Gregory A. Mihailoff

Collateral branches of cerebellopontine axons reach the thalamus, superior colliculus, or inferior olive: a double-fluorescence and combined fluorescence-horseradish peroxidase study in the rat.

Retrograde double-labeling methods that used two different fluorescent dyes or a fluorescent dye in combination with wheat germ agglutinin horseradish peroxidase were used in the rat to study the collateralization of cerebellopontine fibers to the thalamus, the superior colliculus, or the inferior olive. In cases with combined basilar pontine nuclei and thalamus injections, double-labeled neurons were located in the rostral part of the lateral cerebellar nucleus as well as within the interpositus anterior and interpositus posterior nuclei. These cells are medium to large in size and multipolar-shaped. A much smaller number of double-labeled cells was observed in the combined basilar pontine nuclei and superior colliculus injections. In these cases most of the double-labeled cells were intermediate- to large-sized and either bipolar- or multipolar-shaped. Such neurons were distributed throughout the rostrocaudal extent of the lateral cerebellar nucleus, with only a few double-labeled cells located in the interpositus anterior and posterior nuclei. Finally, in the cases with combined basilar pontine nuclei and inferior olive injections, double-labeled cells were located in interpositus anterior and posterior nuclei and the medial portion of the lateral cerebellar nucleus. The double-labeled cells were relatively small in size and most were spindle-shaped. No double-labeled cells were observed in the medial cerebellar nucleus in any of the three injection combinations. Based upon the observation of double-labeled neurons in the deep cerebellar nuclei in each of the three injection combinations involving the basilar pontine nuclei, we conclude that cerebellar projections to the basilar pons arise in part as collaterals of axons that project to the thalamus, superior colliculus, or the inferior olive.

Autoradiographic and electron microscopic degeneration evidence for axonal sprouting in the rat corticopontine system.

Newborn (2 or 3 days postnatal) rats were subjected to unilateral cerebral cortical lesions involving much of the anterior (sensorimotor) cortical surface in the right hemisphere. Three months later, the survivors were divided into two groups, one group receiving a [3H]leucine injection in the left sensorimotor cortex and the other sustaining a lesion involving the left sensorimotor cortex. Routine autoradiographic studies in the first group revealed abnormally dense axonal and terminal labeling in the pontine gray contralateral to the leucine-injected hemisphere, suggesting that much of the label was due to sprouting from intact corticopontine axons into the neonatally deafferented pontine gray. In the group with a second (adult) cortical lesion contralateral to the neonatal ablation, degenerating axons and boutons were abundant in the pontine gray contralateral to the adult lesion and at least some of these were interpreted to represent sprouted corticopontine axons and their terminals.

Principal neurons of the basilar pons as the source of a recurrent collateral system

The basilar pontine nuclei in the opossum are composed of two general categories of neurons, intrinsic cells and the principal or projection neurons. Observations from Golgi material indicate that principal neurons whose primary axons project to the cerebellar cortex may also give rise to recurrent branches distributing within the pontine gray. Such collaterals were observed to arise near the soma and at some distance from the cell body of the parent axon. The electron microscopic correlate of such a system was identified in the basilar pontine neuropil in animals subjected to lesions of the cerebellar cortex. These lesions destroyed mossy terminals and their parent axons and thus initiated a retrograde reaction in basilar pontine projection neurons which manifested itself in the form of morphologic alterations observed in somata, dendrites, and a class of axonal boutons. Similar altered axon terminals were not observed in control material and did not correspond to the terminals of cerebello-pontine axons described in previous work. It was therefore suggested that such boutons represented the terminals of the recurrent collateral system observed in Golgi material.

A double retrograde fluorescent tracing analysis of dorsal column nuclear projections to the basilar pontine nuclei, thalamus, and superior colliculus in the rat

Injections of the fluorescent dyes Nuclear yellow and True blue were used to determine that the dorsal column nuclei project in collateral fashion to the basilar pontine nuclei (BPN) and the ventral posterolateral nucleus of the thalamus or the BPN and the superior colliculus. Results indicated that relatively few dorsal column nuclear cells project to both the basilar pons and the superior colliculus. In contrast, many dorsal column nuclear cells that project to the BPN also give rise to collateral projections to the thalamus. Thus it is suggested that the latter dorsal column-BPN connections might at least represent in part the anatomical substrate for the electrophysiological demonstration that cerebellar granule cells can be activated at relatively short latency by peripheral tactile receptor stimulation.

THE NEURONS AND THEIR POSTNATAL-DEVELOPMENT IN THE BASILAR PONTINE NUCLEI OF THE RAT

Abstract The development of basilar pontine neurons was investigated utilizing a variety of Golgi techniques in female Sprague-Dawley rats ranging in age from 0 (birth) to 120 days. Basilar pontine neurons in 120-day old animals demonstrated many of the characteristic morphological features observed in adult animals. The perikarya of these neurons exhibited a variety of shapes, gave rise to from 2–7 dendrites, and ranged in size from 8–45 μm. Dendritic patterns were generally divisible into four groups. The most common pattern was a multipolar arrangement of relatively long dendrites, the surfaces of which were studded with a variety of protrusions. The next two most common patterns each exhibited a sparse complement of dendritic appendages but differed in other respects. One was characterized by stout, gradually tapering dendrites arising from large somata while the other possessed perikarya of intermediate sizes and long, thin dendrites of uniform caliber. The final and least common pattern was a bipolar configuration comprised of infrequently branching dendrites originating from small, ovoid or spindle-shaped cell bodies. Surface appendages were present only in small numbers on such neurons. Axons of adult basilar pontine neurons were found to arise from either the somata or a proximal dendrite and appeared on occasion to give rise to collaterals within the pontine gray. At birth, the perikarya of basilar pontine cells were small and irregular in shape. Dendrites and axons were thin, short and displayed bulbous growth cones on their terminal segments and along their lengths. By the end of the first postnatal week, the dendrites had become more uniform in diameter, while the cell bodies had increased considerably in size. Surface appendages on somata and dendrites first appeared 10–12 days after birth, differing from the adult in their greater density and wider distribution. The end of the second postnatal week was marked by a major dendritic growth spurt which continued until Day 16. At that time, most basilar pontine neurons attained adult dendritic patterns. A gradual disappearance of proximal dendritic appendages coupled with the persistence of distal surface appendages was also noted during the second postnatal week. This process was completed by postnatal Day 24 when the basilar pontine neurons appeared to attain adult morphology.

Two types of degenerating axon terminals in the basilar pontine nuclei of the opossum following cerebral cortical lesions

Abstract Subsequent to large cerebral cortical lesions in adult opossums, small boutons (less than 2 μm) contacting small dendritic profiles were observed to undergo the dark, electron dense type of degeneration in most cases. When lesions were restricted to post-orbital (sensorimotor) regions, the majority of degenerating boutons were again small and dark. However, if the lesion was restricted to occipital (visual) regions, most degenerating boutons were somewhat larger (1–3 μm), contacted some intermediate and proximal dendrites and underwent an initial filamentous reaction before becoming electron dense. Thus, it was postulated that at least two different systems of corticofugal axons reach the pontine nuclei, one (small dark boutons) perhaps arising as collaterals or corticospinal axons, the other (filamentous boutons) representing a more direct corticobulbar or corticopontine system.

Anatomic evidence suggestive of dendrodendritic synapses in the opossum basilar pons

In Golgi-prepared material, opossum basilar pontine intrinsic neurons measured less than 22 microns and gave origin to only two or three primary dendrites which gradually tapered in diameter, branched infrequently and exhibited few spines or protrusions. Characteristically, such neurons gave rise to several axon-like processes which might take origin from proximal or distal dendrites as well as the soma. Morphologically similar neurons have been observed in several other regions throughout the CNS where they have been shown to be a source of presynaptic dendritic elements. That opossum basilar pontine intrinsic neurons might also give rise to presynaptic dendrites was supported by the following electron microscopic observations. Profiles containing a small cluster of pleomorphic vesicles, occasional ribosomes and numerous microtubules formed synaptic active sites in which the membrane densities were intermediate between Grays type I and type II. Such presumed presynaptic dendritic profiles were observed to participate in serial synaptic arrangements in which they were always postsynaptic to a round vesicle bouton and presynaptic to another dendritic element. The above features are compatible with electron microscopic descriptions of presynaptic dendrites in other CNS regions and thus suggest that basilar pontine intrinsic neurons may represent a source of presynaptic dendritic elements.

Electron microscopic identification of cerebellopontine axon terminals in the opossum

Following unilateral cerebellar nuclear ablation or transection of the brachium conjunctivum, degenerating axon terminals were identified within the pontine nuclei of adult opossums. Most frequently observed was a category of large boutons (1.5-7.5 microns) exhibiting an early filamentous reaction (1-5 days survival) and later becoming electron dense and shrunken (9-12 days survival) while being engulfed by phagocytic elements. Such boutons characteristically were found nestled within a cluster of spine-like projections taking origin from somata as well as proximal and intermediate dendrites. A smaller variety of dark degenerating boutons (0.5-2.0 micron) was observed after survival periods of intermediate length (6-10 days) and although there was some overlap in size with the smallest filamentous boutons, the majority (71%) were clearly less than 1.5 micron in their greatest dimension. The small dark boutons formed synaptic contacts only with the shafts of intermediate and distal dendrites rather than the claw-like dendritic complex apposed to the large filamentous degenerating boutons. Because of this difference in postsynaptic locus and their small size it was suggested that such boutons might represent the terminals of a second population of cerebello-pontine axons. Such observations lead to the hypothesis that the large filamentous endings contacting the distinctive claw-like somal or dendritic projections from axons of relatively large cerebellar nuclear neurons which also project rostrally to the red nucleus and thalamus where they form similar boutons and synaptic complexes. On the other hand, the small dark boutons may have arisen from small projection-type cerebellar nuclear neurons, the majority of whose axons project caudally to the inferior olive after contributing a relativley small number of collateral branches to the pontine nuclei.

Electron microscopic identification of superior colliculo-pontine axon terminals

Synaptic boutons emanating from axons of superior colliculus origin were identified by electron microscopy in the neuropil of the basilar pontine nuclei. Such boutons were relatively small (0.6-2.0 microns) and exhibited electron-dense degeneration within a 1-2 day period following electrolytic lesions which involved much of the superior colliculus. Degenerating boutons were observed in synaptic contact with dendritic shafts and spines as well as neuronal somata. The reactive boutons were rapidly engulfed by phagocytic elements and were no longer visible in the neuropil after 6 days of survival.

Cerebral cortical afferent terminations on identified spiny basilar pontine neurons, a combined Golgi-EM degeneration study

Observations contained in this study suggest that in general, the population of spiny basilar pontine (BP) projection neurons receives synaptic contacts from both small dark and large filamentous degenerating cortical axon terminals. In some instances a single spiny BP neuron was contacted by both types of cortical boutons. Long-Evans hooded rats received unilateral thermocautery lesions involving a large expanse of sensorimotor and visual cortices. Following survival periods ranging from 4-8 days, brains were perfused and processed routinely for rapid-Golgi staining. Selected tissue blocks containing spiny basilar pontine projection neurons isolated within known termination zones of the ablated ipsilateral cortex were removed from the brainstem and the cells drawn in their entirety, gold toned, de-impregnated and processed for electron microscopy. Processes of these neurons identified by their content of electron dense gold particles were then localized in sequences of ultrathin sections taken through the tissue block. All degenerating boutons contacting gold-containing profiles of the identified neuron were categorized as dark or filamentous and their location approximated on the surface of the cell. If the two different varieties of degenerating corticopontine boutons reflects a difference in functional properties, the implications of the present findings are that (1) the population of spiny-type BP projection neurons receive convergent inputs from both corticopontine systems and (2) that some individual spiny BP neurons integrate both types of input.