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Vasoactive intestinal polypeptide (VIP) immunoreactive elements in the caudal ventral striatum of the rat: A light and electron microscopic study
Vasoactive Intestinal Polypeptide (VIP) Immunor~active Elements in the Caudal Ventral Striatum of the Rat: A Light and Electron Microscopic Study H. T. CHANG
Received CHANG, of
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rut:
Il.
T. AND
Q. TIAN.
A li~qht and ekwon
AND Q. TIAN
23 January
I99 I
Vmoactive intestinal po/pq?fide (MP) inrttrunorror,rire ehents in thy ururirri rmrrcd .strirmn~ ~;(.~~~s~~~~?~~ stud!. BRAIN RES BULL 26(6) 937-956. 1991 _--Under the posterior limb of the
anterior commiqsure. a brain region intercalated between the ventral striatum and the ventral pailidum was previously identified as the interstitial nucleus of the posterior limb of the anterior commissure by de Oimoa (7). This region, referred here as the caudnl ventral striatum (VSc), is characterized by a dense plexus of vasoactive intestinal peptide immunoreative (VIP+ ) axons. Doublefluorescence immunocytochemical reactions reveal that the dense VIP-t plexus is found in a region also rich in dopaminergc (i.e.. tyrosme-hydroxylase immunoreactive) fibers but poor in enkephalinergic terminals. The dense plexuses of VIP+ axon5 in VSc appear to be contiguous with those in the bed nucleus of stria terminalis (BNST) and the central nucleus of amygdala (CNA). as proposed recently by Alheid and These results support the notion that this WC region is a part of the “extended amygdala” Heimer (11. and con~rms that its anatomical properties are closer related to the ventral striatum than the ventral palI~d~iln. Electron microscopic analysis reveals that the VIP+ boutons form asymmetrical synapses with dendrites and spines, and symmetrical synapses with somata of unlabeled VSc neurons. The few VIP+ neurons within this area form synapses with many unlabeled axon terminals on both their somata and dendrites. Some VIP + neurons. however. also form axosomatic and axodendritic synapse\ with VIP + boutons. Vasoactive
intestinal
polypeptide
(VIP1
Extended
amygdala
PREVIOUS studies have shown that vasoactive intestinal peptide (VIP), a 28 amino acid polypeptide, is found in very high concentration in several basal forebrain limbic structures (1, 10. 11, 21, 22. 26) that are now considered as components of an “extended amygdala” (2). These regions include the bed nucleus of the stria terminalis (BNST), the central amygdala nucleus (CNA). and the sublenticular part of the substantia innominata (SI). Many authors have also noted that a dense band of VIP immunoreactive (VIP+ ) axons can be found ventral to the posterior limb of the anterior commissure (1. 9. 13. 22). a region that is intercalated between the ventral striatum (VS) and the ventral pallidul~~ (VP). both of which are very poorly inne~ated by VIP+ fibers. As this region innervated by VIP+ fibers is not readily distinguishable from the caudal VS in conventional histological preparations. it is referred here as the caudal VS (VSC). In this stud). we seek to reexamine the distribution of the VIPS fibers in VSc. Using the dense VIP+ axonal plexus as a marker. we aim to ascertain: I ) whether this VSc region shares similar anatomical properties with either ventral striatum or vmtral pallidum. and 2) what are the light (LM) and electron microscopic (EM) morphology of the VIP+ neurnnal elements in VSc.
Ventral
striatum
METHOD
Male adult Long-Evans rats (200~0~ g). anesthetized with urethane, were perfused through the ascending aorta with 200 ml of 0.02 M phosphate buffered saline (PBS. pH 7.4) and followed by one liter of fixative solution containing 3% paraformaldehyde. 0.5% glutaraldehyde. and IS0 ml of saturated picric acid in 0.1 M phosphate buffer (pH 7.3). The brains were removed and postfixed in the same fixative solution overnight at 4°C. The brains were blocked in either frontal or parasagittal planes and sectioned at a thickness of 50 (*m with a Vibratometi. Serial sections were collected in sequential test tubes containing 30% sucrose in PBS. and were frozen in a bath containing a mixture of dry ice and ethanol. The frozen tissue specimens were stored in either -70°C or -20°C freezers until ready for subsequent immunocytochen~ical procedures.
For the visualization
of VIP immunoreactive
elements.
fro-
FIG. 1. Low magnification light micrograph of a parasagittal rat brain section postfixed with osmium tetroxide is shown in A. The ventral striatum is lighter in appearance and is located to the left of the darker staining ventral pallidurn. Boxed area under the anterior commissure (ac) at the caudal part of the ventral striatum is shown at higher magnification in B revealing the dense plexus of i~uno~~xidase reacted VIP + axons and a VIP + neuron.
VIP IN VSc
949
solution containing rabbit antiserum to VIP (INCSTAR, 1: 1000 dilution), 0.3% Triton X-100 and 0.1% sodium azide in PBS for 12 to 24 hours. The sections were incubated in biotinylated secondary antibody solutions without Triton X-100 for 2 hours, and were followed by incubation for another 2 hours in a solution of avidinbiotin complexed with horseradish peroxidase (ABC-HRP, Vector). The peroxidase was visualized by incubating sections in a PBS solution of 0.05% 3,3’-diaminobenzidine (DAB) and 0.005% hydrogen peroxide for 10 to 15 minutes at room temperature. Tissue sections were rinsed with several changes of PBS between successive incubation steps. The secondary antibodies were either biotinylated goat antirabbit (Sigma) or biotinylated donkey anti-rabbit (Jackson). all were used at 1: 100 dilution from the stock solutions. Normal sera from the same species that donated the secondary antibodies was added to a final concentration of 2% into both primary and the secondary antisera incubation solutions to minimize nonspecific binding of the secondary antibodies. All incubations were carried out at room temperature, gently agitated on a laboratory shaker. Following DAB reactions, the tissue sections were postfixed in 0.5% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 hour. rinsed in distilled water, dehydrated in ethanol and propylene oxide, and prepared for LM and EM analysis as described previously (4). Controls
2. A drawing-tube tracing of the VIP+ neuronal elements from the same single section as shown in Fig. 1. Note that the dendrites of the VIP+ neuron are bipolarly oriented, and are found in region less densely innervated by VIP+ axons. FIG.
zen tissue sections were thawed at room temperature, and rinsed thoroughly with PBS until the yellow color of picric acid was completely washed out of the sections. The sections were reacted with 0.2% sodium borohydride in PBS for 30 minutes on a laboratory shaker and rinsed with PBS until all the gas bubbles disappeared from the solutions. Tissue sections were incubated in a
Lateral
Lateral 1.40 mm
The paper spot technique (20) was employed to test the antigenie specificity of the VIP antiserum (INCSTAR). The VIP primary antiserum (1: 1000 dilution) labeled intensely the spot on nitrocellulose paper containing 30 ng (about 10 pmol) of synthetic VIP (human, porcine, rat) (Peninsula Laboratories, Inc.. catalog No. 7161), and lightly with 300 pg of VIP. No crossreactions were detected with synthetic growth hormone releasing factor (100 ng) and somatostatin (10 ng) spotted on the same paper. A separate VIP antisera (Amersham) (1: 1000 dilution) also produced similar staining pattern. Similar test information from the supplier (INCSTAR) indicated that the VIP antisera used in the study showed no cross-reaction with several other proteins and polypeptides. The VIP-like immunoreactive staining pattern in this study is identical with those reported in previous single-labeling immu-
2.90 mm
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,
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I
:
SI
,/
VP
.
RG. 3. Boxed areas in these schematic drawings of the rat brain parasagittal
sections (27) outline the locations of the doublelabeling immunofluorescence micrographs in the following figures (Figs. 4-7). ac: anterior commissure; BNST: bed nucleus of stria terminalis; CNA: central nucleus of amygdala: SI: substantia innominata; VP: ventral pallidurn.
FIG. 4. At 1.4 mm lateral to the midline, the dense VIP-t
plexus (B, D, F) appears to extend from BNST around rhc anterio? commissure (ac) into WC. Double-labeling immunofluorescence reaction reveals that the regions innervated by VIP 4 axons are not densely innervated by ENK+ axon terminals (A, C. EL A and B are low magnification micrographs taken through diffeexmt fluorescence filter sets. Boxed areas in A are shown in higher magnifications in C and E respectively. and identical regions showing VIP i fibers are shown in D and F respectively. Both Scale bars in A and C are 200 pm.
nohistochemical studies using the same VIP antisera (9, 12, 19, 28, 29, 36). Deletion of primary antisera or preadso~tion of
pallidal properties: The “striatai” property was indentified by a dense terminal plexus of dopa~ergic fibers originated from the
primary antisera solution (1:lOOO dilution) with synthetic VIP (3.3 p.g/ml) abolished all VIP-like immunoreactive staining. In this study, all elements displaying VIP-like immunoreactive staining are called as VIP+ elements.
midbrain substantia nigra ventral tegmental area. The “pallidal” property was identified by a dense plexus of enkephalin immunoreactive terminals originated from the striopallidal axons (14). Conventional double-labeling immunofluorescence reactions were performed to determine the relationships of VIP+ plexus with either dopaminergic fibers that are immunoreactive for tyrosine hydroxylase (TH), the rate limiting synthetic enzyme for dopamine, or enkephaiin immunoreactive striopallidal terminals.
Double-Labeling Immunojuorescence Reactions Two lines of tests, complementing each other, were used to determine whether a territory is endowed with either striatal or
VIP IN vsc
FIG. 5. At 2.9 mm lateral to the midline, the dense VIP+ plexus (B. D) is found under the anterior commissure (ac) in a region that is also innervated densely by TH t axons (A, C). Boxed area in A is shown in higher magni~cation~ in C, and the identical region showing VIP+ fibers is shown in D. Both scale bars in A and C are 200 IJ.~.
Mouse monoclonal antibodies to either TH (Boehringer-Mannheim) or leucine-enkephalin (ENK) (Accurate Chemical) were used in combination with the rabbit antiserum to VIP. Instead of biot~nyIated secondary antibodies as described above. VIP+ fibers were revealed with Texas Red labeled secondary antiserum. whereas TH immunoreactive (TH + ) or ENK immunoreactive (END- ) neuronal elements were revealed with fluorescene isothiocyanate (FIT0 labeled secondary antisera. RESULTS Single-Lubelitq
LM Analysis
ofVIP-+Neuronal Elements
Since VS is rostra1 to VP, all figures in this report are from parasagittal sections to better illustrate both VS and VP in single sections. As shown in Figs. 1 and 2. a dense plexus of VIP+ axon terminals is found at the junctional area between VS and VP. The dense VIP+ plexus consists of numerous varicose axons and only a few VIP+ neurons that are usually found at the periphery of the dense VIP+ axonal plexus. The somata of VIP+ neurons (largest diameter, 10 to 15 pm) appear smaller than the neostriatal medium spiny neurons reported in previous studies (6). Two or 3 sparsely branched dendrites radiate bipolarly from the soma. The surfaces of the VIP+ soma and dendrites appear to be aspinous at the light microscopic level. Although most VIP+ dendrites radiate mainly in territories not
densely innervated by the VIP + axons, some VIP+ axon terminals are found in close vicinity of the VIP+ dendrites. Some VIP+ neurons give rise to axon collaterals that can be traced into the dense VIP+ axonal plexus. However. it remains unclear whether all of the dense VIP-t axonal plexus originate only from local collaterals of the VIP + neurons. Double-Labeling
Immunclfluorescence
Reactions
As described in previous studies. the dense VfPt axonal plexus can be found extending mediolaterally along the posterior limb of the anterior commissure from BNST to CNA (Fig. 3). Double-labeling immunofluorescence reactions reveal that the VSc territory innervated by VIP+ axons is poorly innervated by ENK+ fibers (Figs. 4, 6). but densely innervated by dopaminergic TH+ fibers (Figs. 5, 7). This result shows that this region innervated densely by the VIP+ axons is endowed with more striatal than pallidal properties (and thus justifies its designation as VSc). EM Analysis
of’ VIP+
Neuronal
Elements
in VSc
Under electron microscopic examination. the somata of VIP + neurons are often found near a cluster of several unlabeled medium-sized VSc neurons (Fig. 8A). The somatic plasma membrane of VIP+ neurons often fomls apposition with that of neighbor-
PIG. 6. At 3.4 mm lateral to the midline, the small patch of dense VIP+ plexus (B, D) is found just caudal the anterior commissure (ac) in a regu: that is less densely innervated by ENK + axons (A, C) as the ventral pallidurn. Boxed area in A is shown in higher magnifications in C. and the identical region showing VIP+ fibers is shown in D. st: Stria terminalis. Both scale bars in A and C are 200 pm.
ing unlabeled VSc neurons that in turn also form appositions to other unlabeled VSc neurons. Gap junctions were not found in these appositions, however. The VIP+ neurons are characterized by centrally located nuclei that are deeply indented with invaginated nuclear membrane (Fig. 8A). This feature is in contrast to most of its neighboring unlabeled medium-sized VSc neurons that have round, unindented nuclei. The somata and dendrites of VIP+ neurons are richly innervated, forming synapses with mainly unlabeled axon terminals (Fig. 8B, C). Occasionally, small spine-like somatic protrusions unnoticed under the light microscopic examination also form synapses with unlabeled axon terminals. In the surrounding neuropil, many VIP+ axon terminals form synapses with unlabeled neuronal elements. Prominent postsynaptic densities, indicative of asymmetricai synapses, are often found in synapses between small dendrites and VIP+ terminals (Fig. 8D), or those between dendritic spines and VIP+ terminals. On the other hand, despite searching through serial thin sections, some VIP+ terminals appear to form synapses with little postsynaptic densities suggestive of symmetrical synapses. Most of these symmetrical synapses are formed with somata (Fig. 8A, large arrowheads) and large dendrites (Fig. 8E). Infrequently, VIP+ terminals are found to form synapses with VIP+ somata (Fig. 8F) and dendrites. The membrane specializations of these later synapses, however, are difficult to determine due to the presence of dense immunoperoxidase reaction products within both the presynaptic and the postsynaptic eIements. Nevertheless, all VIP + terminals
share a similar property in that they are filled with smafl round vesicles (diameters 30-50 nm). The dis~bution of the postsynaptic elements which form synapses with VIP+ terminals is shown in Fig. 9. Clearly most symmetrical synapses are formed with somata and larger dendrites while asymmetrical synapses are formed with smaller dendrites and dendritic spines. DISCUSSION
Distribution of VIP-t- Plexus and the Extended Amygdala
Our finding confirms results of previous immunocytochemical studies (1,13) that a region in the caudal ventral striatum along the posterior limb of the anterior commissure is densely innervated by VIP+ plexus. This is in contrast to the dorsal striatum that has only a moderate hunt of VIP+ neurons and axons (1, 9, 21, 22, 32, 36). This VSc region apparently corresponds to the interstitial nucleus of the posterior limb of the anterior commissure (7). We have shown in this study that this region has more striatal than pallidal properties because it is innervated richly by dopaminergic fibers that merge imperceptibly with dopaminergic fibers in the rest of the striatum. Furthermore, in contrast to the ventral pallidurn that is densely innervated by enkephalinergic terminals (16,34), this region is comparable to the rest of the striatum in its density of enkephalinergic terminals. The general distribution pattern of VIP+ plexus in the basal
VIP IN WC
953
FIG. 7. At 3.9 mm lateral to the midline, the dense VIP+ plexus (B, D, F) in WC is contiguous with a larger, and denser VIP+ plexus located within the central amygdala nucleus (on the right side in Fig. 7Bl. The VSc region innervated by VIP+ axons is clearly also innervated by TH+ axons (A, C. E). The boxed area in A is shown in higher magnifications in C, and the boxed area in C is enlarged at twice the magnification in E. Identical regions showing VIP+ fibers are shown in B, D, F. respectively. Both scale bars in A and C are 200 pm.
forebrain areas appears to be very similar to those reported previously for fibers immunoreactive for either angiotensin II (2) or calcitonin gene related peptide (CGRP) (5). All have dense fiber plexuses in the VSc region that appear to be contiguous with similarly dense terminal plexuses in BNST and CNA. These observations suggest that this VSc region share very similar anatomical properties with BNST and CNA, and thus may be
considered as one of the components of the “extended amygdala“ (2). This is consistent with the observation (2) that portions of the ventral striatum (nucleus accumbens) have connections that are not typical of “striatal” efferents. but are typical of amygdaloid efferents: some projections have been traced to the hypothalamus. the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus and area surrounding the superior cerebellar
FIG. 8. (A) A low magnification EM micrograph showing the VIPs- neuron near a cluster of unlabeled VSc neurons (Nl, N2, X.31. ‘This VIP+ neuron is me same cell illustrated in Figs. I and 2. Dashed line marks the apposing border between Nl and NZ. Note that the VlP+- neuron ha:, indented nucleus (small arrows), whereas the neigh~ring unlabeled VSc neurons have round, unindented nuclei. Note aiso that two VIP+- terminals (large arrowheads) form axosomatic synapses with one of the neigh~~ng VSc neurons. (B) Two unlabeled terminals form axosomatic synapses (arrows) with a VIP+ neuron. (C) Several unlabeled terminals form axodendritic synapses (arrows) with a VIP+ dendrite. (D) An example of an asymmetrical synapse (arrow) between a VIP+ terminal and an unlabeled dendrite. (El An example of a symmetnical synapse (arrow1 between a VIP+ terminal and the shaft of an unlabeled dendrite tbat gives rise to a spine (s). (F) An exampfe of a synapse (arrow) between a VIP f rerminal and a VIP -I- soma. B, C, D. E, and F are at the same magn~~cation, scale bars in A and B are 2 pm and 0.5 I.~rn_respectively.
peduncle (15, 24, 25, 33). Moreover, this VSc region appears to be the only part of the striatum innervated by noradrenergic fibers immunoreactive for dopamine P-hydroxylase (unpublished observation), which densely innervate all parts of the extended
amygdaia, including BNST, CNA and sublenticufar substantia i~omina~ (SI) (4). These observations together show that this VSc region along the posterior limb of the anterior commissure may be considered as a kind of striatal-amygdala transition area
955
VIP IN VSc
parts of the striatum. and thus may be considered as a peculiar “patch” of the ventral striatum distinguished by its “amygdaloid” type of immunohistochemical properties. The unlabeled VSc neurons within the clusters exhibit morphology typical of striatal medium spiny neurons in that they have round unindented nuclei, a narrow rim of perikaryal cytoplasm, and very few axosomatic synapses. However. it remains to be ascertained whether any of the unlabeled neurons within the cluster are medium spiny neurons. As the somata of the VSc neurons within the clusters are tightly apposed to each other. it is likelv that neurons within each cluster share very similar afferent input, including the dopaminergic fibers shown in this study,
Postsynaptic
FIG. 9. Histogram
Dendritic
showing the distribution terminals.
postsynaptic
to VIP+
possessing
both striatal and amygdaloid
Mameter
(~1
of the dendritic
diameters
properties.
The smallish somatic size piaces the VIP-f- neurons at the lower end of the striatal medium-sized neurons category. The aspinous dendritic surfaces of VIP + neurons correspond broadly to the morphological characteristics of striatal medium aspiny neurons. However. since there are only a few VIP+ neurons in the striatum (1. 26. 36). it is not surprising that smallish medium-sized striatal neurons with bipolarly arranged dendritic fields similar to the VIP+ neurons have been observed only rarely in previous Golgi studies (6). This situation is in contrast to that in the cerebral cortex in which the relationships between the numerous VIP+ neurons and the bipolar neurons stained in Golgi studies have been analyzed in detail (X,29). Nevertheless, as more and more striatal neuron types are identified by immunocytochemical means, the anatomical and functional organization of the striatal ahpiny neurons will become better understood. The ultrastructural features of VIP+ neurons in VSc are similar to those described previously for VIP+ neurons in the striatum (36) and CNA (13). Their deeply indented nuclei are typical of the striatal medium aspiny neurons described in previous EM studies (3, 8, 35). We have found in this study that individual VIP+ neurons often form tight appositions with clusters of several (3 to 3) unlabeled medium-sized VSc neurons. The areas covered by these VSc neuronal clusters are smaller than the typical neuronal “patches” or “striosomes” in most of the striatum in which larger groups of striatal neurons, from tens to hundreds. are associated in “patches” distinguished by differential histochemical or immunocytochemical reactivity (14.17). Indeed. the entire VSc outlined by the dense VIP+ plexus has roughly the same size as a typical “patch” found in the other
Since the surrounding neuropil is densely innervated by VIP + axons. it is not surprising that many VIP+ axons form synapses with both VIP+ and unlabeled neurons within the clusters. However. how VIP regulates the functions of these clusters of VSc neurons is not clear. The few VIP+ medium-sized neurons found next to the dense VIP+ plexus seem too few to account for the heavy VIP+ terminal plexus in VSc. Several lines of evidence suggest that this dense plexus of VIP i- axon terminaIs arises from two different sources: 1) Our EM analysis has revealed that most VIP+ axon terminals form asyI~~]netrical synapses with mostly small dendrites and dendritic spines, and the rest form symmetrical synapses with mainly somata and larger dendrites (Fig. 9). This distribution pattern is consistent with that in the striatum in which the extrinsic afferent axons f’orm mainly asymmetrical synapses on small dendrites and spines while intrinsic axons form symmetrical synapses with mainly larger dendrites and somata ( 18). 2) Typical striatal VIP+ neuron5 are associated with very sparse VIP+ axon plexuses. which form symmetrical synapses u ith either VIP + somata or neighboring neurons (36). 3) Only one type of VIP+ neurons could be distinguished in VSc. and they have similar morphology as those found in the other parts of the ctriatum. 3) Previous studies (9, ‘23. 30. 3 I ) have shown that there are at least two sources of VIP-+ axons that innervated the extended amygdala: A) eutrinsic VIP+ neurons in the brain stem nuclei, and B) intrinsic VIP+ neurons located within the extended amygdaia. These observations suggest that the dense VIP+ plexuses in VSc also consist of both intrinsic and extrinsic fibers. Given the intimate relationship of VSc with the “extended amygdala.” it is very likely that the extrinsic VIP+ fibers in VSc arise from the same sources that innervate the other parts of the extended amygdala. Whether the extrinsic or the intrinsic VIP+ fibers are associated with different types of VIP receptors or different synaptic specializations remains to be determined.
This study was supported by VSPHS Grant AG059-U, NS2 1003. Biomedical Research Support Grant RR05423. a grant from the Aizheimer‘s Disease and Related Disorders Association, and a postdoctoral fellowship from the Neuroscience Center of Excellence of The linkersity of Tennessee. Memphis.
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104:101-112; 1989. 5. Chang, H. T.; Kuo, H. Calcitonin gene-related peptide (CGRP) in the rat substantia innominata and globus pallidus: A light and electron microscopic immunocytochemical study. Brain Res. 495: 167172; 1989. 6. Chang, H. T.; Wilson, C. J.; Kitai, S. T. A Golgi study of rat neostiatal neurons: Light microscopic analysis. J. Comp. Neurol. 208: 107-126; 1982. 7. de Olmos, J. S. The amygdaloid projection field in the rat as studied with the cupric-silver method. In: Eleftheriou, B. E., ed. The neurobiology of the amygdala. New York: Plenum Press; 1972:145204. 8. DiFiglia, M.; Pasik, P.; Pasik, T. Ultrastructure of Golgi-impregnated and gold-toned spiny and aspiny neurons in the monkey neostriatum. J. Neurocytol. 9:471492; 1980. 9. Eiden, L. E.; Hokfelt, T.; Brownstein, M. J.; Palkovits, M. Vasoactive intestinal polypeptide afferents to the bed nucleus of the stria terminalis in the rat: An immunohistochemical and biochemical study. Neuroscience 15:999-1013; 1985. 10. Ftlxe, K.; Hofelt, T.; Said, S. I.; Mutt, V. Vasoactive intestinal polypeptide and the nervous system: immunohistochemical evidence for localization in central and peripheral neurons, particularly intracortical neurons of the cerebral cortex. Neurosci. Len 5:241-246; 1977. 11. Giachetti, A.; Said, S. I.; Reynolds, R. C.; Koniges, F. C. Vasoactive intestinal polypeptide in brain. Localization in the release from isolated nerve terminals. Proc. Natl. Acad. Sci. USA 74:3424-3428; 1977. 12. Giesler, G. J., Jr.; Elde, R. P. Immunocytochemical studies of the peptidergic content of fibers and terminals within the lateral spinal and lateral cervical nuclei. J. Neurosci. 5:1833-1841; 1985. E. A.; 13. Gray, T. S.; Cassell, M. D.; Nilaver, G.; Zimmerman, Williams, T. H. The distribution and ultrastructure of VIP-immunoreactivity in the central nucleus of the rat amygdala. Neuroscience 11:399-408; 1984. 14. Graybiel, A. M.; Ragsdale, C. W., Jr. Biochemical anatomy of the striatum. In: Emson, P.C., ed. Chemical neuroanatomy. New York: Raven Press; 1983:427-504. 15. Groenewegen, H. J.; Russchen, F. T. Organization of the efferent projections of the nucleus accumbens to pallidal, hypothalamic, and mesencephalic StNCNreS: A tracing and immunohistochemical study in the cat. J. Comp. Neurol. 223:347-367; 1984. 16. Haber, S. N.; Nauta, W. J. H. Ramifications of the globus pallidus in the rat as indicated by patterns of immunohistochemistry. Neuroscience 9:245-260; 1983. 17. Herkenham, M.; Moon Edley, S.; Stuart, J. Cell clusters in the nucleus accumbens of the rat, and the mosaic relationship of opiate receptors, acetylcholinesterase. and subcortical afferent terminations. Neuroscience 11561-593; 1984. of the 18. Kemp, J. M.; Powell, T. P. S. The synaptic organization caudate nucleus. Philos. Trans. R. Sot. Ser. B. 262:403412; 1971. analysis of vasoactive intestinal poly19. Kohler, C. A morphological peptide (VIP)-like innumoreactive neurons in the area dentata of the rat brain. J. Comp. Neurol. 221:247-262; 1983.
<:HANCi
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Received CHANG, of
r/w
rut:
Il.
T. AND
Q. TIAN.
A li~qht and ekwon
AND Q. TIAN
23 January
I99 I
Vmoactive intestinal po/pq?fide (MP) inrttrunorror,rire ehents in thy ururirri rmrrcd .strirmn~ ~;(.~~~s~~~~?~~ stud!. BRAIN RES BULL 26(6) 937-956. 1991 _--Under the posterior limb of the
anterior commiqsure. a brain region intercalated between the ventral striatum and the ventral pailidum was previously identified as the interstitial nucleus of the posterior limb of the anterior commissure by de Oimoa (7). This region, referred here as the caudnl ventral striatum (VSc), is characterized by a dense plexus of vasoactive intestinal peptide immunoreative (VIP+ ) axons. Doublefluorescence immunocytochemical reactions reveal that the dense VIP-t plexus is found in a region also rich in dopaminergc (i.e.. tyrosme-hydroxylase immunoreactive) fibers but poor in enkephalinergic terminals. The dense plexuses of VIP+ axon5 in VSc appear to be contiguous with those in the bed nucleus of stria terminalis (BNST) and the central nucleus of amygdala (CNA). as proposed recently by Alheid and These results support the notion that this WC region is a part of the “extended amygdala” Heimer (11. and con~rms that its anatomical properties are closer related to the ventral striatum than the ventral palI~d~iln. Electron microscopic analysis reveals that the VIP+ boutons form asymmetrical synapses with dendrites and spines, and symmetrical synapses with somata of unlabeled VSc neurons. The few VIP+ neurons within this area form synapses with many unlabeled axon terminals on both their somata and dendrites. Some VIP + neurons. however. also form axosomatic and axodendritic synapse\ with VIP + boutons. Vasoactive
intestinal
polypeptide
(VIP1
Extended
amygdala
PREVIOUS studies have shown that vasoactive intestinal peptide (VIP), a 28 amino acid polypeptide, is found in very high concentration in several basal forebrain limbic structures (1, 10. 11, 21, 22. 26) that are now considered as components of an “extended amygdala” (2). These regions include the bed nucleus of the stria terminalis (BNST), the central amygdala nucleus (CNA). and the sublenticular part of the substantia innominata (SI). Many authors have also noted that a dense band of VIP immunoreactive (VIP+ ) axons can be found ventral to the posterior limb of the anterior commissure (1. 9. 13. 22). a region that is intercalated between the ventral striatum (VS) and the ventral pallidul~~ (VP). both of which are very poorly inne~ated by VIP+ fibers. As this region innervated by VIP+ fibers is not readily distinguishable from the caudal VS in conventional histological preparations. it is referred here as the caudal VS (VSC). In this stud). we seek to reexamine the distribution of the VIPS fibers in VSc. Using the dense VIP+ axonal plexus as a marker. we aim to ascertain: I ) whether this VSc region shares similar anatomical properties with either ventral striatum or vmtral pallidum. and 2) what are the light (LM) and electron microscopic (EM) morphology of the VIP+ neurnnal elements in VSc.
Ventral
striatum
METHOD
Male adult Long-Evans rats (200~0~ g). anesthetized with urethane, were perfused through the ascending aorta with 200 ml of 0.02 M phosphate buffered saline (PBS. pH 7.4) and followed by one liter of fixative solution containing 3% paraformaldehyde. 0.5% glutaraldehyde. and IS0 ml of saturated picric acid in 0.1 M phosphate buffer (pH 7.3). The brains were removed and postfixed in the same fixative solution overnight at 4°C. The brains were blocked in either frontal or parasagittal planes and sectioned at a thickness of 50 (*m with a Vibratometi. Serial sections were collected in sequential test tubes containing 30% sucrose in PBS. and were frozen in a bath containing a mixture of dry ice and ethanol. The frozen tissue specimens were stored in either -70°C or -20°C freezers until ready for subsequent immunocytochen~ical procedures.
For the visualization
of VIP immunoreactive
elements.
fro-
FIG. 1. Low magnification light micrograph of a parasagittal rat brain section postfixed with osmium tetroxide is shown in A. The ventral striatum is lighter in appearance and is located to the left of the darker staining ventral pallidurn. Boxed area under the anterior commissure (ac) at the caudal part of the ventral striatum is shown at higher magnification in B revealing the dense plexus of i~uno~~xidase reacted VIP + axons and a VIP + neuron.
VIP IN VSc
949
solution containing rabbit antiserum to VIP (INCSTAR, 1: 1000 dilution), 0.3% Triton X-100 and 0.1% sodium azide in PBS for 12 to 24 hours. The sections were incubated in biotinylated secondary antibody solutions without Triton X-100 for 2 hours, and were followed by incubation for another 2 hours in a solution of avidinbiotin complexed with horseradish peroxidase (ABC-HRP, Vector). The peroxidase was visualized by incubating sections in a PBS solution of 0.05% 3,3’-diaminobenzidine (DAB) and 0.005% hydrogen peroxide for 10 to 15 minutes at room temperature. Tissue sections were rinsed with several changes of PBS between successive incubation steps. The secondary antibodies were either biotinylated goat antirabbit (Sigma) or biotinylated donkey anti-rabbit (Jackson). all were used at 1: 100 dilution from the stock solutions. Normal sera from the same species that donated the secondary antibodies was added to a final concentration of 2% into both primary and the secondary antisera incubation solutions to minimize nonspecific binding of the secondary antibodies. All incubations were carried out at room temperature, gently agitated on a laboratory shaker. Following DAB reactions, the tissue sections were postfixed in 0.5% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 hour. rinsed in distilled water, dehydrated in ethanol and propylene oxide, and prepared for LM and EM analysis as described previously (4). Controls
2. A drawing-tube tracing of the VIP+ neuronal elements from the same single section as shown in Fig. 1. Note that the dendrites of the VIP+ neuron are bipolarly oriented, and are found in region less densely innervated by VIP+ axons. FIG.
zen tissue sections were thawed at room temperature, and rinsed thoroughly with PBS until the yellow color of picric acid was completely washed out of the sections. The sections were reacted with 0.2% sodium borohydride in PBS for 30 minutes on a laboratory shaker and rinsed with PBS until all the gas bubbles disappeared from the solutions. Tissue sections were incubated in a
Lateral
Lateral 1.40 mm
The paper spot technique (20) was employed to test the antigenie specificity of the VIP antiserum (INCSTAR). The VIP primary antiserum (1: 1000 dilution) labeled intensely the spot on nitrocellulose paper containing 30 ng (about 10 pmol) of synthetic VIP (human, porcine, rat) (Peninsula Laboratories, Inc.. catalog No. 7161), and lightly with 300 pg of VIP. No crossreactions were detected with synthetic growth hormone releasing factor (100 ng) and somatostatin (10 ng) spotted on the same paper. A separate VIP antisera (Amersham) (1: 1000 dilution) also produced similar staining pattern. Similar test information from the supplier (INCSTAR) indicated that the VIP antisera used in the study showed no cross-reaction with several other proteins and polypeptides. The VIP-like immunoreactive staining pattern in this study is identical with those reported in previous single-labeling immu-
2.90 mm
:......~........... . ,
: :
I
,
c kr : ,/ i/ i
I
:
SI
,/
VP
.
RG. 3. Boxed areas in these schematic drawings of the rat brain parasagittal
sections (27) outline the locations of the doublelabeling immunofluorescence micrographs in the following figures (Figs. 4-7). ac: anterior commissure; BNST: bed nucleus of stria terminalis; CNA: central nucleus of amygdala: SI: substantia innominata; VP: ventral pallidurn.
FIG. 4. At 1.4 mm lateral to the midline, the dense VIP-t
plexus (B, D, F) appears to extend from BNST around rhc anterio? commissure (ac) into WC. Double-labeling immunofluorescence reaction reveals that the regions innervated by VIP 4 axons are not densely innervated by ENK+ axon terminals (A, C. EL A and B are low magnification micrographs taken through diffeexmt fluorescence filter sets. Boxed areas in A are shown in higher magnifications in C and E respectively. and identical regions showing VIP i fibers are shown in D and F respectively. Both Scale bars in A and C are 200 pm.
nohistochemical studies using the same VIP antisera (9, 12, 19, 28, 29, 36). Deletion of primary antisera or preadso~tion of
pallidal properties: The “striatai” property was indentified by a dense terminal plexus of dopa~ergic fibers originated from the
primary antisera solution (1:lOOO dilution) with synthetic VIP (3.3 p.g/ml) abolished all VIP-like immunoreactive staining. In this study, all elements displaying VIP-like immunoreactive staining are called as VIP+ elements.
midbrain substantia nigra ventral tegmental area. The “pallidal” property was identified by a dense plexus of enkephalin immunoreactive terminals originated from the striopallidal axons (14). Conventional double-labeling immunofluorescence reactions were performed to determine the relationships of VIP+ plexus with either dopaminergic fibers that are immunoreactive for tyrosine hydroxylase (TH), the rate limiting synthetic enzyme for dopamine, or enkephaiin immunoreactive striopallidal terminals.
Double-Labeling Immunojuorescence Reactions Two lines of tests, complementing each other, were used to determine whether a territory is endowed with either striatal or
VIP IN vsc
FIG. 5. At 2.9 mm lateral to the midline, the dense VIP+ plexus (B. D) is found under the anterior commissure (ac) in a region that is also innervated densely by TH t axons (A, C). Boxed area in A is shown in higher magni~cation~ in C, and the identical region showing VIP+ fibers is shown in D. Both scale bars in A and C are 200 IJ.~.
Mouse monoclonal antibodies to either TH (Boehringer-Mannheim) or leucine-enkephalin (ENK) (Accurate Chemical) were used in combination with the rabbit antiserum to VIP. Instead of biot~nyIated secondary antibodies as described above. VIP+ fibers were revealed with Texas Red labeled secondary antiserum. whereas TH immunoreactive (TH + ) or ENK immunoreactive (END- ) neuronal elements were revealed with fluorescene isothiocyanate (FIT0 labeled secondary antisera. RESULTS Single-Lubelitq
LM Analysis
ofVIP-+Neuronal Elements
Since VS is rostra1 to VP, all figures in this report are from parasagittal sections to better illustrate both VS and VP in single sections. As shown in Figs. 1 and 2. a dense plexus of VIP+ axon terminals is found at the junctional area between VS and VP. The dense VIP+ plexus consists of numerous varicose axons and only a few VIP+ neurons that are usually found at the periphery of the dense VIP+ axonal plexus. The somata of VIP+ neurons (largest diameter, 10 to 15 pm) appear smaller than the neostriatal medium spiny neurons reported in previous studies (6). Two or 3 sparsely branched dendrites radiate bipolarly from the soma. The surfaces of the VIP+ soma and dendrites appear to be aspinous at the light microscopic level. Although most VIP+ dendrites radiate mainly in territories not
densely innervated by the VIP + axons, some VIP+ axon terminals are found in close vicinity of the VIP+ dendrites. Some VIP+ neurons give rise to axon collaterals that can be traced into the dense VIP+ axonal plexus. However. it remains unclear whether all of the dense VIP-t axonal plexus originate only from local collaterals of the VIP + neurons. Double-Labeling
Immunclfluorescence
Reactions
As described in previous studies. the dense VfPt axonal plexus can be found extending mediolaterally along the posterior limb of the anterior commissure from BNST to CNA (Fig. 3). Double-labeling immunofluorescence reactions reveal that the VSc territory innervated by VIP+ axons is poorly innervated by ENK+ fibers (Figs. 4, 6). but densely innervated by dopaminergic TH+ fibers (Figs. 5, 7). This result shows that this region innervated densely by the VIP+ axons is endowed with more striatal than pallidal properties (and thus justifies its designation as VSc). EM Analysis
of’ VIP+
Neuronal
Elements
in VSc
Under electron microscopic examination. the somata of VIP + neurons are often found near a cluster of several unlabeled medium-sized VSc neurons (Fig. 8A). The somatic plasma membrane of VIP+ neurons often fomls apposition with that of neighbor-
PIG. 6. At 3.4 mm lateral to the midline, the small patch of dense VIP+ plexus (B, D) is found just caudal the anterior commissure (ac) in a regu: that is less densely innervated by ENK + axons (A, C) as the ventral pallidurn. Boxed area in A is shown in higher magnifications in C. and the identical region showing VIP+ fibers is shown in D. st: Stria terminalis. Both scale bars in A and C are 200 pm.
ing unlabeled VSc neurons that in turn also form appositions to other unlabeled VSc neurons. Gap junctions were not found in these appositions, however. The VIP+ neurons are characterized by centrally located nuclei that are deeply indented with invaginated nuclear membrane (Fig. 8A). This feature is in contrast to most of its neighboring unlabeled medium-sized VSc neurons that have round, unindented nuclei. The somata and dendrites of VIP+ neurons are richly innervated, forming synapses with mainly unlabeled axon terminals (Fig. 8B, C). Occasionally, small spine-like somatic protrusions unnoticed under the light microscopic examination also form synapses with unlabeled axon terminals. In the surrounding neuropil, many VIP+ axon terminals form synapses with unlabeled neuronal elements. Prominent postsynaptic densities, indicative of asymmetricai synapses, are often found in synapses between small dendrites and VIP+ terminals (Fig. 8D), or those between dendritic spines and VIP+ terminals. On the other hand, despite searching through serial thin sections, some VIP+ terminals appear to form synapses with little postsynaptic densities suggestive of symmetrical synapses. Most of these symmetrical synapses are formed with somata (Fig. 8A, large arrowheads) and large dendrites (Fig. 8E). Infrequently, VIP+ terminals are found to form synapses with VIP+ somata (Fig. 8F) and dendrites. The membrane specializations of these later synapses, however, are difficult to determine due to the presence of dense immunoperoxidase reaction products within both the presynaptic and the postsynaptic eIements. Nevertheless, all VIP + terminals
share a similar property in that they are filled with smafl round vesicles (diameters 30-50 nm). The dis~bution of the postsynaptic elements which form synapses with VIP+ terminals is shown in Fig. 9. Clearly most symmetrical synapses are formed with somata and larger dendrites while asymmetrical synapses are formed with smaller dendrites and dendritic spines. DISCUSSION
Distribution of VIP-t- Plexus and the Extended Amygdala
Our finding confirms results of previous immunocytochemical studies (1,13) that a region in the caudal ventral striatum along the posterior limb of the anterior commissure is densely innervated by VIP+ plexus. This is in contrast to the dorsal striatum that has only a moderate hunt of VIP+ neurons and axons (1, 9, 21, 22, 32, 36). This VSc region apparently corresponds to the interstitial nucleus of the posterior limb of the anterior commissure (7). We have shown in this study that this region has more striatal than pallidal properties because it is innervated richly by dopaminergic fibers that merge imperceptibly with dopaminergic fibers in the rest of the striatum. Furthermore, in contrast to the ventral pallidurn that is densely innervated by enkephalinergic terminals (16,34), this region is comparable to the rest of the striatum in its density of enkephalinergic terminals. The general distribution pattern of VIP+ plexus in the basal
VIP IN WC
953
FIG. 7. At 3.9 mm lateral to the midline, the dense VIP+ plexus (B, D, F) in WC is contiguous with a larger, and denser VIP+ plexus located within the central amygdala nucleus (on the right side in Fig. 7Bl. The VSc region innervated by VIP+ axons is clearly also innervated by TH+ axons (A, C. E). The boxed area in A is shown in higher magnifications in C, and the boxed area in C is enlarged at twice the magnification in E. Identical regions showing VIP+ fibers are shown in B, D, F. respectively. Both scale bars in A and C are 200 pm.
forebrain areas appears to be very similar to those reported previously for fibers immunoreactive for either angiotensin II (2) or calcitonin gene related peptide (CGRP) (5). All have dense fiber plexuses in the VSc region that appear to be contiguous with similarly dense terminal plexuses in BNST and CNA. These observations suggest that this VSc region share very similar anatomical properties with BNST and CNA, and thus may be
considered as one of the components of the “extended amygdala“ (2). This is consistent with the observation (2) that portions of the ventral striatum (nucleus accumbens) have connections that are not typical of “striatal” efferents. but are typical of amygdaloid efferents: some projections have been traced to the hypothalamus. the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus and area surrounding the superior cerebellar
FIG. 8. (A) A low magnification EM micrograph showing the VIPs- neuron near a cluster of unlabeled VSc neurons (Nl, N2, X.31. ‘This VIP+ neuron is me same cell illustrated in Figs. I and 2. Dashed line marks the apposing border between Nl and NZ. Note that the VlP+- neuron ha:, indented nucleus (small arrows), whereas the neigh~ring unlabeled VSc neurons have round, unindented nuclei. Note aiso that two VIP+- terminals (large arrowheads) form axosomatic synapses with one of the neigh~~ng VSc neurons. (B) Two unlabeled terminals form axosomatic synapses (arrows) with a VIP+ neuron. (C) Several unlabeled terminals form axodendritic synapses (arrows) with a VIP+ dendrite. (D) An example of an asymmetrical synapse (arrow) between a VIP+ terminal and an unlabeled dendrite. (El An example of a symmetnical synapse (arrow1 between a VIP+ terminal and the shaft of an unlabeled dendrite tbat gives rise to a spine (s). (F) An exampfe of a synapse (arrow) between a VIP f rerminal and a VIP -I- soma. B, C, D. E, and F are at the same magn~~cation, scale bars in A and B are 2 pm and 0.5 I.~rn_respectively.
peduncle (15, 24, 25, 33). Moreover, this VSc region appears to be the only part of the striatum innervated by noradrenergic fibers immunoreactive for dopamine P-hydroxylase (unpublished observation), which densely innervate all parts of the extended
amygdaia, including BNST, CNA and sublenticufar substantia i~omina~ (SI) (4). These observations together show that this VSc region along the posterior limb of the anterior commissure may be considered as a kind of striatal-amygdala transition area
955
VIP IN VSc
parts of the striatum. and thus may be considered as a peculiar “patch” of the ventral striatum distinguished by its “amygdaloid” type of immunohistochemical properties. The unlabeled VSc neurons within the clusters exhibit morphology typical of striatal medium spiny neurons in that they have round unindented nuclei, a narrow rim of perikaryal cytoplasm, and very few axosomatic synapses. However. it remains to be ascertained whether any of the unlabeled neurons within the cluster are medium spiny neurons. As the somata of the VSc neurons within the clusters are tightly apposed to each other. it is likelv that neurons within each cluster share very similar afferent input, including the dopaminergic fibers shown in this study,
Postsynaptic
FIG. 9. Histogram
Dendritic
showing the distribution terminals.
postsynaptic
to VIP+
possessing
both striatal and amygdaloid
Mameter
(~1
of the dendritic
diameters
properties.
The smallish somatic size piaces the VIP-f- neurons at the lower end of the striatal medium-sized neurons category. The aspinous dendritic surfaces of VIP + neurons correspond broadly to the morphological characteristics of striatal medium aspiny neurons. However. since there are only a few VIP+ neurons in the striatum (1. 26. 36). it is not surprising that smallish medium-sized striatal neurons with bipolarly arranged dendritic fields similar to the VIP+ neurons have been observed only rarely in previous Golgi studies (6). This situation is in contrast to that in the cerebral cortex in which the relationships between the numerous VIP+ neurons and the bipolar neurons stained in Golgi studies have been analyzed in detail (X,29). Nevertheless, as more and more striatal neuron types are identified by immunocytochemical means, the anatomical and functional organization of the striatal ahpiny neurons will become better understood. The ultrastructural features of VIP+ neurons in VSc are similar to those described previously for VIP+ neurons in the striatum (36) and CNA (13). Their deeply indented nuclei are typical of the striatal medium aspiny neurons described in previous EM studies (3, 8, 35). We have found in this study that individual VIP+ neurons often form tight appositions with clusters of several (3 to 3) unlabeled medium-sized VSc neurons. The areas covered by these VSc neuronal clusters are smaller than the typical neuronal “patches” or “striosomes” in most of the striatum in which larger groups of striatal neurons, from tens to hundreds. are associated in “patches” distinguished by differential histochemical or immunocytochemical reactivity (14.17). Indeed. the entire VSc outlined by the dense VIP+ plexus has roughly the same size as a typical “patch” found in the other
Since the surrounding neuropil is densely innervated by VIP + axons. it is not surprising that many VIP+ axons form synapses with both VIP+ and unlabeled neurons within the clusters. However. how VIP regulates the functions of these clusters of VSc neurons is not clear. The few VIP+ medium-sized neurons found next to the dense VIP+ plexus seem too few to account for the heavy VIP+ terminal plexus in VSc. Several lines of evidence suggest that this dense plexus of VIP i- axon terminaIs arises from two different sources: 1) Our EM analysis has revealed that most VIP+ axon terminals form asyI~~]netrical synapses with mostly small dendrites and dendritic spines, and the rest form symmetrical synapses with mainly somata and larger dendrites (Fig. 9). This distribution pattern is consistent with that in the striatum in which the extrinsic afferent axons f’orm mainly asymmetrical synapses on small dendrites and spines while intrinsic axons form symmetrical synapses with mainly larger dendrites and somata ( 18). 2) Typical striatal VIP+ neuron5 are associated with very sparse VIP+ axon plexuses. which form symmetrical synapses u ith either VIP + somata or neighboring neurons (36). 3) Only one type of VIP+ neurons could be distinguished in VSc. and they have similar morphology as those found in the other parts of the ctriatum. 3) Previous studies (9, ‘23. 30. 3 I ) have shown that there are at least two sources of VIP-+ axons that innervated the extended amygdala: A) eutrinsic VIP+ neurons in the brain stem nuclei, and B) intrinsic VIP+ neurons located within the extended amygdaia. These observations suggest that the dense VIP+ plexuses in VSc also consist of both intrinsic and extrinsic fibers. Given the intimate relationship of VSc with the “extended amygdala.” it is very likely that the extrinsic VIP+ fibers in VSc arise from the same sources that innervate the other parts of the extended amygdala. Whether the extrinsic or the intrinsic VIP+ fibers are associated with different types of VIP receptors or different synaptic specializations remains to be determined.
This study was supported by VSPHS Grant AG059-U, NS2 1003. Biomedical Research Support Grant RR05423. a grant from the Aizheimer‘s Disease and Related Disorders Association, and a postdoctoral fellowship from the Neuroscience Center of Excellence of The linkersity of Tennessee. Memphis.
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<:HANCi
AND
‘fL4h
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