Neuroscience Research 55 (2006) 105–115 www.elsevier.com/locate/neures
Review article
Towards the classification of subpopulations of layer V pyramidal projection neurons Zolta´n Molna´r *, Amanda F.P. Cheung Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, University of Oxford, South Parks Road, Oxford OX1 3QX, UK Received 29 November 2005; accepted 8 February 2006 Available online 15 March 2006
Abstract The nature of cerebral cortical circuitry has been increasingly clarified by markers for the identification of precise cell types with specific morphology, connectivity and distinct physiological properties. Molecular markers are not only helpful in dissecting cortical circuitry, but also give insight into the mechanisms of cortical neuronal specification and differentiation. The two principal neuronal types of the cerebral cortex are the pyramidal and GABAergic cells. Pyramidal cells are excitatory and project to distant targets, while GABAergic neurons are mostly inhibitory non-pyramidal interneurons. Reliable markers for specific subtypes of interneurons are available and have been employed in the classification and functional analysis of cortical circuitry. Until recently, cortical pyramidal neurons have been considered a homogeneous class of cells. This concept is now changing as the powerful tools of molecular biology and genetics identify molecular tags for subtypes of pyramidal cells such as: Otx-1 [Frantz, G.D., Bohner, A.P., Akers, R.M., McConnell, S.K., 1994. Regulation of the POU domain gene SCIP during cerebral cortical development. J. Neurosci. 14, 472–485; Weimann, J.M., Zhang, Y.A., Levin, M.E., Devine, W.P., Brulet, P., McConnell, S.K., 1999. Cortical neurons require Otx1 for the refinement of exuberant axonal projections to subcortical targets. Neuron 24, 819–831]; SMI-32, N200 and FNP-7 [Voelker, C.C., Garin, N., Taylor, J.S., Gahwiler, B.H., Hornung, J.P., Molna´r, Z., 2004. Selective neurofilament (SMI-32, FNP-7 and N200) expression in subpopulations of layer V pyramidal neurons in vivo and in vitro. Cereb. Cortex 14, 1276–1286]; ER81 [Hevner, R.F., Daza, R.A., Rubenstein, J.L., Stunnenberg, H., Olavarria, J.F., Englund, C., 2003. Beyond laminar fate: toward a molecular classification of cortical projection/pyramidal neurons. Dev. Neurosci. 25 (2–4), 139–151; Yoneshima, H., Yamasaki, S., Voelker, C., Molna´r, Z., Christophe, E., Audinat, E., Takemoto, M., Tsuji, S., Fujita, I., Yamamoto, N., 2006. ER81 is expressed in a subpopulation of layer 5 projection neurons in rodent cerebral cortices. Neuroscience, 137, 401–412]; Lmo4 [Bulchand, S., Subramanian, L., Tole, S., 2003. Dynamic spatiotemporal expression of LIM genes and cofactors in the embryonic and postnatal cerebral cortex. Dev. Dyn. 226, 460–469; Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., Macklis, J.D., 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45 (2), 207–221]; CTIP2 [Arlotta, P., Molyneaux, B.J., Chen, J., Inoue, J., Kominami, R., Macklis, J.D., 2005. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo. Neuron 45 (2), 207– 221]; Fez1 [Molyneaux, B.J., Arlotta, P., Hirata, T., Hibi, M., Macklis, J.D., 2005. Fez1 is required for the birth and specification of corticospinal motor neurons. Neuron 47 (6), 817–831; Chen, B., Schaevitz, L.R., McConnell, S.K., 2005. Fez1 regulates the differentiation and axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proc. Natl. Acad. Sci. U.S.A. 102 (47), 17184–17189]. These genes outline the numerous subtypes of pyramidal cells and are increasingly refining our previous classifications. They also indicate specific developmental programs operate in cell fate decisions. This review will describe the progress made on the correlation of these markers to each other within a specific subtype of layer V neurons with identified, stereotypic projections. Further work is needed to link these data with observations on somatodendritic morphology and physiological properties. The integrated molecular, anatomical and physiological characterisation of pyramidal neurons will lead to a much better appreciation of functional cortical circuits. # 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Pyramidal neurons; ER81; OTX-1; FNP-7; N200; SMI-32; Fez1; Callosum; Corticospinal projections
* Corresponding author. Tel.: +44 1865 282 664; fax: +44 1865 272 420. E-mail address:
[email protected] (Z. Molna´r). 0168-0102/$ – see front matter # 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2006.02.008
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Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Layer V pyramidal cells: an accessible model for the study of target selection, dendritic and physiological differentiation. The general neurobiological relevance of the model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The diversity of somatodendritic morphology of layer V neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Electrophysiological classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Layer V marker gene expression pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Do GABAergic projection neurons contribute to subcortical or callosal connectivity? . . . . . . . . . . . . . . . . . . . . . . . . . . Transgenic mice expressing fluorescent proteins could provide useful tools to characterise layer V subclasses . . . . . . . . . Co-localisation studies suggest the existence of several subclasses of layer V neurons . . . . . . . . . . . . . . . . . . . . . . . . . . Dual projections can be established in numerous combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The original classification of most cerebral cortical neurons originated in the previous century. Neurons were characterised based on their size, shape and dendritic branching pattern as they appear on Golgi-stained preparations (Golgi, 1886; Ramo´n y Cajal, 1911; Lorente de No, 1949). The basic terminology for the identification of cortical neurons (e.g. pyramidal, stellate or granular cell, etc.) is still in use today. It was not until the advent of reliable tracing methods that connectivity became a pertinent and useful criterion. The principal neuronal types of the cerebral cortex are the excitatory pyramidal cells, which project to distant targets, and the inhibitory non-pyramidal cells, which are the cortical interneurons (Peters and Jones, 1985). These different classes of neurons originate from distinct regions. Pyramidal neurons are generated in the cortical neuroepithelium and migrate radially to reach the cortex following an inside–out gradient (Rakic, 1988), whereas most of the interneurons originate from the basal telencephalon and migrate to the cortex through tangential migration (Parnavelas, 2000; Marı´n and Rubenstein, 2001). Histochemical and immunohistochemical analysis revealed further details especially of the different types of interneurons (Markram et al., 2004). A list of relatively simple but reliable markers aided the identification of different interneuron classes, and these markers are still used today (Somogyi and Klausberger, 2004). Projection neurons have been further classified by the laminar position of their cell body, morphology, electrophysiology and hodology (Toyama et al., 1974; Peters and Jones, 1985), but there are relatively few neurochemical markers available for their identification. Classification of central nervous system (CNS) regions has been advanced in recent years by exploiting modern mouse molecular genetics. Markers are useful for classification, but also they are equally interesting for understanding development. The specific combination of transcription factors defines neuronal fate, and their combinatorial expression pattern closely correlates with neuronal diversity (Gray et al., 2004). Genes which regulate the production of cortical cell types have been identified (Guillemot et al., 2006; Wu et al., 2005; Hevner et al.,
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2006), however, the molecular profile within pyramidal cell populations in the cortex remains relatively undeveloped. The reason behind this might be the lack of integrated approaches that utilise several of the classification criteria (hodology, morphology and physiology) synchronously. Approaches employing methods where all these components are viewed in conjunction have yielded the greatest progress (Migliore and Shepherd, 2005; Nelson, 2005; Sugino et al., 2006). We wish to give an update on the efforts made in this field by reviewing recent studies on subtypes of layer V pyramidal neurons in the rodent cerebral cortex. Pyramidal cells of this class provide an exceptional model system to test ideas on cell classification and to study neuronal specification within the same cortical lamina. 2. Layer V pyramidal cells: an accessible model for the study of target selection, dendritic and physiological differentiation Within layer Vof the adult rodent cortex, pyramidal neurons with different soma size can be distinguished. Neurons with smaller sized soma tend to occupy the lower portion of layer V (Vb), whereas cells with larger soma tend to reside within the upper sector (Va). However, the location is not an absolute predictor since the two types of somas mingle with one another and there is a considerable overlap between them within layer V. The different soma sizes can be linked to distinctions in their projection site, somatodendritic morphology and physiological characteristics in the adult (Klein et al., 1986; Larkman and Mason, 1990; Hallman et al., 1990). Pyramidal neurons of layer V of the adult rodent cortex fall into two major classes that can be distinguished on the basis of their projection site, morphology and physiological properties (Hallman et al., 1990; Klein et al., 1986; Larkman and Mason, 1990). Type I cells project to the superior colliculus, spinal cord or basal pons, they are characterised by thick tufted apical dendrites, and bursts firing pattern (Kasper et al., 1994). Type II layer V projection neurons project their axons to the contralateral hemisphere or to the ipsilateral striatum, their apical dendrite is slender with fewer oblique branches that end without terminal tufts, usually in the upper part of layer II/III, and never fire
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Fig. 1. Layer V pyramidal neurons fall into two major classes that can be distinguished on the basis of their projection site, morphology and physiological properties in the adult. The diagram summarises the developmental sequence leading to target selection, somatodendritic differentiation and emergence of distinct physiological properties.
bursts (Kasper et al., 1994). Since these differences emerge sequentially within the very same layer (Fig. 1), the differentiation of these distinct projection neurons provides a unique model system to study cortical neuron specification. The majority of pyramidal neurons of layer V in the rat are born between embryonic days (E) 15 and 16 (Bayer and Altman, 1991), migrate to layer V by E19–20 (Miller, 1998) and appear indistinguishable when they first migrate into position (Koester and O’Leary, 1992). At this stage all have stout apical dendrites reaching layer I with terminal tufts (Koester and O’Leary, 1992), and none of them fire bursts after the injection of depolarising currents and recording with sharp electrode (Kasper et al., 1994) or with whole cell patchclamp method (Christophe et al., 2005). During or shortly after they have migrated into the cortical plate, the axons of these two classes of cells start to grow towards their appropriate targets. Initially all type I cells start to extend simple unbranched axons into the spinal cord through striatum, internal capsule, cerebral peduncle and basal pons. During early postnatal stages these axons develop branches to pons, superior colliculus, striatum and cerebellar nuclei (Stanfield and O’Leary, 1985). The pattern of selective elimination and maintenance of these projections is dependent on the particular cortical area, e.g. occipital cortex will loose spinal cord projections, but will maintain connections with superior colliculus (O’Leary and Stanfield, 1989; Koester and O’Leary, 1993). Type II cells extend processes to the contralateral hemisphere with an ipsilateral projection to the striatum. They reach their targets around birth, but do not invade target regions until early postnatal period P0–5. The end of this period correlates with the divergence of these two types of morphologies (Koester and O’Leary, 1992, 1993). Type I cells with axons to the superior colliculus start to have larger soma and they maintain their thick apical dendrites which terminate in a large tuft within layer I. Type II cells with axons to the contralateral hemisphere have smaller soma and slender apical dendrites with fewer oblique branches that end without terminal tufts (usually in the upper
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part of layer II/III). The divergence in the somatodendritic morphology of the type II from type I neurons is due to the substantial remodelling of the initially undistinguishable tufted morphology of the type II cells (Koester and O’Leary, 1992). The molecular regulation of this process is not well known. It has been demonstrated that the transformation from the tufted into the non-tufted morphology in a significant portion of type II projection neurons could be delayed with the postnatal application of MK-801, a NMDA receptor antagonists during the first 9 postnatal days (Molna´r et al., 1997). Larkman and Mason (1990) demonstrated that in adult rat visual cortex these two groups differ in other intrinsic physiological properties such as input resistance and time constant. From the end of the second postnatal week the two classes begin to differ in their physiological characteristics. Type I layer V neurons begin to fire bursts, whereas type II do not show this behaviour (Kasper et al., 1994). The divergence of these cells into two distinct classes provides an excellent model system to study the contribution of genetic and epigenetic factors during cortical development and provides numerous technical advantages. The value of this system is that the two classes can be distinguished before the somatodendritic transformation or the acquisition of the characteristic physiological behaviour through labelling of their specific projections (Koester and O’Leary, 1992; Kasper et al., 1994). It is relatively easy to selectively label each subclass in living or fixed tissue through their specific projections with fluorescent latex microspheres or carbocyanine dyes. This can be exploited in several developmental or adult studies. Layer V pyramidal cells are among the largest cells in the cerebral cortex, which is of considerable advantage for anatomical, electrophysiological recording and single cell RT-PCR studies (Christophe et al., 2005). This permits the selective identification of the different cells in combination with immunohistochemistry, in situ hybridisation, single cell recording or FAC sorting (Frantz et al., 1994; Weimann et al., 1999; Hevner et al., 2003; Voelker et al., 2004; Christophe et al., 2005; Arlotta et al., 2005; Molyneaux et al., 2005). The molecular changes can be monitored in identified cell populations through backlabelling during the early stages of somatodendritic differentiation and the development since the subclasses reach their specific targets before any distinctions can be observed in their morphology or physiology. In spite of the advantages of this system, it seems that our current knowledge on interneuron population is much more developed (Cauli et al., 1997; Markram, 2004) compared to pyramidal cells. Specific markers for classes and subclasses of pyramidal neurons are needed to both advance our efforts to understand development, but also facilitate our studies on functional cortical circuits. 3. The general neurobiological relevance of the model An obvious question is whether this system is general across different cerebral cortical areas in rodent, and whether it can be found in various different species including primates. While most experiments were performed in rat visual cortex, the existence of two similar types of layer V neurons were
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demonstrated in auditory, motor and somatosensory regions as well (Chagnac-Amitai et al., 1990; Games and Winter, 1988). In the adult rat cortex, the particular cortical area determines the permanent projection site of the type I neurons (motor cortex to spinal cord or basal pons and visual cortex to superior colliculus). Similar projection patterns have been revealed within the mouse cortex (Mitchell and Macklis, 2005; Spires et al., 2005) suggesting that the two major types of layer V projections are a general feature of rodent cortex. However, the mouse and rat similarities should not be taken for granted. We believe that there might be some interesting differences between the two species. While studying the somatodendritic development of the callosally projecting layer V neurons in the mouse, we observed that the loss of the apical tufts occur later (since we have observed tufted layer V cells projecting to the callosum even at P14; Spires et al., 2005) or it does not occur in such a clear cut fashion as it has been reported in rat visual cortex (Kasper et al., 1994) or in rat somatosensory cortex (Koester and O’Leary, 1992). It is not known whether the somatodendritic transformation of callosally projecting layer V neurons is a general phenomenon across various species. A very similar pattern of somatodendritic development has been reported for callosally projecting layer IV neurons in kitten (Vercelli et al., 1992, 1997). The presence of the apical tufts is essential for the type I class to access the input from the superficial layers. Only the tufted cells can receive feedback projections from higher cortical regions, since the type II neurons rarely extend beyond layer III. The two types of layer V projection neurons integrate into the extracortical circuitry in a distinct manner, they possess different striatal terminals, suggesting specific functional properties (Reiner et al., 2003). 4. The diversity of somatodendritic morphology of layer V neurons Although pyramidal cells share numerous common features within layer V, they are very heterogeneous in their somatodendritic morphology (Schofield et al., 1987; Hallman et al., 1990). The basic classification of type I (tufted) and type II (non-tufted) has proven to be helpful but oversimplified. At least two clear subclasses of tufted populations have been identified. One subclass possessed a simple main apical dendrite reaching layer I and terminating in a single tuft, while another has side branches with a more elaborated tuft (Angulo et al., 2003). These morphological differences were reflected in their physiological characteristics (Angulo et al., 2003). Yuste and co-workers used multidimensional cluster analysis of a series of dendritic morphological variables on biocytin-filled layer V pyramidal cells in mouse primary visual cortex (Tsiola et al., 2003). The advantage of this approach is that it is unbiased and enables a more precise description of new classes of neurons. Layer V neurons have been shown to be divided into five major classes. These emerging specific morphological types will have to be specifically allocated and integrate the new hodological and molecular data. One possible explanation for incongruence between these morphological subtypes and molecular characteristics is the lack of retrograde labelling
method which could provide reliable information on the entire somatodendritic morphology. Only selected regions (superior colliculus, spinal cord and contralateral hemisphere) have been explored with the combination of retrograde labelling and cell filling (Kasper et al., 1994), while other regions (e.g. striatum, thalamus, basal pons or the homotopic and heterotopic callosal and intracortical projections) have been entirely neglected in this respect. Very few studies examine the relationship between target and somatodendritic morphology of specific classes of layer V neurons. However, this information is essential for the understanding of functional cortical circuits (Thomson and Bannister, 2003). The apical dendrites of individual layer V pyramidal neurons are known to form closely associated radial bundles (minicolumns) in the upper layers (Peters and Walsh, 1972). The close association of dendrites of projection neurons sharing the same targets has been demonstrated by Vercelli et al. (2004). For example, neurons projecting to the superior colliculus form minicolumns with neurons projecting to the striatum, but not with those projecting to the ipsi- or contralateral cortex. The functional significance of these close interactions (microcolumns) remains unclear (Rockland and Ichinohe, 2004). It is interesting to note here that some developmental transcription factors exhibit ‘‘minicolumn-like’’ expression in the neonatal period, such as Id2 (Rubenstein et al., 1999), and they might provide the developmental basis for the organization of projections. 5. Electrophysiological classification The electrophysiological distinctions between the two major types of layer V neurons have been described by Kasper et al. (1994) using sharp electrode recording. Prelabelling with fluorescent latex microspheres enabled Kasper et al. (1994) to record from neurons with identified projections and correlate the electrophysiological parameters to the somatodendritic morphology and projection target. The development of subthreshold and action potential properties of layer V neurons recently have been definitively characterised with patch electrode recording (Christophe et al., 2005). This latter study also revealed very specific effect of in vivo anaesthesia on the action potential firing pattern of layer V pyramidal cells in vitro. Prolonged in vitro anaesthesia with pentobarbital or ketamine–xylazine consistently elicited burst firing pattern in layer V neurons projecting to superior colliculus, whereas this behaviour has not been observed in the population projecting to the contralateral hemisphere. The factors controlling the development of burst firing pattern and the relationship to prolonged in vitro anaesthesia are currently not known. 6. Layer V marker gene expression pattern The quest of finding neurochemical markers for layer V projection neurons started at the time when layer V neurons were being classified into type I subcortically projecting and type II callosally projecting neurons (Stanfield and Jacobowitz,
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Fig. 2. Schematic comparison of the timing of the specific gene expression patterns in three classes of layer V projection neurons. CSMN, corticospinal motor neuron; CTN, corticotectal neuron; CCN, corticocortical neuron (see text for references).
1990; Larkman and Mason, 1990). There are numerous layer V-specific protein markers. To integrate with existing classification schemes, here we describe the markers that have been demonstrated to be type-specific by combining retrograde labelling with in situ hybridisation or immunohistochemistry (Fig. 2). The recent breakthrough in the pursuit of markers began when a specific population of backlabelled (Arlotta et al., 2005; Molyneaux et al., 2005) or GFP positive cell bodies from layer V transgenic lines (Feng et al., 2000) were sorted and their gene expression profile analyzed by microarray (Sugino et al., 2006). Single cell RT-PCR methods also give extra information on physiological properties and if combined with cell filling, on somatodendritic morphology. Single cell RT-PCR has proven very useful for studying the expression of already known genes in specific cell populations (Yoneshima et al., 2006), and further examination of the possible co-localisation of genes expressed in single cell PCR matrix data at different ages (Christophe et al., 2005). While this approach is yet to reveal novel markers, selective gene expression could elucidate developmental mechanisms. Neurochemical markers are instrumental for further subdivision of the layer V neurons into stage-, target- or even regionspecific subtypes. By looking into these markers at specific periods of differentiation, we hope to gain insight into the mechanisms controlling developmental decision making (Guillemot et al., 2006). This information is crucial for further investigating recruitment and manipulation of endogenous stem cells for use in clinical therapy (Gates et al., 2000; Hevner et al., 2006). The following list summarises the currently available markers and genes which have specific expression patterning in layer V cell subpopulations. Protein 35 and protein 36: These two antibodies were found to be the first to exclusively label type I subcortically projecting neurons but not type II (Stanfield and Jacobowitz, 1990). The human homologue of protein 35 plays a role in Fas-mediated cell death (Schweitzer et al., 2002).
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Glutamate and aspartate: Dori et al. (1992) described that glutamate (Glu) or aspartate (Asp) are the two major transmitters used by corticofugal efferent neurons. This study drew the attention to the morphological and molecular heterogeneity of the projection neurons providing the various efferent pathways from the rat visual cortex. It has been demonstrated that the distribution, morphology and proportions of Glu- and Asp-containing neurons in the efferent pathways from the rat visual cortex vary according to the target (corticotectal, corticopontine, corticofugal or callosal). Injections of WGA-HRP into the superior colliculus backlabelled large and medium-sized pyramidal neurons in the upper portion of layer V of the visual cortex. Forty-six percent of the backlabelled cells were stained for glutamate and 66% for aspartate. Similar labelling from the pontine nuclei revealed cells in the deeper part of cortical layer V, which were also immunoreactive for Glu (42%) or Asp (51%). Injections of WGA-HRP into the contralateral visual cortex retrogradely labelled small and medium-sized pyramidal cells throughout layers II–VI of the visual cortex and 38% of these neurons were also labelled for Glu while 49% were also Asp-immunoreactive. Unfortunately WGAHRP tracing do not provide sufficient detail on the somatodendritic morphology for detailed somatodendritic classification, but it would be of interest to determine the possible morphological differences between the Glu and Asp populations with identical projection sites. Otx-1: Otx-1 is first described as a transcription factor specifically expressed in the ventricular zone, and later in development, in layer VI and V neurons (corticotectal, corticospinal and corticobulbar; Frantz et al., 1994). Otx-1 is required for the refinement of layer V connections to appropriate subcortical targets. In Otx-1 null mice, the normally transient pattern of exuberant connections is retained into adulthood (Weimann et al., 1999). Deletion of the Otx-1 gene also influences development in all cortical layers, most noticeably a decrease of neurons expressing parvalbumin and calbindin in layer V. This suggests Otx-1 is involved in the development and differentiation of neurons expressing calcium-binding proteins (Panto et al., 2004). N200/SMI-32/FNP-7: Neurofilament proteins have been found in cells with well-myelinated axons (Kirkcaldie et al., 2002) and it is thought to be related to fast conduction of axons (Lawson and Waddell, 1991). N200, SMI-32 and FNP-7 are neurofilament proteins that are expressed in several layers of the cortex (including layers II/III, V and VI). Within layer V, they have been shown to express exclusively in type I neurons (Voelker et al., 2004; Fig. 3). Er81: ER81, is a transcription factor of the ETS family (de Launoit et al., 1997). It is expressed in motorneurons in the spinal cord and it plays a role in the formation of their functional connections with Ia sensory afferents (Lin et al., 1998; Arber et al., 2000). ER81 is expressed almost exclusively in a subset of cortical layer V projection neurons across various cortical areas (Xu et al., 2000; Sugitani et al., 2002; Hevner et al., 2003; Beggs et al., 2003; Hasegawa et al., 2004; Gray et al., 2004). Recent study by Yoneshima et al.
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Fig. 3. SMI-32, N200 and FNP-7 are markers of type I layer V projection neurons. Confocal microscopic images of layer V pyramidal neurons showing that the three neurofilament antibodies (red) specifically stained neurons that project subcortically, but they did not stain cells with projections to the contralateral cortex (all populations contain green microspheres). SMI-32 (A and B), N200 (D and E) and FNP-7 (G and H) were specifically located in the spinal cord (A, D and G) and superior colliculus (B, E and H) projecting layer V neurons (arrows indicate examples of double-labelling). (C, F and I) contralateral cortex projecting neurons (green microspheres indicated by arrowheads), did not express SMI-32 (C), N200 (F) or FNP-7 (I). The open arrow heads indicate neurons expressing the neurofilaments, but do not contain green microspheres and thus do not project to the contralateral cortex. Scale bar = 20 mm. Reproduced with permission from Voelker et al. (2004).
(2006) further examined the developmental expression and the projection targets of Er81-expressing neurons and demonstrated that ER81 was expressed in a large proportion of spinal cord and superior colliculus projecting neurons while only one-third of the layer V neurons with contralateral projections expressed the protein (Yoneshima et al., 2006). This study also revealed that Er81 was expressed in various cortical areas specifically in layer V in infant Japanese monkey. Ctip2: COUP-TF interacting protein 2 (Ctip2) is a zinc finger protein thought to be associated with the extension, fasciculation and path-finding of subcortically projecting neurons. CTIP2, a gene of yet unkown function is expressed in layer V of numerous cortical regions. It has strong expression in corticospinal and corticotectal neurons, but not in layer V neurons with callosal projections (Arlotta et al., 2005). In Ctip2 null mice, corticospinal axons failed to extend beyond the pons, and some cases even extend towards ectopic targets (Arlotta et al., 2005). Fez1: Fasciculation and elongation protein zeta 1 (Fez1) is a zinc finger type transcription factor, expressed mainly in
plasma membrane. It is involved in axonal outgrowth and fasciculation. Fez1 forms complexes with cytoskeletal proteins (e.g. F-actin and kinesin), that may be involved in organelle transport during axonal elongation (Fujita et al., 2004). Fez1 is a general identity gene for corticospinal neurons that is expressed from E13 onwards, and is required for the specification of subcortical projection neurons (Arlotta et al., 2005). The corticospinal tract was absent in Fez1 null mice, whereas the corticotectal and pontine projections were severely reduced (Molyneaux et al., 2005; Chen et al., 2005). The Ctip2 and Fez1 null mice show very similar phenotype, which suggests that these two genes may act in a common pathway. It has been proposed that Fez1 acts upstream of Ctip2 to control layer V cortical motorneuron fate determination (Hirata et al., 2004; Chen et al., 2005). Lmo4: LIM domain only 4 (Lmo4) is a transcription factor, which together with Clim, forms complexes that regulate gene expression by associating with DNA-binding proteins (Bulchand et al., 2003). Lmo4 knockout die shortly after
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birth with defects in neural tube closure. Lmo4 is expressed exclusively in type II layer V pyramidal neurons (Arlotta et al., 2005). Calretinin: The calcium-binding proteins are considered as markers for GABAergic interneurons. However, evidence suggests that some calcium-binding proteins, such as parvalbumin, calbindin and calretinin may also be expressed in layer V pyramidal cells (Jinno and Kosaka, 2004). Study from our group has suggested that although calretinin and parvalbumin were expressed in layer V pyramidal neurons, only calretinin was selectively expressed in a small subpopulation of callosally projecting neurons (Mitchell et al., 2006). Nogo-A: Nogo-A is a major neurite growth inhibitor for CNS neurons. It is expressed in oligodendrocytes and neurons. Recent immunohistochemical study showed that Nogo-A is predominately expressed in layer V pyramidal neurons that have a larger soma and thicker apical dendrites than Nogo-A negative neurons (Shin et al., 2006). This suggests that Nogo-A positive neurons are type I intrinsically bursting neurons that project axons to subcortical regions. Clim1, Diap3, S100a10, encephalopsin, Crim1,and Mucrystallin: These markers emerged from the recent report where a specific population of backlabelled layer V neurons were isolated and their gene expression profile analyzed by microarray (Arlotta et al., 2005). Cofactors of LIM homeodomain protein 1 (Clim1) is a transcriptional activator that associates with the LIM homeoproteins and coordinates transcription. Clim1 mRNA is expressed only in early development across layer V. Encephalopsin is involved in phototransduction in the brain. It labels type I corticospinal motor neurons, however, its role in the cortex is unknown. Cysteine-rich motor neuron 1 (Crim1) showed strong expression in the hindbrain, midbrain and corticospinal motor neurons in layer V. Diaphanous protein homologue 3 (Diap3), S100 calcium-binding protein A10 (S100a10) and Mu-crystallin are also expressed in type I corticospinal motor neurons of layer V only, but their function remains unknown. Unfortunately most of these molecules have not been integrated into a global molecular network. Co-localisation studies of two or more of these markers on type-specific layer V population would be very important. Moreover, the areaspecificity of marker expression (or expression regulation by area-specific cortical genes) is currently not known. 7. Do GABAergic projection neurons contribute to subcortical or callosal connectivity? It has been shown that mature GABAergic neurons can develop long range projections intracortically (Fabri and Manzoni, 1996), and the vast majority are immunoreactive for somatostatin, neuropeptide Y and nitric oxide synthase (Tomioka et al., 2005). In developing rat neocortex, GABAergic neurons can even travel across the corpus callosum (Kimura and Baughman, 1997). At present, the general view is that
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roughly 1% of the callosally projecting neurons are GABAergic in the mature nervous system of rats and cats (Gonchar et al., 1995; Fabri and Manzoni, 2004). The introduction of GFP transgenic animals has allowed us to readdress the issue with increased accuracy. We determined whether the callosally projecting layer V neurons also contain a subpopulation of GABAergic cells by using young postnatal GAD65-eGFP transgenic mice (Lo´pez-Bendito et al., 2004) with DiI placed in the corpus callosum. DiI-labelled cells were distributed mostly in layers V, II/III and least in layers IV and VI. Of the 1174 DiIlabelled cells examined, only one cell in layer II/III was doublelabelled with GAD65-eGFP. We conclude that although GABAergic neurons with contralateral projections do exist, their number is extremely low in postnatal development (A.F.P. Cheung and Z. Molna´r, unpublished observations, 2005). This suggests that there might be a difference between species. It is still not known if GABAergic neurons can develop long range projections to subcortical targets. The function for these transient callosally projecting GABAergic neurons remains unknown, but possible roles in path-finding, neuronal differentiation and neurite outgrowth have been suggested (Shatz et al., 1990; Behar et al., 1994; Ben-Ari et al., 1994). 8. Transgenic mice expressing fluorescent proteins could provide useful tools to characterise layer V subclasses GABA-containing interneurons are well-defined by proteins such as calbindin, calretinin, parvalbumin, neuropeptide Y, vasoactive intestinal peptide, somatostatin and cholecystokinin (Markram et al., 2004, for review). Although no specific type of interneuron can be defined by a single marker, some types express specific combinations of different markers. With the continuous isolation of molecular tags specific for layer V pyramidal neurons, we can refine the classification of projection neurons further by using transgenic mice that express fluorescent proteins under promoters that are specific to cell types (Feng et al., 2000; Sugino et al., 2006). Moreover, we can also more readily relate molecular markers to somatodendritic morphology in fluorescent protein expressing models. A recent study of the potassium channel Kv3.1-eYFP transgenic mice revealed that eYFP positive cells were found in layer V motor and somatosensory cortices (Akemann et al., 2004). The eYFP positive cells have a smaller number of apical oblique dendrites and feature a steady spike frequency adaptation (eYFP negative cells did not). There are more transgenic mice available (e.g. M-line with GFP tagged to Thy.1; Feng et al., 2000), but further characterisation is needed to fully correlate different markers to specific projection neurons. 9. Co-localisation studies suggest the existence of several subclasses of layer V neurons An important aim of this study is to unify molecular classification with other aspects of layer V neuronal classification in adult and during development. It is important to correlate the combination of expressed genes with projection
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Fig. 4. By combining different neurochemical markers, it is feasible to reveal distinct subpopulations within each type of layer V pyramidal neurons. Although ER81 expresses in both subcortically and contralaterally projecting neurons, type I-specific markers such as N200, SMI-32 and OTX-1 are very rarely expressed with ER81 within the same neuron (Hevner et al., 2003; Rolph et al., 2005; A.F.P. Cheung and Z. Molna´r, unpublished observations, 2005). It still remains unclear if other markers such as Ctip2, Lmo4 and calretinin also represent distinct subpopulations.
targets and specific somatodendritic morphology. Co-localisation studies on OTX-1 and ER81 indicate that the two markers are not expressed within the same postnatal layer V neurons (Hevner et al., 2003). Using retrograde labelling and immunohistochemistry, we have also shown that although ER81 and N200 or SMI-32 are expressed in type I layer V neurons, they never co-express in the same projection neurons (Rolph et al., 2005; A. Cheung, C. Voelker, R. Rolph and Z. Molna´r, unpublished observations, 2005). This suggests that there are at least two distinct neurochemical subpopulations within type I layer V pyramidal cells. It is imperative that as more and more markers become available, their possible colocalisation is investigated (Fig. 4). It is conceivable that the correlation between neurochemical characteristics and hodology will require the consideration of multiple rather than single targets. 10. Dual projections can be established in numerous combinations Within any single layer of the cortex, there are numerous morphological subtypes with different cortical (intracortical or intercortical) and subcortical connectivity (Thomson and Bannister, 2003). While it is clear that there are stereotypic patterns in the connections from the earliest stages of development, it is also clear that the idea of single destination has to be abandoned. Throughout the developing CNS, promiscuous initial connections are pruned back to few targets or eliminated entirely (Innocenti and Price, 2005). It has been demonstrated that target refinement is a general feature of layer V corticofugal projections (O’Leary and Stanfield, 1985, 1986). Some of the early projections (e.g. corticospinal projections from the occipital cortex) are transient and are replaced with permanent corticotectal projections (O’Leary et al., 1981; Stanfield et al., 1982; Stanfield and O’Leary, 1985). However, there are numerous surprising examples of permanent dual projections involving both types of neurons in the adult rat and
mouse brains. Examples include the homotopic interhemispheric projecting layer V neurons which also develop an ipsilateral and contralateral projection to the striatum in the rat somatosensory cortex (Reiner et al., 2003), and the simultaneous callosal and frontal projections maintained by layer V neurons in the mouse somatosensory cortex (Mitchell and Macklis, 2005). Somatodendritic subtype and neurochemical markers have not been allocated to these layer V pyramidal neurons. Currently we do not fully understand the distinctions between layer V neurons with single, dual or multiple projections. It is conceivable that the morphology or neurochemical properties are different. It is possible that there are more layer V neurons with dual projections in the adult rodent brain to be discovered. The detection of dual projections requires studying labelling patterns from broad areas rather than individual focal injections, consideration of regionspecificity and the age of the animal. Cortical architecture is reshaped considerably during the first few postnatal weeks. We do not know the significance of specific sets of transient projections and the corresponding gene expression pattern or somatodendritic changes in the corresponding set of cells. How these different classes of layer V projection neurons fit into the broader picture of feed forward and backward projections to subserve sensorimotor integration remains a major question. 11. Summary Our challenge is now to understand the combinatorial effect of lineage- and area-specific gene expression profiles. These fundamental components drive neurogenesis, differentiation and regional cortical connectivity. Potential molecular markers for layer V projection neurons are continually being found. Correlation of these markers with other aspects of neuronal phenotype will offer a more comprehensive classification of layer V neurons. More importantly, markers will reveal mechanisms by which cortical differentiation occurs within the developing mammalian neocortex. The markers will not only help in the dissection of functional cortical circuits, but will eventually lead to the means of restoring pathological conditions. Acknowledgements The review is based on a talk given by ZM at the meeting held on Neuronal Differentiation in Cortical Development at Icho-kaikan in Osaka University, Suita, Osaka, in September 16–17, 2005. This meeting was held to mark the end of the Human Frontiers Science Program Grant (RG 107/2001) by Nobuhiko Yamamoto, Etienne Audinat, Daniel Lavery and Zolta´n Molna´r. Consortium members very much valued the interactions during the period of the grant which resulted in development of numerous collaborative projects. We are very grateful for Nobuhiko Yamamoto and his group for their exceptional hospitality in Osaka. Thanks goes to Jamin DeProto for discussions and for his critical reading of an earlier version of this manuscript.
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