Development and Plasticity in Sensory Thalamus

Development and Plasticity in Sensory Thalamus and Cortex 1 Pioneer Neurons and Interneurons in the Developing Subplate: Molecular Markers, Cell Birthdays, and Neurotransmitters Robert F. Hevner and Nada Zecevic 1. Abstract Subplate neurons are essential for the development of cortical axon pathways, including thalamocortical innervation as well as the formation of some cortico-cortical and descending cortical efferent connections. Previous evidence suggests that the critical subplate neurons are early- born “pioneer’’ neurons, which extend the first axons out of the cortex to subcortical forebrain regions. However, pioneer neurons are not the only type of neuron in the subplate layer. The subplate contains both glutamatergic and GABAergic neurons, some of which are tran- sitory due to either ongoing cell migration or subsequent cell death. We have studied the cellular composition of the subplate in developing mouse and human cortex by retrograde axon tracing, cell birth- dating, and immunohistochemical analysis of specific markers. Our results indicate that pioneer neurons are early-born glutamatergic neurons that express transcription factor Tbr1, transgene golli-lacZ, and other markers. In contrast, GABAergic interneurons in the subplate do not make subcortical (pioneer) axon projections, but instead migrate tangentially and radially through the subplate layer, express transcription factor Dlx, and are born both early and late in corticogenesis. Subplate neurons are essential in development of the initial cortical connectiv- ity, and it is thus important to distinguish between the different cell types present in this compartment, using molecular markers. The subplate in humans appears to contain a similar diversity of neuron types as in mice, but is markedly thicker than in mice, as confirmed by the broad band of Tbr1 expression extending below the cortical plate in humans. Keywords: Cerebral cortex, Dlx, GABA, golli-lacZ, reeler, Tbr1 2 Robert F. Hevner and Nada Zecevic 2. Introduction The subplate is a neuronal layer in the developing cerebral cortex, located deep to the cortical plate and superficial to the intermediate zone (reviewed by Allendoerfer and Shatz, 1994). The subplate is present in all mammals, though its thickness, distinctness as a morphological layer, and persistence in adulthood vary among species. In rodents, the subplate is relatively thin and morphologically distinct, and many subplate neurons persist into adulthood as layer VIb of the mature cortex. At the opposite end of the spectrum, in primates, the subplate is relatively thick (up to four times thicker than the cortical plate in human somatosensory cortex at 22 gestational weeks) and indistinct, blurring with the cortical plate above and the intermediate zone below (Kostovic and Rakic, 1990). Also, relatively few subplate neurons persist to adulthood in primates, mainly as “interstitial neurons’’ in the white matter (Kostovic and Rakic, 1980). Even in simpler mammalian species where the subplate forms a distinct layer, “subplate neurons’’ are also recog- nized in the embryonic intermediate zone, and are thought to persist in small numbers into adulthood as interstitial neurons. Subplate neurons are important because they play an essential role in the development of cortical axon connections. They give rise to the first (“pioneer’’) efferent axons out of the cortex, and provide the first postsy- naptic targets for afferent thalamocortical axons (McConnell et al., 1989; Molnar´ et al., 1998a). Ablation of subplate neurons causes localized ab- sence of thalamocortical innervation, as well as defects of some cortical efferent pathways (Ghosh et al., 1990; Ghosh et al., 1993; McConnell et al., 1994). The mechanisms by which subplate neurons mediate development of these several axon pathways are not all clear. In the case of reciprocal thalamocortical and corticothalamic connections, early inter- actions between cortical pioneer axons and thalamic axons in the internal capsule may be critical for guiding both sets of axons. This mechanism is known as the “handshake hypothesis’’(Molnar´ and Blakemore, 1995). Efforts to characterize subplate neurons have been difficult because the subplate and intermediate zone contain heterogeneous types of neurons. In addition to pioneer neurons, which are glutamatergic and are produced from progenitors in the cortical neuroepithelium, the subplate also contains numerous interneurons, which are produced mainly from progenitors in the regions of the basal forebrain known as the me- dial ganglionic eminence (MGE), caudal ganglionic eminence (CGE), and lateral ganglionic eminence (LGE) (Anderson et al., 1997; Wichterle et al., 2001; Mar´ın and Rubenstein, 2001; Nery et al., 2002; Xu et al., 2004). Many immature interneurons migrate tangentially and radially through the developing subplate at the same time as pioneer axons project to the internal capsule. Also, some interneurons cease migration in the subplate, and account for a proportion of neurons in adult layer 6b and white matter (Fairen´ et al., 1986). Proposed markers of subplate pioneer neurons include p75 neu- rotrophin receptor (p75NTR), kynurenine aminotransferase (KAT), 1 Pioneer Neurons and Interneurons in the Developing Subplate 3 golli-lacZ transgene, transcription factor Tbr1, SP-1 antibody to cytosolic peptide, and others (Antonini and Shatz, 1990; Allendoerfer et al., 1990; Allendoerfer and Shatz, 1994; Bicknese et al., 1994; Dunn et al., 1995; Landry et al., 1998; Hevner et al., 2001; Csillik et al., 2002; Heuer et al., 2003). With regard to cell birthdays, many subplate neurons, especially the pioneer neurons, are produced at the earliest stages of neurogenesis (Allendoerfer and Shatz, 1994). However, our own studies in mice sug- gest that a significant minority of subplate interneurons are produced later in neurogenesis (Hevner et al., 2004). In primates, early neurons in the preplate contain both GABAergic and non-GABAergic (presum- ably glutamatergic) neurons (Zecevic and Milosevic, 1997; Zecevic et al., 1999). After the primate cortical plate is formed, the subplate grows in size by late generated subplate neurons that probably originate in the cortical subventricular zone (Smart et al., 2002; Zecevic et al., 1999, 2005). Previous work by Antonini and Shatz (1990) showed that interneurons and pioneer projection neurons are distinct sets of neurons. In this chapter, we report further studies of the developing mouse and human cortex, in which we show that subplate pioneer neurons and interneurons can be distinguished on the basis of axonal projections, cell birthdays, and—most conveniently—molecular markers. In addition, we show that the same neuron populations can be distinguished in the malformed (overall inverted) cortex of reeler mice, in which subplate pioneer neurons and interneurons occupy abnormal positions in superficial cortex (“superplate’’). 3. Identifying Subplate Neurons Using Markers and Cell Birthdays 3.1. Transcription Factors Tbr1 and Dlx in the Subplate The subplate is the main location of cortical “pioneer’’ neurons, de- fined as neurons that send the earliest axon projections out of the cortex to the internal capsule (McConnell et al., 1989). Our goal was to determine molecular characteristics that distinguish subplate pioneer neurons from cortical interneurons. We hypothesized that the pioneer neurons are glutamatergic projection neurons, which form distinct sub- sets from GABAergic interneurons and their precursors. To identify pioneer neurons in preparation for immunohistochemistry with glutamatergic and GABAergic markers, we placed crystals of DiI (a fluo- rescent retrograde and anterograde axon tracer) in the internal capsule of fixed E16.5 mouse forebrains (Fig. 1A and B). As predicted from previous studies, many subplate neurons were labeled retrogradely, as well as scattered neurons in the intermediate zone and cortical plate (Fig. 1B). The DiI also labeled axon bundles in the subplate and intermediate zone of the cortex, and several neurons in the dorsal thalamus (Fig. 1A). Sec- tions from the DiI-labeled tissues were used for immunohistochemistry to detect Tbr1, a marker of the glutamatergic lineage, and Dlx, a marker of the GABAergic lineage (Hevner et al., 2001; Anderson et al, 1997; 4 Robert F. Hevner and Nada Zecevic Figure 1 Pioneer neurons, labeled retrogradely with DiI, express Tbr1 but not Dlx in E16.5 mouse cortex. A, Pioneer neurons in the subplate of the cortex (ctx) were labeled retrogradely with DiI placed in the internal capsule (asterisk) of fixed E16.5 mouse forebrain. Cells in the dorsal thalamus (dt) were also labeled retrogradely. B, Within the cortex, retrogradely labeled cells were located mainly in the subplate (sp), as well as the deep cortical plate (cp) and intermediate zone (iz). Rarely, the marginal zone (mz) contained retrogradely labeled cells (not shown). C–D, DiI-labeled cells (red) expressed Tbr1 (green). Arrows indicate double labeled cells. (C) shows standard epifluorescence microscopy and (D) shows a confocal image. E–F , DiI-labeled cells (red) did not express Dlx (green). Both (E) and (F ) are confocal images. Scale bar: 1 mm in A;40µmin B–D;80µminE;20µminF . Stuhmer¨ et al., 2002). We found that the DiI-labeled subplate pioneer neurons expressed Tbr1 (Fig. 1C and D) but not Dlx (Fig. 1E and F). These results supported our hypothesis and revealed that within the subplate, Tbr1+ pioneer neurons and Dlx+ interneurons are different neuron types,

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