Lauren R. Marotte

”Rodent-like” and ”primate-like” types of astroglial architecture in the adult cerebral cortex of mammals: a comparative study

Previous observations disclosed that astroglia with interlaminar processes were present in the cerebral cortex of adult New and Old World monkeys, but not in the rat, and scarcely in the prosimian Microcebus murinus. The present report is a more systematic and comprehensive comparative analysis of the occurrence of such processes in the cerebral cortex of several mammalian species. Brain samples were obtained from adult individuals from the following orders: Carnivora (canine), Rodentia (rat and mouse), Marsupialia (Macropus eugenii), Artiodactyl (bovine and ovine), Scandentia (Tupaia glis), Chiroptera (Cynopteris horsfieldii and C. brachyotis), and Primate: Prosimian (Eulemur fulvus), non-human primate species (Cebus apella, Saimiri boliviensis, Callithrix, Macaca mulatta, Papio hamadryas, Macaca fascicularis, Cercopithecus campbelli and C. ascanius) and from a human autopsy. Tissues were processed for immunocytochemistry using several antibodies directed against glial fibrillary acidic protein (GFAP), with or without additional procedures aimed at the retrieval of antigens and enhancement of their immunocytochemical expression. The cerebral cortex of non-primate species had an almost exclusive layout of stellate astrocytes, with only the occasional presence of long GFAP-IR processes in the dog that barely crossed the extent of lamina I, which in this species had comparatively increased thickness. Species of Insectivora and Chiroptera showed presence of astrocytes with long processes limited to the ventral basal cortex. Interlaminar GFAP-IR processes were absent in Eulemur fulvus, at variance with their limited presence and large within- and inter-individual variability as reported previously in Microcebus murinus. In New World monkeys such processes were absent in Callithrix samples, at variance with Cebus apella and Saimirí boliviensis. Overall, the expression of GFAP-IR interlaminar processes followed a progressive pattern: bulk of non-primate species (lack of interlaminar processes) –Chiroptera and Insectivora (processes restricted to allocortex) <strepsirhini <haplorhini (platirrhini<catarrhini). This trend is suggestive of the emergence of new evolutionary traits in the organization of the cerebral cortex, namely, the emergence of GFAP-IR long, interlaminar processes in the primate brain. Interlaminar processes may participate in a spatially restricted astroglial role, as compared to the one provided by the astroglial syncytium. It is proposed that the widely accepted concept of an exclusively astroglial syncytium is probably linked with a specific laboratory animal species (”rodent-type” or, rather, ”general mammalian-type” model) that misrepresents the astroglial architecture present in the cerebral cortex of most anthropoid adult primates (”primate-type” model), including man.

Australian marsupials as models for the developing mammalian visual system

This article makes two points. First, the diprotodont marsupials, including the kangaroos, wallabies and the Australian possum are not primitive mammals, and their brains make as good a general model of the higher mammals such as monkeys and humans as do those of the more common laboratory mammals such as cats and rats. Second, the peculiarities of marsupial reproduction, which comprises a very short period of intrauterine development, followed by a relatively protracted period of development in the pouch, provide unparalleled advantages for research into mammalian neuroembryology. Examples will be provided of how such research has made a contribution to our understanding of neural development, concentrating primarily on the visual system.

Ontogeny of the Projection Tracts and Commissural Fibres in the Forebrain of the Tammar Wallaby (Macropus eugenii): Timing in Comparison with Other Mammals

The sequence of appearance of major forebrain projection and commissural fibre bundles in the tammar wallaby (Macropus eugenii) during development was examined with the aid of silver and haematoxylin stained material. At the time of birth (P0), the cerebral cortex is unformed, but two prominent fibre bundles are apparent in the forebrain: the medial forebrain bundle and the stria medullaris thalami. There is also an unidentified tract (possibly thalamostriate or striothalamic), which appears to be transient, in that it cannot be identified at P8. By P2 the posterior commissure, fasciculus retroflexus and mammillothalamic tract have appeared. Fibres of the fornix were first visible at P8. Cortical projection fibres (internal and external capsular fibres) were first noted at P10 and the anterior commissure at P14. It was not until P18 that the cortical commissural bundle unique to diprotodontid metatherians, namely the fasciculus aberrans, was first seen. The hippocampal commissure was seen to develop relatively late, at P35. The sequence and tempo of development of these tracts has been compared in metatherian and eutherian forebrains. The sequence is similar in the two groups of mammals with one exception: isocortical commissural connections appear to develop considerably earlier in diprotodontid metatherians than in eutherians. With regard to the tempo of forebrain tract development, mammals with r selection reproductive patterns (large litter sizes, many litters per reproductive lifetime, rapid development of offspring, e.g. polyprotodontid metatherians, rodents) appear to have forebrain tract development occupying a relatively greater proportion of the period from conception to the attainment of behavioural autonomy than do those animals with K selection reproductive patterns (few offspring per reproductive lifetime, relatively prolonged development of offspring, e.g. diprotodontid metatherians, primates). This difference is irrespective of whether a mammal is metatherian or eutherian, independent of encephalization, and probably reflects the greater time allocated to aspects of brain development occurring after initial tract formation (elaboration of cortical and forebrain circuitry, dendritic tree growth, synapse overproduction and elimination) among selection mammals.

Timecourse of development of the wallaby trigeminal pathway: III. thalamocortical and corticothalamic projections

The development of trigeminal projections between the thalamus and cortex has been investigated in the marsupial mammal, the wallaby, by using a carbocyanine dye, horseradish peroxidase conjugated to wheat germ agglutinin (WGA‐HRP), Neurobiotin, and biocytin as pathway tracers. The appearance of whisker‐related patterns in the cortex in relation to their appearance in the brainstem and thalamus was examined, as was the presence or absence of a waiting period for thalamocortical afferents and the identity of the first cortical cells to project to the thalamus. Thalamic afferents first reached the cortex at postnatal day (P) 15 and were distributed up to the deep edge of the compact cell zone in the superficial cortical plate throughout development, in both dye and WGA‐HRP labelled material, with no evidence of a waiting period. The initial corticothalamic projection, detected by retrograde transport of WGA‐HRP from the thalamus, occurred at P60 from layer 5 cells. This was confirmed by labelling of corticothalamic axons after cortical injections of Neurobiotin and biocytin. Scattered, labelled cells seen before P60 after dye labelling from the thalamus presumably resulted from transcellular labelling via thalamic afferents. Clustering of afferents in layer 4 and cell bodies and their dendrites in layers 5 and 6 first occurred simultaneously at P76. There is a sequential onset of pattern formation, first in brainstem, then in thalamus, and finally in cortex, with a long delay between afferent arrival and pattern formation at each level. Independent regulation at each level, likely depending on target maturation, is suggested. J. Comp. Neurol. 387:194–214, 1997.

Development of whisker representation in the cortex of the tammar wallaby Macropus eugenii

The somatosensory cortex associated with the whiskers has been studied in adult tammar wallabies (Macropus eugenii) and in pouch young from 60-120 days of pouch life. The time course of anatomical changes examined with succinic dehydrogenase (SDH) histochemistry and Nissl staining has been correlated with the maturation of electrically evoked cortical responses to stimulation of the whisker follicles. The earliest signs of aggregates of SDH reaction product in layer IV of the cortex were seen at 85 days, coincident with the first recordings of an immature cortical evoked potential. Aggregates, in a pattern corresponding to that of the facial whiskers, were most clearly seen from 90-140 days. At later stages, and in the adult, they were present but their arrangement was less clearly seen. By 186 days the electrical activity resembled the mature pattern. Patches of SDH activity in layer IV were not associated with changes in soma density characteristic of true barrels.

Two stages in the development of a mammalian retinocollicular projection.

The retinocollicular projection in the marsupial mammal the wallaby Macropus eugenii, has been investigated anatomically to determine the order in the developing projection and electrophysiologically to determine the time of onset of synaptic transmission by recording evoked potentials in the colliculus in response to stimulation of the optic nerve. There are two clear stages: a protracted period when retinal axons grow into the colliculus in coarse retinotopic order with no recordable electrical activity followed by the formation of terminal zones in retinotopically correct positions, the loss of more widely distributed axons and the onset of evoked potentials. The two stages are not seen in non-mammalian vertebrates where the projection is functional from the beginning.

Development of the Olfactory System in a Wallaby (Macropus eugenii)

We used carbocyanine dye tracing techniques in conjunction with hematoxylin and eosin staining, immunohistochemistry for GAP-43, and tritiated thymidine autoradiography to examine the development of the olfactory pathways in early pouch young tammar wallabies (Macropus eugenii). The overarching aim was to test the hypothesis that the olfactory system of newborn tammars is sufficiently mature at birth to contribute to the guidance of the pouch young to the nipple. Although GAP-43 immunoreactive fibers emerge from the olfactory epithelium and enter the olfactory bulb at birth, all other components of the olfactory pathway in newborn tammars are very immature at birth, postnatal day (P0). In particular, maturation of the vomeronasal organ and its projections to the accessory olfactory bulb appears to be delayed until P5 and the olfactory bulb is poorly differentiated until P12, with glomerular formation delayed until P25. The lateral olfactory tract is also very immature at birth with pioneer axons having penetrated only the most rostral portion of the piriform lobe. Interestingly, there were some early (P0) projections from the olfactory epithelium to the medial septal region and lamina terminalis (by the terminal nerve) and to olfactory tubercle and basal forebrain. The former of these is presumably serving the transfer of LHRH+ neurons to the forebrain, as seen in eutherians, but neither of these very early pathways is sufficiently robust or connected to the more caudal neuraxis to play a role in nipple finding. Tritiated thymidine autoradiography confirmed that most piriform cortex pyramidal neurons are generated in the first week of life and are unlikely to be able to contribute to circuitry guiding the climb to the pouch. Our findings lead us to reject the hypothesis that olfactory projections contribute to guidance of the newborn tammar to the pouch and nipple. It appears far more likely that the trigeminal pathways play a significant role in this behavior because the central projections of the trigeminal nerve are more mature at birth in this marsupial.

ANTERIOR COMMISSURE OF THE WALLABY (MACROPUS EUGENII) : ADULT MORPHOLOGY AND DEVELOPMENT

In metatheria, all neo‐ and paleo‐cortical commissural connections are made by the anterior commissure. We have examined the adult morphology of this commissure and its development in a diprotodontid metatherian, the wallaby (Macropus eugenii), at both the light and electron microscope level. The total number of axons in the adult anterior commissure was 21.7 million, of which 55–62% were myelinated. The dorsal two thirds of the commissure, containing neocortical commissural axons, showed a higher percentage of larger, myelinated axons than the ventral one third, which contains paleocortical commissural axons. The commissure also showed a topographical gradient, with cells in the dorsal cortex projecting through the dorsal region of the commissure, the fasciculus aberrans. In the rostrocaudal axis, axons from the frontal cortex tended to pass more anteriorly through the commissure and those from the occipital more posteriorly, but there was extensive overlap of projections from different areas.

Cyto- and Chemoarchitecture of the Cortex of the Tammar Wallaby (Macropus eugenii): Areal Organization

We have examined the cyto- and chemoarchitecture of the isocortex of a diprotodontid marsupial, the tammar wallaby (Macropus eugenii), using Nissl staining in combination with enzyme histochemical (acetylcholinesterase – AChE, NADPH-diaphorase – NADPHd, cytochrome oxidase) and immunohistochemical (non-phosphorylated neurofilament – SMI-32) markers. The primary sensory cortex showed distinctive patterns of reactivity in cytochrome oxidase, acetylcholinesterase and NADPH diaphorase. For example, in AChE material, S1 showed a heterogeneous appearance, with regions exhibiting a double layer of AChE activity (layers II and IV) adjacent to poorly reactive regions. In NADPHd preparations, activity in S1 was strongest in layers I to IV although, as in AChE material, there were consistent patches of reduced NADPHd activity which corresponded to poorly reactive regions in the AChE sections. Each of the primary sensory areas of the isocortex showed a different pattern of distribution of SMI-32+ neurons. In V1, SMI-32+ neurons were distributed in two layers (III and V) throughout the tangential extent of that region. In S1, SMI-32+ neurons were concentrated in layer V, but large and discrete patches within S1 had additional SMI-32+ neurons in layer III. In primary auditory cortex there was a dense band of SMI-32+ neurons in layer V, with only occasional labeled pyramidal neurons in layer III. In the secondary sensory areas (V2 and S2) SMI-32+ neurons were either distributed in layers III and V (V2) or solely within layer V (S2). The tangential and laminar distribution of Type I reactive NADPH diaphorase neurons in the tammar wallaby cortex was more like that seen in eutheria than in polyprotodontid metatheria.

Retinotopic order in the optic nerve and superior colliculus during development of the retinocollicular projection in the wallaby (Macropus eugenii)

Abstract Retinotopic order of optic axons in the optic nerve and superior colliculus of the marsupial mammal, the wallaby (Macropus eugenii), has been examined and compared during development of the retinocollicular projection to investigate the role of order in the nerve in map formation. Small groups of axons from different retinal quadrants were labelled in vivo with a carbocyanine dye from just after axons first reached the colliculus to when the projection was mature. The distribution and branching patterns of axons and their arbors on the colliculus were assessed quantitatively during this period, as was the degree of order in the nerve. Initially, axons accumulated in coarse retinotopic order in the colliculus, with little branching and no sign of arborization to form terminal zones. Axons labelled from deposits covering a mean of 2.2% of the retina reached a mean collicular coverage of around 30% at 41–47 days, at which time they began arborizing in their retinotopically correct positions. By 55 days axons from all retinal quadrants had formed terminal zones in their retinotopically correct positions. Axons did not arborise in incorrect positions as has been reported in the rat. By 61–68 days coverage had decreased to around 10%. By 90–95 days only axons suppying terminal zones were present and terminal zones were smaller. In the nerve, axons showed a coarse and consistent order throughout development. This order was retinotopic only immediately behind the eye. Temporal and nasal axons occupied corresponding halves of the nerve along its course. Axons from dorsal and ventral retina shifted from dorsal and ventral positions in the nerve, respectively, to opposite sides of the nerve just before the chiasm. This would assist in positioning them in the appropriate lateral and medial optic tracts, respectively, in the positions they occupied as they approached the colliculus. However, the position in the nerve was not related to the ability to arborize in the correct collicular position. In particular, the increase in retinotopic order in the colliculus late in development was not accompanied by an increase in order in the nerve. Since the final organization in the colliculus shows greater order than is ever seen in the nerve, additional mechanisms must be involved in the maturation of the collicular map.