Circuitry Underlying Experience-Dependent Plasticity in the Mouse Visual System.

Since the discovery of ocular dominance plasticity, neuroscientists have understood that changes in visual experience during a discrete developmental time, the critical period, trigger robust changes in the visual cortex. State-of-the-art tools used to probe connectivity with cell-type-specific resolution have expanded the understanding of circuit changes underlying experience-dependent plasticity. Here, we review the visual circuitry of the mouse, describing projections from retina to thalamus, between thalamus and cortex, and within cortex. We discuss how visual circuit development leads to precise connectivity and identify synaptic loci, which can be altered by activity or experience. Plasticity extends to visual features beyond ocular dominance, involving subcortical and cortical regions, and connections between cortical inhibitory interneurons. Experience-dependent plasticity contributes to the alignment of networks spanning retina to thalamus to cortex. Disruption of this plasticity may underlie aberrant sensory processing in some neurodevelopmental disorders.

Refinement of Spatial Receptive Fields in the Developing Mouse Lateral Geniculate Nucleus Is Coordinated with Excitatory and Inhibitory Remodeling.

Receptive field properties of individual visual neurons are dictated by the precise patterns of synaptic connections they receive, including the arrangement of inputs in visual space and features such as polarity (On vs Off). The inputs from the retina to the lateral geniculate nucleus (LGN) in the mouse undergo significant refinement during development. However, it is unknown how this refinement corresponds to the establishment of functional visual response properties. Here we conducted in vivo and in vitro recordings in the mouse LGN, beginning just after natural eye opening, to determine how receptive fields develop as excitatory and feedforward inhibitory retinal afferents refine. Experiments used both male and female subjects. For in vivo assessment of receptive fields, we performed multisite extracellular recordings in awake mice. Spatial receptive fields at eye-opening were >2 times larger than in adulthood, and decreased in size over the subsequent week. This topographic refinement was accompanied by other spatial changes, such as a decrease in spot size preference and an increase in surround suppression. Notably, the degree of specificity in terms of On/Off and sustained/transient responses appeared to be established already at eye opening and did not change. We performed in vitro recordings of the synaptic responses evoked by optic tract stimulation across the same time period. These recordings revealed a pairing of decreased excitatory and increased feedforward inhibitory convergence, providing a potential mechanism to explain the spatial receptive field refinement.SIGNIFICANCE STATEMENT The development of precise patterns of retinogeniculate connectivity has been a powerful model system for understanding the mechanisms underlying the activity-dependent refinement of sensory systems. Here we link the maturation of spatial receptive field properties in the lateral geniculate nucleus (LGN) to the remodeling of retinal and inhibitory feedforward convergence onto LGN neurons. These findings should thus provide a starting point for testing the cell type-specific plasticity mechanisms that lead to refinement of different excitatory and inhibitory inputs, and for determining the effect of these mechanisms on the establishment of mature receptive fields in the LGN.

Developmental changes in medial auditory thalamic contributions to associative motor learning.

Eyeblink conditioning (EBC) was used in the current study to examine the mechanisms underlying the ontogeny of associative motor learning in rats. Eyeblink conditioning emerges ontogenetically between postnatal day 17 (P17) and P24 in rats. Previous studies used electrical stimulation to show that the ontogeny of EBC is influenced by developmental changes in input from the medial auditory thalamus to the pontine nuclei, which in turn affects input to the cerebellum. The current study used tetrode recordings to examine the ontogeny of medial auditory thalamic sensory responses to the conditioned stimulus (CS) and learning-related activity during EBC. Rat pups were implanted with multiple tetrodes in the medial nucleus of the medial geniculate (MGm) and suprageniculate (SG) and trained on delay EBC on P17-P19, P24-P26, or P31-P33 while recording spike activity. Developmental changes in MGm and SG sensory-related activity were found during a pretraining session with unpaired presentations of the auditory CS and periorbital stimulation unconditioned stimulus (US). Substantial developmental changes were observed in learning-related activity in the MGm and SG during CS-US paired training. The ontogenetic changes in learning-related activity may be related to developmental changes in input to the medial auditory thalamus from the amygdala and cerebellum. The findings suggest that the ontogeny of associative motor learning involves developmental changes in sensory input to the thalamus, amygdala input to the thalamus, thalamic input to the pontine nuclei, and cerebellar feedback to the thalamus.

Neuronal pentraxins mediate silent synapse conversion in the developing visual system.

Neuronal pentraxins (NPs) are hypothesized to play important roles in the recruitment of AMPA receptors (AMPARs) to immature synapses, yet a physiological role for NPs at nascent synapses in vivo has remained elusive. Here we report that the loss of NP1 and NP2 (NP1/2) leads to a dramatic and specific reduction in AMPAR-mediated transmission at developing visual system synapses. In thalamic slices taken from early postnatal mice (

Differential gene expression in the developing lateral geniculate nucleus and medial geniculate nucleus reveals novel roles for Zic4 and Foxp2 in visual and auditory pathway development.

Primary sensory nuclei of the thalamus process and relay parallel channels of sensory input into the cortex. The developmental processes by which these nuclei acquire distinct functional roles are not well understood. To identify novel groups of genes with a potential role in differentiating two adjacent sensory nuclei, we performed a microarray screen comparing perinatal gene expression in the principal auditory relay nucleus, the medial geniculate nucleus (MGN), and principal visual relay nucleus, the lateral geniculate nucleus (LGN). We discovered and confirmed groups of highly ranked, differentially expressed genes with qRT-PCR and in situ hybridization. A functional role for Zic4, a transcription factor highly enriched in the LGN, was investigated using Zic4-null mice, which were found to have changes in topographic patterning of retinogeniculate projections. Foxp2, a transcriptional repressor expressed strongly in the MGN, was found to be positively regulated by activity in the MGN. These findings identify roles for two differentially expressed genes, Zic4 and Foxp2, in visual and auditory pathway development. Finally, to test whether modality-specific patterns of gene expression are influenced by extrinsic patterns of input, we performed an additional microarray screen comparing the normal MGN to "rewired" MGN, in which normal auditory afferents are ablated and novel retinal inputs innervate the MGN. Data from this screen indicate that rewired MGN acquires some patterns of gene expression that are present in the developing LGN, including an upregulation of Zic4 expression, as well as novel patterns of expression which may represent unique processes of cross-modal plasticity.

Development of auditory thalamocortical connections in the pallid bat, Antrozous pallidus.

Auditory thalamocortical connections are organized as parallel pathways originating in various nuclei of the medial geniculate body (MGB). The development of these pathways has not been studied. Therefore it remains unclear whether thalamocortical connections segregate before the onset of hearing or whether refinement of exuberant thalamocortical connections occurs following hearing onset. We studied this issue in the pallid bat. In adult pallid bats, parallel thalamocortical pathways represent two different sounds used in two different behaviors. The suprageniculate (SG) nucleus of the dorsal division of the MGB (MGBd) projects to a high-frequency cortical region selective for the echolocation calls, but not to a low-frequency cortical region sensitive to noise transients used in the localization of prey. Conversely, the ventral division (MGBv) projects to the low-frequency, but not the high-frequency, cortical region. Here we studied the development of these parallel pathways. Based on retrograde tracer injections in electrophysiologically characterized cortical loci, we show that there is an asymmetrical overlap in projection patterns from postnatal (P) day 15-60. The low-frequency region receives extensive input from both the SG and the MGBv. In contrast, the high-frequency region receives the great majority of its input from the SG, as in adults, whereas projections from the MGBv appear to make only a minor contribution, if any. By P150, these pathways are segregated and adult-like. These data suggest that these anatomically segregated pathways arise through postnatal refinement of initially overlapping connections.

Development of the diencephalic relay structures of the visual thalamofugal system in pigeons.

To compare the developmental pattern of the visual tecto- and thalamofugal pathways in the altricial pigeon, we examined the posthatch differentiation of the retinothalamic system. Choleratoxin was injected into the left and right eye to visualize the retinal innervation pattern of the lateral geniculate nucleus of the thalamus (GLd). The calcium-binding proteins parvalbumin and calbindin and GABA(Abeta) receptors were used as indicators for the functional development of the GLd. Although all retinorecipient thalamic target structures were invaded by retinal fibers directly after hatching, density of the projection increased during the first week. While the adult GLd was characterized by a substantial number of cells displaying calbindin-immunoreactivity and by a sparse innervation by parvalbumin-immunoreactive fibers, after hatching no labelling for calcium-binding proteins could be detected. Calbindin-immunoreactivity appeared not before posthatching day 7, while parvalbumin-immunoreactive fibers were detected only after the third week. In contrast, a dense but diffuse GABA(Abeta) receptor-labelling was present from hatching onwards that decreased during development. The delayed expression of calbindin as well as changes in the density of GABA(Abeta) receptors indicate that maturation of GLd neurons extends long into the posthatch period. It is likely that the GABAergic interneurons mainly develop within this posthatch timeframe. Combined with the delayed development of the parvalbumin-positive innervation, the developmental pattern of GLd neurons suggests that the thalamofugal networks are immature after hatching and therefore still sensitive to modulations of posthatch visual experience.

Developmental refinement in the mammalian thalamus.

The sensory relay synapses in the thalamus undergo extensive refinement during early life. Disruptions of spontaneous activity, but not sensory deprivation, can induce large-scale re-organization of neuronal connections in the thalamus. Recent studies also reveal an extended period of synaptic refinement in the visual and somatosensory relay synapses, where sensory deprivation produces some unexpected effects on synaptic remodeling. This article aims to provide a brief overview of recent findings and current ideas about the refinement of relay synapses in the thalamus.

Metallothionein I and II mitigate age-dependent secondary brain injury.

Both the immediate insult and delayed apoptosis contribute to functional deficits after brain injury. Secondary, delayed apoptotic death is more rapid in immature than in adult CNS neurons, suggesting the presence of age-dependent protective factors. To understand the molecular pathobiology of secondary injury in the context of brain development, we identified changes in expression of oxidative stress response genes during postnatal development and target deprivation-induced neurodegeneration. The antioxidants metallothionein I and II (MT I/II) were increased markedly in the thalamus of adult C57BL/6 mice compared to mice <15 days old. Target deprivation generates reactive oxygen species that mediate neuronal apoptosis in the central nervous system; thus the more rapid apoptosis observed in the immature brain might be due to lower levels of MT I/II. We tested this hypothesis by documenting neuronal loss after target-deprivation injury. MT I/II-deficient adult mice experienced greater thalamic neuron loss at 96 hr after cortical injury compared to that in controls (80 +/- 2% vs. 57 +/- 4%, P < 0.01), but not greater overall neuronal loss (84 +/- 4% vs. 79 +/- 3%, MT I/II-deficient vs. controls). Ten-day-old MT I/II-deficient mice, however, experienced both faster onset of secondary neuronal death (30 vs. 48 hr) and greater overall neuronal loss (88 +/- 2% vs. 69 +/- 4%, P = 0.02). MT I/II are thus inhibitors of age-dependent secondary brain injury, and the low levels of MT I/II in immature brains explains, in part, the enhanced susceptibility of the young brain to neuronal loss after injury. These findings have implications for the development of age-specific therapeutic strategies to enhance recovery after brain injury.

Diverse receptive fields in the lateral geniculate nucleus during thalamocortical development.

Most models of thalamocortical development in the visual system assume a homogeneous population of thalamic inputs to the cortex, each with concentric on- or off-center receptive fields. To test this, we made high-resolution spatial maps of receptive fields in the developing ferret lateral geniculate nucleus (LGN). Developing receptive fields (RFs), had a variety of shapes: some concentric, others elongated (like adult cortical receptive fields) and some with 'hot spots' of sensitivity. These receptive fields seemed to arise from convergence of multiple retinal afferents onto LGN neurons. We present a Hebbian model whereby imprecise retinogeniculate connections help refine geniculocortical connections, sharpening both thalamocortical topography and perhaps orientation selectivity.

Correlational structure of spontaneous neuronal activity in the developing lateral geniculate nucleus in vivo.

The properties of spontaneous activity in the developing visual pathway beyond the retina are unknown. Multielectrode recordings in the lateral geniculate nucleus (LGN) of awake behaving ferrets, before eye opening, revealed patterns of spontaneous activity that reflect a reshaping of retinal drive within higher visual stages. Significant binocular correlations were present only when cortico-thalamic feedback was intact. In the absence of retinal drive, cortico-thalamic feedback was required to sustain correlated LGN bursting. Activity originating from the contralateral eye drove thalamic activity far more strongly than that originating from the ipsilateral eye. Thus, in vivo patterns of LGN spontaneous activity emerge from interactions between retina, thalamus, and cortex.

Pattern formation by retinal afferents in the ferret lateral geniculate nucleus: developmental segregation and the role of N-methyl-D-aspartate receptors.

The projection from the retina to the lateral geniculate nucleus (LGN) in ferrets segregates during development into eye-specific layers and ON/OFF sublayers. The projection pattern and the morphology of single axons was examined at several postnatal ages. The axons progress from a simple, sparsely branched morphology at birth to crude arbors at postnatal day 7 (P7). At P14-P15, axons have terminal arbors that span one eye-specific layer. By P19-P21, retinal afferents in the A layers have segregated into inner and outer sublaminae that correspond to ON- and OFF-center cells. Sublaminae form mainly by directed growth of terminal arbors in appropriately positioned regions of the LGN, along with elimination of extraneous branches in inappropriate regions. From P28 to P35, the LGN assumes an adult-like shape, and retinogeniculate axons form terminal boutons on branch endings. During the period between P14 and P21, when retinogeniculate axons segregate into ON/OFF sublaminae, N-methyl-D-aspartate (NMDA) receptors were blocked with chronic infusion of specific antagonists into the LGN. NMDA receptor blockade prevents the retinal afferent segregation into ON/OFF sublaminae. Some individual retinogeniculate axons have arbors that are not restricted appropriately, and most are restricted in size but are located inappropriately within the eye-specific laminae. Thus, NMDA receptor blockade prevents the positioning of retinogeniculate arbors that lead to the formation of ON/OFF sublaminae in the LGN. These results indicate that the activity of postsynaptic cells, and the activation of NMDA receptors in particular, can influence significantly the patterning of inputs and the structure of presynaptic afferents during development.

Postnatal cytoarchitecture of the rat medial geniculate body.

The medial geniculate body (MGB) is a thalamic structure that provides vital information flow to the forebrain for complex acoustic processing. The development of cytoarchitectural features of the MGB was examined in rat to identify age-related patterns of growth in major geniculate compartments that have been described previously (Clerici and Coleman [1990] J. Comp. Neurol. 297:14-31; Clerici et al. [1990] J. Comp. Neurol. 297:32-54): the ventral (MGv), dorsal (MGd), and medial (MGm) divisions. Results show that, on the day of parturition, all major nuclei of each division are characterized, including the ovoid (OV) and ventral (LV) nuclei of MGv; the dorsal, deep dorsal (DD), caudodorsal, limitans, and suprageniculate nuclei of MGd; and the MGm. The MGv and MGd, which display comparable areas at birth, show rapid growth to postnatal day 7 (PND7), which then slows until PND11, around the time of ear canal opening; subsequently, MGv accelerates growth to reach larger adult size. From PND11 to PND16, thionin facilitates parcellation by extensive staining of dendritic processes of MGd, MGm, and lateral posterior nucleus neurons but not neurons of the MGv or the dorsal lateral geniculate nucleus. Golgi stains after birth reveal restricted dendritic arborizations in MGv cells and dichotomous branching patterns of MGd neurons. Somal size in MGB increases dramatically subsequent to afferent innervation and again following onset of auditory function. Somal growth occurs between all postnatal age groups tested for OV, LV, and DD nuclei, although LV segments related to high and low frequencies do not differ. Cell packing density predicts the expanse of major MGB divisions better than somal size. These results demonstrate the integrity and growth patterns of MGB nuclei and divisions from nascence and provide a substrate for subsequent study of anatomical and physiological development of the MGB.

Nitric oxide synthase in the adult and developing thalamus: histochemical and immunohistochemical study in the rat.

The distribution of neuronal elements that express nitric oxide synthase (NOS), the synthetic enzyme of the free radical nitric oxide, was investigated in the adult and developing rat thalamus by means of NADPH-diaphorase (NADPH-d) histochemistry, which is a marker of NOS. Immunocytochemistry was also used to confirm the equivalence between the histochemical pattern of staining and the distribution of the expression of the neuronal NOS isoform. In the adult thalamus, NADPH-d-positive and NOS-immunoreactive perikarya were selectively concentrated along the midline (in the paraventricular, rhomboid, and central medial nuclei) and in the dorsal and ventral lateral geniculate nuclei. Isolated clusters of stained neurons were also observed in the lateral posterior nucleus, in the dorsal part of the medial geniculate nucleus, and in the ventromedial nucleus. Positive perikarya were either absent or very sparse in the other thalamic nuclei. Many thalamic domains were, however, characterized by distinct patterns of NADPH-d-positive fibers, preterminal and terminal-like elements. The highest density of stained neuropil was observed in the anteroventral and anteromedial nuclei, in several of the midline nuclei, in the anterior intralaminar nuclei, and in the lateral and medial geniculate nuclei. Although histochemical reactivity was observed in the thalamus at birth, the intensity and the pattern of distribution of staining observed in adulthood was not achieved until the end of the third postnatal week. The NADPH-d histochemical positivity followed discrete developmental schedules in various thalamic domains, and different areas reached a mature pattern at different ages. In addition, populations of transiently stained neuronal cell bodies were observed in the medial thalamus during the first two postnatal weeks. These results show discrete patterns of expression of NOS in the adult and developing thalamus and suggest that nitric oxide may be involved in selected physiological and developmental roles in different thalamic domains.

Postnatal development of membrane properties and delta oscillations in thalamocortical neurons of the cat dorsal lateral geniculate nucleus.

The development of membrane properties, firing patterns, and delta oscillations in neurons of the cat dorsal lateral geniculate nucleus (dLGN) was investigated in vitro during the first 7 postnatal weeks. Compared with adult neurons, the resting membrane potential was more depolarized at postnatal days 1-9 (P1-P9), the input resistance was higher at P1-P7, and action potentials had a higher threshold and a smaller amplitude at P1-P3 and a longer duration at P1-P9. At P1-P3 trains longer than 200 msec were rarely observed, and trains with more than three action potentials were only present in 41% of the neurons, whereas at P1-P7 the normalized slope of the instantaneous frequencies at the first five interspike intervals was smaller than in the adult. A long-lasting (up to 6 sec) afterhyperpolarization followed a short train of action potentials in 88 and 30% of neurons at P1-P3 and P30-P32, respectively, but it was rarely observed in the adult. The low-threshold Ca2+ potential could evoke a burst of action potentials since P1. However, at P1-P7 the number of action potentials per burst was smaller (range, one to five), and at P1-P9 their maximum instantaneous frequency was lower (<190 Hz) than in the adult (range, six to eight, and 344 Hz, respectively). No delta oscillations were observed until P17, and their frequency (0.36 Hz) was lower than that in the adult (1.8 Hz). The percentage of neurons displaying delta oscillations and their frequency reached adult values by the end of the seventh postnatal week, i.e., well after the maturation of the membrane properties and firing patterns (second postnatal week). In conclusion, the maturation of the electrophysiological properties of thalamocortical neurons in the cat dLGN is completed later than the retinogeniculate axon segregation (Shatz CJ, 1983), and the immaturity of the oscillatory, and not of the burst-firing, activity is a limiting factor in the development of delta waves.

Retinal activity regulates developmental switches in functional properties and ifenprodil sensitivity of NMDA receptors in the lateral geniculate nucleus.

Previous studies have shown that marked changes occur in the kinetic properties of N-methyl-D-aspartate (NMDA) receptors during development of the visual pathways. In the lateral geniculate nucleus (LGN) of the ferret, excitatory postsynaptic currents (EPSCs) induced by activation of NMDA receptors display a very slow decay time during the first postnatal month, then become shorter in duration following eye-opening (around postnatal day 32; P32). In view of the critical role that NMDA receptors play in activity-dependent refinement of visual connections during development, we have examined the mechanisms that underlie these changes and how they are regulated. To examine the role of retinal activity, whole-cell recordings were conducted in the LGN slice preparation obtained from normal ferrets and ferrets treated with continuous intraocular application of tetrodotoxin (TTX) from P25 until the time of recording. Blockade of ganglion cell activity with TTX prevented the changes in decay rate of the postsynaptic current induced by NMDA receptors. Treated animals older than P40 had NMDA-EPSCs markedly longer in duration than normal animals at a similar age, resembling responses present in normal newborn animals. To examine whether changes in subunit composition of the NMDA receptor may contribute to the maturation of its kinetic properties, we used the antagonist ifenprodil, which produces selective inhibition of heteromeric NMDA receptors containing the NR-2B subunit. Ifenprodil induced profound inhibition of NMDA receptor activity in normal young animals and TTX-treated mature animals, but substantially less inhibition in normal mature animals. These findings indicate that retinal activity is required for the developmental switch from a juvenile form of the NMDA receptor to a more mature form, possibly affecting NR2 subunit expression.

Transient expression of the serotonin transporter in the developing mouse thalamocortical system.

The serotonin transporter was visualized in sections through the developing mouse thalamus by autoradiography of [3H]citalopram binding. In late gestation, a high density of transporter expression appeared in the ventrobasal thalamic complex and medial geniculate body. During the first postnatal week, binding in these areas decreased to low levels. A similar pattern of transient [3H]citalopram binding was observed in the somatosensory cortex, although the rise and decline of labeling occurred some days later. The density of the serotonergic innervation in the ventrobasal thalamic complex is known to be very low during the entire developmental period. Therefore, these data suggest that the serotonin transporter may be expressed transiently by thalamic neurons projecting to the cerebral cortex (as a "heterocarrier") which are capable of taking up serotonin in the somatosensory cortex. We propose that serotonin may act temporarily as a "false" transmitter in thalamocortical axons.

Specialized neuronal and glial contributions to development of the hamster lateral geniculate complex and circadian visual system.

The intergeniculate leaflet (IGL) is an integral part of the adult circadian visual system. It is characterized by the presence of retinal afferents and peptidergic cells projecting via a geniculohypothalamic tract (GHT) to the suprachiasmatic nucleus (SCN), site of the mammalian circadian clock. The adult IGL also contains abundant reactive astrocytes immunoreactive to GFAP. Because glia have a large role in brain development, we examined the ontogeny of the hamster IGL with respect to both glial and neuronal markers. Neuropeptide Y-immunoreactive (NPY-IR) cells destined for the IGL appear on embryonic day 11 (E11) in a matrix of vimentin (VIM)-IR radial glia. Migratory ellipsoid NPY-IR cells with long leading and trailing processes become oriented between the reticular thalamic neuroepithelial lobule, and the developing IGL. Most NPY-IR cells arrive in the IGL by E14 and extend axons ventrally into the GHT. These penetrate the SCN at P3 and arborize to an adult-like stage by P10. A specialized GFAP-IR radial glial path coinciding with the migratory route of NPY-IR cells appears by E14. As early as E15, cells contributing to this path are found displaced away from the ventricle. As the glial path disappears from the maturing brain, the entire length of the IGL becomes filled with GFAP-IR astrocytes. These features are consistent with translocation and transformation of a specialized set of radial glia into IGL astrocytes. The results demonstrate that the IGL is a large, developmentally important, feature of the lateral geniculate complex that is embryologically distinct from adjacent dorsal and ventrolateral geniculate nuclei.

Ultrastructural evidence for synaptic interactions between thalamocortical axons and subplate neurons.

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.