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Developmental profiles of SMI-32 immunoreactivity in monkey striate cortex
Developmental Brain Research 119 Ž2000. 85–95 www.elsevier.comrlocaterbres
Research report
Developmental profiles of SMI-32 immunoreactivity in monkey striate cortex Cary S. Kogan ) , Shahin Zangenehpour, Avi Chaudhuri Department of Psychology, McGill UniÕersity, 1205 Dr. Penfield AÕenue, Montreal, QC, Canada H3A 1B1 Accepted 12 October 1999
Abstract A monoclonal antibody that recognizes a nonphosphorylated epitope on the medium and high molecular weight subunits of neurofilament ŽNF. proteins was used to investigate laminar and cell morphology changes in monkey striate cortex during post-natal development. Six cortices were obtained from monkeys of a variety of ages: five from developing animals with ages spanning the critical period and one adult. At post-natal day ŽPD. 0, immunohistochemistry with the SMI-32 antibody revealed immunoreactive ŽIR. cells in layer IVB and in infragranular layer VI. Early in the critical period ŽPD 7., these layers become more defined with an increase in the density of immunopositive cells. At the height of the critical period ŽPD 30 and 42., a drastic increase in the density of SMI-32 labelled pyramidal neurons in layers V and VI was observed. Similarly, layer IVC showed an abundance of dendritic fragments and dendrites that appeared to originate from the infragranular layers. At the end of the critical period ŽPD 103., a trend toward morphological maturation for individual neurons found within each layer was observed. During any developmental time point, neurons at first appearance tended to show an immature morphology with staining largely restricted to the cell bodies. As such, the characteristic arborizations common to mature pyramidal and multipolar cells was not evident. We propose that the staining pattern seen in this study is consistent with the idea that layers anatomically associated with the magnocellular ŽM. pathway develop earlier than their parvocellular ŽP. counterparts. q 2000 Elsevier Science B.V. All rights reserved. Keywords: SMI-32; Monkey striate cortex; Post-natal development; Neurofilament; Immunohistochemistry; Magnocellular vs. parvocellular
1. Introduction Neurofilaments ŽNFs. are strands of protein that belong to one of the three major cytoskeletal elements found in neurons. They are formed as a consequence of the polymerization of three different subunits — NFH Ž200 kDa., NFM Ž168 kDa., and NFL Ž68 kDa.. These subunits differ only in the length of their respective C-terminal domains. NFs are thought to serve an important structural role in neurons. Phosphorylated NFs regulate the conformational states of other cytoskeletal proteins, leading to changes in the dynamic interactions that exist between cytoskeletal and cytoplasmic organelles within neurons w12x. NFs are assembled in the cell body of neurons, transported down the length of the axon, and degraded in the nerve terminal. The dynamic nature of NFs implicates these proteins in the process of neuronal growth and development w16,17,19,22,23x. )
Corresponding author. Fax: q 1-514-398-4896; e-mail: [email protected]; URL: www.psych.mcgill.carlabsrcvl
There is evidence that the number and composition of NFs determine axon calibre w4x, which in turn is known to affect transmission speed. The NFH subunit appears to be especially important for the development of large calibre neurons w4x. The fact that it is NFH that appears to give additional stability and integrity to neurons w5x is crucial because it suggests that those cells expressing higher levels of this protein are likely representative of connections that have reached their developmental end-point w8x. It has been speculated that the quantity and distribution of NFs found in a particular neocortical region may correlate with a unique function for that area w6x. The monoclonal antibody SMI-32 recognizes an epitope present in NFs in the nonphosphorylated state. Previous immunostaining studies in monkey striate cortex Žarea V1. have shown that cell bodies and dendrites of neurons with pyramidal morphology are especially immunoreactive ŽIR., along with multipolar cells within layer IVB. It has also been shown that SMI-32 immunostaining can delineate cortical areas on the basis of differences in the pattern of staining. One idea is that NFs are expressed in greater
0165-3806r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 1 6 2 - 5
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abundance within the magnocellular ŽM. visual pathway w3,6,7x. Indeed, those cortical areas that have a functional association with the M pathway show immunostaining profiles that are distinguishable from those areas linked to the parvocellular ŽP. pathway. We now know that the neocortex undergoes an intense series of developmental changes during the critical period that involves cell growth, migration, synaptogenesis, and pruning. Given the dynamic properties of NFs and their intracellular function, SMI-32 immunostaining may provide a suitable means for tracking developing neurons in primary visual cortex. Furthermore, given that the two functional pathways diverge in early embryological development w11x, SMI-32 staining may be used to document the fates of neurons belonging to these two separate streams. And, finally, SMI-32 staining profiles that change in a correlated manner with known developmental milestones may be used to chart functional segregation within cortical compartments of the visual brain. A previous study of SMI-32 staining in human striate cortex showed clear changes in NF expression profiles during development w1x. In addition to histological differences across laminae, the pattern of staining at the cellular level became increasingly more complex with age. Initially, apical dendrites of SMI-32 IR neurons stained only near the cell soma. Coincident with maturation of the cortex, neurons showed progressive SMI-32 IR beginning at the soma and later radiating into the dendrites. The staining progression observed across developmental ages in the human was generally consistent with the overall developmental progression of the laminae in area V1. This pattern could also be attributed to a preference for the early development of neurons with large cell bodies, since morphological maturation may occur more rapidly in larger cells w9x. This was supported by the observation in human preparations that the largest cells of V1, Meynert cells found at the border of layers V and VI, are SMI-32 IR as early as at birth w1x. To date, there have been no studies that have examined the developmental sequence of NF expression in monkey visual cortex. One reason for undertaking such a study is that the anatomical correlates of M and P functionality have been well documented in monkey visual cortex. Furthermore, monkey striate cortex serves as an excellent model for charting the developmental sequence of neurochemical changes during the critical period. In this paper, we report the SMI-32 staining profiles that emerge at distinct time points during the critical period of development in the monkey.
2. Materials and methods A total of six vervet monkeys Ž Cercopithicus aethiops . were used in this study. One adult animal was used along with five developing monkeys from the following time
points: post-natal day ŽPD. 0, 7, 30, 42 and 103. All animals were initially anesthetized with ketamine hydrochloride Ž10 mgrkg, i.m.., removed from their cages, euthanized with an overdose of sodium pentobarbital Ž2 mgrkg, i.v.., and perfused transcardially with 0.1 M PBS until completely exsanguinated. The externalized brain was blocked along the midline followed by a coronal block along the lunate sulcus and flash frozen in a liquid nitrogenrisopentane bath. One block from each animal was fixed in 4% paraformaldehyde for 24 h followed by cryoprotection in graded Ž15% and 30%. sucrose. This was followed by paraffin embedding. Sections were cut from either frozen Žcryostat. or paraffin Žsliding microtome. blocks at a thickness of 20 mm. Paraffinized sections were air dried at room temperature, baked overnight on a slide warmer at 588C, de-paraffinized according to standard procedures, and pressure cooked for 1 min in antigen unmasking buffer according to the manufacturer’s protocol ŽVector Labs, Burlingame, CA.. Frozen sections were air dried and maintained at y808C until histological processing. The monoclonal antibody SMI-32 ŽSternberger Monoclonals, Baltimore, MD. known to recognize nonphosphorylated epitopes on the medium and high molecular weight subunits of the NF protein w20x served as the primary antibody. Sections were incubated initially for 30 min in 0.1 M PBSr5% normal horse serum followed by an overnight incubation with mild agitation at 48C in the primary antibody solution Ž1r5000 dilution of antibody in 0.1 M PBSr5% normal horse serum.. The sections were then washed three times in PBS, each wash lasting 10 min. This was followed by incubation for 2 h at room temperature in biotinylated anti-mouse secondary antibody raised in horse Ž1r500 dilution in 0.1 M PBS. or in Alexa-594 conjugated anti-mouse secondary antibody. After a further set of washes in PBS, sections processed with biotinylated secondary antibody were placed in a solution of avidin– biotin conjugated horseradish peroxidase complex ŽABC, Vector Labs. for 1 h at room temperature. After a further set of three washes in PBS, these sections were developed with a Vector w VIP peroxidase substrate ŽVector Labs. according to the manufacturer’s specifications. The reaction produced a purple stain exclusive to SMI-32 IR neurons. The sections processed with fluorophore-conjugated secondary antibody were immediately mounted in water-soluble anti-fade mounting medium for those sections intended for fluorescent visualization ŽMolecular Probes, Eugene, OR.. The laminar distribution patterns of SMI-32 IR were determined by examination of adjacent Nissl-stained sections, performed according to standard protocols. The delineation of laminar boundaries was followed after Lund w24x. Digital images were captured with a DAGE-MTI cooled color CCD camera and a Scion Series 7.0 three-chip frame grabber. Adobe Photoshope 5.0 for the Macintosh was used in image processing.
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3. Results The changing pattern of SMI-32 IR in area V1 of the developing vervet monkey was clearly distinguishable, especially upon comparison with adjacent sections stained for Nissl substance. SMI-32 staining was of high intensity within the cell body. The extent of staining within processes depended on the developmental age of the animal. In general, we observed that neuronal staining at early developmental time points was largely confined to the cell body with minimal dendritic staining. With increasing age, however, the immunopositive neurons began to display a large arborized pattern of staining. None of the sections inspected showed staining of cell nuclei nor of dendritic spines. We outlined the details of this observation within the context of the laminar staining profiles that were evident at each of the time points. In all cases, the laminar boundaries were established by comparison with adjacent Nissl-stained sections. SMI-32 immunostaining in area V1 at birth ŽPD 0. ŽFig. 1. showed scattered multipolar cells and infrequently, large pyramidal neurons in layer VI ŽFig. 1B.. These cells stood in stark contrast to the negligibly stained neuropil of these layers. Layer IVB contained lightly stained multipolar neurons with a weakly stained neuropil ŽFig. 1C.. Of those infragranular pyramidal cells inspected at high magnification, branching in the dendritic tree that is characteristic of mature neurons, was absent ŽFig. 1D.. Layer IVC was devoid of staining with the exception of sparsely stained
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dendritic fragments. Layers I, II, and III were devoid of staining. In the PD 7 animal, layers V and VI were seen to contain greater numbers of stained multipolar neurons than at PD 0 ŽFig. 2.. However, as before, many of these neurons had immature morphology, as evidenced by the confinement of SMI-32 immunostaining to the cell body ŽFig. 2F.. As such, they appeared more spherical than a typical multipolar cell with less complexity in their dendritic fields. Some small pyramidal cells were also stained in these layers and were apparent against a background of moderately stained neuropil. Layer IVB multipolar cells were clearly visible against a moderately dense background of tangentially arrayed neuropil ŽFig. 2E.. A few weakly stained cells were visible in the upper portion of layer IVCa ŽFig. 2D.. There were no stained cells in layer IVCb. With the exception of scattered dendritic fragments, the neuropil of layer IVC was not stained. This animal also showed very weak staining for pyramidal neurons in layer IIrIII ŽFig. 2C.. Those cells that were visible, however, were largely confined to the lower margin of layer III, near the border with layer IVCa. There were no stained cells visible in layer I nor was there any evidence of neuropil staining. The SMI-32 immunostaining pattern changed considerably as developing monkeys reached the height of the critical period. In the PD 30 animal, there was an abundance of IR pyramidal neurons in layers V and VI ŽFig. 3.. These cells projected strongly stained apical dendrites that
Fig. 1. Laminar expression of NF protein in area V1 at PD 0. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. SMI-32 IR identified pyramidal cells Žarrowhead. and scattered lightly stained multipolar cells in the infragranular layer VI Žopen arrowhead. and in layer IVB. The neuropil was negligibly stained throughout all layers of the cortex. Neurons visualized at high magnification ŽC, D. exhibited little dendritic arborization with immunostaining largely restricted to the cell body. Scale bars: 500 mm ŽA, B., 50 mm ŽC, D..
88 C.S. Kogan et al.r DeÕelopmental Brain Research 119 (2000) 85–95 Fig. 2. Laminar expression of NF protein in area V1 at PD 7. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. Lightly stained small pyramidal neurons appear in layer IIrIII ŽC.. A few weakly stained cells are visible in layer IVCa Žindicated by an arrow.. While layer IVC is largely devoid of cells, intensely stained dendritic fragments Žindicated by an open arrow. appear throughout ŽD.. Multipolar neurons of layer IVB are more intensely stained than previously and appear interspersed amongst small pyramidal neurons ŽE.. These cells appear more complex in their dendritic fields as compared to the PD 0 animal. SMI-32 IR large pyramidal neurons were found in layers V and VI as in the PD 0 animal, however, the apical and basal dendritic fields appear more developed ŽF.. Scale bars: 500 mm ŽA, B., 50 mm ŽC–F..
C.S. Kogan et al.r DeÕelopmental Brain Research 119 (2000) 85–95 Fig. 3. Laminar expression of NF protein in area V1 at PD 30. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. Layer IIrIII pyramidal neurons are visible. These cells have few arborizations in the basal fields ŽC.. Many intensely stained pyramidal neurons are visible at the lower margin of layer IIrIII where it borders layer IV ŽD.. Multipolar neurons of layer IVB are intensely stained and project numerous intralaminar dendrites ŽE.. These cells appear interspersed amongst small pyramidal neurons. Again, weakly stained cells are visible in layer IVCa. Layer IVC is largely devoid of cells, although many intensely stained dendritic fragments appear throughout ŽF.. The infragranular layers V and VI are densely packed with pyramidal neurons. Multipolar neurons are sparsely found throughout these layers and have complex dendritic fields Žindicated by an arrow head. ŽG.. Apical dendrites of the pyramidal neurons appear to be projecting toward the supragranular layers. Scale bars: 500 mm ŽA, B., 50 mm ŽC–G..
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90 C.S. Kogan et al.r DeÕelopmental Brain Research 119 (2000) 85–95 Fig. 4. Laminar expression of NF protein in area V1 at PD 42. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. For this time point, there is a marked increase in the number of cells stained in each layer and likewise an increase in the complexity of the dendritic fields for these neurons. Layer IIrIII neurons have intensely stained pyramidal neurons throughout. These neurons are mature with respect to the extent of their dendritic complexity ŽC.. Layer IVB contains a dense neuropil with numerous multipolar cells and pyramidal cells with dendrites that appear to project towards the pial surface ŽD.. Layer IVC shows an abundance of dendritic fragments and dendrites that appear to originate from the infragranular layers. The infragranular in turn, are very densely packed with pyramidal neurons whose apical dendrites project towards the supragranular layers ŽE.. Scale bars: 500 mm ŽA, B., 50 mm ŽC, D..
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were observed to course through layer IVC ŽFig. 3F.. We also observed numerous multipolar cells that were interspersed among the pyramidal neurons and located mainly in the upper segment of layer V ŽFig. 3G.. A few Meynert cells with tangential and diagonal intralaminar projections could also be observed. A significant new feature of this time point was the abundance of dendritic fragments that were found in layer IVC ŽFig. 3B.. Otherwise, as at earlier time points, this layer was largely devoid of cell bodies with the exception of some weakly stained cells in the upper margin of IVCa. Layer IVB was observed to contain many multipolar neurons and scattered pyramidal neurons, whose apical dendrites in some cases projected as far as layer I ŽFig. 3E.. As before, there was intense neuropil staining which was generally organized in a tangential manner. The staining pattern in layer IIrIII offers another defining feature of this time point because of the presence of moderate numbers of pyramidal neurons with immature morphology ŽFig. 3D, upper portion.. SMI-32 IR in these cells was confined to the cell body. Layer I did not contain any SMI-32 IR neurons. The PD 42 animal showed a very high density of stained pyramidal neurons in layers V and VI ŽFig. 4.. These cells extended large numbers of intensely stained apical dendrites towards the pial surface and complex basal fields that projected tangentially and diagonally within the infragranular layers ŽFig. 4E.. Large multipolar neurons in the infragranular layers were found mainly in the upper part of layer V. These cells possessed tangential and diagonal intralaminar projections. Meynert cells were also present at the border of layers V and VI. The intensity of staining and extent of arborization suggested that neurons in these layers have become morphologically mature by this stage. In layer IVC, an abundance of dendritic fragments and dendrites that appear to originate from the infragranular layers were visible. Otherwise, this layer is again largely devoid of cell bodies with the exception of some weakly stained cells in the upper margin of layer
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IVCa. Layer IVCb is devoid of cell staining. Layer IVB contains numerous multipolar neurons and pyramidal cells with intralaminar projections towards the pial surface ŽFig. 4D.. The neuropil in this layer was strongly stained. Layer IIrIII showed numerous pyramidal cells with strongly stained apical dendrites ŽFig. 4C.. Layer I was devoid of cellular staining but for this animal began to show weakly stained neuropil. The major feature in the PD 103 animals was a trend toward morphological maturation for individual neurons found within each layer ŽFig. 5.. Otherwise, the staining pattern was unremarkable. However, an especially prominent feature at this time point was the further maturation of the large pyramidal neurons in layers V and VI. This was evident in a high magnification capture of immunofluorescent staining ŽFig. 5C.. The dendritic branches of these neurons were greater in diameter and appeared more heavily stained. Multipolar neurons of layer IVB extended many reciprocal branches that appeared at high magnification to contact neighboring cells within this layer ŽFig. 5B.. In the adult animal, most of the neurons possessed fully developed apical and basal dendrites throughout all layers of area V1 ŽFig. 6.. The neurons were highly developed and exhibited prominent dendritic projections. The basal dendritic field of these cells was broad and extensive while the apical dendrite was far reaching and often extended well into the supragranular layers. The density of pyramidal neurons in layers V and VI was reduced in comparison to PD 42 ŽFig. 6H.. Another remarkable feature of this layer was the greatly reduced number of stained dendrites projecting out of the infragranular layers in comparison to the animals at the height of the critical period. Layer IVC contained sparsely stained cells that were largely confined to IVCa but at this time point, there are also a few stained cells in layer IVCb ŽFig. 6F and G.. Layer IVB contained intensely stained multipolar neurons that had many complex branches ŽFig. 6E.. Finally, layer IIrIII shows an
Fig. 5. Expression of NF protein in area V1 at PD 103. Layer IIrIII neurons continue to mature in complexity at this time point ŽA. while the multipolar cells of layer IVB appear to extend many reciprocal branches to neighboring cells ŽB.. Further maturation of SMI-32 IR large pyramidal neurons in layers V and VI is visible in this high magnification capture of an immunofluorescent stained neuron ŽC.. Scale bar: 50 mm ŽA, B..
92 C.S. Kogan et al.r DeÕelopmental Brain Research 119 (2000) 85–95 Fig. 6. Laminar expression of NF protein in area V1 in the adult. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. Layer IIrIII neurons have intensely stained pyramidal neurons throughout with highly arborized dendritic fields ŽC, D.. Layer IVB contains a dense neuropil with numerous multipolar cells and pyramidal cells with dendrites that appear to project towards the pial surface ŽE.. Some IR cells were visible in the geniculorecipient layers IVCa ŽF. and IVCb ŽG.. However, more immunopositive cells appear in the former, a layer known to receive input from the M layers of the lateral geniculate nucleus. Relatively fewer pyramidal cells as compared with the PD 30, 42, and 103 animals are visible in the infragranular layers V and VI although those cells present are more complex in their dendritic fields ŽH.. The apical dendrites of these neurons are observed to project to the supragranular layers. Pyramidal neurons in this preparation appear more complex in their dendritic fields. Stained neurons are overall more numerous throughout all layers. Scale bars: 500 mm ŽA, B., 50 mm ŽC–H..
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abundance of SMI-32 IR pyramidal neurons with apical dendrites that terminated within this layer and in layer I ŽFig. 6C and D.. Mature multipolar neurons also appeared sparsely within layer IIrIII.
4. Discussion The results from this study show the general developmental progression of SMI-32 staining in area V1 during the critical period of development. The time points were selected to reflect important milestones in development. This includes the following — one shortly after birth in which the animal had very little visual exposure ŽPD 0., one animal at an early part of the critical period ŽPD 7., two at the approximate height of the critical period ŽPD 30 and 42., and one at the end of the critical period ŽPD 103.. 4.1. Laminar staining profile We have found that the overall pattern of SMI-32 IR in area V1 shows considerable change over the first 15 weeks of post-natal life. SMI-32 IR is confined at birth to the large pyramidal and multipolar neurons in the infragranular layers. During development, there is a progressive increase in the number of pyramidal and multipolar neurons in these layers. Accompanying this is the remarkable intensification in dendritic projection out of the infraganular layers by the height of the critical period. This is followed by a reduction in the number of stained neurons so that by the end of the critical period, only a moderate number of stained cells and dendritic projections are visible. It is possible that the SMI-32 staining under-represents the actual developmental changes in dendritic arborization. We assume however, that there is a correlation between SMI-32 expression and actual dendritic changes. Concurrent with the overall laminar changes in SMI-32 IR during the post-natal developmental period, maturational advances of the dendritic arbors become evident. More dendrites appear SMI-32 IR and the overall complexity of these dendrites increases with age. This process of maturation appears to follow a gradient with those cells in the infragranular layers maturing before those cells in the supragranular layers. This finding is supported by Golgi studies that indicate that neurons in the deep layers develop more substantial networks of dendritic arbors before those in more superficial layers w21x. By virtue of the progression of neuronal development described in these Golgi studies, it is safe to assume that the staining seen in the present study is accurately reflecting the actual changes that occur in the developing dendritic arbor and the neuropil in general. Layer IVC remains poorly stained throughout most of development, except for some weakly stained cells in IVCa that appear in early development and persist to adulthood. Layer IVCb remains unstained except for a few
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weakly stained cells that are visible in the adult animal. Layer IVB is distinguishable at birth by its greater staining relative to neighboring layers. This includes scattered multipolar cells that appear on a background of moderately stained neuropil. A few pyramidal neurons are seen by PD 7. In general, the developmental sequence for this layer is unremarkable except for an increase in cell number and the complexity of dendritic fields by the height of the critical period followed by a discernible reduction in cell number by adulthood. Layer IVB neurons are known to project to area MT and as such, are considered part of the M pathway. Layer IIrIII shows almost no cellular staining at birth and remains poorly stained during the early part of the critical period ŽPD 7. and confined largely to the lower margin of layer III. In contrast to layers IVB and VrVI, layer IIrIII stained neurons only appear in significant numbers at the height of the critical period. It is interesting to note that neurons in layer IIrIII are known to extend projections to the association cortices such as areas V2 and V3. We also observed a progressive laminar appearance so that the first stained cells were observed along the lower margins of layer III followed by appearance at higher levels within layer II. By adulthood, many pyramidal and, for the first time, multipolar cells are visible throughout these two layers. 4.2. Morphological maturity of stained neurons We observed a striking difference in the staining pattern at the individual cellular level throughout development. At early time point, and especially at birth, staining in SMI-32 immunopositive neurons was largely confined to the cell body with only marginal extension into the dendritic processes. The microscopic appearance of these stained cells showed them to be neurons with immature morphology. As Lund et al. w9x noted, post-natal maturity in area V1 pyramidal neurons shows progressive changes in dendritic arborization. The confined staining of NF protein that we observed in area V1 at birth may reflect the immature morphological state of neurons in general, and pyramidal neurons in particular. The changes may be also be accounted for by a lower level of NF expression in these neurons. At greater developmental time points, the SMI-32 staining revealed neurons with more extensive and far-reaching dendritic processes. The progression of cellular staining from confined to extensive patterns with developmental time was a common feature of all layers. 4.3. Comparison to human neocortical deÕelopment An earlier study by Ang et al. w1x explored the significant events in the SMI-32 developmental sequence in human visual cortex. The results of their study showed the same general pattern of staining across developmental time points. That is, Ang et al. w1x found that SMI-32 im-
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munopositive neurons were present at birth in the infraganular layers though there is no evidence of such staining in the striated layer ŽIVB.. With increasing developmental time, the human cortex shows a generalized increase in SMI-32 staining that encompasses greater numbers of neurons in both supragranular and infragranular layers. There appear to be many more stained neurons in the supragranular layers of adult humans in comparison to our adult monkey. Campbell and Morrison w2x also found a relative reduction in SMI-32 stained neurons in adult monkey supragranular layers in comparison to human. Despite this difference, the same layers and more specifically, the same type of neurons are SMI-32 immunopositive in area V1 of humans and monkeys. The early maturation of SMI-32 immunostaining in the infragranular layers may be linked to the normal sequence of cortical development. It is known that deeper layer cells mature initially followed by a developmental progression from infragranular to supragranular layers w13x. Yet, another explanation might be that the pattern of staining is attributed to a preference for the early development of large neurons rather than the generation time, arrival at a final laminar position, or cell type. It has been shown, for example, that morphological maturation occurs more rapidly in larger cells w9x. This idea is supported by the results presented here and the observation in human preparations that the Meynert cells, which are found at the border of layers V and VI, are the most prominent SMI-32 IR cells at birth w1x.
develops earlier than its P counterpart. Further support for this notion comes from work by Mates and Lund w10x that suggests a developmental lag in the initial accumulation of type 2 dendritic spines for the P pathway geniculorecipient layer IVCa. Although SMI-32 IR does not permit visualization of dendritic spines, it is interesting to note that dendritic spine formation, an indicator of synaptic formation or loss, exhibits differences for the M and P pathways, respectively. Cells of layer IVCb Ža P recipient layer. show a slower initial accumulation of type 2 contacts compared to neurons of layer IVCa Ža M recipient layer., or to pyramidal cells of layer VI. Furthermore, type 2 synapses were more susceptible to reduction in layer IVCa and layer VI than in layer IVCb after an 8-week visual deprivation period w10x. Other evidence of M and P pathway developmental differences is supported by tracing studies in the monkey LGN w14,15x. The results indicate that the LGN M layers develop earlier than their P counterparts. Although this may not extend to the cortical areas as has been previously suggested by Lund w24x, it is not unreasonable to assume that input from lower levels of the visual system shape higher-order processes. Finally, it has been observed that retinal ganglion cells diverge into their respective M or P specialized functions soon after their last mitotic division, suggesting that the developmental fates of these groups of cells are regulated separately and therefore may develop at different rates w11x.
4.4. Relationship to deÕelopment of parallel Õisual pathways
Acknowledgements
Prior studies have shown the utility of SMI-32 IR in preferentially staining the M visual pathway w3,6,7x. Chaudhuri et al. w3x, for instance, describe the pattern of SMI-32 staining at the level of the lateral geniculate nucleus ŽLGN., where M and P layers are clearly distinguishable. They show that the two M layers of the LGN are heavily stained in comparison to the four P layers. Furthermore, SMI-32 immunostaining has been previously shown to be intense in layers IVB and VI of area V1, areas known to be anatomically related to the M pathway w6x. It has also been suggested that the ratio of the number of cells stained by SMI-32 in infragranular layers compared to supragranular layers can define different hierarchical levels of cortical processing w6x. Hence, areas belonging to the M pathway have a different distribution of SMI-32 immunoreactivity. It is tempting to speculate that the developmental results presented here represent early maturation of the M pathway in area V1. We observed that layers IVB and VI show stained cells earlier than other layers. Together with evidence implicating NFs in determining the end-point of connectional maturation for neurons w1,2x it appears as if the M pathway, presumed to be a phylogenically older pathway w18x,
This work was supported by grants from the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council of Canada to Avi Chaudhuri. The authors thank Fariborz Rahbar-Dehghan for technical assistance.
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patterns of cortical organization in the macaque monkey visual system: a quantitative immunohistochemical analysis, J. Comp. Neurol. 352 Ž1995. 161–186. P.R. Hof, L.G. Ungerleider, M.J. Webster, R. Gattass, M.M. Adams, C.A. Sailstad, J.H. Morrison, Neurofilament protein is differentially distributed in subpopulations of corticocortical projection neurons in the macaque monkey visual pathways, J. Comp. Neurol. 376 Ž1996. 112–127. Y. Liu, R. Dyck, M. Cynader, The correlation between cortical neuron maturation and neurofilament phosphorylation: a developmental study of phosphorylated 200 kDa neurofilament protein in cat visual cortex, Brain Res. Dev. Brain Res. 81 Ž1994. 151–161. J.S. Lund, R.G. Boothe, R.D. Lund, Development of neurons in the visual cortex Žarea 17. of the monkey Ž Macaca nemestrina.: a Golgi study from fetal day 127 to post-natal maturity, J. Comp. Neurol. 176 Ž1977. 149–188. S.L. Mates, J.S. Lund, Developmental changes in the relationship between type 2 synapses and spiny neurons in the monkey visual cortex, J. Comp. Neurol. 221 Ž1983. 98–105. C. Meissirel, K.C. Wikler, L.M. Chalupa, P. Rakic, Early divergence of magnocellular and parvocellular functional subsystems in the embryonic primate visual system, Proc. Natl. Acad. Sci. U.S.A. 94 Ž1997. 5900–5905. R.A. Nixon, R.K. Sihag, Neurofilament phosphorylation: a new look at regulation and function, Trends Neurosci. 14 Ž1991. 501–506. P. Rakic, Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition, Science 183 Ž1974. 425–427. P. Rakic, Timing of major ontogenetic events in the visual cortex of the rhesus monkey, UCLA Forum Med. Sci. Ž1975. 3–40. P. Rakic, Prenatal development of the visual system in rhesus monkey, Philos. Trans. R. Soc. London, Ser. B 278 Ž1977. 245–260.
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w16x B.M. Riederer, R. Porchet, R.A. Marugg, Differential expression and modification of neurofilament triplet proteins during cat cerebellar development, J. Comp. Neurol. 364 Ž1996. 704–717. w17x L.A. Sawant, N.N. Hasgekar, L.S. Vyasarayani, Developmental expression of neurofilament and glial filament proteins in rat cerebellum, Int. J. Dev. Biol. 38 Ž1994. 429–437. w18x P.H. Schiller, N.K. Logothetis, E.R. Charles, Functions of the colour-opponent and broad-band channels of the visual system wsee commentsx, Nature 343 Ž1990. 68–70. w19x R. Steinschneider, P. Delmas, J. Nedelec, M. Gola, D. Bernard, J. Boucraut, Appearance of neurofilament subunit epitopes correlates with electrophysiological maturation in cortical embryonic neurons cocultured with mature astrocytes, Brain Res. Dev. Brain Res. 95 Ž1996. 15–27. w20x L.A. Sternberger, N.H. Sternberger, Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ, Proc. Natl. Acad. Sci. U.S.A. 80 Ž1983. 6126–6130. w21x J. Tigges, M. Tigges, S. Anschel, N.A. Cross, W.D. Letbetter, R.L. McBride, Areal and laminar distribution of neurons interconnecting the central visual cortical areas 17, 18, 19, and MT in squirrel monkey Ž Saimiri ., J. Comp. Neurol. 202 Ž1981. 539–560. w22x N. Ulfig, J. Nickel, J. Bohl, Monoclonal antibodies SMI-311 and SMI-312 as tools to investigate the maturation of nerve cells and axonal patterns in human fetal brain, Cell Tissue Res. 291 Ž1998. 433–443. w23x Z. Xu, J.R. Marszalek, M.K. Lee, P.C. Wong, J. Folmer, T.O. Crawford, S.T. Hsieh, J.W. Griffin, D.W. Cleveland, Subunit composition of neurofilaments specifies axonal diameter, J. Cell Biol. 133 Ž1996. 1061–1069. w24x J.S. Lund, Organization of neurons in the visual cortex, area 17, of the monkey Ž Macaca mulatta., J. Comp. Neurol. 147 Ž1973. 455– 496.
Research report
Developmental profiles of SMI-32 immunoreactivity in monkey striate cortex Cary S. Kogan ) , Shahin Zangenehpour, Avi Chaudhuri Department of Psychology, McGill UniÕersity, 1205 Dr. Penfield AÕenue, Montreal, QC, Canada H3A 1B1 Accepted 12 October 1999
Abstract A monoclonal antibody that recognizes a nonphosphorylated epitope on the medium and high molecular weight subunits of neurofilament ŽNF. proteins was used to investigate laminar and cell morphology changes in monkey striate cortex during post-natal development. Six cortices were obtained from monkeys of a variety of ages: five from developing animals with ages spanning the critical period and one adult. At post-natal day ŽPD. 0, immunohistochemistry with the SMI-32 antibody revealed immunoreactive ŽIR. cells in layer IVB and in infragranular layer VI. Early in the critical period ŽPD 7., these layers become more defined with an increase in the density of immunopositive cells. At the height of the critical period ŽPD 30 and 42., a drastic increase in the density of SMI-32 labelled pyramidal neurons in layers V and VI was observed. Similarly, layer IVC showed an abundance of dendritic fragments and dendrites that appeared to originate from the infragranular layers. At the end of the critical period ŽPD 103., a trend toward morphological maturation for individual neurons found within each layer was observed. During any developmental time point, neurons at first appearance tended to show an immature morphology with staining largely restricted to the cell bodies. As such, the characteristic arborizations common to mature pyramidal and multipolar cells was not evident. We propose that the staining pattern seen in this study is consistent with the idea that layers anatomically associated with the magnocellular ŽM. pathway develop earlier than their parvocellular ŽP. counterparts. q 2000 Elsevier Science B.V. All rights reserved. Keywords: SMI-32; Monkey striate cortex; Post-natal development; Neurofilament; Immunohistochemistry; Magnocellular vs. parvocellular
1. Introduction Neurofilaments ŽNFs. are strands of protein that belong to one of the three major cytoskeletal elements found in neurons. They are formed as a consequence of the polymerization of three different subunits — NFH Ž200 kDa., NFM Ž168 kDa., and NFL Ž68 kDa.. These subunits differ only in the length of their respective C-terminal domains. NFs are thought to serve an important structural role in neurons. Phosphorylated NFs regulate the conformational states of other cytoskeletal proteins, leading to changes in the dynamic interactions that exist between cytoskeletal and cytoplasmic organelles within neurons w12x. NFs are assembled in the cell body of neurons, transported down the length of the axon, and degraded in the nerve terminal. The dynamic nature of NFs implicates these proteins in the process of neuronal growth and development w16,17,19,22,23x. )
Corresponding author. Fax: q 1-514-398-4896; e-mail: [email protected]; URL: www.psych.mcgill.carlabsrcvl
There is evidence that the number and composition of NFs determine axon calibre w4x, which in turn is known to affect transmission speed. The NFH subunit appears to be especially important for the development of large calibre neurons w4x. The fact that it is NFH that appears to give additional stability and integrity to neurons w5x is crucial because it suggests that those cells expressing higher levels of this protein are likely representative of connections that have reached their developmental end-point w8x. It has been speculated that the quantity and distribution of NFs found in a particular neocortical region may correlate with a unique function for that area w6x. The monoclonal antibody SMI-32 recognizes an epitope present in NFs in the nonphosphorylated state. Previous immunostaining studies in monkey striate cortex Žarea V1. have shown that cell bodies and dendrites of neurons with pyramidal morphology are especially immunoreactive ŽIR., along with multipolar cells within layer IVB. It has also been shown that SMI-32 immunostaining can delineate cortical areas on the basis of differences in the pattern of staining. One idea is that NFs are expressed in greater
0165-3806r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 9 . 0 0 1 6 2 - 5
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abundance within the magnocellular ŽM. visual pathway w3,6,7x. Indeed, those cortical areas that have a functional association with the M pathway show immunostaining profiles that are distinguishable from those areas linked to the parvocellular ŽP. pathway. We now know that the neocortex undergoes an intense series of developmental changes during the critical period that involves cell growth, migration, synaptogenesis, and pruning. Given the dynamic properties of NFs and their intracellular function, SMI-32 immunostaining may provide a suitable means for tracking developing neurons in primary visual cortex. Furthermore, given that the two functional pathways diverge in early embryological development w11x, SMI-32 staining may be used to document the fates of neurons belonging to these two separate streams. And, finally, SMI-32 staining profiles that change in a correlated manner with known developmental milestones may be used to chart functional segregation within cortical compartments of the visual brain. A previous study of SMI-32 staining in human striate cortex showed clear changes in NF expression profiles during development w1x. In addition to histological differences across laminae, the pattern of staining at the cellular level became increasingly more complex with age. Initially, apical dendrites of SMI-32 IR neurons stained only near the cell soma. Coincident with maturation of the cortex, neurons showed progressive SMI-32 IR beginning at the soma and later radiating into the dendrites. The staining progression observed across developmental ages in the human was generally consistent with the overall developmental progression of the laminae in area V1. This pattern could also be attributed to a preference for the early development of neurons with large cell bodies, since morphological maturation may occur more rapidly in larger cells w9x. This was supported by the observation in human preparations that the largest cells of V1, Meynert cells found at the border of layers V and VI, are SMI-32 IR as early as at birth w1x. To date, there have been no studies that have examined the developmental sequence of NF expression in monkey visual cortex. One reason for undertaking such a study is that the anatomical correlates of M and P functionality have been well documented in monkey visual cortex. Furthermore, monkey striate cortex serves as an excellent model for charting the developmental sequence of neurochemical changes during the critical period. In this paper, we report the SMI-32 staining profiles that emerge at distinct time points during the critical period of development in the monkey.
2. Materials and methods A total of six vervet monkeys Ž Cercopithicus aethiops . were used in this study. One adult animal was used along with five developing monkeys from the following time
points: post-natal day ŽPD. 0, 7, 30, 42 and 103. All animals were initially anesthetized with ketamine hydrochloride Ž10 mgrkg, i.m.., removed from their cages, euthanized with an overdose of sodium pentobarbital Ž2 mgrkg, i.v.., and perfused transcardially with 0.1 M PBS until completely exsanguinated. The externalized brain was blocked along the midline followed by a coronal block along the lunate sulcus and flash frozen in a liquid nitrogenrisopentane bath. One block from each animal was fixed in 4% paraformaldehyde for 24 h followed by cryoprotection in graded Ž15% and 30%. sucrose. This was followed by paraffin embedding. Sections were cut from either frozen Žcryostat. or paraffin Žsliding microtome. blocks at a thickness of 20 mm. Paraffinized sections were air dried at room temperature, baked overnight on a slide warmer at 588C, de-paraffinized according to standard procedures, and pressure cooked for 1 min in antigen unmasking buffer according to the manufacturer’s protocol ŽVector Labs, Burlingame, CA.. Frozen sections were air dried and maintained at y808C until histological processing. The monoclonal antibody SMI-32 ŽSternberger Monoclonals, Baltimore, MD. known to recognize nonphosphorylated epitopes on the medium and high molecular weight subunits of the NF protein w20x served as the primary antibody. Sections were incubated initially for 30 min in 0.1 M PBSr5% normal horse serum followed by an overnight incubation with mild agitation at 48C in the primary antibody solution Ž1r5000 dilution of antibody in 0.1 M PBSr5% normal horse serum.. The sections were then washed three times in PBS, each wash lasting 10 min. This was followed by incubation for 2 h at room temperature in biotinylated anti-mouse secondary antibody raised in horse Ž1r500 dilution in 0.1 M PBS. or in Alexa-594 conjugated anti-mouse secondary antibody. After a further set of washes in PBS, sections processed with biotinylated secondary antibody were placed in a solution of avidin– biotin conjugated horseradish peroxidase complex ŽABC, Vector Labs. for 1 h at room temperature. After a further set of three washes in PBS, these sections were developed with a Vector w VIP peroxidase substrate ŽVector Labs. according to the manufacturer’s specifications. The reaction produced a purple stain exclusive to SMI-32 IR neurons. The sections processed with fluorophore-conjugated secondary antibody were immediately mounted in water-soluble anti-fade mounting medium for those sections intended for fluorescent visualization ŽMolecular Probes, Eugene, OR.. The laminar distribution patterns of SMI-32 IR were determined by examination of adjacent Nissl-stained sections, performed according to standard protocols. The delineation of laminar boundaries was followed after Lund w24x. Digital images were captured with a DAGE-MTI cooled color CCD camera and a Scion Series 7.0 three-chip frame grabber. Adobe Photoshope 5.0 for the Macintosh was used in image processing.
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3. Results The changing pattern of SMI-32 IR in area V1 of the developing vervet monkey was clearly distinguishable, especially upon comparison with adjacent sections stained for Nissl substance. SMI-32 staining was of high intensity within the cell body. The extent of staining within processes depended on the developmental age of the animal. In general, we observed that neuronal staining at early developmental time points was largely confined to the cell body with minimal dendritic staining. With increasing age, however, the immunopositive neurons began to display a large arborized pattern of staining. None of the sections inspected showed staining of cell nuclei nor of dendritic spines. We outlined the details of this observation within the context of the laminar staining profiles that were evident at each of the time points. In all cases, the laminar boundaries were established by comparison with adjacent Nissl-stained sections. SMI-32 immunostaining in area V1 at birth ŽPD 0. ŽFig. 1. showed scattered multipolar cells and infrequently, large pyramidal neurons in layer VI ŽFig. 1B.. These cells stood in stark contrast to the negligibly stained neuropil of these layers. Layer IVB contained lightly stained multipolar neurons with a weakly stained neuropil ŽFig. 1C.. Of those infragranular pyramidal cells inspected at high magnification, branching in the dendritic tree that is characteristic of mature neurons, was absent ŽFig. 1D.. Layer IVC was devoid of staining with the exception of sparsely stained
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dendritic fragments. Layers I, II, and III were devoid of staining. In the PD 7 animal, layers V and VI were seen to contain greater numbers of stained multipolar neurons than at PD 0 ŽFig. 2.. However, as before, many of these neurons had immature morphology, as evidenced by the confinement of SMI-32 immunostaining to the cell body ŽFig. 2F.. As such, they appeared more spherical than a typical multipolar cell with less complexity in their dendritic fields. Some small pyramidal cells were also stained in these layers and were apparent against a background of moderately stained neuropil. Layer IVB multipolar cells were clearly visible against a moderately dense background of tangentially arrayed neuropil ŽFig. 2E.. A few weakly stained cells were visible in the upper portion of layer IVCa ŽFig. 2D.. There were no stained cells in layer IVCb. With the exception of scattered dendritic fragments, the neuropil of layer IVC was not stained. This animal also showed very weak staining for pyramidal neurons in layer IIrIII ŽFig. 2C.. Those cells that were visible, however, were largely confined to the lower margin of layer III, near the border with layer IVCa. There were no stained cells visible in layer I nor was there any evidence of neuropil staining. The SMI-32 immunostaining pattern changed considerably as developing monkeys reached the height of the critical period. In the PD 30 animal, there was an abundance of IR pyramidal neurons in layers V and VI ŽFig. 3.. These cells projected strongly stained apical dendrites that
Fig. 1. Laminar expression of NF protein in area V1 at PD 0. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. SMI-32 IR identified pyramidal cells Žarrowhead. and scattered lightly stained multipolar cells in the infragranular layer VI Žopen arrowhead. and in layer IVB. The neuropil was negligibly stained throughout all layers of the cortex. Neurons visualized at high magnification ŽC, D. exhibited little dendritic arborization with immunostaining largely restricted to the cell body. Scale bars: 500 mm ŽA, B., 50 mm ŽC, D..
88 C.S. Kogan et al.r DeÕelopmental Brain Research 119 (2000) 85–95 Fig. 2. Laminar expression of NF protein in area V1 at PD 7. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. Lightly stained small pyramidal neurons appear in layer IIrIII ŽC.. A few weakly stained cells are visible in layer IVCa Žindicated by an arrow.. While layer IVC is largely devoid of cells, intensely stained dendritic fragments Žindicated by an open arrow. appear throughout ŽD.. Multipolar neurons of layer IVB are more intensely stained than previously and appear interspersed amongst small pyramidal neurons ŽE.. These cells appear more complex in their dendritic fields as compared to the PD 0 animal. SMI-32 IR large pyramidal neurons were found in layers V and VI as in the PD 0 animal, however, the apical and basal dendritic fields appear more developed ŽF.. Scale bars: 500 mm ŽA, B., 50 mm ŽC–F..
C.S. Kogan et al.r DeÕelopmental Brain Research 119 (2000) 85–95 Fig. 3. Laminar expression of NF protein in area V1 at PD 30. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. Layer IIrIII pyramidal neurons are visible. These cells have few arborizations in the basal fields ŽC.. Many intensely stained pyramidal neurons are visible at the lower margin of layer IIrIII where it borders layer IV ŽD.. Multipolar neurons of layer IVB are intensely stained and project numerous intralaminar dendrites ŽE.. These cells appear interspersed amongst small pyramidal neurons. Again, weakly stained cells are visible in layer IVCa. Layer IVC is largely devoid of cells, although many intensely stained dendritic fragments appear throughout ŽF.. The infragranular layers V and VI are densely packed with pyramidal neurons. Multipolar neurons are sparsely found throughout these layers and have complex dendritic fields Žindicated by an arrow head. ŽG.. Apical dendrites of the pyramidal neurons appear to be projecting toward the supragranular layers. Scale bars: 500 mm ŽA, B., 50 mm ŽC–G..
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90 C.S. Kogan et al.r DeÕelopmental Brain Research 119 (2000) 85–95 Fig. 4. Laminar expression of NF protein in area V1 at PD 42. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. For this time point, there is a marked increase in the number of cells stained in each layer and likewise an increase in the complexity of the dendritic fields for these neurons. Layer IIrIII neurons have intensely stained pyramidal neurons throughout. These neurons are mature with respect to the extent of their dendritic complexity ŽC.. Layer IVB contains a dense neuropil with numerous multipolar cells and pyramidal cells with dendrites that appear to project towards the pial surface ŽD.. Layer IVC shows an abundance of dendritic fragments and dendrites that appear to originate from the infragranular layers. The infragranular in turn, are very densely packed with pyramidal neurons whose apical dendrites project towards the supragranular layers ŽE.. Scale bars: 500 mm ŽA, B., 50 mm ŽC, D..
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were observed to course through layer IVC ŽFig. 3F.. We also observed numerous multipolar cells that were interspersed among the pyramidal neurons and located mainly in the upper segment of layer V ŽFig. 3G.. A few Meynert cells with tangential and diagonal intralaminar projections could also be observed. A significant new feature of this time point was the abundance of dendritic fragments that were found in layer IVC ŽFig. 3B.. Otherwise, as at earlier time points, this layer was largely devoid of cell bodies with the exception of some weakly stained cells in the upper margin of IVCa. Layer IVB was observed to contain many multipolar neurons and scattered pyramidal neurons, whose apical dendrites in some cases projected as far as layer I ŽFig. 3E.. As before, there was intense neuropil staining which was generally organized in a tangential manner. The staining pattern in layer IIrIII offers another defining feature of this time point because of the presence of moderate numbers of pyramidal neurons with immature morphology ŽFig. 3D, upper portion.. SMI-32 IR in these cells was confined to the cell body. Layer I did not contain any SMI-32 IR neurons. The PD 42 animal showed a very high density of stained pyramidal neurons in layers V and VI ŽFig. 4.. These cells extended large numbers of intensely stained apical dendrites towards the pial surface and complex basal fields that projected tangentially and diagonally within the infragranular layers ŽFig. 4E.. Large multipolar neurons in the infragranular layers were found mainly in the upper part of layer V. These cells possessed tangential and diagonal intralaminar projections. Meynert cells were also present at the border of layers V and VI. The intensity of staining and extent of arborization suggested that neurons in these layers have become morphologically mature by this stage. In layer IVC, an abundance of dendritic fragments and dendrites that appear to originate from the infragranular layers were visible. Otherwise, this layer is again largely devoid of cell bodies with the exception of some weakly stained cells in the upper margin of layer
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IVCa. Layer IVCb is devoid of cell staining. Layer IVB contains numerous multipolar neurons and pyramidal cells with intralaminar projections towards the pial surface ŽFig. 4D.. The neuropil in this layer was strongly stained. Layer IIrIII showed numerous pyramidal cells with strongly stained apical dendrites ŽFig. 4C.. Layer I was devoid of cellular staining but for this animal began to show weakly stained neuropil. The major feature in the PD 103 animals was a trend toward morphological maturation for individual neurons found within each layer ŽFig. 5.. Otherwise, the staining pattern was unremarkable. However, an especially prominent feature at this time point was the further maturation of the large pyramidal neurons in layers V and VI. This was evident in a high magnification capture of immunofluorescent staining ŽFig. 5C.. The dendritic branches of these neurons were greater in diameter and appeared more heavily stained. Multipolar neurons of layer IVB extended many reciprocal branches that appeared at high magnification to contact neighboring cells within this layer ŽFig. 5B.. In the adult animal, most of the neurons possessed fully developed apical and basal dendrites throughout all layers of area V1 ŽFig. 6.. The neurons were highly developed and exhibited prominent dendritic projections. The basal dendritic field of these cells was broad and extensive while the apical dendrite was far reaching and often extended well into the supragranular layers. The density of pyramidal neurons in layers V and VI was reduced in comparison to PD 42 ŽFig. 6H.. Another remarkable feature of this layer was the greatly reduced number of stained dendrites projecting out of the infragranular layers in comparison to the animals at the height of the critical period. Layer IVC contained sparsely stained cells that were largely confined to IVCa but at this time point, there are also a few stained cells in layer IVCb ŽFig. 6F and G.. Layer IVB contained intensely stained multipolar neurons that had many complex branches ŽFig. 6E.. Finally, layer IIrIII shows an
Fig. 5. Expression of NF protein in area V1 at PD 103. Layer IIrIII neurons continue to mature in complexity at this time point ŽA. while the multipolar cells of layer IVB appear to extend many reciprocal branches to neighboring cells ŽB.. Further maturation of SMI-32 IR large pyramidal neurons in layers V and VI is visible in this high magnification capture of an immunofluorescent stained neuron ŽC.. Scale bar: 50 mm ŽA, B..
92 C.S. Kogan et al.r DeÕelopmental Brain Research 119 (2000) 85–95 Fig. 6. Laminar expression of NF protein in area V1 in the adult. Nissl staining of an adjacent section ŽA. was used to identify layers in the SMI-32 immunostained preparation ŽB.. Layer IIrIII neurons have intensely stained pyramidal neurons throughout with highly arborized dendritic fields ŽC, D.. Layer IVB contains a dense neuropil with numerous multipolar cells and pyramidal cells with dendrites that appear to project towards the pial surface ŽE.. Some IR cells were visible in the geniculorecipient layers IVCa ŽF. and IVCb ŽG.. However, more immunopositive cells appear in the former, a layer known to receive input from the M layers of the lateral geniculate nucleus. Relatively fewer pyramidal cells as compared with the PD 30, 42, and 103 animals are visible in the infragranular layers V and VI although those cells present are more complex in their dendritic fields ŽH.. The apical dendrites of these neurons are observed to project to the supragranular layers. Pyramidal neurons in this preparation appear more complex in their dendritic fields. Stained neurons are overall more numerous throughout all layers. Scale bars: 500 mm ŽA, B., 50 mm ŽC–H..
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abundance of SMI-32 IR pyramidal neurons with apical dendrites that terminated within this layer and in layer I ŽFig. 6C and D.. Mature multipolar neurons also appeared sparsely within layer IIrIII.
4. Discussion The results from this study show the general developmental progression of SMI-32 staining in area V1 during the critical period of development. The time points were selected to reflect important milestones in development. This includes the following — one shortly after birth in which the animal had very little visual exposure ŽPD 0., one animal at an early part of the critical period ŽPD 7., two at the approximate height of the critical period ŽPD 30 and 42., and one at the end of the critical period ŽPD 103.. 4.1. Laminar staining profile We have found that the overall pattern of SMI-32 IR in area V1 shows considerable change over the first 15 weeks of post-natal life. SMI-32 IR is confined at birth to the large pyramidal and multipolar neurons in the infragranular layers. During development, there is a progressive increase in the number of pyramidal and multipolar neurons in these layers. Accompanying this is the remarkable intensification in dendritic projection out of the infraganular layers by the height of the critical period. This is followed by a reduction in the number of stained neurons so that by the end of the critical period, only a moderate number of stained cells and dendritic projections are visible. It is possible that the SMI-32 staining under-represents the actual developmental changes in dendritic arborization. We assume however, that there is a correlation between SMI-32 expression and actual dendritic changes. Concurrent with the overall laminar changes in SMI-32 IR during the post-natal developmental period, maturational advances of the dendritic arbors become evident. More dendrites appear SMI-32 IR and the overall complexity of these dendrites increases with age. This process of maturation appears to follow a gradient with those cells in the infragranular layers maturing before those cells in the supragranular layers. This finding is supported by Golgi studies that indicate that neurons in the deep layers develop more substantial networks of dendritic arbors before those in more superficial layers w21x. By virtue of the progression of neuronal development described in these Golgi studies, it is safe to assume that the staining seen in the present study is accurately reflecting the actual changes that occur in the developing dendritic arbor and the neuropil in general. Layer IVC remains poorly stained throughout most of development, except for some weakly stained cells in IVCa that appear in early development and persist to adulthood. Layer IVCb remains unstained except for a few
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weakly stained cells that are visible in the adult animal. Layer IVB is distinguishable at birth by its greater staining relative to neighboring layers. This includes scattered multipolar cells that appear on a background of moderately stained neuropil. A few pyramidal neurons are seen by PD 7. In general, the developmental sequence for this layer is unremarkable except for an increase in cell number and the complexity of dendritic fields by the height of the critical period followed by a discernible reduction in cell number by adulthood. Layer IVB neurons are known to project to area MT and as such, are considered part of the M pathway. Layer IIrIII shows almost no cellular staining at birth and remains poorly stained during the early part of the critical period ŽPD 7. and confined largely to the lower margin of layer III. In contrast to layers IVB and VrVI, layer IIrIII stained neurons only appear in significant numbers at the height of the critical period. It is interesting to note that neurons in layer IIrIII are known to extend projections to the association cortices such as areas V2 and V3. We also observed a progressive laminar appearance so that the first stained cells were observed along the lower margins of layer III followed by appearance at higher levels within layer II. By adulthood, many pyramidal and, for the first time, multipolar cells are visible throughout these two layers. 4.2. Morphological maturity of stained neurons We observed a striking difference in the staining pattern at the individual cellular level throughout development. At early time point, and especially at birth, staining in SMI-32 immunopositive neurons was largely confined to the cell body with only marginal extension into the dendritic processes. The microscopic appearance of these stained cells showed them to be neurons with immature morphology. As Lund et al. w9x noted, post-natal maturity in area V1 pyramidal neurons shows progressive changes in dendritic arborization. The confined staining of NF protein that we observed in area V1 at birth may reflect the immature morphological state of neurons in general, and pyramidal neurons in particular. The changes may be also be accounted for by a lower level of NF expression in these neurons. At greater developmental time points, the SMI-32 staining revealed neurons with more extensive and far-reaching dendritic processes. The progression of cellular staining from confined to extensive patterns with developmental time was a common feature of all layers. 4.3. Comparison to human neocortical deÕelopment An earlier study by Ang et al. w1x explored the significant events in the SMI-32 developmental sequence in human visual cortex. The results of their study showed the same general pattern of staining across developmental time points. That is, Ang et al. w1x found that SMI-32 im-
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munopositive neurons were present at birth in the infraganular layers though there is no evidence of such staining in the striated layer ŽIVB.. With increasing developmental time, the human cortex shows a generalized increase in SMI-32 staining that encompasses greater numbers of neurons in both supragranular and infragranular layers. There appear to be many more stained neurons in the supragranular layers of adult humans in comparison to our adult monkey. Campbell and Morrison w2x also found a relative reduction in SMI-32 stained neurons in adult monkey supragranular layers in comparison to human. Despite this difference, the same layers and more specifically, the same type of neurons are SMI-32 immunopositive in area V1 of humans and monkeys. The early maturation of SMI-32 immunostaining in the infragranular layers may be linked to the normal sequence of cortical development. It is known that deeper layer cells mature initially followed by a developmental progression from infragranular to supragranular layers w13x. Yet, another explanation might be that the pattern of staining is attributed to a preference for the early development of large neurons rather than the generation time, arrival at a final laminar position, or cell type. It has been shown, for example, that morphological maturation occurs more rapidly in larger cells w9x. This idea is supported by the results presented here and the observation in human preparations that the Meynert cells, which are found at the border of layers V and VI, are the most prominent SMI-32 IR cells at birth w1x.
develops earlier than its P counterpart. Further support for this notion comes from work by Mates and Lund w10x that suggests a developmental lag in the initial accumulation of type 2 dendritic spines for the P pathway geniculorecipient layer IVCa. Although SMI-32 IR does not permit visualization of dendritic spines, it is interesting to note that dendritic spine formation, an indicator of synaptic formation or loss, exhibits differences for the M and P pathways, respectively. Cells of layer IVCb Ža P recipient layer. show a slower initial accumulation of type 2 contacts compared to neurons of layer IVCa Ža M recipient layer., or to pyramidal cells of layer VI. Furthermore, type 2 synapses were more susceptible to reduction in layer IVCa and layer VI than in layer IVCb after an 8-week visual deprivation period w10x. Other evidence of M and P pathway developmental differences is supported by tracing studies in the monkey LGN w14,15x. The results indicate that the LGN M layers develop earlier than their P counterparts. Although this may not extend to the cortical areas as has been previously suggested by Lund w24x, it is not unreasonable to assume that input from lower levels of the visual system shape higher-order processes. Finally, it has been observed that retinal ganglion cells diverge into their respective M or P specialized functions soon after their last mitotic division, suggesting that the developmental fates of these groups of cells are regulated separately and therefore may develop at different rates w11x.
4.4. Relationship to deÕelopment of parallel Õisual pathways
Acknowledgements
Prior studies have shown the utility of SMI-32 IR in preferentially staining the M visual pathway w3,6,7x. Chaudhuri et al. w3x, for instance, describe the pattern of SMI-32 staining at the level of the lateral geniculate nucleus ŽLGN., where M and P layers are clearly distinguishable. They show that the two M layers of the LGN are heavily stained in comparison to the four P layers. Furthermore, SMI-32 immunostaining has been previously shown to be intense in layers IVB and VI of area V1, areas known to be anatomically related to the M pathway w6x. It has also been suggested that the ratio of the number of cells stained by SMI-32 in infragranular layers compared to supragranular layers can define different hierarchical levels of cortical processing w6x. Hence, areas belonging to the M pathway have a different distribution of SMI-32 immunoreactivity. It is tempting to speculate that the developmental results presented here represent early maturation of the M pathway in area V1. We observed that layers IVB and VI show stained cells earlier than other layers. Together with evidence implicating NFs in determining the end-point of connectional maturation for neurons w1,2x it appears as if the M pathway, presumed to be a phylogenically older pathway w18x,
This work was supported by grants from the Medical Research Council of Canada and the Natural Sciences and Engineering Research Council of Canada to Avi Chaudhuri. The authors thank Fariborz Rahbar-Dehghan for technical assistance.
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