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Differential distribution of diagonal band afferents to subnuclei of the interpeduncular nucleus in rats
Neuroscience Letters, 48 (1984) 43-48 Elsevier Scientific Publishers Ireland Ltd.
43
NSL 02755
DIFFERENTIAL DISTRIBUTION OF DIAGONAL BAND AFFERENTS TO SUBNUCLEI OF THE INTERPEDUNCULAR NUCLEUS IN RATS
GEOFFREY S. HAMILL 1'* and BARRY FASS2
tNIMH, Laboratory of Clinical Science, Bldg. 10, Rm. 3D48, Bethesda, MD 20205, and 2Department of Neurosurgery, University of Viriginia School of Medicine, Box 420, Charlottesville, VA 22908 (U.S.A.) (Received January 23rd, 1984; Accepted March 7th, 1984)
Key words: interpeduncular nucleus - nucleus of diagonal band - autoradiography - anterograde tract-tracing
Previous research has demonstrated a subnuclear organization within the interpeduncular nucleus (IPN) based upon cytoarchitecture, synaptology, and the distribution of biogenic amines and peptides. To determine whether individual subnuclei of IPN can be further differentiated with regard to their afferents, we investigated the distribution of inputs from the nucleus of the diagonal band. Autoradiographic analyses demonstrated a diagonal band projection to IPN which is not homogeneously distributed among individual subnuclei. The greatest density of silver grains was located over the dorsal subnucleus, followed in order of diminishing density by the rostral, central, intermediate and lateral subnuclei. These findings confirm the existence of a projection from the nucleus of the diagonal band to the IPN, and support the concept that individual subnuclei within the IPN may be further differentiated on the basis of their afferent input.
The interpeduncular nucleus (IPN) in rats can be conceptualized as a heterogeneous structure. Our recent studies have demonstrated that the IPN is organized into seven subnuclei, o f which three are located on the midline (i.e. the rostral, central and dorsal subnuclei) and four are bilateral (i.e. the intermediate, lateral, interstitial and dorsal lateral subnuclei) [7]. These subnuclei differ with regard to their internal structure (i.e. cytoarchitecture and synaptology) [7] and distribution of putative neurotransmitters [8,10,12,16]. Although there is evidence for a topographic organization o f some afferents to the IPN (see e.g. refs. 2, 11, and 14), the possibility that they might be differentially distributed across individual subnuclei has not been studied systematically. To test this possibility, we reexamined the pathway from the nucleus of the diagonal band (NDB) to the IPN [1,2,5]. The subjects were 8 adult male Sprague-Dawley rats (250-300 g; Dominion Labs); 5 were intact, and 3 had sustained unilateral lesions of the entorhinal cortex * Present address: Dept. of Anatomy, The Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, U.S.A.
0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
44
as part of another experiment. The NDB in each rat was injected with 15-20/~Ci of [3H]proline (New England Nuclear; 20-40 Ci/mmol; 10 #Ci/0.10/~1 of deionized water) using a microliter syringe at a 14 ° angle toward the midline (AP, 2.0 mm; L, 1.5; V, - 6.0). After a 3-4 day survival interval, the rats were perfused intracardially with 10% neutral buffered formalin. Their brains were processed for autoradiography [3]. Emulsion-coated slides with brain sections were exposed for 2-10 weeks, developed in D-19, and counter-stained with cresyl violet. The distribution of NDB afferents to the IPN was analyzed qualitatively by rating the relative density of silver grains over each subnucleus compared to background on each section, at various rostrocaudal levels of the IPN. We used the following scale of ratings: intense, heavy, moderate and sparse labeling. Autoradiograms with an unacceptably high background were excluded from the analysis. Since our two sets of ratings were highly correlated, a mean rating was calculated for each subnucleus. To confirm these ratings quantitatively, we evaluated the density of silver grains over IPN subnuclei in a representative brain. Using dark-field illumination at × 250 magnification, each of the authors independently counted silver grains over each subnucleus at four representative rostrocaudal levels of IPN. The counts were obtained from a selected 4800/zm 2 area within each subnucleus, and a mean value then was calculated. A typical injection site is illustrated in Fig. 1. In almost all cases, the site of injection was centered on the midline and primarily labeled the NDB (vertical limb). The [3H]proline did spread into the ventromedial margins of the medial, lateral and A
B
C
Fig. 1. The illustrations show the most rostral (A), middle (B) and most caudal (C) levels of a typical injection of [3Hlproline into the nucleus of the diagonal band (NDB). Redrawn from ref. 15. Abbreviations: AC, anterior commissure; acb, nucleus accumbens; CPu, caudate-putamen; f, fornix; LS, lateral septal nucleus; MS, medial septal nucleus; VDBD, nucleus of the vertical limb of the diagonal band (dorsal); VDBV, nucleus of the vertical limb of the diagonal band (ventral).
45
Fig. 2. Dark-field photomicrographs of two levels of interpeduncular nucleus (IPN). The heterogeneous pattern of silver grains over IPN reflects the subnuclear distribution of NDB afferents. × 135. A: at this level of IPN (corresponding to level B in Fig. 3), silver grains are most densely concentrated in a band (Re) along the ventrolateral margins of the rostral subnucleus. These bands help to define the outline of two ovoid-shaped regions (white arrows) over which there was only sparse labeling. The next highest density of grains was observed over the central subnucleus (C), followed in order of diminishing density by the intermediate (I), and lateral (L) subnuclei. B: at a more caudal level of IPN (corresponding to level C in Fig. 3), silver grains are most densely concentrated throughout the dorsal subnucleus (D), followed in order o f diminishing density by central (C), lateral (L) and intermediate (I) subnuclei. (Publisher's reproduction factors: A, 0.48; B, 0.48.)
46 triangular septal nuclei, in addition to the precommissural fornix, anterior commissure, and medial preoptic nucleus. In one exceptional case, the injection site was predominantly unilateral and included the NDB (horizontal limb), medial edge o f the nucleus accumbens and bed nucleus of the stria terminalis. An examination of sections at the level of the midbrain revealed that the greatest density of silver grains was concentrated over the IPN. By contrast, the density over most other structures and o f f the sections was negligible (except for a modest band o f grains over the superior colliculus and very dense bands oriented tangentially, dorsolateral to the margin of the IPN bilaterally). The most striking feature of the labeling over the IPN was its differential distribution across individual subnuclei. The greatest density of grains was observed over the dorsal subnucleus, followed in order of diminishing density by the rostral, central, intermediate and lateral subnuclei (Fig. 2). The density of grains over the interstitial and dorsal lateral subnuclei appeared no greater than background and was not further analyzed. Our qualitative impressions and ratings were confirmed by the quantitative analyses; the results obtained at four rostrocaudal levels of the IPN from a representative case are summarized in Fig. 3. At the pontine level of the IPN (see Fig. 1D in ref. 7), for example, the relative density of grains over the dorsal subnucleus was more than two-fold greater than that over the rostral and central subnuclei and 4-fold greater than over the remaining subnuclei (Fig. 3C). By contrast, the density over the rostral and central subnuclei was approximately two,fold greater than over the intermediate and lateral subnuclei. There were several interesting features to the pattern of labeling over particular subnuclei of the IPN. The distribution of grains over the central subnucleus demonstrated a topographic organization such that the highest concentration of grains was found at middle to caudal levels o f the IPN (Fig. 3). We also observed a horizontal lamination in the distribution of grains over the central subnucleus at several levels of the IPN. At the midlevel of IPN (Fig. 3B), a heavy band of silver grains was distributed over the ventrolateral margins of the rostral subnucleus. This band defined the outline of two ovoid-shaped regions, over which there was only sparse labeling. Rostral to this level of the IPN, silver grains were distributed uniformly over the rostral subnucleus. Silver grains also were uniformly distributed over the lateral subnucleus, except for a conspicuous absence of grains over the ventrolateral corners of this subnucleus (Fig. 3D). The results of this study provide support for the existence of a projection from the nucleus of the diagonal band to the IPN in rats, which has been demonstrated with retrograde labeling and biochemical techniques [2,5]. The existence of this pathway was also reported by others [1,18]; but, until now, its complex distribution within the IPN has not been fully appreciated (cf. refs. 9, 11 and 14). Our findings suggest that IPN receives NDB afferents which are both topographically and heterogeneously distributed across individual subnuclei. Previous studies have demonstrated a subnuclear organization within the IPN
47
D
-!
O 70" O 00 60-
D
50'
Z m ,¢ 40n" 30"
R
::1:1= 2O' 10 A
B L E V E L S OF
ii
C
D
IPN
Fig. 3. Density of silver grains over individual subnuclei of IPN at four rostrocaudal levels (A-D) from an individual case. The greatest density of grains was located over the dorsal subnucleus (D), followed in order of diminishing density by the rostral (R), central (C), intermediate (I) and lateral (L) subnuclei. At level B, a dense band of grains located along the ventrolateral margins of the rostral subnucleus (Re) helped to define the outline of two ovoid-shaped regions (R) over which there was only a moderate density of grains. NDB afferents also appear to be topographically distributed to mid (B) and caudal (C) levels of the central subnucleus.
based upon cytoarchitecture, synaptology and the distribution of putative neurotransmitters [7-10,16]. The present results indicate that IPN subnuclei may be further differentiated on the basis of their afferent input. NDB inputs are most heavily concentrated in the dorsal subnucleus, followed in order of diminishing density by the rostral, central, intermediate and lateral subnuclei. Preliminary results obtained in other laboratories support the present concept that afferents to IPN are not homogeneously distributed [6]. The putative neurotransmitters of NDB afferents to the IPN are not known at present. However, enkephalin (Enk)-, cholecystokinin (CCK)-, and LH-RH-reactive perikarya have been demonstrated in the septal forebrain of rats [4,13,17,19]. Using immunocytochemical techniques, we have found a similarity between the distribution of Enk- and CCK-containing processes in the rostral subnucleus [8] and inputs from the NDB described in this report. Therefore, the possibility exists that a forebrain limbic projection from the NDB to the IPN may be peptidergic. Studies currently are in progress to explore this possibility. This research was supported by NSF Grant BNS76-17750 and NIH Grant NS12333 to Oswald Steward. Dr. David M. Jacobowitz kindly provided use of the
48 microscope
facilities in the Laboratory
of Clinical Science at NIMH.
We are
g r a t e f u l t o J a y G i l l e n w a t e r , R e b e c c a O g l e , F. L e e S n a v e l y a n d D r . J u l i o R a m i r e z for their assistance. 1 Conrad, L.C.A. and Pfaff, D.W., Efferents from medial basal forebrain and hypothalamus in the rat. I. An autoradiographic study of the medial preoptic area, J. comp. Neurol., 169 (1976) 185-220. 2 Contestabile, A. and Flumerfelt, B.A., Afferent connections of the interpeduncular nucleus and the topographic organization of the habenulointerpeduncular pathway: an HRP study in the rat, J. comp. Neurol., 196 (1981) 253-270. 3 Fass, B. and Steward, O., Increases in protein-precursor incorporation in the denervated neuropil of the dentate gyrus during reinnervation, Neuroscience, 9 (1983) 653-664. 4 Finley, J.C.W., Maderdrut, J.L. and Petrusz, P., The immunocytochemical localization of enkephalin in the central nervous system of the rat, J. comp. Neurol., 198 (1981) 541-565. 5 Gottesfeld, Z. and Jacobowitz, D.M., Cholinergic projection of the diagonal band to the interpeduncular nucleus of the rat brain, Brain Res., 156 (1978) 329-332. 6 Groenewegen, H.J., Kowall, N.W. and Nauta, W.H.J., Efferent connections of the dorsal tegmental region in the rat, Soc. Neurosci. Abstr., 13 (1983) 516. 7 Hamill, G.S. and Lenn, N.J., The subnuclear organization of the rat interpeduncular nucleus: a light and electron microscopy study, J. comp. Neurol., 222 (1984) 396-408. 8 Hamill, G.S., Olschowka, J.A., Lenn, N.J. and Jacobowitz, D.M., The subnuclear distribution of substance P, cholecystokinin, vasoactive intestinal peptide, somatostatin, Leu-enkephalin, dopamineB-hydroxylase, and serotonin in the rat interpeduncular nucleus, J. comp. Neurol., in press. 9 Hayakawa, T., Seki, M. and Zyo, K., Studies on the efferent projections of the interpeduncular complex in cats, Okajimas Folia Anat. Jpn., 58 0981) 1-16. 10 Hemmendinger, L.M. and Moore, R.Y., The interpeduncular nucleus of the rat: cytoarchitecture and cytochemistry, Soc. Neurosci. Abstr., 8 (1982) 665. ll Herkenham, M. and Nauta, W.J.H., Efferent connections of the habenular nuclei in the rat, J. comp. Neurol., 187 (1979) 19-48. 12 Houser, C.R., Crawford, G., Anderson, L., Barber, R., Salvaterra, P.M. and Vaughn, J.E., lmmunocytochemical localization of cholinergic neurons with a monoclonal antibody to choline acetyltransferase, Soc. Neurosci. Abstr., 8 (1982) 662. 13 Khachaturian, H., Lewis, M.E., Hollt, V. and Watson, S.J., Telencephalic enkephalinergic systems in the rat brain, J. Neurosci., 3 (1983) 844-855. 14 Marchand, E.R., Riley, J.N. and Moore, R.Y., Interpeduncular nucleus afferents in the rat, Brain Res., 193 (1980) 339-352. 15 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 16 Rotter, A. and Jacobowitz, D.M., Colocalization of substance P, acetylcholinesterase, muscarinic receptors and alpha-bungarotoxin binding sites in the rat interpeduncular nucleus, Soc. Neurosci. Abstr., 8 (1982) 118. 17 Silverman, A.J. and Krey, L.C., The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea pig brain. I. Intra- and extra-hypothalamic projections, Brain Res., 157 (1978) 233-246. 18 Swanson, L.W. and Cowan, W.M., The connections of the septal region in the rat, J. comp. Neurol., 156 (1979) 621-656. 19 Woodhams, P.L., Roberts, G.W., Polak, J.M. and Crow, T.J., Distribution of neuropeptides in the limbic system of the rat: the bed nucleus of the stria terminalis, septum and preoptic area, Neuroscience, 8 (1983) 677-703.
43
NSL 02755
DIFFERENTIAL DISTRIBUTION OF DIAGONAL BAND AFFERENTS TO SUBNUCLEI OF THE INTERPEDUNCULAR NUCLEUS IN RATS
GEOFFREY S. HAMILL 1'* and BARRY FASS2
tNIMH, Laboratory of Clinical Science, Bldg. 10, Rm. 3D48, Bethesda, MD 20205, and 2Department of Neurosurgery, University of Viriginia School of Medicine, Box 420, Charlottesville, VA 22908 (U.S.A.) (Received January 23rd, 1984; Accepted March 7th, 1984)
Key words: interpeduncular nucleus - nucleus of diagonal band - autoradiography - anterograde tract-tracing
Previous research has demonstrated a subnuclear organization within the interpeduncular nucleus (IPN) based upon cytoarchitecture, synaptology, and the distribution of biogenic amines and peptides. To determine whether individual subnuclei of IPN can be further differentiated with regard to their afferents, we investigated the distribution of inputs from the nucleus of the diagonal band. Autoradiographic analyses demonstrated a diagonal band projection to IPN which is not homogeneously distributed among individual subnuclei. The greatest density of silver grains was located over the dorsal subnucleus, followed in order of diminishing density by the rostral, central, intermediate and lateral subnuclei. These findings confirm the existence of a projection from the nucleus of the diagonal band to the IPN, and support the concept that individual subnuclei within the IPN may be further differentiated on the basis of their afferent input.
The interpeduncular nucleus (IPN) in rats can be conceptualized as a heterogeneous structure. Our recent studies have demonstrated that the IPN is organized into seven subnuclei, o f which three are located on the midline (i.e. the rostral, central and dorsal subnuclei) and four are bilateral (i.e. the intermediate, lateral, interstitial and dorsal lateral subnuclei) [7]. These subnuclei differ with regard to their internal structure (i.e. cytoarchitecture and synaptology) [7] and distribution of putative neurotransmitters [8,10,12,16]. Although there is evidence for a topographic organization o f some afferents to the IPN (see e.g. refs. 2, 11, and 14), the possibility that they might be differentially distributed across individual subnuclei has not been studied systematically. To test this possibility, we reexamined the pathway from the nucleus of the diagonal band (NDB) to the IPN [1,2,5]. The subjects were 8 adult male Sprague-Dawley rats (250-300 g; Dominion Labs); 5 were intact, and 3 had sustained unilateral lesions of the entorhinal cortex * Present address: Dept. of Anatomy, The Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, U.S.A.
0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
44
as part of another experiment. The NDB in each rat was injected with 15-20/~Ci of [3H]proline (New England Nuclear; 20-40 Ci/mmol; 10 #Ci/0.10/~1 of deionized water) using a microliter syringe at a 14 ° angle toward the midline (AP, 2.0 mm; L, 1.5; V, - 6.0). After a 3-4 day survival interval, the rats were perfused intracardially with 10% neutral buffered formalin. Their brains were processed for autoradiography [3]. Emulsion-coated slides with brain sections were exposed for 2-10 weeks, developed in D-19, and counter-stained with cresyl violet. The distribution of NDB afferents to the IPN was analyzed qualitatively by rating the relative density of silver grains over each subnucleus compared to background on each section, at various rostrocaudal levels of the IPN. We used the following scale of ratings: intense, heavy, moderate and sparse labeling. Autoradiograms with an unacceptably high background were excluded from the analysis. Since our two sets of ratings were highly correlated, a mean rating was calculated for each subnucleus. To confirm these ratings quantitatively, we evaluated the density of silver grains over IPN subnuclei in a representative brain. Using dark-field illumination at × 250 magnification, each of the authors independently counted silver grains over each subnucleus at four representative rostrocaudal levels of IPN. The counts were obtained from a selected 4800/zm 2 area within each subnucleus, and a mean value then was calculated. A typical injection site is illustrated in Fig. 1. In almost all cases, the site of injection was centered on the midline and primarily labeled the NDB (vertical limb). The [3H]proline did spread into the ventromedial margins of the medial, lateral and A
B
C
Fig. 1. The illustrations show the most rostral (A), middle (B) and most caudal (C) levels of a typical injection of [3Hlproline into the nucleus of the diagonal band (NDB). Redrawn from ref. 15. Abbreviations: AC, anterior commissure; acb, nucleus accumbens; CPu, caudate-putamen; f, fornix; LS, lateral septal nucleus; MS, medial septal nucleus; VDBD, nucleus of the vertical limb of the diagonal band (dorsal); VDBV, nucleus of the vertical limb of the diagonal band (ventral).
45
Fig. 2. Dark-field photomicrographs of two levels of interpeduncular nucleus (IPN). The heterogeneous pattern of silver grains over IPN reflects the subnuclear distribution of NDB afferents. × 135. A: at this level of IPN (corresponding to level B in Fig. 3), silver grains are most densely concentrated in a band (Re) along the ventrolateral margins of the rostral subnucleus. These bands help to define the outline of two ovoid-shaped regions (white arrows) over which there was only sparse labeling. The next highest density of grains was observed over the central subnucleus (C), followed in order of diminishing density by the intermediate (I), and lateral (L) subnuclei. B: at a more caudal level of IPN (corresponding to level C in Fig. 3), silver grains are most densely concentrated throughout the dorsal subnucleus (D), followed in order o f diminishing density by central (C), lateral (L) and intermediate (I) subnuclei. (Publisher's reproduction factors: A, 0.48; B, 0.48.)
46 triangular septal nuclei, in addition to the precommissural fornix, anterior commissure, and medial preoptic nucleus. In one exceptional case, the injection site was predominantly unilateral and included the NDB (horizontal limb), medial edge o f the nucleus accumbens and bed nucleus of the stria terminalis. An examination of sections at the level of the midbrain revealed that the greatest density of silver grains was concentrated over the IPN. By contrast, the density over most other structures and o f f the sections was negligible (except for a modest band o f grains over the superior colliculus and very dense bands oriented tangentially, dorsolateral to the margin of the IPN bilaterally). The most striking feature of the labeling over the IPN was its differential distribution across individual subnuclei. The greatest density of grains was observed over the dorsal subnucleus, followed in order of diminishing density by the rostral, central, intermediate and lateral subnuclei (Fig. 2). The density of grains over the interstitial and dorsal lateral subnuclei appeared no greater than background and was not further analyzed. Our qualitative impressions and ratings were confirmed by the quantitative analyses; the results obtained at four rostrocaudal levels of the IPN from a representative case are summarized in Fig. 3. At the pontine level of the IPN (see Fig. 1D in ref. 7), for example, the relative density of grains over the dorsal subnucleus was more than two-fold greater than that over the rostral and central subnuclei and 4-fold greater than over the remaining subnuclei (Fig. 3C). By contrast, the density over the rostral and central subnuclei was approximately two,fold greater than over the intermediate and lateral subnuclei. There were several interesting features to the pattern of labeling over particular subnuclei of the IPN. The distribution of grains over the central subnucleus demonstrated a topographic organization such that the highest concentration of grains was found at middle to caudal levels o f the IPN (Fig. 3). We also observed a horizontal lamination in the distribution of grains over the central subnucleus at several levels of the IPN. At the midlevel of IPN (Fig. 3B), a heavy band of silver grains was distributed over the ventrolateral margins of the rostral subnucleus. This band defined the outline of two ovoid-shaped regions, over which there was only sparse labeling. Rostral to this level of the IPN, silver grains were distributed uniformly over the rostral subnucleus. Silver grains also were uniformly distributed over the lateral subnucleus, except for a conspicuous absence of grains over the ventrolateral corners of this subnucleus (Fig. 3D). The results of this study provide support for the existence of a projection from the nucleus of the diagonal band to the IPN in rats, which has been demonstrated with retrograde labeling and biochemical techniques [2,5]. The existence of this pathway was also reported by others [1,18]; but, until now, its complex distribution within the IPN has not been fully appreciated (cf. refs. 9, 11 and 14). Our findings suggest that IPN receives NDB afferents which are both topographically and heterogeneously distributed across individual subnuclei. Previous studies have demonstrated a subnuclear organization within the IPN
47
D
-!
O 70" O 00 60-
D
50'
Z m ,¢ 40n" 30"
R
::1:1= 2O' 10 A
B L E V E L S OF
ii
C
D
IPN
Fig. 3. Density of silver grains over individual subnuclei of IPN at four rostrocaudal levels (A-D) from an individual case. The greatest density of grains was located over the dorsal subnucleus (D), followed in order of diminishing density by the rostral (R), central (C), intermediate (I) and lateral (L) subnuclei. At level B, a dense band of grains located along the ventrolateral margins of the rostral subnucleus (Re) helped to define the outline of two ovoid-shaped regions (R) over which there was only a moderate density of grains. NDB afferents also appear to be topographically distributed to mid (B) and caudal (C) levels of the central subnucleus.
based upon cytoarchitecture, synaptology and the distribution of putative neurotransmitters [7-10,16]. The present results indicate that IPN subnuclei may be further differentiated on the basis of their afferent input. NDB inputs are most heavily concentrated in the dorsal subnucleus, followed in order of diminishing density by the rostral, central, intermediate and lateral subnuclei. Preliminary results obtained in other laboratories support the present concept that afferents to IPN are not homogeneously distributed [6]. The putative neurotransmitters of NDB afferents to the IPN are not known at present. However, enkephalin (Enk)-, cholecystokinin (CCK)-, and LH-RH-reactive perikarya have been demonstrated in the septal forebrain of rats [4,13,17,19]. Using immunocytochemical techniques, we have found a similarity between the distribution of Enk- and CCK-containing processes in the rostral subnucleus [8] and inputs from the NDB described in this report. Therefore, the possibility exists that a forebrain limbic projection from the NDB to the IPN may be peptidergic. Studies currently are in progress to explore this possibility. This research was supported by NSF Grant BNS76-17750 and NIH Grant NS12333 to Oswald Steward. Dr. David M. Jacobowitz kindly provided use of the
48 microscope
facilities in the Laboratory
of Clinical Science at NIMH.
We are
g r a t e f u l t o J a y G i l l e n w a t e r , R e b e c c a O g l e , F. L e e S n a v e l y a n d D r . J u l i o R a m i r e z for their assistance. 1 Conrad, L.C.A. and Pfaff, D.W., Efferents from medial basal forebrain and hypothalamus in the rat. I. An autoradiographic study of the medial preoptic area, J. comp. Neurol., 169 (1976) 185-220. 2 Contestabile, A. and Flumerfelt, B.A., Afferent connections of the interpeduncular nucleus and the topographic organization of the habenulointerpeduncular pathway: an HRP study in the rat, J. comp. Neurol., 196 (1981) 253-270. 3 Fass, B. and Steward, O., Increases in protein-precursor incorporation in the denervated neuropil of the dentate gyrus during reinnervation, Neuroscience, 9 (1983) 653-664. 4 Finley, J.C.W., Maderdrut, J.L. and Petrusz, P., The immunocytochemical localization of enkephalin in the central nervous system of the rat, J. comp. Neurol., 198 (1981) 541-565. 5 Gottesfeld, Z. and Jacobowitz, D.M., Cholinergic projection of the diagonal band to the interpeduncular nucleus of the rat brain, Brain Res., 156 (1978) 329-332. 6 Groenewegen, H.J., Kowall, N.W. and Nauta, W.H.J., Efferent connections of the dorsal tegmental region in the rat, Soc. Neurosci. Abstr., 13 (1983) 516. 7 Hamill, G.S. and Lenn, N.J., The subnuclear organization of the rat interpeduncular nucleus: a light and electron microscopy study, J. comp. Neurol., 222 (1984) 396-408. 8 Hamill, G.S., Olschowka, J.A., Lenn, N.J. and Jacobowitz, D.M., The subnuclear distribution of substance P, cholecystokinin, vasoactive intestinal peptide, somatostatin, Leu-enkephalin, dopamineB-hydroxylase, and serotonin in the rat interpeduncular nucleus, J. comp. Neurol., in press. 9 Hayakawa, T., Seki, M. and Zyo, K., Studies on the efferent projections of the interpeduncular complex in cats, Okajimas Folia Anat. Jpn., 58 0981) 1-16. 10 Hemmendinger, L.M. and Moore, R.Y., The interpeduncular nucleus of the rat: cytoarchitecture and cytochemistry, Soc. Neurosci. Abstr., 8 (1982) 665. ll Herkenham, M. and Nauta, W.J.H., Efferent connections of the habenular nuclei in the rat, J. comp. Neurol., 187 (1979) 19-48. 12 Houser, C.R., Crawford, G., Anderson, L., Barber, R., Salvaterra, P.M. and Vaughn, J.E., lmmunocytochemical localization of cholinergic neurons with a monoclonal antibody to choline acetyltransferase, Soc. Neurosci. Abstr., 8 (1982) 662. 13 Khachaturian, H., Lewis, M.E., Hollt, V. and Watson, S.J., Telencephalic enkephalinergic systems in the rat brain, J. Neurosci., 3 (1983) 844-855. 14 Marchand, E.R., Riley, J.N. and Moore, R.Y., Interpeduncular nucleus afferents in the rat, Brain Res., 193 (1980) 339-352. 15 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. 16 Rotter, A. and Jacobowitz, D.M., Colocalization of substance P, acetylcholinesterase, muscarinic receptors and alpha-bungarotoxin binding sites in the rat interpeduncular nucleus, Soc. Neurosci. Abstr., 8 (1982) 118. 17 Silverman, A.J. and Krey, L.C., The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea pig brain. I. Intra- and extra-hypothalamic projections, Brain Res., 157 (1978) 233-246. 18 Swanson, L.W. and Cowan, W.M., The connections of the septal region in the rat, J. comp. Neurol., 156 (1979) 621-656. 19 Woodhams, P.L., Roberts, G.W., Polak, J.M. and Crow, T.J., Distribution of neuropeptides in the limbic system of the rat: the bed nucleus of the stria terminalis, septum and preoptic area, Neuroscience, 8 (1983) 677-703.