Parcellation of the mammalian cerebral cortex into distinct areas is essential for proper cortical function; however, the developmental program that results in the genesis of distinct areas is not fully understood. We examined the expression of members of the EphA family-the EphA receptor tyrosine kinases and the ephrin-A ligands-within the developing mouse cerebral cortex, with the aim of characterizing this component of the molecular landscape during cortical parcellation. We found that specific embryonic zones, such as the ventricular, subventricular, intermediate, subplate, and marginal zones, as well as the cortical plate, were positive for particular EphA genes early in corticogenesis (E12-E15). Along with this zone-selective expression, several genes (EphA3, EphA4, EphA5) were evenly expressed along the axes of the developing cortex, whereas one family member (EphA7) was expressed in a distinct anteroposterior pattern. Later in corticogenesis (E16-E18), other EphA family members became selectively expressed, but only within the cortical plate: EphA6 was present posteriorly, and ephrin-A5 was expressed within a middle region. At birth, patterning of EphA gene expression was striking. Thus, we found that the expression of a single EphA gene or a combination of family members can define distinct embryonic zones and anteroposterior regions of the neocortex during development. To examine whether cellular context affects the patterning of EphA expression, we examined gene expression in embryonic cortical cells grown in vitro, such that all cellular contacts are lacking, and in Mash-1 mutant mice, in which thalamocortical connections do not form. We found that the expression patterns of most EphA family members remained stable in these scenarios, whereas the pattern of ephrin-A5 was altered. Taken together, this work provides a comprehensive picture of EphA family expression during mouse corticogenesis and demonstrates that most EphA expression profiles are cell intrinsically based, whereas ephrin-A5 is plastically regulated.

Visual cortex in mammals is composed of many distinct areas that are linked by reciprocal connections to form a multilevel hierarchy. Ascending information is sent via forward connections from lower to higher areas and is thought to contribute to the emergence of increasingly complex receptive field properties at higher levels. Descending signals are transmitted via feedback connections from higher to lower areas and are believed to provide information about the context in which a stimulus appears, to contribute to modulation of visual responses by attention, and to play a role in memory processes. To determine whether forward and feedback pathways in rat visual cortex constitute distinct intracortical circuits, we have studied the distribution of reciprocal corticocortical inputs to pyramidal cells and gamma-aminobutyric acid (GABA)ergic interneurons. For this purpose, we chose forward and feedback connections between primary visual cortex and the secondary extrastriate lateromedial (LM) area as a model system. Pathways were traced with the axonal marker phaseolus vulgaris-leucoagglutinin. Labeled terminals were identified in the electron microscope, and GABA immunocytochemistry was used to identify the postsynaptic dendritic shafts of GABAergic interneurons. In both pathways, inputs to pyramidal cells were directed preferentially to dendritic spines and not to shafts. In the forward pathway, 90% of labeled inputs were distributed to pyramidal cells and 10% to interneurons. This proportion was similar to that of nearby unlabeled connections in the neuropil, indicating that forward connections are not selective for pyramidal cells or interneurons. In sharp contrast, feedback connections were significantly different from the unlabeled connections and supplied almost exclusively pyramidal cells (98%). Feedback inputs to GABAergic neurons were five times weaker (2%) relative to the forward direction. These structural differences suggest that disynaptic GABAergic inhibition is much stronger in forward than in feedback pathways. Recent physiological experiments have confirmed this prediction (Shao et al. [1995] Soc. Neurosci. Abstr., 21:1274) and we, therefore, conclude that relatively small anatomical differences in the microcircuitry can have important functional consequences. It remains an open question whether generally reciprocal interareal circuits at all levels of the cortical hierarchy are organized in similar fashion.

Feedback connections from extrastriate cortex to primary visual cortex (V1) in the primate may provide "top-down" information that plays a role in visual attention and object recognition. Our work in a rodent model of corticocortical circuitry demonstrates that feedback pathways synapse preferentially with pyramidal cells in V1 (Johnson and Burkhalter, 1996) and favor excitation over inhibition in cortical microcircuits .To investigate the polysynaptic circuits activated by feedback inputs, we studied chains of neurons postsynaptic to feedback connections using a combination of axonal tract tracing and anterograde degeneration. This approach enabled independent labeling of local collaterals of forward-projecting neurons in V1 and feedback connections from extrastriate lateromedial (LM) visual area to V1. Postsynaptic targets were identified in the electron microscope after retrograde transport of biotinylated dextran amine (BDA) to identify dendrites of forward-projecting neurons (i.e., from V1 to LM) and postembedding immunogold labeling to identify GABAergic interneurons.The results show that feedback connections provide strong monosynaptic input to forward-projecting neurons in V1. These neurons in turn make local connections that preferentially form synapses with other pyramidal cells (ϳ97%), many of which were identified as forward-projecting neurons. This indicates that feedback pathways provide input directly to neurons which make the reciprocal forward connection, and that feedbackrecipient forward-projecting neurons are strongly interconnected. The function of these excitatory networks within V1 may be to amplify feedback activity and provide a circuit for modulation of striate cortical activity by top-down influences.

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Changes in N-methyl-D-aspartate (NMDA) receptor expression may represent a molecular substrate for differences in synaptic plasticity between early postnatal and adult brains (Fox and Zahs [1994] Curr. Opinion Neurobiol. 4:112-119). We have, therefore, examined the regional and laminar distribution of NR1, the essential subunit of the NMDA receptor, in two regions in which synaptic plasticity has been most thoroughly studied: primary visual cortex and hippocampus. To study NR1 expression at the light and electron microscopic levels we have used a new antiserum (NR1-C1; Sheng et al. [1994] Nature 368:144-147) directed against a differentially spliced C-terminal exon ("C1"). The most striking result was that the pattern of NR1-C1 labeling in the adult was more restricted than that of previously published NR1-specific antibodies. Specifically, NR1-C1 did not label cells in the CA3, dentate gyrus or subicular regions of the hippocampus or in layer 4 of the visual cortex. Quantitative ultrastructural analysis revealed that these differences were paralleled by differential expression of NR1-C1 at synapses. In sharp contrast to the pattern in the adult, NR1-C1 immunoreactivity was distributed more widely in the developing brain. At postnatal day 11, NR1-C1 splice variants were expressed in all layers of the visual cortex and in all regions of the hippocampus. The transient expression of NR1-C1 splice variants in layer 4 of visual cortex suggests that NR1-C1 may play a role in determining the critical period for binocular plasticity. Continued expression of NR1-C1 in upper and lower layers of the adult cortex and in CA1 of the hippocampus may provide a substrate for plasticity in corticocortical connections and Schaffer collateral synapses beyond the critical period. In addition to abundant postsynaptic staining, NR1-C1 immunoreactivity was found in a large number of axon terminals in the dorsal subiculum, but in very few terminals in visual cortex. This strongly suggests that presynaptic NMDA receptors play a major role in neuronal processing of hippocampal output through the subiculum, but play a relatively minor role in visual processing.

It is a commonly accepted notion that cells which make projections between the multiple cortical areas found in the mammalian visual system are excitatory, but there is little direct evidence that this is the case. Here we demonstrate using retrograde tracing with D-[3H]aspartate that connections in the rat which project from lower to higher visual areas (i.e. forward) and those which project from higher to lower areas (i.e. feedback) may use excitatory amino acid neurotransmitters. Following injection into the primary visual cortex, clusters of retrogradely labelled cells were found in several extrastriate areas within the cytoarchitectonic subdivisions 18a ('areas' LM, AL, PX, FLX, RL, AX) and 18b ('area' MX), and in the retrosplenial cortex. In all of these areas D-[3H]aspartate-labelled cells were surrounded by diffuse label which may represent anterograde labelling of axon terminals. This suggests that both legs of reciprocal intracortical circuits have similar chemospecificity. To directly demonstrate excitatory amino acid localization in forward projections, D-[3H]aspartate was injected into extrastriate area LM. As expected, the results revealed retrogradely labelled neurons within area 17. Outside area 17, LM injections labelled neurons in AL, PX, FLX, RL, AX and MX. Taken in the context of the hierarchy of areas in rat cerebral cortex (Coogan and Burkhalter, J. Neurosci., 13, 3749-3772, 1993), these results show that D-[3H]aspartate labels: (1) forward connections from area 17 to LM, AL, PX, RL, AX and MX, (2) feedback connections from LM, AL, FLX, PX, RL, AX and MX to area 17, (3) feedback connections from AL, PX, RL, AX and MX to LM, and (4) lateral connections between FLX and LM. These findings strongly indicate that both forward and feedback connections as well as lateral connections at several different levels of the cortical hierarchy use excitatory amino acid neurotransmitters.