Brain Research, 516 (1990) 141-146 Elsevier

141

BRES 24071

Evidence for the presence of presynaptic dendrites and GABA-immunogold labeled synaptic boutons in the monkey basilar pontine nuclei Gregory A. Mihailoff1'2 and Barbara G. Border 2 Departments of 1Cell Biology and Neuroscience and 2Neurology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235-9039 (U.S.A.)

(Accepted 23 January 1990) Key words: Cerebellum; Corticopontine; Interneuron; Immunocytochemistry; Brainstem

Two general categories of GABA-immunoreactive (GABA-Ir) boutons are present in the monkey basilar pontine nuclei (BPN). One type characterized by a pale or lucent appearance is involved both in glomerular arrangements that include serial, triadic synapses, and in non-glomerular synapses. The second type of GABA-Ir bouton exhibits a wide variety in size and shape and contains a greater complement of synaptic vesicles than the first type, giving it a darker appearance in comparison to the pale GABA-Ir boutons. Such boutons participate only in non-glomerular synapses. It is suggested that the pale GABA-Ir boutons arise from the intrinsic population of GABA neurons, while the darker appearing boutons might take origin from one of the GABA-ergic afferent systems that reach the BPN. Recent light microscopic (LM) studies have demonstrated the presence of both glutamic acid decarboxylaseimmunoreactive and GABA-immunoreactive (GABAIr) neurons and axon terminals in the basilar pontine nuclei (BPN) of the rat 2, cat 4 and primate 4'22. The implication is that these G A B A - I r neurons represent a population of BPN interneurons or local circuit neurons and such a suggestion is supported by the observation that in double-labeling experiments, the BPN G A B A - I r neurons are not retrogradely labeled by injections of W G A - H R P in the cerebellar cortex 4. Subsequently, electron microscopic (EM) immunochemical studies have confirmed the presence of G A B A - I r somata, dendrites, axons, and axon terminals in the BPN of the rat 3. However, in the rat, double-labeling experiments involving the retrograde transport of H R P in combination with G A B A immunochemistry indicated that BPN afferent systems originating in the zona incerta, deep cerebellar nuclei, anterior pretectal nucleus, perirubral area, and medullary reticular formation are, at least in part, composed of G A B A neurons 1. Thus, G A B A - I r synaptic boutons in the BPN are likely derived from both the intrinsic population of basilar pontine G A B A neurons as well as the various GABAergic afferent systems mentioned above. In the present studies, our objective was to use electron microscopy to explore the involvement of G A B A - I r bout0ns in the synaptic circuitry of the monkey

BPN. Previous EM studies of the BPN in the monkey 5 reported the presence of serial synaptic arrangements involving synapses between two vesicle-containing profiles similar to those observed in other brain regions known to contain local circuit neurons. Our expectation that such glomerular complexes would contain a G A B A Ir bouton was confirmed in the present studies, while the involvement of G A B A - I r boutons at other synaptic loci in the BPN became evident as well. Three adult cynomolgous monkeys were utilized in these studies. Each animal was sacrificed and transcardially perfused with fixative in the Animal Resource Center under the direct supervision of a technician specifically trained in the care of non-human primates. After being immobilized with ketamine, each monkey was intubated, placed under general anesthesia with Nembutal, and continuously ventilated using a standard hand-held air bag connected to the endotracheal tube. Once stabilized, the animal received an intravenous injection of the vasodilating agent Diamox (acetazolamide; 1 ml/kg). After 15 min the thorax was opened, the pericardium incised, and the vascular system perfused through the left ventricle with 300 ml of warm saline followed by a two-stage fixative consisting of 1 liter of 1% paraformaldehyde and 1% glutaraidehyde in 0.1 M sodium phosphate buffer (pH 7.2) which was in turn followed by 1.5 liters of a solution containing 2%

Correspondence: G.A. Mihailoff, Dept. of Cell Biology and Neuroscience, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75235-9039, U.S.A.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

142 paraformaldehyde, and 2% glutaraldehyde in the same buffer. Total perfusion time was 35-40 rain, whereupon the brain was immediately removed from the skull and immersed overnight in the more concentrated fixative. On the following morning, the basilar pons was excised from the brainstem and divided into small (1 × 2 mm) tissue blocks which were then prepared for electron microscopy in a routine manner as previously described 13. A post-embedding immunogold method was used to identify GABA-Ir profiles within the BPN. Briefly, after collecting ultrathin sections on nickel grids, the tissue was exposed to 1% periodic acid (4 min), 1% sodium metaperiodate (4 min), and 5% normal goat serum (NGS, 45 min) with gentle washing in distilled water between each step. The tissue was then incubated in rabbit anti-GABA 1° diluted 1:1000 with 1% NGS containing 0.3% Triton X-100 for 1.5 h. Following 3 washes (5 min each) in a solution composed of 10 ml 0.05 M Tris containing 5 mg polyethylene glycol, the tissue was incubated in goat anti-rabbit IgG conjugated to 15 nm colloidal gold particles (Janssen; 1:20 dilution) for 1.5 h. The sections were then gently rinsed with 4 changes (5 min each) of distilled water and stained with uranyl acetate (20 min) and lead citrate (20 min). Although there was no attempt to rigorously quantify the number of gold particles overlying a given profile, the extent of potential non-specific labeling was assessed by noting the number of gold particles present over the lumen of blood vessels and the compact portion of axonal myelin. Such non-specific or background labeling was extremely sparse. Presynaptic profiles immunoreactive for GABA were present in tissue collected from all BPN locations but were observed with greatest frequency in lateral and medial tissue blocks. Two general categories of GABA-Ir boutons are discernable and are termed pale and dark. One of the most characteristic ultrastructural features of the monkey BPN neuropil is a curious synaptic glomerulus partially enclosed by glial processes (Fig. 1A). In this glomerular arrangement, a relatively large, non-immunoreactive central bouton contains spheroidal synaptic vesicles and forms asymmetric synaptic contact with several dendritic and synaptic vesicle-containing profiles on its perimeter. Dendritic profiles most frequently observed in the glomerulus are small non-

immunoreactive spine-like dendritic protrusions, although occasionally intermediate-sized dendritic shafts are apposed to the central bouton as well. The peripheral vesicle-containing profiles are GABA-Ir (Fig. 1B) and frequently are similar in size to the neighboring small peripheral dendritic protrusions. In many instances such boutons exhibit a characteristic lucent or pale matrix and contain a relatively small number of synaptic vesicles, most of which are round although some tend to be discoid in shape and there is considerable variability in the size of the vesicles. The small peripheral dendritic spine-like profiles that do not contain synaptic vesicles, never exhibit immunolabeling. The direction of impulse flow in the glomerulus appears to be dictated by the fact that the central bouton forms asymmetric membrane specializations at each of its active sites including the peripheral vesicle-containing profiles, and is always in a presynaptic position. The small, peripheral GABA-Ir profiles form symmetric synaptic membrane specializations with adjacent dendritic profiles at which the immunolabeled profile is presynaptic (Fig. 1B). The small dendritic profile that is postsynaptic to the pale GABA-Ir bouton is also postsynaptic to the central axon terminal, thus resulting in the formation of a triadic, serial synaptic complex. A slight variant of this synaptic arrangement is occasionally observed and is also characterized by the presence of a synapse between two vesicle-containing profiles. Typically, in this situation, a total of 3 synaptic elements are involved. A small, round vesicle bouton is presynaptic to a pale GABA-Ir bouton that is in turn presynaptic to a small dendritic shaft or spine-like protrusion (Fig. 1E). Rarely is the positioning of the profiles such that the round vesicle bouton is presynaptic to both the dendritic element and the pale GABA-Ir bouton. In some instances, the pale GABA-Ir bouton is readily distinguished by its relatively large size compared to the adjacent round vesicle bouton. Also, the pale GABA-Ir boutons often exhibit a distinct spheroidal shape and appear to be linked to one another by a thin connecting stalk (Fig. 1C,D). Occasionally, a pale GABA-Ir bouton is postsynaptic to another GABA-Ir bouton (Fig. 2B) or presynaptic to an immunolabeled dendrite (Fig. 2C). Although the precise origin of pale GABA-Ir boutons remains uncertain, it is significant that presynaptic dendrites (Fig. 2A) can be observed in the

Fig. 1. Shown in A is a BPN synaptic giomerulus (non-immunostained material) that consists of a central bouton (Ax) surrounded by multiple dendritic (Dd) structures and two vesicle-containing profiles (*). At higher magnification in B, a similar glomerulus includes a triadic synaptic array composed of a central axon terminal (Ax), a GABA-Ir bouton (block arrow), and a dendritic spine (Dd). Thin arrows indicate synaptic polarity. Round pale GABA-Ir boutons (block arrows) are shown in C and two such boutons are linked by a thin stalk (curved arrows). Similarly in D, an en passage pale GABA-Ir bouton (*) exhibiting two thin stalks (block arrows) can be seen adjacent to an immunolabeled dendrite (Dd). In E, a non-glomerular serial synapse consists of a pale GABA-Ir bouton (*) that is postsynaptic to a small unlabeled bouton (open arrow) and presynaptic to a dendritic spine (block arrow). In A, C, and D, bar = 1.0/tm; in B and E, bar = 0.5/~m.

144 B P N n e u r o p i l . T h u s it is likely that the p r e s y n a p t i c d e n d r i t e s o r i g i n a t e from B P N local circuit n e u r o n s whose

dendritic a p p e n d a g e s t h e n are r e p r e s e n t e d by the pale GABA-Ir boutons.

Fig. 2. A: transversely sectioned BPN dendrite (Dd) in non-immunostained material that is presynaptic at 3 locations (thin arrows). Dense core vesicles (block arrows) are also present. Shown in B is a serial synapse that includes a pale GABA-Ir bouton (solid block arrow) that is postsynaptic to a dark GABA-Ir bouton (open block arrow) and presynaptic to a dendritic spine (thin arrow). In C, a pale (3ABA-Ir bouton (block arrow) is presynaptic to a GABA-positive dendrite (*) exhibiting a distinct subsynaptic specialization (curved arrow). Examples of small rounded (D) and large irregularly shaped (E) dark GABA-Ir boutons are also illustrated. In A, B, C, and E, bar = 1.0 #m; in D, bar = 0.5 ]zm.

145 The dark type of GABA-Ir bouton characteristically exhibits a dense accumulation of round synaptic vesicles that nearly occupies the entirety of the bouton. The most common variety of dark GABA-Ir bouton is typically a relatively small profile with a fairly uniform rounded or bulbous shape that forms a symmetrical active site with a single dendritic element or soma (Fig. 2D). The postsynaptic dendritic profile is frequently small in size and most likely represents a dendritic protrusion or a distal dendritic shaft. Only rarely is a large (proximal) dendritic shaft observed in contact with a small dark GABA-Ir bouton. This type of bouton does not participate in glomerular arrays but is involved in serial synapses in which it is presynaptic to a pale GABA-Ir bouton that is in turn presynaptic to a dendritic spine (Fig. 2B). A second type of dark GABA-Ir presynaptic profile is encountered less frequently than the first variety and is shown in Fig. 2E. These boutons are typically larger than the other dark GABA-Ir boutons, exhibit an irregular shape, and contain round synaptic vesicles as well as an occasional dense core vesicle. Observations in the present study are consistent with ongoing EM studies of the rat BPN which have illustrated the presence of two basic types of GABA-Ir boutons 3. In contrast, however, no glomerular triadic synaptic arrangements with GABA-Ir components have as yet been observed in the rat, although the presence of nonglomerular serial synaptic arrays has been reported 12. Moreover, the present findings, which illustrate several varieties of GABA-Ir boutons in the monkey BPN, confirm and extend previous light microscopic findings that demonstrated the presence of a rather substantial population of GABA-Ir neuronal somata and axons within the basilar pontine nuclei of the monkey4'22. The work of Thier and Koehler 2~ illustrated intensely stained GABA-Ir neurons in the monkey BPN that appeared to give rise to bulbous dendritic protrusions with thin stalks as well as multiple thin beaded processes that frequently originated from dendritic locations. Such morphology is consistent with earlier Golgi studies of the BPN in the monkey 6 as well as observations in the opossum 12 and rat 14 that described neurons with similar dendritic protrusions and thin, beaded axon-like processes. Morphological features of this type have come to be regarded as characteristic for Golgi type II local circuit neurons, particularly as a result of studies of the dorsal lateral geniculate nucleus (DLG) where it has been shown that dendritic protrusions arising from local circuit neurons exhibit a distinctive pale appearance and participate in triadic, serial synaptic complexes termed glomeruli in which they form both pre- and postsynaptic elements 7' 8,14-19. Also, the dendritic shafts of DLG local circuit neurons form both pre- and postsynaptic contacts but

these types of synapses are located outside the glomeruli. The present findings in the monkey BPN correlate with these observations in the DLG and reveal that glomerular synaptic arrangements in the BPN contain GABA-Ir boutons that form one component of a triadic serial array in which the labeled boutons are both presynaptic to a dendritic profile, and postsynaptic to a central bouton, presumably an afferent axon terminal. On the basis of these studies in the DLG, the description of BPN G A B A neurons provided by Thier and Koehler22, and the ultrastructural observations in the present study, it is suggested that the glomerular GABA-Ir boutons are presynaptic dendritic protrusions. The morphology of BPN local circuit neurons that emerges from the present study is compatible with previous EM studies of the monkey BPN 5 which illustrated a distinctive category of pale vesicle-containing profiles (but not glomerular arrangements) that participate in serial synapses where they are postsynaptic to other synaptic boutons and presynaptic to dendrites. Similarly, ultrastructural studies of the BPN in the opossum 11'12 and r a t 13 have illustrated serial synaptic arrays and suggested the presence of presynaptic dendrites. In the cat BPN, Hollander et al. 9 provided a drawing of a similar glomerular synaptic arrangement but in the text did not specifically refer to the presence of a small peripheral bouton in synaptic contact with the central axon terminal. These authors did report the existence of axo-axonic synapses but did not provide any illustrations of such contacts or a description of the profiles involved. Thus it seems reasonable to conclude that local circuit type neurons are present in the BPN of each of the experimental animals investigated to date with light and electron microscopy. What remains unclear at present is whether the differences in synaptic organization observed in the BPN are the result of actual species differences or whether for example, the lack of glomerular synaptic arrangements in the opossum and rat is simply a sampling problem and a reflection of the small number of G A B A neurons in the BPN of these species. Whether BPN local circuit neurons give rise to a conventional axon must remain an open question. In the DLG it appears that some, but not all, local circuit neurons exhibit an axon that forms non-glomerular axodendritic synapses 16'zl. In BPN Golgi preparations, some local circuit neuron somata give rise to a thin, beaded and branched process that has the typical morphology of an axon, while other cells of this same type exhibit one or several of these thin, beaded strands that originate from an intermediate or distal dendrite 6'14. In addition, the work of Ramon y Cajal 2° illustrates a heavily spine-laden type of cell in the BPN which gives rise to an axon-like process that originates from a

146 d e n d r i t e and appears to collateralize in the vicinity of the cell body. The particular point of origin certainly does not preclude that such processes are indeed axons but there is as yet no ultrastructural evidence that would answer this question for the BPN. In the present studies, the presence of the non-glomerular, dark type of G A B A Ir boutons lends itself to the interpretation that one type might take origin from the axon of a local circuit neuron. Alternatively, as has been r e p o r t e d for the rat 1 (but not yet described in the m o n k e y ) , axon terminals derived from a B P N G A B A e r g i c afferent system might represent a n o t h e r source for the dark G A B A - I r boutons. Essentially, however, there is as yet no direct evidence that would associate the non-glomerular varieties of dark G A B A - I r boutons with either G A B A afferents to the B P N or the intrinsically derived axon-like processes of G A B A local circuit neurons. Finally, similar to the work in the rat BPN 3, this study documents the existence of synaptic contacts linking two G A B A - I r elements in the m o n k e y BPN. In one example of this sort, a dark G A B A - I r b o u t o n is presynaptic to a pale G A B A - I r b o u t o n which is in turn presynaptic to a

Supported by USPHS Grant NS-12644. The technical assistance provided by Mr. K.W. Bourell is gratefully acknowledged.

1 Border, B.G, Kosinski, R.J., Azizi, S.A. and Mihailoff, G.A., Certain basilar pontine afferent systems are GABAergic: combined HRP and immunocytochemical studies in th~ rat, Brain Res. Bull., 17 (1986) 169-179. 2 Border, B.G. and Mihailoff, G.A., GAD-immunoreactive neural elements in the basilar pontine nuclei and nucleus reticularis tegmenti pontis of the rat. I. Light microscopic studies, Exp. Brain Res., 59 (1985) 600-614. 3 Border, B.G. and Mihailoff, G.A., GABAergic neural elements in the rat basilar pons: electron microscopic immunochemistry, J. Comp, Neurol., in press. 4 Brodal, P., Mihailoff, G.A., Border, B.G., Ottersen, O.E and Storm-Mathison, J., GABA-containing neurons in the pontine nuclei of the rat, cat, and monkey. An immunocytochemical study, Neuroscience, 25 (1988) 27-45. 5 Cooper, M.H. and Beal, J.A., The neurons and synaptic endings in the primate basilar pontine gray, J. Comp. Neurol., 180 (1978) 17-42. 6 Cooper, M.H. and Fox, C.A., The basilar pontine gray in the adult monkey (Macaca mulatta): a Golgi study, J. Comp. Neurol., 168 (1976) 145-174. 7 Famiglieni, E.V. and Peters, A., The synaptic glomerulus and the intrinsic neuron in the dorsal lateral geniculate nucleus of the cat, J. Comp. Neurol., 144 (1972) 285-334. 8 Hamos, J.E., Van Horn, S.C., Raczkowski, D., Uhlrich, D.J. and Sherman, S.M., Synaptic connectivity of a local circuit neuron in the lateral geniculate nucleus of the cat, Nature (Lond.), 317 (1985) 618-621. 9 Hollander, H., Brodal, E and Walberg, E, Electron microscopic observations on the structures of the pontine nuclei and the mode of termination of the corticopontine fibers. An experimental study in the cat, Exp. Brain Res., 7 (1969) 95-110. 10 Maley, B. and Newton, B.W., Immunohistochemistry of gamma-aminobutyric acid in the cat nucleus tractus solitarius, Brain Research, 330 (1985) 364-368. 11 Mihailoff, G.A., Anatomic evidence suggestive of dendrodendritic synapses in the basilar pons, Brain Res. Bull., 3 (1978)

333-340. 12 Mihailoff, G.A. and King, J.S., The basilar pontine gray of the opossum; a correlated light and electron microscopic analysis, J. Comp. Neurol., 159 (1975) 521-552. 13 Mihailoff, G.A. and McArdle, C.B., The cytoarchitecture, cytology and synaptic organization of the basilar pontine nuclei in the rat. II. Electron microscopic studies, J. Comp. Neurol., 195 (1981) 203-219. 14 Mihailoff, G.A., McArdle, C.B. and Adams, C.E., The cytoarchitecture, cytology and synaptic organization of the basilar pontine nuclei in the rat. I. Nissl and Golgi studies, J. Comp. Neurol., 195 (1981) 181-201. 15 Montero, V.M., Localization of GABA in type 3 cells and demonstration of their sourceto F2 terminals in the cat lateral geniculate nucleus: a Golgi electron microsopic GABA-immunocytochemical study, J. Comp. Neurol., 254 (1986) 228-245. 16 Montero, V.M., Ultrastructural identification of synaptic terminals from the axon of type 3 interneurons in the cat lateral geniculate nucleus, J. Comp. Neurol., 264 (1987) 268-283. 17 Morest, D.K., Dendrodendritic synapses of cells that have axons; the fine structure of the Golgi type II cell in the medial geniculate body of the cat, Z. Anat. Entwicklungsgesh., 133 (1971) 216-246. 18 Pasik, P., Pasik, T., Hamori, J. and Szentagothai, J., Golgi type II interneurons in the neuronal circuit of the monkey lateral geniculate nucleus, Exp. Brain Res., 17 (1973) 18-34. 19 Rafols, J.A. and Vaiverde, T., The structure of the dorsal lateral geniculate nucleus in the mouse, J. Comp. Neurol., 150 (1973) 303-332. 20 Ramon y Cajal, S., Histologie du Systdme Nerveux de l'Homme et des Vertdbr~s, Vol. 1, Paris, Maloine, 1911, pp. 960-978. 21 Sherman, S.M. and Friedlander, M.J., Identification of X versus Y properties for interneurons in the A-laminae of the cat's lateral geniculate nucleus, Exp. Brain Res., 73 (1988) 384-392. 22 Thier, P. and Koehler, W., Morphology, number and distribution of putative GABAergic neurons in the pontine basilar grey of the monkey, J. Comp. Neurol., 265 (1987) 311-322.

dendritic spine. Such circuitry might represent the substrate for a disinhibitory mechanism that involves G A B A afferent-mediated inhibition of an inhibitory B P N local circuit neuron. The G A B A afferent system might be driven by cerebral cortical input (i.e. cortex to zona incerta) and thus could result in the prolongation or e n h a n c e m e n t of a direct cortical excitatory input to the BPN. In the other example of this type, a pale G A B A - I r b o u t o n is presynaptic to a G A B A - I r dendrite. This suggests that B P N local circuit neurons have the potential to interact with one another, thus forming a second type of disinhibitory mechanism that p e r h a p s is related to the selection of specific subsets of p o n t o c e r e b e l l a r projections neurons by various B P N afferent projections. Thus, although m a n y details regarding the synaptic circuitry within the BPN have yet to be described, the picture beginning to evolve is one that portrays a far m o r e complex functional role for the B P N than has been depicted in earlier studies.