Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer

The multiplication of cells in the proliferative zone of the external germinal layer and the early steps in the differentiation of basket, stellate and granule cells were studied in the cerebellar cortex of rats aged 0, 3, 5, 7, 10, 12, 15, 21 and 30 days with histological, histochemical, autoradiographic and electron microscopic techniques. Between 0–9 days the proliferative zone has a constant depth of four to five cells; the bipolar cells in the underlying premigratory zone increase in depth during this period from 0–6 cells. Thereafter, there is a decline in the cell depth of both zones. In the premigratory zone there is a gradient in the length of the extruded processes of the bipolar cells (concentration of such profiles), the future parallel fibers. Presumably when the latter reach their final length, the cell body migrates downward and the parallel fiber becomes part of the upper zone of the molecular layer. Thus, parallel fibers are progessively formed on the surface of others from the bottom upward by a stacking process and the external germinal layer, as a consequence, is continually pushed upward. This design makes possible the assembly of a matrix of very long, thin and straight beams of horizontally oriented parallel fibers which pile up vertically according to their age. When synaptogenesis starts during the second week in the molecular layer the enlarging junctional processes produce a spurt in the growth of this layer. Basket cells which are formed in the pyramis on days 6–7 are arrested in the formative molecular layer because their processes are oriented at a right angle to the underlying bed of parallel fibers. Therefore, there is also a stacking of the cells of the molecular layer from the bottom upward as a function of time of onset of their differentiation. Parallel fiber synapses may be seen on differentiating basket cells as early as the seventh day, forming connections with these inhibitory interneurons before they synapse with spines of Purkinje cells.     

Postnatal development of the cerebellar cortex in the rat: V. Spatial organization of Purkinje cell perikarya

The development of the spatial organization of Purkinje cell perikarya was examined in the rat cerebellum from birth to adulthood. Dispersion of the perikarya following birth is made possible by the rapid expansion of the cortical surface. Their subsequent regular monocellular alignment is ensured by mechanical factors, the pressure exerted from below by the expanding granular layer and the barrier formed above by the pile of parallel fibers which prevent the penetration of the bulky perikarya into the molecular layer. The perikarya remain in this position even after the slender stem dendrite pierces the molecular layer along the descending axons of basket cells. The increase in interperikaryal distance between Purkinje cells is rapid up to day 12, then declines. This is temporally associated with the growth of the basket cell plexus and glial envelope around the perikaryon. The increase in perikaryal size continues up to day 30. This may be temporally associated with the growth of the Purkinje cell dendritic arbor as reflected by the expansion of the molecular layer up to day 30. The spatial arrangement of Purkinje cells within the monocellular sheet was graphically displayed with computer aid. In the adult cerebellum a hexagonal arrangement could be recognized in a proportion of “near-neighborhoods,” consisting of about six Purkinje cells and their neighbors. When the neighborhoods were extended with fixed orientation with respect to the axis of the folium, the hexagonal arrangement disappeared. When orientation was ignored, the superimposed nearneighborhoods could be rotated to produce a hexagonal pattern. In the infant cerebellum the hexagonal arrangement could not be demonstrated before the alignment of Purkinje cells in a monolayer. Thereafter there appeared to be an increase with age in the proportion of hexagonally arranged near-neighborhoods. It was concluded that in the monocellular ganglionic layer Purkinje cells are not aligned in regular rows with respect to the geometrically arranged elements of the supraganglionic layer. The formation of an imprecise hexagonal pattern, like the alignment of Purkinje cells in a monolayer, was attributed to mechanical factors.     

Postnatal development of the cerebellar cortex in the rat. 3. Maturation of the components of the granular layer

The migration of granule cells and the maturation of the various elements of the granular layer were studied in the cerebellar cortex of rats aged 0, 3, 5, 7, 10, 12, 15, 21 and 30 days with histological, histochemical, autoradiographic and electron microscopic techniques. The bulk of the granule cells are formed during the second week, but due to the time required for their migration and the lag in the formation of dendrites, few glomerular synapses are formed with mossy fibers before the beginning of the third week and the process is still in progress at 30 days, long after the dissolution of the external germinal layer. The maturation of Golgi cells is a protracted process. Their axons synapse with granule cell dendrites as soon as the glomeruli begin to mature. Evidence was obtained that mossy fibers synapse with the dendrites of Golgi cells. Towards the end of the second week the Lugaro cells are formed and synapses appear on their somata during the third week. Among these synapses the recurrent collaterals of Purkinje cell axons were identified. The Lugaro cells may be the primary targets of the infra and supraganglionic plexuses formed by these collaterals. In conclusion it was suggested that there are three major, successive stages in the neurogenesis of the cerebellar cortex, the morphogenic, synaptogenic and gliogenic. However, in the large Purkinje cell the synaptic maturation of one region (the soma) may begin before the morphogenic and synaptogenic maturation of the entire cell (the dendrites) is completed.     

Postnatal development of the chemosensitivity of rat cerebellar Purkinje cells to excitatory amino acids. An in vitro study

In vitro sagittal slices of immature rat cerebellum were used to study the development of the sensitivity of Purkinje cells (PCs) to L-aspartate (L-Asp), L-glutamate (L-Glu) and related derivatives. As early as postnatal day 0 all PCs already displayed clear excitatory responses to short iontophoretic applications of L-Asp, L-Glu and quisqualate while in the same conditions no effect of N-methyl-D,L-aspartate (NMDLA) was detected. By postnatal day 5, i.e. after the onset of the synaptogenesis, the sensitivity of PCs to L-Asp, L-Glu and quisqualate significantly increased up to values similar to those recorded in adult rat cerebellum and surprisingly nearly all (87%) the recorded cells now also displayed excitatory responses to NMDLA. Although this sensitivity of PCs to NMDLA was significantly lower than that observed with the other drugs, it persisted until the end of the first postnatal month when the adult type of connectivity is already well established but at this stage only 30 per cent of the tested cells were still sensitive to the agonist. During this period, excitatory responses elicited by NMDLA were selectively antagonized by 2-amino-5-phosphonovalerate (2-APV), suggesting that during postnatal development, NMDA receptor types are transiently expressed on PCs membranes since in the adult, NMDLA no longer had an excitatory effect. Instead, this drug now exerted a preferential antagonistic action on the excitatory response elicited by L-Asp. Also in the adult, no major changes occurred in the sensitivity of PCs to L-Asp, L-Glu and quisqualate when these drugs were ejected at a dendritic site whereas, when ejected at the somatic level, the sensitivity of the cell appeared 2-3 times lower.     

Developmental dynamics of Purkinje cells and dendritic spines in rat cerebellar cortex

Quantitative morphological changes of the developing Purkinje cells were studied from 6 to 90 postnatal (PN) days in the IVth lobule of vermis in the cerebellum of rats. The soma size (mean diameter) of Purkinje cells increased rapidly between 6 PN (on average 10 μm) and 18 PN (about 17 μm) days; it did not change between 18 and 25 PN days, but increased moderately again between 25 and 48 PN days (22–23 μm) and stabilized on the same value. In contrast, the number of Purkinje cells/100 μm (the “linear density”) decreased rapidly from 6 to 18 PN days. The molecular layer area belonging to 1 Purkinje cell increased rapidly from 6 to 25 PN days (from about 370 to 6,200 μm²) and less rapidly between PN days 30 to 48 (up to 9,300 μm²), followed by a moderate decrease at PN day 90 (about 6,600 μm²). The volume belonging to 1 Purkinje cell dendritic arbor was about 5,500 μm³ at PN day 6,93,000 μm³ at PN day 25, and 100,000 μm³ at PN day 90. The numerical density of dendritic spines in the molecular layer showed a biphasic curve: a rapid increase from PN days 6 to 21 followed by a significant but short decrease at PN day 25, moderate rise from PN days 25 to 48, and a subsequent decline between PN days 48 and 90. The number of spines belonging to 1 Purkinje cell showed two developmental “peaks”: the first peak at 21 PN days was moderate (5.6 × 10⁴ spines/Purkinje cell) while the second maximum at 48 PN days was more significant (1.2 × 10⁵ spines/Purkinje cell), which then declined to 6.3 × 10⁴ spines/Purkinje cell at PN day 90. It is suggested that the temporary overproduction and the following decline in the number of Purkinje dendritic spines during the development of the cerebellar cortex may be the morphological indicator of the dynamics of synaptogenetic and of synaptic stabilization processes. © 1994 Wiley-Liss, Inc.     

Postnatal development of unipolar brush cells in the cerebellar cortex of cat

The postnatal developmental distribution pattern of metabotropic glutamate receptor (mGluR1a) immunoreactive unipolar brush cells (UBCs) was studied in the cerebellar cortex of kittens. On the day of birth (P0) UBCs are already present in the white matter in lobule X of the vermis, but only a few of these cell seemed to migrate to the deeper region of the internal granular layer. By the end of the first week (P8) UBCs were seen to invade the white matter + internal granular layer of lobules IX, VIII, I, and II of the vermis, and they spread further in the transitory area medio-laterally from the vermis toward the cerebellar hemispheres. By P15, UBCs appeared in lobules III and VII of the vermis, as well as in corresponding lobules of the neocerebellum, with especially high numbers in lobule VII. By P22, UBCs migrated further after their medio-lateral course in the neocerebellum, and began to invade lobules V and VI. At P62 the amount of UBCs in midsagittal planes of early developing vermal lobules (I, II, VII-X) resembled the P132 or adult pattern. The medio-lateral migration and incorporation of UBCs into the late-developing cerebellar lobules V and VI was completed only by P132, when the spatial distribution of UBCs in both the vermal and neocerebellar lobules was comparable to that seen in the 1 year old young adult cat. Although by P132 the postnatal migration of the vast majority of UBCs seemed to be completed, in the cerebellum of adult cats a few migrating UBCs could still be observed in the white matter of the cerebellar lobules, and beneath the ependyma of the fourth ventricle. It is concluded that during ontogenesis the migration course of UBCs follows essentially the developmental sequence of cerebellar lobules, although the incorporation of UBCs into the internal granular layer continues until 4 months postnatally, i.e., much beyond the apparent completion (about two months postnatally) of cytoarchitectonic built up of the cerebellar cortex of kittens.     

Quantitative study of granule and Purkinje cells in the cerebellar cortex of the rat

The numerical densities of granule and Purkinje cells in the cerebellar cortex of the rat were determined by stereological methods. The density of Purkinje cells in our fixed material was 1,018 +/- 39 per mm2 (mean +/- s.e.m.) of Purkinje cell layer and that of granule cells 1.92 +/- 0.03 x 10(6) per microliter of granular layer. The total area of Purkinje cell layer was 332 mm2 and the volume of granular layer was 48 microliters. The rat cerebellum therefore contains 3.38 x 10(5) Purkinje cells and 9.2 x 10(7) granule cells, from which there are 274 granule cells for each Purkinje cell. The density of granule cells and the density of parallel fibers in the molecular layer observed in a companion study indicate that the average length of a parallel fiber is around 5 mm.     

Morphological development of dendritic spines on rat cerebellar Purkinje cells

The posterior cerebellum is strongly involved in motor coordination and its maturation parallels the development of motor control. Climbing and mossy fibers from the spinal cord and inferior olivary complex, respectively, provide excitatory afferents to cerebellar Purkinje neurons. From post-natal day 19 climbing fibers form synapses with thorn-like spines located on the lower primary and secondary dendrites of Purkinje cells. By contrast, mossy fibers transmit synaptic information to Purkinje cells trans-synaptically through granule cells. This communication occurs via excitatory synapses between the parallel fibers of granule cells and spines on the upper dendritic branchlets of Purkinje neurons that are first evident at post-natal day 21. Dendritic spines influence the transmission of synaptic information through plastic changes in their distribution, density and geometric shape, which may be related to cerebellar maturation. Thus, spine density and shape was studied in the upper dendritic branchlets of rat Purkinje cells, at post-natal days 21, 30 and 90. At 90 days the number of thin, mushroom and thorn-like spines was greater than at 21 and 30 days, while the filopodia, stubby and wide spines diminished. Thin and mushroom spines are associated with increased synaptic strength, suggesting more efficient transmission of synaptic impulses than stubby or wide spines. Hence, the changes found suggest that the development of motor control may be closely linked to the distinct developmental patterns of dendritic spines on Purkinje cells, which has important implications for future studies of cerebellar dysfunctions.     

Postnatal development of the cerebellar cortex in the rat. IV. Spatial organization of bipolar cells, parallel fibers and glial palisades.

The ontogeny of the spatial organization of some components of the molecular layer was investigated in cerebella sectioned systematically in the sagittal, coronal and horizontal planes. There is no discernible organization in the distribution of cells of the proliferative zone of the external germinal layer (EGL) but from birth the differentiating bipolar cells of the subproliferative zone are aligned parallel to the surface and to the long axis of the folium. While they are still in or at the base of the EGL, the bipolar cells emit long processes, the future parallel fibers. The next step is the outgrowth of a vertical process which may reach the base of the molecular layer before the granule cell nucleus becomes translocated. The idea that the cell body truly migrates through the molecular layer is not supported by the observations. Bergmann glia cells are frequently seen in Golgi material in neonates but they are probably less numerous than in older infants and their processes are not as well aligned. It is only gradually that the EGL is perforated by flial endfeet which in older infants are occasionally organized into longitudinal rows. In mature cerebella the parallel fibers are separated by thin and relatively narrow, unstained spaces which are oriented in the longitudinal plane and can be traced from the pial surface to a zone just above the layer of Purkinje cells. It is postulated that these spaces are occupied by glial palisades formed by apposed thin vertical processes to which many Bergmann glia cells contribute. The alignment of these palisades is dependent on the orientation of parallel fibers. When the parallel fibers are reoriented by X-irradiation the glial palisades become correspondingly realigned. These observation indicate that the oriented growth of parallel fibers, which follows the polarization of bipolar cells, determines the spatial organization of the glial framework of the molecular layer. They also suggest that the glial palisades mediate functions that are not primarily developmental in nature.     

Focal axonal swellings in rat cerebellar Purkinje cells during normal development

Focal axonal swellings are characteristic of a wide range of neuropathies. Three neuron-specific monoclonal antibodies have been used to identify focal axonal swellings in the normal developing rat cerebellar cortex. Between 7 and 15 days postnatal, swellings are a common feature of the granular layer and white matter tracts. Using a Purkinje cell-specific antibody, the majority of swellings were shown to occur in Purkinje cell axons. Focal axonal swellings therefore seem to be a normal adjunct of Purkinje cell maturation.     

Postnatal maturation of rat Purkinje cells cultivated in the absence of two afferent systems: an ultrastructural study.

Organized cultures of newborn rat cerebellum were established in Maximow chambers in order to study the maturation of Purkinje cells in absence of afferent systems. In the first model, standard cultures were devoid of extracerebellar afferents mossy and climbing fibers. Despite this absence, somatic spines appeared upon Purkinje cells during the first week in vitro and maturation proceeded normally except for the almost absence of spiny branchlets. Large dendritic trunks were studded with numerous spines, some of which were naked, a few bearing isolated post-synaptic densities and others occupied by boutons of parallel fibers. Stellate and basket axons made synapses upon the smooth portions of dendrites and soma. In a second model, the cultures were fed the antimitotic drug methylazoxymethanol (MAM) to prevent multiplication of granule cell precursors. Despite the absence of climbing and parallel fibers, the elongation of Purkinje dendrites was not prevented, but again the dendritic arbor consisted of large trunks studded with spines; somatic as well as dendritic spines were contacted by large boutons identified as Purkinje recurrent collaterals (PRC). It is concluded that the Purkinje cell possesses a large autonomy from afferent systems as to the growth of soma and dendrites. Conversely, the geometry of the dendrite and especially the spiny branchlets depend on the presence of both climbing and parallel fibers. One may conclude from the above experiments that specificity of synaptic contacts is maintained as long as postsynaptic sites are not devoid of their normal afferents. Heterologous synapses are formed when postsynaptic sites are present, their normal afferents absent and aberrant ones increasing by collateral sprouting. Such is probably the case in the second model of this study.     

Dendritic development of Purkinje and granule cells in the cerebellar cortex of rats treated postnatally with alpha-difluoromethylornithine

Postnatal administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), is known to curtail replication of granule cells in the cerebellum of the rat, but its effects on post-proliferative neurons is unknown. This Golgi study assessed the influence of daily postnatal injections of DFMO on dendritic development of the Purkinje and granule cells. Although the overall appearance of the Purkinje cells was normal, DFMO affected morphogenesis by causing: 1) a reduction in somatic growth, 2) an elongation of the primary dendrite, 3) a lengthening of the distance between the soma and the pial surface, 4) a reduction in the number of tertiary dendrites, and 5) a disruption of the distribution of the tertiary branches. Many of these effects of DFMO may be mediated via the reduction of granule cells rather than representing a direct effect on dendritogenesis. In addition to reducing the number of granule cells, the distribution and morphology of these cells in the DFMO-treated rats were severely affected. Many granule cells did not complete their migration and remained trapped in the molecular layer, and many of those trapped had greatly elongated dendrites that extended into the internal granule cell layer. These results suggest that ornithine decarboxylase is involved in the postproliferative processes of neuronal migration and differentiation.     

The development of the Purkinje cell in the cerebellar cortex of the opossum

Premotor neurons sending their axons to the trigeminal motor nucleus were observed in the cat by light and electron microscopy after labeling the neurons retrogradely or anterogradely with horseradish peroxidase (HRP). After HRP injection into the trigeminal motor nucleus, retrogradely labeled neurons were seen most frequently in the parvocellular reticular formation bilaterally. Many labeled neurons were also seen contralaterally in the intermediate zone at the rostralmost levels of the cervical cord and its rostral extension into the caudalmost levels of the medulla oblongata. Additionally, some neurons were labeled ipsilaterally in the mesencephalic tri-geminal nucleus, contralaterally in the main sensory trigeminal nucleus and the trigeminal motor nucleus, and bilaterally in the oral and interpolar sub-nuclei of the spinal trigeminal nucleus. Only a few labeled neurons were seen in the confines of the gigantocellular reticular formation. All labeled neurons were small or of medium size; no large neurons were labeled. After HRP injection into the regions around the trigeminal motor nucleus or the parvocellular reticular formation, axodendritic terminals containing HRP granules were found contralaterally within the trigeminal motor nucleus. Some of these labeled terminals were filled with round synap-tic vesicles and others contained pleomorphic synaptic vesicles. The varied morphology of labeled axon terminals was considered to reflect the functional heterogeneity of the premotor neurons for the trigeminal motor nucleus.     

Voltage-dependent calcium currents in Purkinje cells from rat cerebellar vermis.

Whole-cell patch clamp recording was used to characterize calcium currents in Purkinje cells dissociated from the cerebellar vermis of 1-3-week postnatal rats. A subset of Purkinje cells had a low-threshold, transient current similar to the T-type current in peripheral neurons. All Purkinje cells had a high-threshold, slowly inactivating current. Only a small component of the high-threshold current was sensitive to dihydropyridine (DHP) antagonists or to the dihydropyridine agonist BAY K8644. omega-Conotoxin had very little effect on the high-threshold current. The results suggest that these Purkinje cells have at least three types of calcium channels: T-type channels (present in only a fraction of cells), DHP-sensitive L-type channels (contributing a small fraction of the high-threshold current), and a predominant type of high-threshold channel that is pharmacologically distinct from L-type and N-type channels characterized in peripheral neurons.     

Adenosine deaminase-'like' immunoreactivity in cerebellar Purkinje cells of rat

The localization of adenosine deaminase (ADA) throughout the rat CNS was investigated immunohistochemically with 5 different affinity purified polyclonal antibody preparations against ADA. Except in the cerebellum, identical immunostaining patterns of ADA-immunoreactivity were observed with all 5 antibodies. In the cerebellum, Purkinje cells and presumptive swellings on their axons were immunostained by only one of the 5 antibodies. Preabsorption with purified ADA abolished immunostaining with all 5 antibodies. Biochemical tests showed that the Km for substrate, the specific activity of immunoprecipitable enzyme and the isozymic characteristics of cerebellar ADA was not different from ADA in whole brain or several other brain regions examined. The atypical immunohistochemical behavior of cerebellar ADA, despite its biochemical similarity to ADA elsewhere in the brain, suggests that the enzyme in the cerebellum has some unique features which must be taken into account when considering its possible role in regulating the neuromodulatory actions of adenosine in the cerebellar cortex.     

Postnatal volumetric development of the prefrontal cortex in the rat

The medial and orbital parts of the prefrontal cortex (PFC) increase in volume during the first weeks of postnatal life. At the end of this period, however, the volumes of both parts of the PFC reach a significantly higher value than in adulthood. Subsequently the volumes decrease until the adult volume is attained. The three subareas of the medial PFC (i.e., the medial precentral area, the dorsal anterior cingulate, and the prelimbic area) reach a maximum volume around day 24, while the two orbital PFC subareas (i.e., the dorsal and ventral agranular insular areas) attain their maximum value around day 30. The differences found in the growth pattern of the five PFC subareas, which are innervated by specific subnuclei of the mediodorsal nucleus of the thalamus, suggest a role of these subnuclei in the PFC development.     

Serotoninergic innervation of the ferret cerebral cortex. II. Postnatal development.

We have investigated the serotoninergic innervation of the ferret cortex from the day of birth to adulthood with immunohistochemical techniques. Due to the premature birth of ferrets, this period spans the entire generation of cells located within the upper cortical layers and their subsequent migration to their final positions. Already at birth, serotoninergic fibers innervate the developing cortex. This innervation is most dense within the marginal zone, the subplate region, and the lower portion of the cortical plate. As long as cell migration continues, serotoninergic fibers enter the expanding portions of the cortex. Only the region just below the marginal zone where newly arriving cells are added to the cortical plate is not innervated by the ingrowing fibers. When the bulk of cell migration ceases, during the third postnatal week, this gap disappears and the fibers gradually form a continuous innervation from the pia to the ventricle. As the cortex matures, the serotoninergic fibers become successively confined to the upper layers, to generate the adult pattern. In the adult ferret cortex, the highest innervation density is found within layers 1, 2, and 3, with a much sparser innervation within the lower layers (Voigt and de Lima, J. Comp. Neurol. 314:403-414, 1991). The dense innervation in the deep cortical layers is only transient, virtually disappearing toward adulthood. These results suggest that serotoninergic axons innervate cortical layers as soon as newly arriving cells reach their final positions within the cortex. This early innervation lends support to the idea that serotonin may play a role during development of the cerebral cortex.     

Postnatal development of dye-coupling among astrocytes in rat visual cortex.

Intercellular coupling among astrocytes was studied in rat visual cortex slices from animals aged 1 week to 4 months. Cell coupling via gap junctions was determined by the dye spread of the low molecular weight dye Lucifer Yellow CH injected into electrophysiologically identified cells to adjacent cells. Coupling among glial cells was first detected at postnatal day 11 and was thereafter consistently observed until adulthood. Dye spread was observed up to 300 microns radially from the injected cell covering multiple cortical layers. Following dye injection into a single cell up to several hundred Lucifer Yellow-positive cells could be observed. Quantitative analysis revealed a similar extent of dye spread at different developmental stages including a quite constant number of dye-coupled astrocytes from the end of the second postnatal week to adulthood. Double labelling of Lucifer Yellow-filled cells with an antiserum against the glial fibrillary acidic protein confirmed the astrocytic nature of the injected and coupled cells. Comparison of the density of dye-coupled cells in a given area and the total number of astrocytes as revealed by immunocytochemical staining suggests that dye-coupling includes the entire local astrocytic population. It is concluded that coupling among astrocytes via gap junctions in rat visual cortex occurs shortly after birth and reflects one of the first steps in astroglial maturation.