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Identification of pontocerebellar axon collateral synaptic boutons in the rat cerebellar nuclei
BRAIN RESEARCH ELSEVIER
Brain Research 648 (1994) 313-318
Identification of pontocerebellar axon collateral synaptic boutons in the rat cerebellar nuclei Gregory A. M i h a i l o f f * Departments of Anatomy and Neurology, Uniuersity of Mississippi Medical Center, Jackson, MS 39216-4505, USA (Accepted 22 March 1994)
Abstract
Injections of the orthogradely transported tracer PHA-L into the the basilar pontine nuclei or reticulotegmental nucleus of hooded rats produced labeling of pontocerebellar axons that distributed to the cerebellar cortex and nuclei. EM examination of the lateral and interposed cerebellar nuclei revealed that labeled pontocerebellar axon terminals formed synaptic boutons in the cerebellar nuclei that were morphologically different from the characteristic mossy fiber terminals observed in the cerebellar cortex.
Key words: Cerebellum; Basilar pons; Cerebellar afferent; Axonal tracing
That sensorimotor areas of the cerebral cortex influence the activity of the lateral and interposed cerebellar nuclei in a relatively direct manner was demonstrated using electrophysiological recording techniques in the cat [7,10] and monkey [1,2]. The latter authors suggested that an early excitatory response recorded from monkey dentate and interpositus neurons following sensorimotor cortical stimulation was mediated by the pontocerebellar mossy fiber afferent system. Similarly in the cat, Giuffrida and co-workers [7] reported that stimulation of the medullary pyramid evoked activity in the lateral cerebellar nucleus that was subsequently abolished when the brachium pontis was sectioned. These observations imply that the basilar pontine nuclei (BPN) can mediate cerebral cortical input to the cerebellar nuclei (CN). The work of Shinoda et al. [10] demonstrated very clearly that dentate neurons receive monosynaptic excitatory inputs following stimulation in the nucleus reticularis tegementi pontis (NRTP) or BPN. In contrast to these electrophysiological findings, anatomical studies concerned with the possible distribution of pontocerebellar axon collaterals in the CN are contradictory. Using autoradiographic methods in
* Fax: (1) (601) 984-1655. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 3 6 0 - 0
the cat, Gerrits and Voogd [6] showed a modest input primarily to the dentate nucleus arising mainly in N R T P and to a lesser extent in the BPN. However, well-documented axonal tracing studies in the cat, employing either anterograde [3] or retrograde [5] transport of wheat germ agglutinin horseradish peroxidase ( W G A H R P ) concluded that only a few, if any, pontocerebellar axons gave rise to collateral terminations in the lateral or interposed cerebellar nuclei. More recent light microscopic studies using intracellular recording and filling with horseradish peroxidase [11] or axonal transport of the lectin Phaseolus uulgaris leucoagglutinin (PHA-L) [8] are in agreement with the earlier cited physiology studies and have shown that axons originating from cells in the N R T P or BPN do reach the CN enroute to their termination in the cerebellar cortex. In addition, the work of Shinoda et al. [11] reported that most, if not all, labeled axons of BPN origin that enter the CN are collateral branches of fibers that continue on to the cerebellar cortex to terminate as mossy fibers. The work of Shinoda et al. [11] and Mihailoff [8] also showed that pontocerebellar axons which terminate in the lateral and interposed nuclei appear to be much thinner, and their terminal boutons smaller than typical mossy fibers and terminals in the cerebellar cortex. Perhaps this narrow caliber explains why this system of axons eluded identification in earlier anatomical studies.
314
G.A. Mihaih~[.[/ Brain Research 648 (1994) 313 318
A
R-2282
B
\
4
Fig. 1. Illustrated in A are four (1-4) transverse sections through the PHA-L injection site in the BPN in case R-2282. Indicated in each section is the area of light brown diffuse stain (shading) which contains densely filled, immunostained neuronal somata (black dots), all of which are limited to portions of the BPN. In B, a view of the ventral surface of the basilar pons illustrates the rostro-caudal extent of the injection site in this case and the approximate level of the four transverse sections shown in A.
The present studies were undertaken to confirm that pontocerebellar axons do establish synaptic contacts in the CN. The identity of pontocerebellar axon terminals in the CN of L o n g - E v a n s hooded rats was
determined using the axonal transport of PHA-L and elecron microscopy. Relatively large injections of PHAL, some restricted primarily to the BPN and others involving portions of both N R T P and the BPN were
Fig. 2. Shown in A is a transverse brainstem section at a mid-pontine level illustrating a representative PHA-[, injection site containing densely immunostained neuronal somata (arrowheads). The injection in this case (R-2282) involves only the ventral BPN and the dorsal peduncular (Dpn) subdivision. Also indicated are the descending cortical axons of the cerebral peduncle (ped), the ascending fibers of the medial lemniscus (ml), and the brachium pontis (bp) which conveys pontocerebellar axons into the cerebellum. For orientation, many of the same structures (including the NRTP) are illustrated in B, a Nissl-stained section at slightly lower magnification than A. Scale bars: A, 0.5 ram: B, 1.0 ram.
G.A. Mihailoff /Brain Research 648 (1994) 313-318
achieved in eight animals in a manner similar to that employed in previous studies [8]. Briefly, each animal was anesthetized (8% chloral hydrate, 0.5 c c / 1 0 0 g i.p.) and electrophoretic placements of 2.5% P H A - L (7-9 t-~A, pulsed, 30-45 min) were made at two differ-
!!
i ~ :i~ii!i¸~
~ !iiii?~ !ilZi:i?~i++++~+ii~:~¸!! ;~ ii~ ~
315
ent locations along the rostro-caudal axis of the basilar pons and NRTP. Following a 7 - 1 2 day survival period, each animal was adminstered a lethal dose of Nembutal (50 m g / m l , 0.1 cc/100 g), artificially respirated, and perfused through the left ventricle with a mixture of 1.5% paraformaldehyde and 0 . 5 - 2 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2). A tissue block containing the pontine portion of the brainstem and cerebellum was sectioned transversely at a thickness of 80 ~ m on a vibratome and the sections reacted with biotin-avidin immunohistochemistry to visualize P H A - L as previously described [8]. Transverse sections through the CN were observed under a dissecting microscope and areas of the lateral or interposed nuclei that exhibited P H A - L labeled axons and terminals were trimmed into small pieces consisting only of those areas that contained PHA-L-labeled pontocerebellar fibers. These pieces of tissue were then osmicated, dehydrated and embedded in plastic in a routine manner for EM examination. A series of four representative transverse sections through the injection site in a case (R-2282) in which P H A - L filled somata were restricted to the BPN is shown in Fig. 1A. Densely labeled somata mostly occupied the ventral and ventral peduncular subdivisions of the BPN, while a small number were located in the interval between the dorsal surface of the cerebral peduncle and the medial lemniscus. The rostro-caudal extent of this injection site is shown in Fig. 1B which depicts a view of the ventral surface of the basilar pons. Also indicated here are the approximate levels of transverse sections 1-4. Shown in Fig. 2A is a tissue section from case R-2282 that corresponds approximately to section 3 of Fig. 1A. The arrowheads indicate some of the BPN somata that are densely filled with P H A - L reaction product. Although some heavily stained somata were located in the region between the medial lemniscus (ml) and the descending cortical axons of the cerebral peduncle (ped), such neurons are considered to be part of the dorsal peduncular subdivision (Dpn) of the BPN and thus no labeled cells were present within the N R T P in this case. At high magnification, labeled axons were followed from the injection
Fig. 3. In A, a Nissl-stained, transverse section through the brainstem and cerebellum illustrates the location of the caudal portion of the posterior interpositus nucleus (PIP). Shown in B is an immunostained section through the cerebellum at about the same level, and with the same orientation as the section shown in A. Indicated are PHA-L labeled pontocerebellar axons (filled arrow) entering and arborizing within the PIP. The trapezoidal area enclosed by the dotted line illustrates how a portion of the PIP containing labeled axons and terminals was trimmed for subsequent electron microscopic processing and observation. The arborization of labeled pontocerebellar axon collaterals within the PIP is shown at higher magnification in C and the inset of C. Scale bars: A,B, 1.0 ram; C, 0.25 ram, inset, 50 u.m.
316
G.A. Mihailoff /Brain Research 648 (1994) 313-318
Fig. 4. Shown in A and B are electron micrographs illustrating PHA-L labeled pontocerebellar axon terminals (filled block arrows) observed in the CN in case R-2282. These boutons contain spheroidal synaptic vesicles and form asymmetric membrane specializations (thin arrows) at their synaptic active sites. Illustrated in C is a representative PHA-L labeled pontocerebellar mossy fiber terminal found in the cerebellar cortex in this same case, while shown in D are examples of labeled mossy fiber terminals observed with light microscopy in case R-2282. Scale bars: A - C , 1.0 ~zm; D, 50 p,m.
G.A. Mihailoff / Brain Research 648 (1994) 313-318
site into either the contralateral or ipsilateral brachium pontis (bp) and then into the cerebellum. For orientation, a Nissl-stained section (Fig. 1B) taken at lower magnification, from a level of the pontine gray comparable to that shown in A, serves to more clearly illustrate the cytoarchitectural details of this portion of the BPN. In this report, we describe labeled axons and terminals in the lateral cerebellar nucleus and in the posterior interpositus nucleus as representative of the system of pontocerebellar axon collaterals that distribute to the CN. In A of Fig. 3, a Nissl-stained transverse section through the cerebellum illustrates the location of the posterior interpositus nucleus (PIP). An immunostained section through a comparable level of the PIP in a PHA-L injected animal is shown in B. Notice the labeled pontocerebellar axons entering the lateral aspect of the PIP from the parafloccular stalk (Fig. 3B, filled block arrow). At higher magnification in C (and inset), PHA-L-labeled axons can be seen as they arborize within the PIP. As described in an earlier report [8], these axons are much thinner, and their swellings (boutons) much smaller than mossy fibers and terminals observed in the cerebellar cortex in these same cases. Vibratome sections containing the PIP (and lateral nucleus) that exhibited labeled axons and terminals were isolated under a dissecting microscope, trimmed as indicated in Fig. 3B to include only areas of labeled axons and terminals, and subsequently processed and embedded in plastic for EM observation. Representative examples of PHA-L labeled boutons observed in the CN in case R-2282 are shown in Fig. 4A,B. Labeled pontocerebellar axon terminals are intermediate in size relative to other presynaptic profiles observed in the CN, and as illustrated in Fig. 4A, contain spheroidal synaptic vesicles and exhibit asymmetric membrane specializations (thin arrow) at their synaptic active sites. Labeled axon terminals most frequently form synaptic contact with dendritic shafts (Fig. 4A,B), and occasionally with spine-like protrusions, but not with neuronal somata or other vesiclecontaining profiles. It was common to find a single dendritic shaft in contact with a labeled bouton and several unlabeled axon terminals. None of the various postsynaptic profiles contacted by labeled boutons exhibited distinctive morphological features that would facilitate the association of these profiles with a specific CN cell type. Also in this case, labeled pontocerebellar boutons were observed in the cerebellar cortex (Fig. 4C,D). With both light and electron microscopy, such boutons clearly exhibited the characteristic features of mossy fiber terminals, which is in contrast to those labeled boutons observed within the CN. Observations presented in this report confirm that pontocerebellar axons arising from neurons located in the BPN (or NRTP) form synaptic contacts in the
317
lateral or interposed CN. This study also offers new evidence that terminal boutons of pontocerebellar axons identified in the interposed and lateral CN are morphologically different from the typical mossy fiber terminals described in the cerebellar cortex [9]. The labeled boutons in the present study are, on average, much smaller than mossy fiber terminals and are not surrounded and indented by multiple postsynaptic profiles in a glomerular fashion as are the mossy terminals in the cerebellar cortex. However, in the CN, unlabeled boutons that exhibited some of the characteristic features of mossy fiber terminals were occasionally observed. This suggests that some mossy fiber systems might give rise to CN collaterals that are similar to the typical cerebellar cortical mossy fiber terminals. These findings correlate with light microscopic observations from an earlier study [8] (see also inset of Figs. 3C,4D) which clearly indicated that PHA-L labeled pontocerebellar axon collaterals in the CN are much thinner than mossy fibers in the cerebellar cortex, and their synaptic boutons much smaller and less elaborately arborized than mossy terminals. The labeled boutons in the present report do share some morphological similarities with axon terminals described by other investigators as mossy fiber collaterals in the rat lateral cerebellar nucleus [4]. The existing literature appears to contain only one other report that does specifically identify a pontocerebellar axon terminal in the CN and that study involved mossy fiber inputs to the fastigial nucleus in the cat [12]. Boutons were visualized by electron microscopic autoradiography following injection of radioisotope in the lateral reticular nucleus, medial vestibular nucleus or the NRTP. One bouton from an N R T P case was illustrated (their Fig. 10), and similar to the present observations, it exhibited little resemblance to a typical cerebellar cortical mossy fiber terminal. The illustrated bouton is, however, morphologically similar to the labeled bouton shown in Fig. 4B of the present report. Both are relatively small and irregular in shape as they insinuate themselves among several postsynaptic profiles. It remains for future studies to clarify what specific CN neuronal elements might receive input via the system of pontocerebellar axon collaterals and, what functional role is played by this circuitry which apparently links the cerebral cortex and the cerebellar nuclei via one intervening synapse in the BPN or NRTP.Support for this work was provided by USPHS Grant NS12644. Appreciation is extended to D. Holmes and W. Dorsett-Martin for their technical contributions. The assistance of D.E. Haines in editing an early version of the manuscript is also acknowledged. [1] Alien, G.I., Gilbert, P.F.C., Marini, R., Schultz, W. and Yin, T.C.T., Integration of cerebral and peripheral inputs by interpositus neurons in monkey, Exp. Brain Res., 27 (1977) 81-99.
318
G.A. Mihailoff / Brain Research 648 (1994) 313-318
[2] Allen, G.I., Gilbert, P.F.C. and Yin, T.C.T., Convergence of cerebral inputs onto dentate neurons in the monkey, Exp. Brain Res., 32 (1978) 151-170. [3] Brodal, P., Dietrichs, E. and Walberg, F., Do pontocerebellar mossy fibers give off collaterals to the cerebellar nuclei? An experimental study in the cat with implantations of crystalline HRP-WGA, Neurosci. Res., 4 (1986) 12-24. [4] Chan-Palay, V., Cerebellar Dentate Nucleus, Springer, New York, NY, 1977, pp. 280-283. [5] Dietrichs, E., Bjaalie, J.G. and Brodal, P., Do pontocerebellar fibers send collaterals to the cerebellar nuclei? Brain Res., 259 (1983) 127-131. [6] Gerrits, N.M. and Voogd, J., The projection of the nucleus reticularis tegmenti pontis and adjacent regions of the pontine nuclei to the central cerebellar nuclei in the cat, J. Comp. Neurol., 258 (1987) 52-69. [7] Giuffrida, R., Licata, F., Volsi, Li, Perciavalle, V. and Urbano,
[8]
[9] [10]
[1 I]
[12]
A., Pyramidal input to the intracerebellar nuclei of the cat, Neuroscience, 9 (1983) 421 -427. Mihailoff, G.A., Cerebellar nuclear projections from the basilar pontine nuclei and nucleus reticularis tegmenti pontis as demonstrated with PHA-L tracing in the rat, J. Comp. Neurol., 3311 (1993) 130-146. Palay, S.L. and Chan-Palay, V., Cerebellar Cortex, Springer, New York, NY, 1974, pp. 143 178. Shinoda, Y., Sugiuchi, Y. and Futami, T., Excitatory inputs to cerebellar dentate nucleus neurons from the cerebral cortex in the cat, Exl>. Brain Res., 67 (1987) 299-315. Shinoda, Y., Sugiuchi, Futami, T. and Izawa, R., Axon collaterals of mossy fibers from the pontine nucleus in the cerebellar dentate nucleus, J. Neurophysiol., 67 (1992) 547-560. Van der Want, J.J.L., Gerrits, N.M. and Voogd, ,I., Autoradiography of mossy fiber terminals in the fastigial nucleus of the cat, J. ~rnp. Neurol., 258 (1987) 70-80.
Brain Research 648 (1994) 313-318
Identification of pontocerebellar axon collateral synaptic boutons in the rat cerebellar nuclei Gregory A. M i h a i l o f f * Departments of Anatomy and Neurology, Uniuersity of Mississippi Medical Center, Jackson, MS 39216-4505, USA (Accepted 22 March 1994)
Abstract
Injections of the orthogradely transported tracer PHA-L into the the basilar pontine nuclei or reticulotegmental nucleus of hooded rats produced labeling of pontocerebellar axons that distributed to the cerebellar cortex and nuclei. EM examination of the lateral and interposed cerebellar nuclei revealed that labeled pontocerebellar axon terminals formed synaptic boutons in the cerebellar nuclei that were morphologically different from the characteristic mossy fiber terminals observed in the cerebellar cortex.
Key words: Cerebellum; Basilar pons; Cerebellar afferent; Axonal tracing
That sensorimotor areas of the cerebral cortex influence the activity of the lateral and interposed cerebellar nuclei in a relatively direct manner was demonstrated using electrophysiological recording techniques in the cat [7,10] and monkey [1,2]. The latter authors suggested that an early excitatory response recorded from monkey dentate and interpositus neurons following sensorimotor cortical stimulation was mediated by the pontocerebellar mossy fiber afferent system. Similarly in the cat, Giuffrida and co-workers [7] reported that stimulation of the medullary pyramid evoked activity in the lateral cerebellar nucleus that was subsequently abolished when the brachium pontis was sectioned. These observations imply that the basilar pontine nuclei (BPN) can mediate cerebral cortical input to the cerebellar nuclei (CN). The work of Shinoda et al. [10] demonstrated very clearly that dentate neurons receive monosynaptic excitatory inputs following stimulation in the nucleus reticularis tegementi pontis (NRTP) or BPN. In contrast to these electrophysiological findings, anatomical studies concerned with the possible distribution of pontocerebellar axon collaterals in the CN are contradictory. Using autoradiographic methods in
* Fax: (1) (601) 984-1655. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 3 6 0 - 0
the cat, Gerrits and Voogd [6] showed a modest input primarily to the dentate nucleus arising mainly in N R T P and to a lesser extent in the BPN. However, well-documented axonal tracing studies in the cat, employing either anterograde [3] or retrograde [5] transport of wheat germ agglutinin horseradish peroxidase ( W G A H R P ) concluded that only a few, if any, pontocerebellar axons gave rise to collateral terminations in the lateral or interposed cerebellar nuclei. More recent light microscopic studies using intracellular recording and filling with horseradish peroxidase [11] or axonal transport of the lectin Phaseolus uulgaris leucoagglutinin (PHA-L) [8] are in agreement with the earlier cited physiology studies and have shown that axons originating from cells in the N R T P or BPN do reach the CN enroute to their termination in the cerebellar cortex. In addition, the work of Shinoda et al. [11] reported that most, if not all, labeled axons of BPN origin that enter the CN are collateral branches of fibers that continue on to the cerebellar cortex to terminate as mossy fibers. The work of Shinoda et al. [11] and Mihailoff [8] also showed that pontocerebellar axons which terminate in the lateral and interposed nuclei appear to be much thinner, and their terminal boutons smaller than typical mossy fibers and terminals in the cerebellar cortex. Perhaps this narrow caliber explains why this system of axons eluded identification in earlier anatomical studies.
314
G.A. Mihaih~[.[/ Brain Research 648 (1994) 313 318
A
R-2282
B
\
4
Fig. 1. Illustrated in A are four (1-4) transverse sections through the PHA-L injection site in the BPN in case R-2282. Indicated in each section is the area of light brown diffuse stain (shading) which contains densely filled, immunostained neuronal somata (black dots), all of which are limited to portions of the BPN. In B, a view of the ventral surface of the basilar pons illustrates the rostro-caudal extent of the injection site in this case and the approximate level of the four transverse sections shown in A.
The present studies were undertaken to confirm that pontocerebellar axons do establish synaptic contacts in the CN. The identity of pontocerebellar axon terminals in the CN of L o n g - E v a n s hooded rats was
determined using the axonal transport of PHA-L and elecron microscopy. Relatively large injections of PHAL, some restricted primarily to the BPN and others involving portions of both N R T P and the BPN were
Fig. 2. Shown in A is a transverse brainstem section at a mid-pontine level illustrating a representative PHA-[, injection site containing densely immunostained neuronal somata (arrowheads). The injection in this case (R-2282) involves only the ventral BPN and the dorsal peduncular (Dpn) subdivision. Also indicated are the descending cortical axons of the cerebral peduncle (ped), the ascending fibers of the medial lemniscus (ml), and the brachium pontis (bp) which conveys pontocerebellar axons into the cerebellum. For orientation, many of the same structures (including the NRTP) are illustrated in B, a Nissl-stained section at slightly lower magnification than A. Scale bars: A, 0.5 ram: B, 1.0 ram.
G.A. Mihailoff /Brain Research 648 (1994) 313-318
achieved in eight animals in a manner similar to that employed in previous studies [8]. Briefly, each animal was anesthetized (8% chloral hydrate, 0.5 c c / 1 0 0 g i.p.) and electrophoretic placements of 2.5% P H A - L (7-9 t-~A, pulsed, 30-45 min) were made at two differ-
!!
i ~ :i~ii!i¸~
~ !iiii?~ !ilZi:i?~i++++~+ii~:~¸!! ;~ ii~ ~
315
ent locations along the rostro-caudal axis of the basilar pons and NRTP. Following a 7 - 1 2 day survival period, each animal was adminstered a lethal dose of Nembutal (50 m g / m l , 0.1 cc/100 g), artificially respirated, and perfused through the left ventricle with a mixture of 1.5% paraformaldehyde and 0 . 5 - 2 % glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2). A tissue block containing the pontine portion of the brainstem and cerebellum was sectioned transversely at a thickness of 80 ~ m on a vibratome and the sections reacted with biotin-avidin immunohistochemistry to visualize P H A - L as previously described [8]. Transverse sections through the CN were observed under a dissecting microscope and areas of the lateral or interposed nuclei that exhibited P H A - L labeled axons and terminals were trimmed into small pieces consisting only of those areas that contained PHA-L-labeled pontocerebellar fibers. These pieces of tissue were then osmicated, dehydrated and embedded in plastic in a routine manner for EM examination. A series of four representative transverse sections through the injection site in a case (R-2282) in which P H A - L filled somata were restricted to the BPN is shown in Fig. 1A. Densely labeled somata mostly occupied the ventral and ventral peduncular subdivisions of the BPN, while a small number were located in the interval between the dorsal surface of the cerebral peduncle and the medial lemniscus. The rostro-caudal extent of this injection site is shown in Fig. 1B which depicts a view of the ventral surface of the basilar pons. Also indicated here are the approximate levels of transverse sections 1-4. Shown in Fig. 2A is a tissue section from case R-2282 that corresponds approximately to section 3 of Fig. 1A. The arrowheads indicate some of the BPN somata that are densely filled with P H A - L reaction product. Although some heavily stained somata were located in the region between the medial lemniscus (ml) and the descending cortical axons of the cerebral peduncle (ped), such neurons are considered to be part of the dorsal peduncular subdivision (Dpn) of the BPN and thus no labeled cells were present within the N R T P in this case. At high magnification, labeled axons were followed from the injection
Fig. 3. In A, a Nissl-stained, transverse section through the brainstem and cerebellum illustrates the location of the caudal portion of the posterior interpositus nucleus (PIP). Shown in B is an immunostained section through the cerebellum at about the same level, and with the same orientation as the section shown in A. Indicated are PHA-L labeled pontocerebellar axons (filled arrow) entering and arborizing within the PIP. The trapezoidal area enclosed by the dotted line illustrates how a portion of the PIP containing labeled axons and terminals was trimmed for subsequent electron microscopic processing and observation. The arborization of labeled pontocerebellar axon collaterals within the PIP is shown at higher magnification in C and the inset of C. Scale bars: A,B, 1.0 ram; C, 0.25 ram, inset, 50 u.m.
316
G.A. Mihailoff /Brain Research 648 (1994) 313-318
Fig. 4. Shown in A and B are electron micrographs illustrating PHA-L labeled pontocerebellar axon terminals (filled block arrows) observed in the CN in case R-2282. These boutons contain spheroidal synaptic vesicles and form asymmetric membrane specializations (thin arrows) at their synaptic active sites. Illustrated in C is a representative PHA-L labeled pontocerebellar mossy fiber terminal found in the cerebellar cortex in this same case, while shown in D are examples of labeled mossy fiber terminals observed with light microscopy in case R-2282. Scale bars: A - C , 1.0 ~zm; D, 50 p,m.
G.A. Mihailoff / Brain Research 648 (1994) 313-318
site into either the contralateral or ipsilateral brachium pontis (bp) and then into the cerebellum. For orientation, a Nissl-stained section (Fig. 1B) taken at lower magnification, from a level of the pontine gray comparable to that shown in A, serves to more clearly illustrate the cytoarchitectural details of this portion of the BPN. In this report, we describe labeled axons and terminals in the lateral cerebellar nucleus and in the posterior interpositus nucleus as representative of the system of pontocerebellar axon collaterals that distribute to the CN. In A of Fig. 3, a Nissl-stained transverse section through the cerebellum illustrates the location of the posterior interpositus nucleus (PIP). An immunostained section through a comparable level of the PIP in a PHA-L injected animal is shown in B. Notice the labeled pontocerebellar axons entering the lateral aspect of the PIP from the parafloccular stalk (Fig. 3B, filled block arrow). At higher magnification in C (and inset), PHA-L-labeled axons can be seen as they arborize within the PIP. As described in an earlier report [8], these axons are much thinner, and their swellings (boutons) much smaller than mossy fibers and terminals observed in the cerebellar cortex in these same cases. Vibratome sections containing the PIP (and lateral nucleus) that exhibited labeled axons and terminals were isolated under a dissecting microscope, trimmed as indicated in Fig. 3B to include only areas of labeled axons and terminals, and subsequently processed and embedded in plastic for EM observation. Representative examples of PHA-L labeled boutons observed in the CN in case R-2282 are shown in Fig. 4A,B. Labeled pontocerebellar axon terminals are intermediate in size relative to other presynaptic profiles observed in the CN, and as illustrated in Fig. 4A, contain spheroidal synaptic vesicles and exhibit asymmetric membrane specializations (thin arrow) at their synaptic active sites. Labeled axon terminals most frequently form synaptic contact with dendritic shafts (Fig. 4A,B), and occasionally with spine-like protrusions, but not with neuronal somata or other vesiclecontaining profiles. It was common to find a single dendritic shaft in contact with a labeled bouton and several unlabeled axon terminals. None of the various postsynaptic profiles contacted by labeled boutons exhibited distinctive morphological features that would facilitate the association of these profiles with a specific CN cell type. Also in this case, labeled pontocerebellar boutons were observed in the cerebellar cortex (Fig. 4C,D). With both light and electron microscopy, such boutons clearly exhibited the characteristic features of mossy fiber terminals, which is in contrast to those labeled boutons observed within the CN. Observations presented in this report confirm that pontocerebellar axons arising from neurons located in the BPN (or NRTP) form synaptic contacts in the
317
lateral or interposed CN. This study also offers new evidence that terminal boutons of pontocerebellar axons identified in the interposed and lateral CN are morphologically different from the typical mossy fiber terminals described in the cerebellar cortex [9]. The labeled boutons in the present study are, on average, much smaller than mossy fiber terminals and are not surrounded and indented by multiple postsynaptic profiles in a glomerular fashion as are the mossy terminals in the cerebellar cortex. However, in the CN, unlabeled boutons that exhibited some of the characteristic features of mossy fiber terminals were occasionally observed. This suggests that some mossy fiber systems might give rise to CN collaterals that are similar to the typical cerebellar cortical mossy fiber terminals. These findings correlate with light microscopic observations from an earlier study [8] (see also inset of Figs. 3C,4D) which clearly indicated that PHA-L labeled pontocerebellar axon collaterals in the CN are much thinner than mossy fibers in the cerebellar cortex, and their synaptic boutons much smaller and less elaborately arborized than mossy terminals. The labeled boutons in the present report do share some morphological similarities with axon terminals described by other investigators as mossy fiber collaterals in the rat lateral cerebellar nucleus [4]. The existing literature appears to contain only one other report that does specifically identify a pontocerebellar axon terminal in the CN and that study involved mossy fiber inputs to the fastigial nucleus in the cat [12]. Boutons were visualized by electron microscopic autoradiography following injection of radioisotope in the lateral reticular nucleus, medial vestibular nucleus or the NRTP. One bouton from an N R T P case was illustrated (their Fig. 10), and similar to the present observations, it exhibited little resemblance to a typical cerebellar cortical mossy fiber terminal. The illustrated bouton is, however, morphologically similar to the labeled bouton shown in Fig. 4B of the present report. Both are relatively small and irregular in shape as they insinuate themselves among several postsynaptic profiles. It remains for future studies to clarify what specific CN neuronal elements might receive input via the system of pontocerebellar axon collaterals and, what functional role is played by this circuitry which apparently links the cerebral cortex and the cerebellar nuclei via one intervening synapse in the BPN or NRTP.Support for this work was provided by USPHS Grant NS12644. Appreciation is extended to D. Holmes and W. Dorsett-Martin for their technical contributions. The assistance of D.E. Haines in editing an early version of the manuscript is also acknowledged. [1] Alien, G.I., Gilbert, P.F.C., Marini, R., Schultz, W. and Yin, T.C.T., Integration of cerebral and peripheral inputs by interpositus neurons in monkey, Exp. Brain Res., 27 (1977) 81-99.
318
G.A. Mihailoff / Brain Research 648 (1994) 313-318
[2] Allen, G.I., Gilbert, P.F.C. and Yin, T.C.T., Convergence of cerebral inputs onto dentate neurons in the monkey, Exp. Brain Res., 32 (1978) 151-170. [3] Brodal, P., Dietrichs, E. and Walberg, F., Do pontocerebellar mossy fibers give off collaterals to the cerebellar nuclei? An experimental study in the cat with implantations of crystalline HRP-WGA, Neurosci. Res., 4 (1986) 12-24. [4] Chan-Palay, V., Cerebellar Dentate Nucleus, Springer, New York, NY, 1977, pp. 280-283. [5] Dietrichs, E., Bjaalie, J.G. and Brodal, P., Do pontocerebellar fibers send collaterals to the cerebellar nuclei? Brain Res., 259 (1983) 127-131. [6] Gerrits, N.M. and Voogd, J., The projection of the nucleus reticularis tegmenti pontis and adjacent regions of the pontine nuclei to the central cerebellar nuclei in the cat, J. Comp. Neurol., 258 (1987) 52-69. [7] Giuffrida, R., Licata, F., Volsi, Li, Perciavalle, V. and Urbano,
[8]
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