Molecular markers distinguishing supragranular and infragranular layers in the human prefrontal cortex Dominique Arion, 1 Travis Unger, 1 David A. Lewis 1,2 and Ka ´roly Mirnics 1,3 1 Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA 15261, USA 2 Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15261, USA 3 Department of Psychiatry, Vanderbilt Kennedy Center for Human Development, Vanderbilt University, Nashville, TN 37203, USA Keywords: DNA microarray, gene expression, in situ hybridization, laser capture microscopy Abstract The human neocortex is organized into six layers that are differentiated by the size and packing density of their constituent neurons. The gene products that guide the establishment of this lamination have been studied extensively, but the gene expression gradients present across the layers of the adult human neocortex are mostly unknown. As the supragranular (SG) and infragranular (IG) layers of the human prefrontal cortex (PFC) differ in their connectivity and developmental time course, we hypothesized that the SG and IG layers will show distinct differences in their transcriptomes. To test this prediction, we used laser capture microdissection coupled with DNA microarray transcriptome profiling. Sixty-nine genes exhibited robust and highly consistent expression differences between the SG and IG layers. For six selected markers, in addition to validating the microarray findings, in situ hybridization revealed a complex, subpopulation-specific neuronal distribution. The markers we identified are likely to be related to the functional differences between the SG and IG layers of the human PFC and can be used for assessing alterations in structure and function of this cortical region in human brain disorders. Introduction The human neocortex is organized into six layers that are differen- tiated by the size and packing density of their constituent neurons. These layers arise as the result of neuronal migration in both the tangential (Anderson et al., 1997) and radial (Angevine & Sidman, 1961) directions during cortical development. During embryogenesis, newly born neocortical projection neurons migrate in successive waves along radial glia, ultimately giving rise to the cortical plate in an inside-out fashion: neurons populating the deep layers are the first ones to differentiate; while the last-born neurons populate the most superficial layers (Angevine & Sidman, 1961; Rakic, 1988; Caviness & Takahashi, 1995; Caviness et al., 1995; Rakic & Caviness, 1995; Gupta et al., 2002; Job & Tan, 2003). Cortical layers also have distinct patterns of connectivity (Somogyi et al., 1998; Bannister, 2005). For example, pyramidal neurons in layers II and III project principally to other cortical regions, those in layer V to the striatum and brainstem, and those in layer VI to the thalamus (Lewis et al., 2002; Rockland, 2004; Bannister, 2005). Diseases of the human neocortex often show a restricted, lamina- and ⁄ or cell type-specific pathology. For example, in schizophrenia large pyramidal cells of layer III show reduced cell size (Rajkowska et al., 1998; Sweet et al., 2003, 2004), while reelin expression deficits are primarily found in the most superficial layers (D’Arcangelo, 2006). Furthermore, cortical lamination deficits have been observed in lissencephaly (Dobyns et al., 1993; Dobyns & Truwit, 1995; Lo Nigro et al., 1997; Pilz et al., 1998), different forms of heterotopia (Gupta et al., 2002), autism (Mukaetova-Ladinska et al., 2004) and intractable epilepsy (Bentivoglio et al., 2003; Powell et al., 2003). Thus, identifying molecular markers of specific layers and neuronal subpopulations in the human neocortex is essential for advancing our understanding of these disorders. The differences in neuronal composition, developmental timing and connectivity across cortical layers strongly suggest the existence of a large number of genes with lamina-specific patterns of expression. However, such lamina-specific markers of the adult human neocortex remain mostly unknown at the present time. In this study, we hypothesized that supragranular (SG; laminae II–III) and infragranular layers (IG; laminae V–VI) of area 46 in the human prefrontal cortex (PFC) show marked differences in gene expression patterns. Given the laminar differences in projection targets of pyramidal neurons, we also hypothesized that some of these markers are specifically expressed by pyramidal neurons. Materials and methods Human brain samples This study conforms to The Code of Ethics of the World Medical Association. Brain specimens were obtained during autopsies conducted at the Allegheny County Coroner’s Office (Pittsburgh, PA) after consent was obtained from the next-of-kin using a protocol approved by the University of Pittsburgh’s Institutional Review Board for Biomedical Research. An independent committee of experienced research clinicians made consensus DSMIV (Diagnosis and Statistical Manual of Mental Disorders, 1994) diagnoses for each subject on the basis of medical records and the results of structured interviews conducted with family members of the deceased. Five male post mortem human brains were used in this study (Table 1). Fresh-frozen human tissue was obtained from the University of Pittsburgh’s Center for the Neuroscience of Mental Disorders Brain Bank. Four of the five Correspondence: Dr K. Mirnics, 3 Department of Psychiatry, as above. E-mail: karoly.mirnics@vanderbilt.edu Received 25 September 2006, revised 18 December 2006, accepted 3 January 2007 European Journal of Neuroscience, Vol. 25, pp. 1843–1854, 2007 doi:10.1111/j.1460-9568.2007.05396.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd