body weight, i.p.) and Hypnorm vet. (Mekos) (0.7 ml/100 g body weight, s.c.). The animals were placed in a David Kopf stereotaxic frame for the subsequent experimental manipulation. The stereotaxic coordinates of the Koenig and Klippe123 atlas were used. (1) Bilateral transection of the stria medullaris was performed by lowering small razor blade splits (2 mm wide) to a depth of 6.5 mm below the skull at the A/P coordinate +5800. The razor blade splits were secured to a holder connected to the vertical pillar of the stereotaxic frame. In order to avoid damage of the sinus sagittalis, the medial edge of the blade was placed 0.5 mm lateral to the midline and inserted in the brain at an angle of 10° . Five days after the operation the rats were decapitated, the brains were immediately removed and sliced in a cold room with a Sorvail tissue chopper. The part of the brain rostral to the habenula was transferred to a cryostatic chamber and sectioned for histological confirmation of the lesion. The habenula and nucleus interpeduncularis were dissected from the tissue chopper slices under the stereomicroscope and homogenized in 150 #l (left and right habenula) or 90/zl (nucleus interpeduncularis) of 0.32 M sucrose for enzyme and protein determination. Unoperated animals of the same lots were used as the control. (2) The area of the posterior septum (nucleus triangularis septi and nucleus septo-fimbrialis) was lesioned using a tungsten electrode insulated except at the tip, delivering a current of 5 m A for 20 s at the following coordinates: A/P + 6600, M/L +_ 0.1, D/V + 0.8. Five or 6 days after the operation the animals were killed, areas of interest were dissected for neurochemical analysis and homogenized as specified
above. The rostral part of the brain was sectioned in the cryostat in order to verify the lesion extension. The posterior septum of an additional group of animals was electrolytically lesioned for enzyme determination. The brains of these animals were immediately frozen under a COe stream, transferred to the cryostat, cut at 40/.tm thickness and freeze-dried 11. The habenular region was dissected from the freezedried sections under the streomicroscope, using very small razor blade splits fixed to a holder. The stria medullaris layer overlying the habenula proper was cut and discarded. The medial and lateral habenula were separated, weighed with a fish-pole balance and assayed as previously described 11. For each animal, 18-20 samples of each habenular subdivision were dissected in the area comprised approximately between the coordinates 23 + 3350 and + 4150. The nucleus interpeduncularis was similarly dissected and 12-14 samples were taken from approximately coordinates23 + 1250 and + 1950. The usual dry weight range of individual samples was 0.5 pg for the medial habenula, 2-4~g for the lateral habenula and 3.5-6.5 pg for the nucleus interpeduncularis. As control for both experimental groups, sham-operated animals were used in which the electrode was positioned 0.5 mm above the lesion point and no current was delivered. (3) Kainic acid was injected bilaterally in different brain areas as follows. Posterior septum: 0.5 or 0.75 pg in 0.5/~1 saline at A/P + 6600, M/L + 0.5, D/V + 0.8. Nucleus entopeduncularis: 0.4pg in 0.5 pl saline at A/P + 5200, M/L + 2.4, D / V - - 1.8. Nucleus of the diagonal band of Broca: 1/~g in 0.5 gl saline at A/P + 7600, M/L + 0.5, D / V - - 2.2. For each experimental group controls were animals in which 0.5 pl saline had been injected in the different nuclei. The injections of kainic acid and saline were performed with a stainless steel cannula connected to a 20 gl Hamilton syringe mounted o n a slow-injection apparatus working a t a rate of 0.25 gl/min, over a period of 2 min. The cannula was left in situ for 5 min at the end of each injection. Kainic acid was dissolved in 0.9% NaCi and adjusted to pH 7.2-7.3 with NaOH. Both kainic acid and salinetreated animals were killed and processed for biochemical assays as detailed above. The survival time was 5-6 days.
289 An additional experimental group of rats with kainic acid injections in the nucleus of the diagonal band was used for freeze-dried sections. In this experimental group the medial and lateral habenula were separately dissected and 24 samples were obtained for each habenular subdivision in the area comprised between the coordinates mentioned above. Twelve alternate samples were assayed for choline acetyltransferase (CHAT) and 12 for glutamate decarboxylase (GAD). E n z y m e assays
Homogenates were activated with Triton X-100 (0.3% final concentration) before assays for enzyme activities. ChAT 10, GAD1,11 and acetylcholinesterase (ACHE) 35 were assayed by previously published methods. The activity was expressed either as protein weight26 or as dry weight 11. Histology
The part of the brain in which lesions or kainic acid injections had been performed, was cut in the cryostat at 40/~m thickness. The sections, mounted on slides, were stained with May-Grunwald and Giemsa in order to verify the extension of the lesion or to evaluate the neuronal damage caused by kainic acid. Other rats, in which kainic acid or saline had been injected in the various nuclei, were perfusion fixed with 10% formalin and used for histological purposes. The brains of these rats were cut with the freezing microtome and stained with cresyl violet. RESULTS Lesion experiments
Bilateral transection of the stria medullaris caused
a 50% drop of ChAT in the habenula and nucleus interpeduncularis, and a 65% decrease of G A D in the habenula (Table I). No significant decrease of A C h E was observed in either nuclei. Lesions performed in the area of the posterior septum caused a signicant decrease of ChAT in both the habenula and nucleus interpeduncularis (35% and 40% respectively, Table II). Further experiments carded out with freeze dried sections to allow better dissection (Table II) revealed more than 30% decrease of ChAT in the medial habenula and nucleus interpeduncularis, while the decrease found in the lateral habenula was not statistically significant. No decrease of G A D and AChE was noticed in habenula or nucleus interpeduncularis (Table II). Histological controls showed that differential destruction of the nucleus triangularis septi and nucleus septo-fimbrialis was obtained, while no encroachment of the stria medullaris occurred (Figs. 1 and 2). The necessity to avoid damage of the stria medullaris reduced the extension of the lesions so that in no case was complete destruction of both nuclei of the posterior septum achieved. The nucleus triangularis septi was usually almost completely destroyed, with the exception of its rostralmost and sometimes its caudalmost part (Fig. 1). The medial parts of the nuclei septo-fimbrialis were usually completely destroyed, while the lateral parts were in most cases partially spared (Fig. 1). Kainic acid experiments
Kainic acid was injected in the posterior septum, the nucleus entopeduncularis and the nucleus of the diagonal band as specified in Experimental Procedures No remarkable neurotoxic effect of kainic acid
TABLE I Effect of bilateral transection of the stria medullaris on enzymes in the habenula and nucleus interpeduncularis
Values are expressed as the mean _S.E.; the number of animals is in brackets. Enzyme activities are expressed as/~mol/gprot./h for ChAT and GAD, and mmoi/g prot./h for ACHE. Habenula
ChAT GAD AChE
Nucleus interpeduncularis
Control (8)
Lesioned (8)
Control (8)
Lesioned (8)
201 + 12.7 431 + 23.7 6.28 + 0.25
102 + 4.5** 151 + 3.7** 5.60 + 0.22
1200 + 74.5 451 + 10.3 13.37 + 0.33
646 + 39.1'* 409 + 22.4 13.45 + 0.62
** P < 0.001 (Student's t-test).
290 TABLE II Effect of electrolytic lesions of the posterior septum on enzymes in the habenula and nucleus interpeduncularis Values are expressed as the mean +S.E.; the number of animals is in brackets. Enzyme activities are expressed as/tmol/g prot./h for ChAT and GAD, and mmol/g prot./h for ACHE. For freeze-dried sections ChAT activity is expressed as/*mol/g dry weight/h (see the text for details). (a) Fresh tissue homogenates Nucleus interpeduncularis
Habenula
ChAT GAD AChE
Sham (9)
Lesioned (9)
Sham (9)
Lesioned (9)
184 + 8.7 437 + 15.6 5.69 + 0.39
130 + 5.3** 443 + 17.8 5.75 + 0.38
1098 + 50.9 475 + 27.7 11.28 + 0.64
676 + 35.2** 488 + 20 11.97 _ 0.98
(b) Freeze-dried sections Medial habenula
ChAT
Lateral habenula
Nucleus interpeduncularis
Sham (7)
Lesioned (6)
Sham (7)
Lesioned (6)
Sham (7)
Lesioned (7)
721 + 41.9
500 + 25.9*
105.1 + 8.88
85.3 + 6.86
961 + 44.8
666 + 38.6**
* P < 0.002; ** P < 0.001 (Student's t-test). could be noticed in the nuclei of the posterior septurn. In histological controls the neural damage appeared restricted to a small area a r o u n d the center of the injection site. The majority of the n e u r o n a l population was therefore unaffected by kainic acid in both nuclei of the posterior septum. I n addition, preliminary experiments showed no modification of enzyme activities in the h a b e n u l a and nucleus interpeduncularis after such injections.
Bilateral injections of kainic acid in the nucleus of the diagonal b a n d caused extensive neural damage to the nucleus itself and partially to the adjacent areas (Figs. 3 a n d 4). The vertical limb of the nucleus appeared more sensitive to the neurotoxic effect of kainic acid, since complete n e u r o n a l disappearance was usually observed in this area for all the rostrocaudal extension of the nucleus. The n e u r o n s of the horizontal limb of the nucleus were less sensitive to
TABLE III Effect of bilateral injections of kainic acid (1/~g) in the nucleus of the diagonal band on enzymes in the habenula and nucleus interpeduncularis Values are expressed as the mean +S.E.; the number of animals is in brackets. Enzyme activities are expressed as/~moi/g prot./h for ChAT and GAD and ramol/g prot./h for ACHE. For freeze-dried sections enzyme activities are expressed as/~mol/g dry weight/h. (a) Fresh tissue homogenates Nucleus interpeduncularis
Habenula
ChAT GAD AChE
Saline (11)
Kainic (9)
Saline (11)
Kainic (9)
173 + 5.6 375 + 22.9 5.36 + 0.14
171 + 12 326 + 19.1 5.55 + 0.22
1112 + 44.9 415 _+14.4 10.12 __.0.32
1005 +__36.9 465 + 13.5 10.88 + 0.53
(b) Freeze-dried sections Medial habenula
ChAT GAD
Lateral habenula
Saline (5)
Kainic (5)
Saline (5)
Kainic (5)
750 + 25.8 153.4 + 10.7
749 + 54.7 91.2 + 2.1"*
98.6 + 12.2 640 _+48.3
107.7 + 3.8 617 _+61.3
** P < 0.001 (Student's t-test).
291
O
Fig. 1. Schematic drawing showing the extension of the electrolyric lesions in the area of the posterior septum from the approximate coordinates 6300 (A) to 6900 (D) of the Koenig and Klippel (1963) atlas. Fig. 2. Example of a lesion in the area of the posterior septum at the level of the nucleus triangularis septi. Note the undamaged striae medullaris (arrows). x32. the neurotoxic action. Some of the large neurons of the horizontal limb were usually spared, with some differences among the various cases examined, also depending on the distance from the injection site. After examination of histological controls, only those cases were selected in which at least 70-80% destruction of the horizontal limb could be assessed by light microscopic analysis. To confirm the actual damage of the nucleus, the dorsal hippocampus overlying the habenular nuclei was dissected and assayed for ChAT activity. Kainic acid injections in the diagonal
band nucleus caused a dramatic decrease of ChAT in the dorsal hippocampus (saline-injected animals (11) 66.4 + 1.7/~mol/g prot./h; kainic add-injected animals (9) 18.5 + 2.3/~mol/g prot./h). Kainic acid lesions of the nucleus of the diagonal band which led to a greater than 70% drop of ChAT in the dorsal hippocampus, did not affect ChAT levels in either the habenula or nucleus interpeduncularis (Table III). G A D activity showed a small decrease in the habenula which was, however, not statistically significant (P ~ 0.1). AChE was unaffected in both the habenula and nucleus interpeduncularis. When the analyses were performed on freezedried sections, which enabled us to separate the medial and lateral habenula, a significant decrease of G A D (40%) was observed in the medial habenula, while ChAT remained unaffected (Table III). Bilateral injections of kalnic acid in the nucleus entopeduncularis led to a virtually complete disappearance of the neuronal population (Figs. 5 and 6). The injections also partially affected the adjacent region of the lateral hypothalamus, which showed a less dramatic decrease of neuronal population. Kainic acid injections in the nucleus entopeduncularis caused marked decrease of G A D in the habenula (40%), leaving ChAT unaffected (Table IV). A C h E activity was not affected. Histological controls did not show any evidence of distant neuronal damage in the nucleus entopeduncularis after kainic acid injections in the nucleus of the diagonal band or, conversely, in the nucleus of the diagonal band after injections in the nucleus entopeduncularis. There was no evidence of damage to neurons of the habenula and nucleus interpeduncularis. TABLE IV Effect of bilateral kainic acid (0.4 l~g) injections in the nucleus entopeduncularis on enzymes in the habenula
Values are expressed as the mean +S.E.; the number of animals is in brackets. Enzyme activities are expressed as/~mol/g prot./h for ChAT and GAD and mmol/g prot./h for ACHE.
ChAT GAD AChE
Saline (8)
Kainic (9)
164 + 5.7 384 + 14 4.67 + 0.13
188 + 9.4 236 + 5.8** 4.59 + 0.15
** P < 0.001 (Student's t-test).
292
Figs. 3 and 4. Effect of kainic acid on the nucleus of the diagonal band. In the kainic acid-injected nucleus (Fig. 3) an almost complete disappearance of nerve cell bodies is apparent in comparison with the saline-injected nucleus (Fig. 4). x60. Figs. 5 and 6. Effect of kainic acid on the nucleus entopeduncularis. In the kainic acid- injected nucleus (Fig. 5) a complete loss of nerve cell bodies and a remarkable glial proliferation can be noticed in comparison with the saline-injected nucleus (Fig. 6). x 100.
293 DISCUSSION
The septal cholinergic system to the habenula and nucleus interpeduncularis
Fig. 7. Diagram summarizing the present knowledge on the identity of neurotransmitters involved in the forebrain projections to the habenula and nucleus interpeduncularis and in the connections between the two nuclei. Solid lines indicate projections for which clear evidence of the neurotransmitter involved has been reached. A question mark indicates that the identity of the putative neurotransmitter still raises some doubt. Dashed lines indicate that the neurotransmitter involved in the projection is unknown or that the available information is too scarce. Note that anatomical studies36indicate that the nucleus septo-funbrialis (NSF) only projects to the medial habennla, whereas the nucleus tdangulads septi (NTS) projects to both the medial habenula (MH) and the nucleus interpeduncularis (NIP). Since both nuclei are affected by a decrease of ChAT after lesions of the posterior septum, we assume that the projection from the nucleus triangularis septi is cholinergic; it is likely, but not certain, that also the projection from the nucleus septo-fimbdalis is cholinergic. The projection from the nucleus entopeduncularis (NE) to the lateral habenula (LH) is GABAergic whereas no conclusive data exist for the area of the lateral hypothalamus (LHy). The projection from the nucleus of the diagonal band (NDB) to the medial habenula is GABAergic whereas we could not confirm that the same is true for the lateral habenula. No information is available concerning the connection between the lateral habenula and the nucleus interpeduncularis. The majority of the available anatomical and neurochemical data seems to point at the medial habenula as the source of the cholinergic and a substance P projection to the nucleus interpeduncularis. However, the question cannot be considered completely settled, since selective lesions of the neurons of the medial habenula are at present very difficult to obtain.
The results of the p r e s e n t p a p e r only partially supp o r t the conclusions o f previous studies. The overall picture of the cholinergic input from the septal areas to the h a b e n u l a and nucleus interpeduncularis appears, in fact, rather different as far as the nuclei of origin are concerned. The present results confirm that about 50% of the cholinergic activity in the h a b e n u l a and nucleus interpeduncularis originates from a forebrain innervation conveyed through the stria medullaris13,14. H o w e v e r , the main source of this cholinergic innervation does not a p p e a r to be the nucleus of the diagonal band13,t4 but the nuclei of the posterior septal division (Table II). The lesion o f these nuclei in fact accounts for a conspicuous portion of C h A T decrease in the habenula and nucleus interpeduncularis after stria medullads transection (Table I). M o r e precisely, since the nucleus trangularis septi is known from anatomical study6, 36 to p r o j e c t to both the h a b e n u l a and nucleus interpeduncularis, we m a y assume from o u r results that both projections are cholinergic. T h e nucleus septo-fimbrialis only projects to the medial h a b e n u 1a18,36. O u r results suggest that also this p r o j e c t i o n is cholinergic, but, since no selective lesions of the nucleus septo-fimbrialis were achieved in the present study, this question cannot be considered finally settled. It was unfortunate that in the present study kainic acid did not show appreciable neurotoxic effect towards the neurons of the two nuclei of the posterior septum, since kainic acid lesions allow selective degeneration of neuronal cell bodies sparing fibers of passage. In our cases, however, careful examination of histological controls could exclude lesions of the stria medullaris and, of course, of the nucleus diagonal band which lies very far from the lesioned area. M o r e o v e r , no decrease of G A D was noticed in the habenula, while lesions of the stria medu|laris would have affected G A D levels to a larger extent than C h A T levels (Table I). It should also be stated that the quantitative d a t a of the cholinergic input in the present study are m i n i m u m values, since the lesions e m p l o y e d always s p a r e d part of the septal nuclei involved.
294 The results obtained with kainic acid injections in the nucleus of the diagonal band of Broca gave indirect support to our conclusions. Our results in fact strongly suggest that a major cholinergic innervation to the habenula and nucleus interpeduncularis is unlikely to derive from the nucleus of the diagonal band. Kainic acid lesions caused a very large, even if not complete, neuronal loss in the nucleus of the diagonal band and a substantial drop of ChAT in the hippocampus, which is known to be a major target of cholinergic input from the nucleus of the diagonal band25,27,31. The same animals, however, did not show any decrease of ChAT in the habenula and nucleus interpeduncularis. Moreover the experiments carded out on freeze-dried sections revealed that ChAT levels were unaffected in both the medial and lateral habenula (Table III). Since complete neuronal destruction could not be achieved in the nucleus of the diagonal band by using kainic acid, the neurons projecting to the habenula and nucleus interpeduncular may have been selectively spared. As far as the habenula is concerned, however, the decrease of GAD activity recorded in the medial habenula after kainic acid injection in the nucleus of the diagonal band, indicates that at least some of the neurons of the diagonal band projecting to the habenula do degenerate. Furthermore, electron microscopic observations showed the presence of degenerating axon terminals in the habenula and nucleus interpeduncularis after kainic acid injections in the nucleus of the diagonal band5, 6. On the basis of all the previous considerations, a cholinergic projection from the nucleus of the diagonal band to the habenula and nucleus interpeduncularis must be regarded, if present, as subsidiary to the major cholinergic innervation from the nuclei of the posterior septum demonstrated by the present paper. The previous identification of the nucleus of the diagonal hand as the primary source of the septal cholinergic input to the habenula and nucleus interpeduncularis based on electrolytic lesions13,14 could be the result of encroachment of fibers coming from the posterior septum. The question, however, requires anatomical confirmation since the exact course of fibers coming from the nucleus triangularis septi and nucleus septofimbrialis and joining the stria medullaris is not known. The possibility that our lesions in the posterior sep-
tum could have encroached cholinergic fiber systems from the nucleus of the diagonal band to the habenula and nucleus interpeduncularis, is not supported by the current anatomical literature. There is in fact general agreement that the frimbria and fornix fibers coming from the nucleus of the diagonal band and crossing the area of the posterior septum, only distribute to the hippocampal formation, the entorhinal area and the cingulate gyrusS,37. Fibers coming from interpeduncularis take an entirely different route, entering at first the medial forebrain bundle and subsequently leaving it for stria medullaris at the level of the anterior hypothalamusS. Neither transection of the stria medullaris nor lesions of the posterior septum can account for more than half of the cholinergic activity in the habenula and nucleus interpeduncularis. It is well known that transection of the fasciculus retroflexus or electrolytic destruction of the habenula, leads to almost complete loss of ChAT in the nucleus interpeduncularis7,21. We therefore suggest, in agreement with preVious hypotheses13,14, that the remaining activity in these two nuclei is due to intrinsic cholinergic neurons of the habenula, also projecting to the nucleus interpeduncularis. Habenular injections of kainic acid which selectively and massively destroy neurons of the lateral habenulaS,9,38, do not result in ChAT decrease in the interpeduncular nucleus 38. This negative finding points at the medial habenula, which massively projects to the nucleus interpeduncularis 4,19, as being the source of the habenulo-interpeduncular cholinergic projection. This conclusion is partially supported by immunohistochemical results which showed ChAT-positive neurons in the medial habenula in the guinea pig 17, bur not in the cat 22. As far as AChE is concerned, there is no significant decrease of this enzyme as a consequence of the various lesions performed. This situation suggests that the AChE conveyed by afferent inputs to the habenula and nucleus interpeduncularis is a minor fraction of the enzyme produced in situ, possibly by cholinoceptive neurons. This suggestion is in agreement with the fact that large populations of AChE-positive neurons are demonstrated in the habenula and in the nucleus interpeduncularis by electron microscopic histochemistry (Contestabile, personal observation) and with the observation that complete bilateral habenular lesions result in only minor decrease of
295 AChE stain in the rostralmost tip of the nucleus interpeduncularis, while the histochemical reaction has completely disappeared from the fasciculus retroflexus9. It is interesting to compare the present results with the picture of the nervous connections derived from anatomical observations. As far as habenula is concerned, the posterior septum looks like a better candidate than the nucleus of the diagonal band for the source of the major cholinergic input. The innervation from the nucleus triangularis septi and nucleus septo-fimbrialis is in fact massive and largely concentrated in the medial habenula18, 36, which is the area with the highest ChAT level and is, from the present study, the only habenular subdivision affected by a reduction of ChAT after lesion of the posterior septurn (Table II). On the contrary, the input from the nucleus of the diagonal band of Broca is quantitatively much smaller and is distributed to both the medial and the lateral habenulaS, TM. In addition, the present study helps in solving a major discrepancy from the anatomical literature regarding the nucleus interpeduncularis. The decrease of ChAT in the nucleus interpeduncularis after lesion of the posterior septum, confirms the projection described from anterograde protein transport36 and an electron microscopic studyS,6. The failure of methods based on the retrograde transport of horseradish peroxidase to demonstrate this pathway4,2s, shows that negative results derived from peroxidase studies should be considered with caution~8.
The forebrain GABAergic system to the habenula The use of combined lesion techniques and kainic acid injections partly confirms previous studies based on electrolytic lesions15,16A9 and give additional information on the GABAergic input to the habenula. Bilateral transection of the stria medullaris results in a 65% drop of G A D in the habenula. The destruction by kainic acid of the nucleus entopeduncularis, which is known to project massively to the lateral habenula3,1s,37, accounts for at least a 40% decrease of G A D in the habenula. Since kainic acid virtually destroyed all the neuronal population of the nucleus entopeduncularis, it seems likely that additional GABAergic input(s) does reach the habenula through the stria medullaris. The use of freeze-dried sections and the
separate assay of the medial and lateral habenula, allow us to identify the nucleus of the diagonal band of Broca as the source of a previously unsuspected GABAergic input to the medial habenula, while a possible GABAergic projection from the nucleus of the diagonal band to the lateral habenula 16 was not apparent in the present study. The presence of the GABAergic projection to the medial habenula was not evident using homogenates of the whole habenula, because it was most likely masked by the disproportion of GAD levels between the lateral and the medial habenula. The use of high doses of kainic acid in the nucleus of the diagonal band, necessary to obtain relevant neuronal destruction in this nucleus, may raise the question if some neuronal degeneration could have affected the nucleus entopeduncularis, or even the habenula and nucleus interpeduncularis, by way of the distant damage mechanism recently described in another neural system 4. As reported above, no signs of such neuronal damage were noticed by examination of histological controls. In addition, degeneration of neurons in the nucleus entopeduncularis would have affected GAD levels in the lateral habenula and this was not the case in our experiments. Finally, recent electron microscopic observations5,6 showed terminal degeneration, but not degeneration of intrinsic cell bodies, in the habenula and nucleus interpeduncularis after kainic injections in the nucleus of the diagonal band. A further possible source of GABAergic input to the habenula might be identified in the area of the lateral hypothalamus which lies very close to the ventral tip of the nucleus entopeduncularis and which has been shown to project to the lateral habenula TM. However, due to the close vicinity of the two regions and to the extreme sensitivity of the nucleus entopeduncularis to small doses of kainic acid, it seems very difficult to study possible differential projections from the two areas. Finally, as far as the nucleus interpeduncularis is concerned, the present paper demonstrates that no GABAergic input reaches this nucleus through the stria medullaris system. The diagram of Fig. 7 is an attempt to summarize the most important information of the neurotransmitters involved in the system constituted of the stria medullaris-habenula-nucleus interpeduncularis, in the light of the present results and those of other investigators.
296 ACKNOWLEDGEMENTS
bile was s u p p o r t e d by a t r a i n e e s h i p of the T r a i n i n g P r o g r a m m e in B r a i n a n d B e h a v i o u r R e s e a r c h of the
T h e e x c e l l e n t technical assistance of Mrs. E v y
European Science Foundation.
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