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Developmental profiles of cholinergic activity in the habenulae and interpeduncular nucleus of the rat
hr. 1. Devl.NeurO8CienCC. Vol. 8.No.5. pp.561-5641990. Printed
0736-5748/90$03.00+0.00 Pergamon Press plc 0 1990ISDN
in Great Britain.
DEVELOPMENTAL PROFILES OF CHOLINERGIC ACTIVITY IN THE HABENULAE AND INTERPEDUNCULAR NUCLEUS OF THE RAT A. CONTESTABILE,* M. VIRGILI?
*Department
and 0. BARNABEI~
of Animal Biology, University of Catania and tDepartment
of Biology, University of Bologna, Italy
(Received 18 October 1989; in revised form 17 January 1990; accepted 22 January 1990) Abstract-Choline acetyltransferase (ChAT) was measured in the habenula and in the interpeduncular nucleus of rats from 1 to 12 weeks of age. A remarkable degree of parallelism was shown by the developmental curves in the two nuclei. In both cases the highest level of enzyme activity was reached at 3 weeks of age and was followed by some decrease towards adult values. A statistically highly significant correlation was demonstrated between ChAT levels in the two nuclei at the various developmental stages. The rise of the cholinergic marker was slightly advanced in the habenula in comparison with the interpeduncular nucleus. The present data may be useful for studies focused on neonatal synaptogenesis, plasticity and synaptic neurochemistry of this relatively simple model of brain connections. Key words: habenulae,
interpeduncular
nucleus, development,
cholinergic system.
The habenulo-interpeduncular projection through the fasciculus retroflexus is the axis of an important brain route connecting several forebrain centers with the so-called limbic midbrain area.“j This pathway has been extensively studied from the anatomical’*9~‘2 and neurochemica12~3~5~6~*~22 points of view. Other studies have revealed that this connection is the site of a remarkable plasticity which can be monitored both during normal development and in response to lesions occurring at neonatal stages or in adults.7~‘0,11*‘5 The habenulo-interpeduncular projection almost exclusively originates from neurons of the medial habenula which do not have any other known projection.‘*9 A major component of this projection is constituted of choline@ neurons crowded in the ventral two thirds of the medial subdivision of the habenulae, which project to the central core of the interpeduncular nucleus in a highly organized topographic way. 1*3According to a recent immunocytochemical study, these cholinergic neurons start to show ChAT immunoreactivity at a very early developmental stage in rodents.‘* Additional cholinergic projections, whose exact site of origin has been subject to controversy, reach the habenulae as well as the interpeduncular nucleus from the septal region2.s.” and, according to a recent report, ** also from the latero-dorsal tegmental area of the brain stem. The convergence of these cholinergic projections makes the interpeduncular nucleus the structure possessing the highest brain values for the specific activity of several cholinergic markers. 17,19The synaptic structures for which a remarkable neonatal and adult plasticity has been demonstrated, the so-called crest synapses, have been identified as cholinergic synapses originated from neurons localized in the medial habenulae.13 In view of further research in this field, it was thought interesting to study the developmental profiles for a specific cholinergic marker in the rat habenulae and interpeduncular nucleus. EXPERIMENTAL
PROCEDURES
Female Wistar rats weighing over 300 g were caged together with adult males and isolated when pregnancy became obvious. The offspring was reduced to six pups which were weaned on the evening of postnatal day 21. Animals were killed by decapitation at 1,2, 3,4,6 and 12 weeks of age. Starting from the 4-week stage, experimental groups were approximately composed of the same number of males and females. No obvious sex differences were noticed with respect to the neurochemical marker studied. The brains were sliced in the cold room with a Sorvall tissue chopper, the habenulae and interpeduncular nucleus were dissected under the stereomicroscope Address correspondence 40126 Bologna, Italy.
to: Dr A. Contestabile,
Department
561
of Biology, University of Bologna, Via Belmeloro 8,
A.
562
Contestabile et al.
and homogenized in 0.32 M sucrose to which Triton X-100 was added to a final 0.5% concentration. Aliquots were used for the determination of ChAT4 and of the protein content. ‘J RESULTS Figure 1 shows the temporal profiles of ChAT activity in the habenulae and interpeduncular nucleus at different developmental and adult stages. A striking similarity between the time course of ChAT levels in the two nuclei is readily apparent. Cholinergic activity increases sharply after the low levels measured at 1 week, peaks at 3 weeks of age and then decreases to reach levels similar to those of adult animals at 4-6 weeks of age. The acquisition of cholinergic levels seems accelerated in the habenulae in comparison with the interpeduncular nucleus during the first two postnatal weeks. Indeed, at 2 weeks of age ChAT level has reached about 75% of the peak value in the habenulae while only 55% of the peak value in the interpeduncular nucleus. As shown by the regression line of Fig. 2, the levels of activity reached by ChAT in the two nuclei at the various developmental stages were correlated in a statistically highly significant way.
IPN
3
5
4
6
12
AGE (weeks)
Fig. 1. Developmental profiles for ChAT activity in the habenulae (Hb) and in the interpeduncular nucleus (IPN) of rats from 1 to 12 weeks of age. Results are the mean * S.E. of 6 (l- and 2-week stages) or 10-16 (other stages) experiments.
2h e
$300% ”
r =0.9741 b= 4.68 i 0.54*
Goo. .g * .z ~100t f
I 100
300 ChAT
1 500 activity
I 700 in IPN (pmoles/g
< 900
1100
prot/ h)
Fig. 2. Regression line showing the correlation between ChAT levels in the habenulae and in the interpeduncular nucleus at the various stages: *P
Habenular
and interpeduncular
choline@
development
563
DISCUSSION The present data demonstrate a remarkable parallelism between the developmental profiles of ChAT activity in the habenulae and interpeduncular nucleus. The strict correlation shown by ChAT maturation curves in the two nuclei is very likely due to the progessive maturation of the habenulo-interpeduncular projection itself as well as to the maturation of other choline@ projections shared by the habenulae and the interpeduncular nucleus.2*5,6722 The fact that the maturation of the cholinergic levels seems to occur in the habenulae slightly in advance of those in the interpeduncular nucleus may be related to the obvious temporal gap between the synthesis of the enzyme in the perikarya of the habenulo-interpeduncular cholinergic neurons and the completion of its transport to their synaptic terminals in the interpeduncular nucleus. In addition, the other cholinergic projections shared by the habenulae and the interpeduncular nucleus2,5*6*22 reach the habenulae before traveling back to the interpeduncular nucleus, and this may further contribute to the earlier rise of ChAT in the habenulae. The decrease of ChAT levels after the peak reached around 3 weeks of age has several possible explanations. One possibility is that elimination of some cholinergic synapses (possibly through natural cell death of their parent neurons) takes place. However, a recent study” has demonstrated that at least one type of putative cholinergic synapse in the interpeduncular nucleus,13 the so-called crest synapse, greatly increases in total number between 21 and 90 days of age. While this result does not conclusively argue against possible developmental synaptic elimination, since other types of cholinergic synapses may be eliminated, it clearly does not support the occurrence of such a process in the interpeduncular nucleus. An alternative explanation is that the developmental decrease of the specific ChAT activity in the habenulae and interpeduncular nucleus is due to an earlier development of the cholinergic innervation in comparison with other neurotransmitter systems. In this case, a transient enrichment of the relative density of cholinergic synapses in the interpeduncular nucleus and in the habenulae would later be reversed by the maturation of noncholinergic synapses. It is noteworthy to recall that while similar transient increase of some neurotransmitter markers during development has often been reported, this does not seem to be the case for other cholinergic systems which, in most cases, show a progressive increase of ChAT specific activity from birth to adulthood.21 In conclusion, the present report shows for the first time the full temporal development of cholinergic activity in the rat habenulae and interpeduncular nucleus and confirms the strict functional interrelationship linking these two nuclei. These data may be useful for studies focusing on neonatal synaptogenesis,“’ plasticity’,“.” and experimental alteration of developmental synaptic neurochemistry2” in this relatively simple model of brain connections. Acknowledgements-The present work has been supported through research grants from the Italian Ministry of Education. The skillful technical assistance of Mr Antonio Bentivogli is gratefully acknowledged.
REFERENCES I. Contestabile A. and Flumerfelt B. A. (1981) Afferent connectionsof the interpeduncular nucleusand the topographic organization of the habenulo-interpeduncular pathway: an HRP study in the rat. J. camp. Neurol. I%, 253-270. 2. Contestabile A. and Fonnum F. (1983) Cholinergic and GABAergic forebrain projections to the habenula and the nucleus interpeduncularis: surgical and kainic acid lesions. Bruin Res. 275, 287-297. 3. Contestabile A., Villani L., Faso10 A., Franzoni M. F., Gribaudo L., Oktedalen 0. and Fonnum F. (1987) Topography of cholinergic and substance P pathways in the habenulo-interpeduncular system of the rat. An immunocytochemical and microchemical approach. Neuroscience 21,253-270. 4. Fonnum F. (1975) A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 75,407-409. 5. Gottesfeld Z. and Jacobowitz D. M. (1978) Cholinergic projection of the diagonal band to the interpeduncular nucleus of the rat brain. Bruin Res. 156, 329-332. 6. Gottesfeld Z. and Jacobowitz D. M. (1979) Cholinergic projection from the septal-diagonal band area to the habenular nuclei. Brain Res. 176, 391-394. 7. Hamill G. S. and Lenn N. J. (1983) Synaptic plasticity within the interpeduncular nucleus after unilateral lesion of the habenula in neonatal rats. .I. Neurosci. 3, 2128-2145. 8. Hamill G. S., Olskowka J. A., Lenn N. J. and Jacobowitz D. M. (1984) The subnuclear distribution of substance P, cholecystokynin, vasoactive intestinal peptide, somatostatin, leu-enkephalin, dopamine /3-hydrolase and serotonin in the rat interpeduncular nucleus. J. camp. Neurol. 226,580-596. 9. Herkenham M. and Nauta W. J. H. (1979) Efferent connections of the habenular nuclei in the rat. J. camp. Neural. 187, 19-48.
564
A. Contestabile
et al.
10. Lenn N. J. and Whitmore L. (1989) Modification of left-right pairing during the development of individual crest synapses in the rat interpeduncular nucleus. 1. camp. Neural. 281, 136-142. It. Lenn N. J., Wong V. and Hamill G. S. (1979) Quantitative demonstration of somatic synapse sprouting following dendritic deafferentation in neonatal rat interpeduncular nucleus. Brain Res. Bull. 4, 843-848. 12. Lenn N. J., Wong V. and Hamill G. S. (1983) Left-right pairing at the crest synapses of the interpeduncular nucleus. Neuroscience 9, 383-389. 13. Lenn N. C., Leranth C. S. and Zaborszky L. (1986) Choline acetyltransferase immunoreactivity is localized to four
types of synapses in the rat interpeduncular nucleus. /. Neurocyfol. 14,909-919. 14. Lowry 0. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 15. Murray M., Zimmer J. and Raisman G. (1979) Quantitative electron microscopic evidence for reinnervation in the adult rat interpeduncular nucleus after lesion of the fasciculus retroflexus. J. camp. Neurol. 187, 447-468. 16. Nauta W. J. H. (1958) Hippocampal projections and related neural pathways in the midbrain of the cat. Brain 81, 319-341. 17. Palkovits M., Saavedra J., Kobayashi R. M. and Brownstein M. (1974) Choline acetyltransferase content of limbic nuclei of the rat. Brain Res. 79, 443450. 18. Schambra U. B., Sulik K. K., Petrusz P. and Lauder J. M. (1989) Ontogeny of cholinergic neurons in the mouse forebrain. J. camp. Neurol. 288, 101-122. 19. Sorimachi M. and Kataoka K. (1974) Choline uptake by nerve terminals: a sensitive and specific marker of cholinergic innervation. Brain Res. 72, 3X-353. 20. Virgili M., Barnabei 0. and Contestabile A. (1988) Depletion of cholinergic habenulo-interpeduncular neurons by selectively timed metylazoxymethanol acetate (MAM) treatment during pregnancy. Brain Res. 460, 361-365. 21. Virgili M., Barnabei 0. markers during postnatal 22. Woolf N. J. and Butcher nucleus. Brain Res. Bull.
and Contestabile A. (1990) Regional maturation of neurotransmitter-related and glial development in the rat. I&. J. devl Neurosci. 8, 159-166. L. L. (1985) Cholinergic systems in the rat brain: II. Projections to the interpeduncular 14, 63-83.
0736-5748/90$03.00+0.00 Pergamon Press plc 0 1990ISDN
in Great Britain.
DEVELOPMENTAL PROFILES OF CHOLINERGIC ACTIVITY IN THE HABENULAE AND INTERPEDUNCULAR NUCLEUS OF THE RAT A. CONTESTABILE,* M. VIRGILI?
*Department
and 0. BARNABEI~
of Animal Biology, University of Catania and tDepartment
of Biology, University of Bologna, Italy
(Received 18 October 1989; in revised form 17 January 1990; accepted 22 January 1990) Abstract-Choline acetyltransferase (ChAT) was measured in the habenula and in the interpeduncular nucleus of rats from 1 to 12 weeks of age. A remarkable degree of parallelism was shown by the developmental curves in the two nuclei. In both cases the highest level of enzyme activity was reached at 3 weeks of age and was followed by some decrease towards adult values. A statistically highly significant correlation was demonstrated between ChAT levels in the two nuclei at the various developmental stages. The rise of the cholinergic marker was slightly advanced in the habenula in comparison with the interpeduncular nucleus. The present data may be useful for studies focused on neonatal synaptogenesis, plasticity and synaptic neurochemistry of this relatively simple model of brain connections. Key words: habenulae,
interpeduncular
nucleus, development,
cholinergic system.
The habenulo-interpeduncular projection through the fasciculus retroflexus is the axis of an important brain route connecting several forebrain centers with the so-called limbic midbrain area.“j This pathway has been extensively studied from the anatomical’*9~‘2 and neurochemica12~3~5~6~*~22 points of view. Other studies have revealed that this connection is the site of a remarkable plasticity which can be monitored both during normal development and in response to lesions occurring at neonatal stages or in adults.7~‘0,11*‘5 The habenulo-interpeduncular projection almost exclusively originates from neurons of the medial habenula which do not have any other known projection.‘*9 A major component of this projection is constituted of choline@ neurons crowded in the ventral two thirds of the medial subdivision of the habenulae, which project to the central core of the interpeduncular nucleus in a highly organized topographic way. 1*3According to a recent immunocytochemical study, these cholinergic neurons start to show ChAT immunoreactivity at a very early developmental stage in rodents.‘* Additional cholinergic projections, whose exact site of origin has been subject to controversy, reach the habenulae as well as the interpeduncular nucleus from the septal region2.s.” and, according to a recent report, ** also from the latero-dorsal tegmental area of the brain stem. The convergence of these cholinergic projections makes the interpeduncular nucleus the structure possessing the highest brain values for the specific activity of several cholinergic markers. 17,19The synaptic structures for which a remarkable neonatal and adult plasticity has been demonstrated, the so-called crest synapses, have been identified as cholinergic synapses originated from neurons localized in the medial habenulae.13 In view of further research in this field, it was thought interesting to study the developmental profiles for a specific cholinergic marker in the rat habenulae and interpeduncular nucleus. EXPERIMENTAL
PROCEDURES
Female Wistar rats weighing over 300 g were caged together with adult males and isolated when pregnancy became obvious. The offspring was reduced to six pups which were weaned on the evening of postnatal day 21. Animals were killed by decapitation at 1,2, 3,4,6 and 12 weeks of age. Starting from the 4-week stage, experimental groups were approximately composed of the same number of males and females. No obvious sex differences were noticed with respect to the neurochemical marker studied. The brains were sliced in the cold room with a Sorvall tissue chopper, the habenulae and interpeduncular nucleus were dissected under the stereomicroscope Address correspondence 40126 Bologna, Italy.
to: Dr A. Contestabile,
Department
561
of Biology, University of Bologna, Via Belmeloro 8,
A.
562
Contestabile et al.
and homogenized in 0.32 M sucrose to which Triton X-100 was added to a final 0.5% concentration. Aliquots were used for the determination of ChAT4 and of the protein content. ‘J RESULTS Figure 1 shows the temporal profiles of ChAT activity in the habenulae and interpeduncular nucleus at different developmental and adult stages. A striking similarity between the time course of ChAT levels in the two nuclei is readily apparent. Cholinergic activity increases sharply after the low levels measured at 1 week, peaks at 3 weeks of age and then decreases to reach levels similar to those of adult animals at 4-6 weeks of age. The acquisition of cholinergic levels seems accelerated in the habenulae in comparison with the interpeduncular nucleus during the first two postnatal weeks. Indeed, at 2 weeks of age ChAT level has reached about 75% of the peak value in the habenulae while only 55% of the peak value in the interpeduncular nucleus. As shown by the regression line of Fig. 2, the levels of activity reached by ChAT in the two nuclei at the various developmental stages were correlated in a statistically highly significant way.
IPN
3
5
4
6
12
AGE (weeks)
Fig. 1. Developmental profiles for ChAT activity in the habenulae (Hb) and in the interpeduncular nucleus (IPN) of rats from 1 to 12 weeks of age. Results are the mean * S.E. of 6 (l- and 2-week stages) or 10-16 (other stages) experiments.
2h e
$300% ”
r =0.9741 b= 4.68 i 0.54*
Goo. .g * .z ~100t f
I 100
300 ChAT
1 500 activity
I 700 in IPN (pmoles/g
< 900
1100
prot/ h)
Fig. 2. Regression line showing the correlation between ChAT levels in the habenulae and in the interpeduncular nucleus at the various stages: *P
Habenular
and interpeduncular
choline@
development
563
DISCUSSION The present data demonstrate a remarkable parallelism between the developmental profiles of ChAT activity in the habenulae and interpeduncular nucleus. The strict correlation shown by ChAT maturation curves in the two nuclei is very likely due to the progessive maturation of the habenulo-interpeduncular projection itself as well as to the maturation of other choline@ projections shared by the habenulae and the interpeduncular nucleus.2*5,6722 The fact that the maturation of the cholinergic levels seems to occur in the habenulae slightly in advance of those in the interpeduncular nucleus may be related to the obvious temporal gap between the synthesis of the enzyme in the perikarya of the habenulo-interpeduncular cholinergic neurons and the completion of its transport to their synaptic terminals in the interpeduncular nucleus. In addition, the other cholinergic projections shared by the habenulae and the interpeduncular nucleus2,5*6*22 reach the habenulae before traveling back to the interpeduncular nucleus, and this may further contribute to the earlier rise of ChAT in the habenulae. The decrease of ChAT levels after the peak reached around 3 weeks of age has several possible explanations. One possibility is that elimination of some cholinergic synapses (possibly through natural cell death of their parent neurons) takes place. However, a recent study” has demonstrated that at least one type of putative cholinergic synapse in the interpeduncular nucleus,13 the so-called crest synapse, greatly increases in total number between 21 and 90 days of age. While this result does not conclusively argue against possible developmental synaptic elimination, since other types of cholinergic synapses may be eliminated, it clearly does not support the occurrence of such a process in the interpeduncular nucleus. An alternative explanation is that the developmental decrease of the specific ChAT activity in the habenulae and interpeduncular nucleus is due to an earlier development of the cholinergic innervation in comparison with other neurotransmitter systems. In this case, a transient enrichment of the relative density of cholinergic synapses in the interpeduncular nucleus and in the habenulae would later be reversed by the maturation of noncholinergic synapses. It is noteworthy to recall that while similar transient increase of some neurotransmitter markers during development has often been reported, this does not seem to be the case for other cholinergic systems which, in most cases, show a progressive increase of ChAT specific activity from birth to adulthood.21 In conclusion, the present report shows for the first time the full temporal development of cholinergic activity in the rat habenulae and interpeduncular nucleus and confirms the strict functional interrelationship linking these two nuclei. These data may be useful for studies focusing on neonatal synaptogenesis,“’ plasticity’,“.” and experimental alteration of developmental synaptic neurochemistry2” in this relatively simple model of brain connections. Acknowledgements-The present work has been supported through research grants from the Italian Ministry of Education. The skillful technical assistance of Mr Antonio Bentivogli is gratefully acknowledged.
REFERENCES I. Contestabile A. and Flumerfelt B. A. (1981) Afferent connectionsof the interpeduncular nucleusand the topographic organization of the habenulo-interpeduncular pathway: an HRP study in the rat. J. camp. Neurol. I%, 253-270. 2. Contestabile A. and Fonnum F. (1983) Cholinergic and GABAergic forebrain projections to the habenula and the nucleus interpeduncularis: surgical and kainic acid lesions. Bruin Res. 275, 287-297. 3. Contestabile A., Villani L., Faso10 A., Franzoni M. F., Gribaudo L., Oktedalen 0. and Fonnum F. (1987) Topography of cholinergic and substance P pathways in the habenulo-interpeduncular system of the rat. An immunocytochemical and microchemical approach. Neuroscience 21,253-270. 4. Fonnum F. (1975) A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 75,407-409. 5. Gottesfeld Z. and Jacobowitz D. M. (1978) Cholinergic projection of the diagonal band to the interpeduncular nucleus of the rat brain. Bruin Res. 156, 329-332. 6. Gottesfeld Z. and Jacobowitz D. M. (1979) Cholinergic projection from the septal-diagonal band area to the habenular nuclei. Brain Res. 176, 391-394. 7. Hamill G. S. and Lenn N. J. (1983) Synaptic plasticity within the interpeduncular nucleus after unilateral lesion of the habenula in neonatal rats. .I. Neurosci. 3, 2128-2145. 8. Hamill G. S., Olskowka J. A., Lenn N. J. and Jacobowitz D. M. (1984) The subnuclear distribution of substance P, cholecystokynin, vasoactive intestinal peptide, somatostatin, leu-enkephalin, dopamine /3-hydrolase and serotonin in the rat interpeduncular nucleus. J. camp. Neurol. 226,580-596. 9. Herkenham M. and Nauta W. J. H. (1979) Efferent connections of the habenular nuclei in the rat. J. camp. Neural. 187, 19-48.
564
A. Contestabile
et al.
10. Lenn N. J. and Whitmore L. (1989) Modification of left-right pairing during the development of individual crest synapses in the rat interpeduncular nucleus. 1. camp. Neural. 281, 136-142. It. Lenn N. J., Wong V. and Hamill G. S. (1979) Quantitative demonstration of somatic synapse sprouting following dendritic deafferentation in neonatal rat interpeduncular nucleus. Brain Res. Bull. 4, 843-848. 12. Lenn N. J., Wong V. and Hamill G. S. (1983) Left-right pairing at the crest synapses of the interpeduncular nucleus. Neuroscience 9, 383-389. 13. Lenn N. C., Leranth C. S. and Zaborszky L. (1986) Choline acetyltransferase immunoreactivity is localized to four
types of synapses in the rat interpeduncular nucleus. /. Neurocyfol. 14,909-919. 14. Lowry 0. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 15. Murray M., Zimmer J. and Raisman G. (1979) Quantitative electron microscopic evidence for reinnervation in the adult rat interpeduncular nucleus after lesion of the fasciculus retroflexus. J. camp. Neurol. 187, 447-468. 16. Nauta W. J. H. (1958) Hippocampal projections and related neural pathways in the midbrain of the cat. Brain 81, 319-341. 17. Palkovits M., Saavedra J., Kobayashi R. M. and Brownstein M. (1974) Choline acetyltransferase content of limbic nuclei of the rat. Brain Res. 79, 443450. 18. Schambra U. B., Sulik K. K., Petrusz P. and Lauder J. M. (1989) Ontogeny of cholinergic neurons in the mouse forebrain. J. camp. Neurol. 288, 101-122. 19. Sorimachi M. and Kataoka K. (1974) Choline uptake by nerve terminals: a sensitive and specific marker of cholinergic innervation. Brain Res. 72, 3X-353. 20. Virgili M., Barnabei 0. and Contestabile A. (1988) Depletion of cholinergic habenulo-interpeduncular neurons by selectively timed metylazoxymethanol acetate (MAM) treatment during pregnancy. Brain Res. 460, 361-365. 21. Virgili M., Barnabei 0. markers during postnatal 22. Woolf N. J. and Butcher nucleus. Brain Res. Bull.
and Contestabile A. (1990) Regional maturation of neurotransmitter-related and glial development in the rat. I&. J. devl Neurosci. 8, 159-166. L. L. (1985) Cholinergic systems in the rat brain: II. Projections to the interpeduncular 14, 63-83.