Variable functional recovery and minor cell loss in the ganglion cell layer of the lizard Gallotia galloti after optic nerve axotomy.

The lizard Gallotia galloti shows spontaneous and slow axon regrowth through a permissive glial scar after optic nerve axotomy. Although much of the expression pattern of glial, neuronal and extracellular matrix markers have been analyzed by our group, an estimation of the cell loss in the ganglion cell layer (GCL) and the degree of visual function recovery remained unresolved. Thus, we performed a series of tests indicative of effective visual function (pupillary light reflex, accommodation, visually elicited behavior) in 18 lizards at 3, 6, 9 and 12 months post-axotomy which were then processed for immunohistochemistry for the neuronal markers SMI-31 (neurofilaments), Tuj1 (beta-III tubulin) and SV2 (synaptic vesicles) at the last timepoint. Separately, cell loss in the GCL was estimated by comparative quantitation of DAPI(+) nuclei in control and 12 months experimental lizards. Additionally, 15 lizards were processed for electron microscopy to monitor relevant ultrastructural changes in the GCL, optic nerve and optic tract throughout regeneration. Hypertrophy of RGCs was persistent, morphology of the regenerated nerves varied from narrow to neuroma-like features and larger regenerated axons underwent remyelination by 9 months. The estimated cell loss in the GCL was 27% and two-third of the animals recovered the pupillary light reflex which involves the pretectum. Strikingly, visually elicited behavior involving the tectum was only restored in two specimens, presumably due to the higher complexity of this pathway. These preliminary results indicate that limited functional regeneration occurs spontaneously in the severely injured visual system of the lacertid G. galloti.

Neuronal and glial differentiation during lizard (Gallotia galloti) visual system ontogeny.

We studied the histogenesis of the lizard visual system (E30 to adulthood) by using a selection of immunohistochemical markers that had proved relevant for other vertebrates. By E30, the Pax6(+) pseudostratified retinal epithelium shows few newborn retinal ganglion cells (RGCs) in the centrodorsal region expressing neuron- and synaptic-specific markers such as betaIII-tubulin (Tuj1), synaptic vesicle protein-2 (SV2), and vesicular glutamate transporter-1 (VGLUT1). Concurrently, pioneer RGC axons run among the Pax2(+) astroglia in the optic nerve and reach the superficial optic tectum. Between E30 and E35, the optic chiasm and optic tract remain acellular, but the latter contains radial processes with subpial endfeet expressing vimentin (Vim). From E35, neuron- and synaptic-specific stainings spread in the retina and optic tectum, whereas retinal Pax6, and Tuj1/SV2 in RGC axons decrease. Müller glia and abundant optic nerve glia express a variety of glia-specific markers until adulthood. Subpopulations of optic nerve glia are also VGLUT1(+) and cluster differentiation-44 (CD44)-positive but cytokeratin-negative, unlike the case in other regeneration-competent species. Specifically, coexpression of CD44/Vim and glutamine synthetase (GS)/VGLUT1 reflects glial specialization, insofar as most CD44(+) glia are GS(-). In the adult optic tract and tectum, radial glia and free astroglia coexist. The latter show different immunocharacterization (Pax2(-)/CD44(-) /Vim(-)) compared with that in the optic nerve. We conclude that upregulation of Tuj1 and SV2 is required for axonal outgrowth and search for appropriate targets, whereas Pax2(+) optic nerve astroglia and Vim(+) radial glia may aid in early axonal guidance. Spontaneous axonal regrowth seems to succeed despite the heterogeneous mammalian-like glial environment in the lizard optic nerve.

Expression of BDNF and NT-3 during the ontogeny and regeneration of the lacertidian (Gallotia galloti) visual system.

Retinal ganglion cell (RGC) axons regrow spontaneously after optic nerve (ON) transection in G. galloti. Because brain-derived neurotrophic factor (BDNF) is considered the major neurotrophin participating in vertebrate visual system development and promotes RGC survival, we investigated its distribution using dual-labeling immunohistochemistry for neuronal and glial markers. We examined the developing and regenerating lizard visual system at 1, 3, 6, 9, and 12 months postlesion to comparatively evaluate BDNF expression patterns. BDNF was detected from midembryonic stages (E35) in both retinal plexiform layers, and in radial glial processes in the tectum. Moreover, RGC axon staining was detected at late prenatal stages (E39), showing a transient punctate staining which progressed in a temporo-spatial pattern that was similar to myelination. Strong expression in RGC axons was maintained in adults. However, transient downregulation of BDNF staining occurred on the experimental side one month after ON transection followed by a gradual recovery with extensive punctate/swelling distribution and persistent upregulation at 12 months. Conversely, quantitative PCR analysis for 1 and 12 months regenerate lizards showed downregulation of the ratio of BDNF mRNA expression at 12 months and nonsignificant changes of NT-3 transcripts. In summary, we demonstrate that BDNF and NT-3 are abundantly expressed during lizard visual system ontogeny and regeneration suggesting their participation in the development, maintenance and plasticity of the system.

Expression of neuronal markers, synaptic proteins, and glutamine synthetase in the control and regenerating lizard visual system.

Spontaneous regrowth of retinal ganglion cell (RGC) axons occurs after optic nerve (ON) transection in the lizard Gallotia galloti. To gain more insight into this event we performed an immunohistochemical study on selected neuron and glial markers, which proved useful for analyzing the axonal regrowth process in different regeneration models. In the control lizards, RGCs were beta-III tubulin- (Tuj1) and HuCD-positive. The vesicular glutamate transporter-1 (VGLUT1) preferentially stained RGCs and glial somata rather than synaptic layers. In contrast, SV2 and vesicular GABA/glycine transporter (VGAT) labeling was restricted to both plexiform layers. Strikingly, the strong expression of glutamine synthetase (GS) in both Müller glia processes and macroglial somata revealed a high glutamate metabolism along the visual system. Upregulation of Tuj1 and HuCD in the surviving RGCs was observed at all the timepoints studied (1, 3, 6, 9, and 12 months postlesion). The significant rise of Tuj1 in the optic nerve head and optic tract (OTr) by 1 and 6 months postlesion, respectively, suggests an increase of the beta-III tubulin transport and incorporation into newly formed axons. Persistent Tuj1(+) and SV2(+) puncta and swellings were abnormally observed in putative degenerating/dystrophic fibers. Unexpectedly, neuron-like cells of obscure significance were identified in the control and regenerating ON-OTr. We conclude that: 1) the persistent upregulation of Tuj1 and HuCD favors the long-lasting axonal regrowth process; 2) the latter succeeded despite the ectopia and dystrophy of some regrowing fibers; and 3) maintenance of the glutamate-glutamine cycle contributes to the homeostasis and plasticity of the system.

Distribution of neurotrophin-3 during the ontogeny and regeneration of the lizard (Gallotia galloti) visual system.

We have previously described the spontaneous regeneration of retinal ganglion cell axons after optic nerve (ON) transection in the adult Gallotia galloti. As neurotrophin-3 (NT-3) is involved in neuronal differentiation, survival and synaptic plasticity, we performed a comparative immunohistochemical study of NT-3 during the ontogeny and regeneration (after 0.5, 1, 3, 6, 9, and 12 months postlesion) of the lizard visual system to reveal its distribution and changes during these events. For characterization of NT-3(+) cells, we performed double labelings using the neuronal markers HuC-D, Pax6 and parvalbumin (Parv), the microglial marker tomato lectin or Lycopersicon esculentum agglutinin (LEA), and the astroglial markers vimentin (Vim) and glial fibrillary acidic protein (GFAP). Subpopulations of retinal and tectal neurons were NT-3(+) from early embryonic stages to adulthood. Nerve fibers within the retinal nerve fiber layer, both plexiform layers and the retinorecipient layers in the optic tectum (OT) were also stained. In addition, NT-3(+)/GFAP(+) and NT-3(+)/Vim(+) astrocytes were detected in the ON, chiasm and optic tract in postnatal and adult lizards. At 1 month postlesion, abundant NT-3(+)/GFAP(+) astrocytes and NT-3(-)/LEA(+) microglia/macrophages were stained in the lesioned ON, whereas NT-3 became downregulated in the experimental retina and OT. Interestingly, at 9 and 12 months postlesion, the staining in the experimental retina resembled that in control animals, whereas bundles of putative regrown fibers showed a disorganized staining pattern in the OT. Altogether, we demonstrate that NT-3 is widely distributed in the lizard visual system and its changes after ON transection might be permissive for the successful axonal regrowth.

Radial glial cells, proliferating periventricular cells, and microglia might contribute to successful structural repair in the cerebral cortex of the lizard Gallotia galloti.

Reptiles are the only amniotic vertebrates known to be capable of spontaneous regeneration of the central nervous system (CNS). In this study, we analyzed the reactive changes of glial cells in response to a unilateral physical lesion in the cerebral cortex of the lizard Gallotia galloti, at 1, 3, 15, 30, 120, and 240 days postlesion. The glial cell markers glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), S100 protein, and tomato lectin, as well as proliferating cell nuclear antigen (PCNA) were used to evaluate glial changes occurring because of cortical lesions. A transitory and unilateral upregulation of GFAP and GS in reactive radial glial cells were observed from 15 to 120 days postlesion. In addition, reactive lectin-positive macrophage/microglia were observed from 1 to 120 days postlesion, whereas the expression of S100 protein remained unchanged throughout the examined postlesion period. The matricial zones closest to the lesion site, the sulcus lateralis (SL) and the sulcus septomedialis (SSM), showed significantly increased numbers of dividing cells at 30 days postlesion. At 240 days postlesion, the staining pattern for PCNA, GFAP, GS, and tomato lectin in the lesion site became similar to that observed in unlesioned controls. In addition, ultrastructural data of the lesioned cortex at 240 days postlesion indicated a structural repair process. We conclude that restoration of the glial framework and generation of new neurons and glial cells in the ventricular wall play a key role in the successful structural repair of the cerebral cortex of the adult lizard.

Long-term evolution of local, proximal and remote astrocyte responses after diverse nucleus basalis lesioning (an experimental Alzheimer model): GFAP immunocytochemical study.

A study on long-term astrocytic responses (from 1 day to 20 months after lesioning in 4-month-old rats, and from 1 day to 6 months in 20-month-old rats) to diverse unilateral damage of the nucleus basalis (nbM) by injection of 40 nmol of ibotenic acid, or 50 or 100 nmols of quisqualic acid was performed using a histochemical method (immunoreactivity against the glial fibrillary acidic protein GFAP). Glial reactivity (i.e., isolated or clustered hypertrophic and/or hyper-reactive astrocytes) was evaluated in several ipsilateral and contralateral brain regions: the 'local response' within the damaged nbM region; the 'proximal response' (a new concept proposed by us) in the non-damaged structures neighbouring the nbM; and the 'remote response' in the ipsilateral brain cortex and in the contralateral cortex and nbM. In 4-month-old animals, the remote cortical glial responses, independent of the involution of cortical cholinergic activity and randomly located in layers I-V of motor and somatosensory cortical regions, were similar in appearance over a long period (13-20 months), with the highest reactivity 45 days after lesioning. The proximal response lasted from 1 day to 13 months and afterwards tended to disappear. Contralateral reactivity and ipsilateral cortical scars were observed. The local (nbM) glial response was maintained throughout the period studied. Subsets of astrocytes of different reactivities were observed, most of their elements being highly intermeshed. In 20-month-old animals, nbM lesions produced less positive, but similar, glial reactive patterns. This glial reactivity was superposed onto the glial reactivity of old age. All these results are discussed. The maintenance of reactive astrocytes many months after lesioning suggests the existence of cellular factors other than those produced by damaged nbM neurons. Taking into account the role of glial cells under pathological conditions, it is possible that these reactive astrocytes in humans could promote neurodegenerative processes, such as amyloid plaque formation and neurodegeneration (Alzheimer's disease). Along this line, nbM cholinergic involution could then originate cortical involution through induced reactive astrocytosis.

Distribution of neurofilaments in the telencephalon and mesencephalon of the adult and developing Gallotia galloti lizard.

The location and chronology during development of the immunoreactivity due to the presence of neurofilaments (NF) in telencephalon and mesencephalon of the lizard Gallotia galloti has been studied. For this purpose we have used two antibodies recognizing both phosphorylated and non phosphorylated neurofilaments (NF), a polyclonal Ab (NF 005), and a commercial monoclonal antibody (NF-200). The study was completed by using the Bielschowsky technique. During ontogeny, the anti-NF 005 immunoreactivity appeared at E40 in some tracts in mesencephalon and increased in intensity in isolated nerve fibers, tracts and commissurae till adult. However, a weak staining appeared in some neurons. In telencephalon, the reactivity was detected only in adult specimens. It was clearly more abundant in mesencephalon than in telencephalon, which could indicate that a greater complexity and functional importance exist in the lizard midbrain in relation to other primitive regions as the basal nuclei and cortical areas. In contrast to young specimens, the monoclonal anti-NF 200 was detected in neuronal perikarya, dendrites and axons in adults. Thus, in lizards, both antibodies highly recognized phosphorylated and non-phosphorylated forms of proteins of NF (NF-H). In mammals, these forms of proteins are implicated in axonal maturation. The presence of these NF in reptiles, identified for the first time, proved to be phylogenetically stable. The anti-NF immunoreactivity distribution occurs both caudo-rostrally and from the ventral to the dorsal regions.

Retinal axon regeneration in the lizard Gallotia galloti in the presence of CNS myelin and oligodendrocytes.

Retinal ganglion cell (RGC) axons in lizards (reptiles) were found to regenerate after optic nerve injury. To determine whether regeneration occurs because the visual pathway has growth-supporting glia cells or whether RGC axons regrow despite the presence of neurite growth-inhibitory components, the substrate properties of lizard optic nerve myelin and of oligodendrocytes were analyzed in vitro, using rat dorsal root ganglion (DRG) neurons. In addition, the response of lizard RGC axons upon contact with rat and reptilian oligodendrocytes or with myelin proteins from the mammalian central nervous system (CNS) was monitored. Lizard optic nerve myelin inhibited extension of rat DRG neurites, and lizard oligodendrocytes elicited DRG growth cone collapse. Both effects were partially reversed by antibody IN-1 against mammalian 35/250 kD neurite growth inhibitors, and IN-1 stained myelinated fiber tracts in the lizard CNS. However, lizard RGC growth cones grew freely across oligodendrocytes from the rat and the reptilian CNS. Mammalian CNS myelin proteins reconstituted into liposomes and added to elongating lizard RGC axons caused at most a transient collapse reaction. Growth cones always recovered within an hour and regrew. Thus, lizard CNS myelin and oligodendrocytes possess nonpermissive substrate properties for DRG neurons--like corresponding structures and cells in the mammalian CNS, including mammalian-like neurite growth inhibitors. Lizard RGC axons, however, appear to be far less sensitive to these inhibitory substrate components and therefore may be able to regenerate through the visual pathway despite the presence of myelin and oligodendrocytes that block growth of DRG neurites.

Heterogeneous immunoreactivity of glial cells in the mesencephalon of a lizard: a double labeling immunohistochemical study.

Astrocytes and radial glia coexist in the adult mesencephalon of the lizard Gallotia galloti. Radial glia and star-shaped astrocytes express glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The same cell markers are also expressed by round or pear-shaped cells that are therefore astrocytes with unusual morphology. Other round or pear-shaped cells, also scattered in the tegmentum and the tectum, display only GS. Electron microscopy reveals that these cells may be oligodendrocytes. In this lizard, the GS is expressed in some oligodendrocytes while this does not occur in the central nervous system of mammals in situ. These results confirm that the cellular specificity of GS is different in various species and suggest that ependymal cells are also immunoreactive for GS but they do not contain GFAP.

Myelin and myelinization in the telencephalon and mesencephalon of the lizard Gallotia galloti as revealed by the immunohistochemical localization of myelin basic protein.

We have studied in the telencephalon and mesencephalon of the lizard Gallotia galloti the localization and the chronology of appearance of the immunoreactivity due to the presence of a myelin-specific protein: the Myelin Basic Protein (MBP). MBP-like immunoreactivity was present with different degrees of intensity in many nerve fibers (isolated, in tracts and in commissurae) and it was apparently more abundant in mesencephalon. During ontogeny the earliest MBP-like immunoreactivity was detected at E.36 in few tracts in mesencephalon and appeared at E.40 in telencephalon, proceeding caudo-rostrally and from the ventral (basal) to the dorsal (alar) regions. Accumulation of MBP continued after hatching. Oligodendrocyte cell bodies were not immunopositive, not even at the youngest ages studied.

An ultrastructural study of ependymal cell differentiation during lizard (Gallotia galloti) midbrain development.

Ependymal cell differentiation was examined in the lizard Gallotia galloti from E31 to adult. From E31 to E34 only one type of cell could be identified making up the pseudostratified columnar neuroepithelium but by E35 to E37 three types of ependymal cell were present. The first type was a narrow, elongated, columnar cell containing rough endoplasmic reticulum filled with an amorphous ground substance similar to that of astrocytes. The second type was broader with the nucleus close to the ventricular surface with numerous lipid droplets of varying sizes in the cytoplasm. The third type had an irregularly shaped apical nucleus and a broad basal process probably extending to the pial surface. The process contained numerous microtubules, glycogen granules and a few filaments. From E38 to hatching the ependyma showed marked regional variation. Much of it was formed by a single layer of moderately dark cuboidal cells but parts were made up of a low columnar epithelium in which the cells had elongated nuclei, frequently indented on the ventricular side. Cilia were common and often cells had cytoplasmic protrusions into the ventricle. Lipid was present in the form of small apical droplets or a large basal droplet. Ependymal cells in the region of the sulcus limitans were packed with lipid as were cells of the adjacent subventricular layer. In the adult the ependymal lining varied from cuboidal to low columnar with nuclear chromatin usually arranged in a reticulated pattern. Two types of ependymal process extending to the pia could be identified. One type was packed with microfilaments whilst the other contained a core of microtubules and scattered glycogen granules. Lipid was still present in the cells of the sulcus limitans.

An ultrastructural study of the development of astrocytes in the midbrain of the lizard.

Astrocyte development was investigated electron microscopically in the midbrain of the lizard Gallotia galloti from E32 to adult. At E32 only very immature (early) glioblasts were present in the midbrain and similar cells could be found until hatching. From E34 two other types of glioblast could be identified--dark glioblasts which had a slightly greater amount of cytoplasm than early glioblasts and light glioblasts, in which cytoplasmic organelles were more abundant. Both dark and light glioblasts were present in very small numbers in the adults. Astroblasts, which could be identified by the characteristic appearance of their rough endoplasmic reticulum, first appeared at E35, at which stage a few very immature astrocytes containing small quantities of gliofilaments were also present. With increasing age the quantity of gliofilaments in astrocyte cytoplasm increased. Astrocytes in the adult white matter contained very large amounts of gliofilaments whereas those in grey matter contained many fewer gliofilaments and had glycogen granules in their cytoplasm which were rarely present in mature fibrous astrocytes.

An ultrastructural study of the development of oligodendrocytes in the midbrain of the lizard.

Oligodendrocyte development was investigated in the midbrain of the lizard Gallotia galloti using the electron microscope. Oligodendroblasts, which had a pale cytoplasm containing numerous microtubules in the perikaryon and processes, were present from E35. Active oligodendrocytes had a pale nucleus, usually containing a nucleolus, and an electron-dense cytoplasm with long parallel stacks of rough endoplasmic reticulum. These were present from E37 to hatching which coincides with the period of rapid myelination. The three types of oligodendrocyte (light, medium and dark) first classified by Mori & Leblond (1970) in the rat could be identified in the lizard. Light oligodendrocytes were present at all ages from E37 to adult. Medium oligodendrocytes first appeared at E40 and dark oligodendrocytes were present at all ages from hatching onwards.

Radial glia and astrocytes in developing and adult telencephalon of the lizard Gallotia galloti as revealed by immunohistochemistry with anti-GFAP and anti-vimentin antibodies.

The development of radial glia and astrocytes in the telencephalon of the lizard Gallotia galloti was studied by immunohistochemistry with anti-vimentin and anti-GFAP antibodies. Vimentin appears at embryonic stage 32 (E32) in the proliferative zone of the lateral ventricle and subpial end-feet in the marginal zone. At E34-35 the staining intensity for vimentin in all radial glia is maximal. It then decreases and disappears in most structures in adult animals. GFAP appears at E35 in the end-feet in the marginal zone and its intensity increases until adulthood, particularly in radial and sinuous fibers and in fibers that originate from the sulci and invade the ventral striatum and the septum. In contrast, the reaction is weak in the cortex, in the anterior dorso-ventricular ridge, and in the amygdala nuclei. Radial glia is still present in the adult, and the composition of its intermediate filaments changes during development from vimentin to GFAP. No GFA-positive cell bodies except those of ependymal glia were detected in telencephalon.

Glial fibrillary acidic protein and vimentin immunohistochemistry in the developing and adult midbrain of the lizard Gallotia galloti.

The distribution of glial fibrillary acidic protein (GFAP)- and vimentin-containing cells was studied by immunohistochemistry in the midbrain of the lizard Gallotia galloti. At embryonic stage 32 (E32), vimentin immunoreactivity appeared first in cell bodies located in the ventricular walls, in radial fibers, and subpial end-feet and increased in these structures until E34/E35. Faint GFAP immunoreactivity gradually appeared in the same structures between E34 and E37, and this increased until adulthood, whereas vimentin immunoreactivity decreased after E35, becoming limited to a few end-feet and fibers in the adult, mainly in the tegmentum. Thus, in developing Gallotia midbrain a shift from vimentin-containing to GFAP-containing intermediate filaments begins around E36 or E37. At E40, in addition to the cell bodies in the ependymal area, dispersed GFAP-positive cells, possibly immature astrocytes appeared. These cells showed the same shift. In the adult lizard, GFAP-positive radial glia are still present and coexist with GFAP-positive astrocytes, which are prefentially located in the marginal optic tract and the oculomotor nuclei, but are absent in the fasciculus longitudinalis medialis. Optic tectum, pretectum, tegmentum, and isthmic nuclei are the areas richest in GFAP-positive radial fibers: these were much less abundant in the deep mesencephalic nuclei. Thus, in this lizard, GFAP-positive astrocytes display a clear cut regional distribution: they are present in mesencephalon, whereas they are absent in telencephalon.

Immunohistochemical localization of glutamine synthetase in mesencephalon and telencephalon of the lizard Gallotia galloti during ontogeny.

The immunohistochemical localization of glutamine synthetase, an astrocyte marker in mammals, was determined in the telencephalon and mesencephalon of the lizard Gallotia galloti during development by using an antiserum raised against chicken brain glutamine synthetase. Ependymal glial cells and their radial processes were glutamine synthetase immunoreactive, and they were present also in the adult. Immunoreactivity was also detected in two populations of scattered cell bodies, each preferentially localized in different zones: star-shaped cells morphologically similar to mammalian astrocytes, and ovoid or pear-shaped cell bodies, the processes of which were aligned with radial fibers and formed perivascular end-feet. Both populations displayed ultrastructural characteristics of astrocytes even though a comparison with our previous results (Monzon-Mayor et al., 1989; Yanes et al., 1989) indicated that many of these cells did not react with antibodies directed against the astrocyte-specific glial fibrillary acidic protein. During ontogeny, glutamine synthetase immunoreactivity appeared in radial glial processes and in ependymal glial cells of midbrain at embryonic stage 35 (E35) and of telencephalon at E37; in both regions, immunoreactivity in the radial glia increased until hatching and then decreased until adulthood, but it did not disappear. Labelled scattered cells became progressively more numerous and more immunoreactive. A comparative analysis of the distribution of these cells at different ages tends to suggest that some of the "ovoid" astrocytes originate in, and migrate out from, the proliferative zone of the different sulci, whereas the star-shaped cells appear directly in situ, probably because they begin to express glutamine synthetase after they have reached their final location.

A Nissl, Golgi-Kopsch and ultrastructural study of the Gallotia galloti red nucleus.

The object of this study was to understand the different types of morphological neurons of the Gallotia galloti red nucleus using quantitative and qualitative analysis, under the aspects of optical (Nissl and Golgi-Kopsch) and electronic microscopy. The results from this study and considering the size, the distribution of NISSL bodies and the dendritic arborization, we have identified three neuronal types: ellipsoidal or ovoid (15 microns), triangular (15-35 microns) and polygonal (20-50 microns). In general the polygonal neurons contain a cytoplasm with abundant organelles, well developed Nissl bodies, perinuclear Golgi complex, numerous mitochondria, an ovoid nucleus and multiple dendrites. The triangular neurons have a similar structure, although the dendritic model is less ramified than that of the polygonal and the ellipsoidal neurons contain a smaller cytoplasm with less dendritic ramification. The contact with spines is not very frequent but can be observed in somas and dendrites. The neuronal population is heterogeneous and therefore neither the magnocellular part nor the other parvicellular were observed separately, but a mixture of both.