The neurons of layer 4 in the adult cerebral cortex receive their major ascending inputs from the thalamus. In development, however, thalamic axons arrive at the appropriate cortical area long before their target layer 4 neurons have migrated into the cortical plate. The axons accumulate and wait in the zone below the cortical plate, the subplate, for several weeks before invading the cortical plate. The subplate is a transient zone that contains the first postmitotic neurons of the telencephalon. These neurons mature well before other cortical neurons, and disappear by cell death after the thalamic axons have grown into the overlying cortical plate. The close proximity of growing thalamocortical axons and subplate neurons suggests that they might be involved in interactions important for normal thalamocortical development. Here we show that early in development the deletion of subplate neurons located beneath visual cortex prevents axons from the lateral geniculate nucleus of the thalamus from recognizing and innervating visual cortex, their normal target. In the absence of subplate neurons, lateral geniculate nucleus axons continue to grow in the white matter past visual cortex despite the presence of their target layer 4 neurons. Thus the transient subplate neurons are necessary for appropriate cortical target selection by thalamocortical axons.

If vision in one eye is blurred or occluded during a critical period in postnatal development, neurons in the visual cortex lose their responses to stimulation through that eye within a few days. Anatomical changes in the nerve terminals that provide input to the visual cortex have previously been observed only after weeks of deprivation, suggesting that synapses become physiologically ineffective before the branches on which they sit are withdrawn. Reconstruction of single geniculocortical axonal arbors in the cat after either brief or prolonged monocular occlusion revealed striking axonal rearrangements in both instances. Rapid withdrawal of the branches of deprived-eye arbors suggests that axonal branches bearing synapses respond quickly to changing patterns of neuronal activity.

Much of what is known about activity-dependent plasticity comes from studies of the primary visual cortex and its inputs in higher mammals, but the molecular bases remain largely unknown. Similar functional plasticity takes place during a critical period in the visual cortex of the mouse, an animal in which genetic experiments can readily be performed to investigate the underlying molecular and cellular events. The experiments of this paper were directed toward understanding whether anatomical changes accompany functional plasticity in the developing visual cortex of the mouse, as they do in higher mammals. In normal mice, transneuronal label after an eye injection clearly delineated the monocular and binocular zones of area 17. Intrinsic signal optical imaging also showed monocular and binocular zones of area 17 but revealed no finer organization of ocular dominance or orientation selectivity. In normal animals, single geniculocortical afferents serving the contralateral eye showed great heterogeneity and no clustering consistent with the presence of ocular dominance patches. Growth and elaboration of terminal arbor continues beyond postnatal day 40 (P40), after the peak of the critical period. After prolonged monocular deprivation (MD) from P20 to P60, transneuronal labeling showed that the projection serving the ipsilateral eye was severely affected, whereas the effect on the contralateral eye's pathway was inconsistent. Optical imaging also showed profound effects of deprivation, particularly in the ipsilateral pathway, and microelectrode studies confirmed continued functional plasticity past P40. Reconstruction of single afferents showed that MD from P20 to P40 promoted the growth of the open eye's geniculocortical connections without causing the closed eye's contralateral projection to shrink, whereas MD from P20 to P60 caused an arrest of growth of deprived arbors. Our findings reveal numerous similarities between mouse and higher mammals in development and plasticity, along with some differences. We discuss the factors that may be responsible for these differences.

This study analyzes the morphological changes in geniculocortical axons terminating in the primary visual cortex of the cat, during the period in which, in normal development, the terminals in layer IV undergo an eye-specific segregation. Geniculocortical afferent fibers were filled anterogradely by the Phaseolus lectin (PHA-L) injected into the main laminae of the LGN. After standard immunohistochemical procedures, single axons were serially reconstructed in two or three dimensions. Experiments were performed in normal kittens and in kittens in which retinal activity was continuously blocked by repeated intraocular injections of TTX. In normal kittens, arbors were reconstructed at four different ages (19, 23, 30-31, and 39 days postnatally) spanning the period during which the geniculocortical projection segregates into eye-specific columns in layer IV (LeVay et al., 1978). Results reveal that sparse but widely extending branches characteristic of young arbors are eliminated during normal development at the same time as selected portions of the arbor grow considerably in length and complexity. The terminal arborizations also subdivide into distinct patches of terminals, consistent with the segregation of left and right eye afferents. In TTX-treated animals, axonal arbors reconstructed at postnatal days 23, 29, and 39 show a complexity and extent of terminal arborization similar to that of normal animals, though more variable in size and degree of elaboration. No progressive changes are evident with age. Further, the majority of arbors reconstructed from TTX-treated animals lack the patchy organization typical of normal animals.

Thalamic axons are known to accumulate in the subplate for a protracted period prior to invading the cortical plate and contacting their ultimate targets, the neurons of layer 4. We have examined the synaptic contacts made by visual and somatosensory thalamic axons during the transition period in which axons begin to leave the subplate and invade the cortical plate in the ferret. We first determined when geniculocortical axons leave the subplate and begin to grow into layer 4 of the visual cortex by injecting 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine (Dil) into the lateral geniculate nucleus (LGN). By birth most LGN axons are still confined to the subplate. Over the next 10 days LGN axons grow into layer 4, but many axons retain axonal branches within the subplate. To establish whether thalamic axons make synaptic contacts within the subplate, the anterograde tracer PHA-L was injected into thalamic nuclei of neonatal ferrets between postnatal day 3 and 12 to label thalamic axons at the electron microscope level. The analysis of the PHA-L injections confirmed the Dil data regarding the timing of ingrowth of thalamic axons into the cortical plate. At the electron microscope level, PHA-L-labelled axons were found to form synaptic contacts in the subplate. The thalamic axon terminals were presynaptic primarily to dendritic shafts and dendritic spines. Between postnatal days 12 and 20 labelled synapses were also observed within layer 4 of the cortex. The ultrastructural appearance of the synapses did not differ significantly in the subplate and cortical plate, with regard to type of postsynaptic profiles, length of postsynaptic density or presynaptic terminal size. These observations provide direct evidence that thalamocortical axons make synaptic contacts with subplate neurons, the only cell type within the subplate possessing mature dendrites and dendritic spines; they also suggest that functional interactions between thalamic axons and subplate neurons could play a role in the establishment of appropriate thalamocortical connections.

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Previous anatomic studies of the geniculocortical projection showed that ocular dominance columns emerge by 3 weeks of age in cat visual cortex, but recent optical imaging experiments have revealed a pattern of physiologic eye dominance by the end of the second week of life. We used two methods to search for an anatomic correlate of this early functional ocular dominance pattern. First, retrograde labeling of lateral geniculate nucleus (LGN) inputs to areas of cortex preferentially activated by one eye showed that the geniculocortical projection was already partially segregated by eye at postnatal day 14 (P14). Second, transneuronal label of geniculocortical afferents in flattened sections of cortex after a tracer injection into one eye showed a periodic pattern at P14 but not at P7. In the classic model for the development of ocular dominance columns, initially overlapping geniculocortical afferents segregate by means of an activity-dependent competitive process. Our data are consistent with this model but suggest that ocular dominance column formation begins between P7 and P14, approximately a week earlier than previously believed. The functional and anatomic data also reveal an early developmental bias toward contralateral eye afferents. This initial developmental bias is not consistent with a strictly Hebbian model for geniculocortical afferent segregation. The emergence of ocular dominance columns before the onset of the critical period for visual deprivation also suggests that the mechanisms for ocular dominance column formation may be partially distinct from those mediating plasticity later in life.

During development, the earliest generated neurons of the mammalian telencephalon reside in a region of the white matter, the subplate, just beneath the cortical plate. Neurons in the subplate are only transiently present in the telencephalon: shortly after birth in the cat the majority have disappeared. During their brief life, however, subplate neurons mature; they extend long-distance and local projections, and express immunoreactivity for GABA and several neuropeptides. In the present study we examined the relation between possible transmitter phenotypes of subplate neurons and their connectivity. To do so, we used a double-label technique in which immunohistochemistry for neuropeptide Y (NPY), somatostatin (SRIF) or calbindin (CaBP) was combined with retrograde tracing. Experiments were performed in neonatal cats and in ferret kits at equivalent postconceptional ages, times when subplate neurons are numerous. Subplate neurons immunoreactive for neuropeptides and CaBP could be double-labelled by an injection of retrograde tracer either into the cortical plate or the white matter, indicating that this particular subset of subplate neurons can make local circuit projections. In contrast, peptide or CaBP immunoreactive subplate neurons could never be retrogradely labelled from a tracer injection into the thalamus. Taken together, these observations indicate that subplate neurons immunoreactive for NPY, SRIF and CaBP are likely to be interneurons exclusively. On the other hand, subplate neurons with long-distance projections to the thalamus or the contralateral hemisphere could be labelled by the retrograde transport of d-[3H]aspartate, suggesting that at least some projection subplate neurons might use an excitatory amino acid as a neurotransmitter. These results indicate that there is a defined relationship between the putative transmitter phenotypes of subplate neurons and their patterns of projection. Interneurons of the subplate express peptidergic properties while projection neurons to the thalamus may use an excitatory amino acid. Thus, these basic organizational features of the transient subplate are reminiscent of those found in the adult cortical layers.