Activity-dependent remodeling of connections in the mammalian visual system.

The development of precise connections in the mammalian central nervous system requires neural activity. Synchronous patterns of afferent activity, and coincident afferent and target activity are required for specifying the neuronal connectivity that characterizes the adult visual pathway in mammals. During development, postsynaptic target neurons communicate with presynaptic afferents. Recent evidence suggests that the mechanisms that underlie activity-dependent development of connections in the visual system may share significant similarities with the mechanisms responsible for synaptic plasticity in the adult brain.

Lack of topography in the spinal cord projection to the rabbit soleus muscle.

Several mammalian muscles in the limb and trunk receive topographically organized innervation from spinal cord motor neurons. Some muscles in which topographic innervation has been demonstrated have a sheet-like architecture; others are compartmentalized and/or have more than one origin. An interesting question is whether topography is related to these anatomical features, or whether it occurs as a general consequence of the development of innervation. To address this question, we examined the pattern of projections to the soleus muscle, which lacks these anatomical features. Intracellular recordings of endplate potentials in early and intermediate age rabbits were used to assess the spinal origin of inputs to two distinct regions of the muscle. Both regions were innervated by both rostral and caudal portions of the motor pool. These experiments also showed that individual muscle fibers frequently receive separate inputs arising from widely separated regions of the motor pool. In another set of experiments, physiological measurements of tension overlap in young, polyinnervated muscles showed that the relative positions of motor neurons in the spinal cord do not correlate with the extent to which motor units share muscle fibers. In a third set of experiments, motor neurons were retrogradely labeled following local injections of tracer into muscle. Small and large local injections resulted in comparably dispersed labeling of motor neurons within the motor pool. Moreover, the rostrocaudal position of labeled neurons was not correlated with the position of the injection site within the muscle. Together, these results provide evidence that the soleus muscle is not topographically innervated. Furthermore, an examination of several age groups suggests that the innervation pattern in this muscle is not altered by postnatal synapse elimination.

Transient expression of NADPH-diaphorase in the lateral geniculate nucleus of the ferret during early postnatal development.

Retinogeniculate projections in the ferret are refined during postnatal development so that inputs from the two eyes become segregated into eye-specific laminae, and each eye-specific lamina is further divided into sublaminae containing inputs from on-center or off-center afferents. Segregation into eye-specific laminae and on/off sublaminae is dependent on neuronal activity; sublamination depends on activation of N-methyl-d-aspartate (NMDA) receptors. By analogy with the suggested role of nitric oxide in NMDA-mediated long-term potentiation in the hippocampus, we investigated a possible role for nitric oxide in ferret retinogeniculate development. The expression of NADPH-diaphorase, a nitric oxide synthase, was examined histologically in the lateral geniculate nucleus of ferrets at several postnatal ages. At birth, neuropil is labeled in the nucleus, although no cell bodies are visible. After the first postnatal week, some labeled cells appear, predominantly in the C laminae. By three postnatal weeks, cell bodies are clearly labeled in all geniculate laminae. Staining reaches a peak in density at about four postnatal weeks, then declines such that by six postnatal weeks labeled cells are no longer visible. This transient expression of NADPH-diaphorase activity is consistent with a role for nitric oxide in the development of mature connections within the ferret lateral geniculate nucleus.

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.

Developmental regulation of EphA4 expression in the chick auditory brainstem.

The avian auditory brainstem nuclei nucleus magnocellularis (NM) and nucleus laminaris (NL) display highly precise patterns of neuronal connectivity. NM projects tonotopically to the dorsal dendrites of ipsilateral NL neurons and to the ventral dendrites of contralateral NL neurons. The precision of this binaural segregation is evident at the earliest developmental stage at which connections can be observed. We have begun to examine the possibility that Eph receptor tyrosine kinase signaling is involved in establishing these spatially segregated connections. The expression of the EphA4 tyrosine kinase was examined at several developmental stages. EphA4 is expressed in rhombomere 5, which contains progenitors for both NM and NL. In this rhombomere, the labeling becomes striped during the time that precursor cells migrate to the auditory anlage. At the precise time when NM-NL projections are forming, EphA4 expression in NL is asymmetric, with markedly higher expression in the dorsal NL neuropil than in the ventral neuropil, suggesting a possible role in guiding growing axons to the appropriate region. At later embryonic ages EphA4 expression is symmetric around NL, and is absent in NM. As auditory function matures, EphA4 expression decreases so that by 4 days after hatch no EphA4 antibody labeling is evident in the auditory brainstem nuclei.

Nitric oxide as a signaling molecule in visual system development.

The lateral geniculate nucleus (LGN) of the ferret is characterized by the readily discernible anatomical patterning of afferent terminations from the retina into both eye-specific layers and On/Off sublaminae. The eye-specific layers form during the first post-natal week, and On/Off sublaminae become apparent during the third to fourth post-natal weeks. The post-natal appearance of these patterns thus provides an advantageous model for the study of the mechanisms of activity-dependent development. The second phase of pattern formation, the appearance of On/Off sublaminae, involves the elaboration of appropriately placed axonal terminals and the restriction (or retraction) of inappropriately placed terminals. Previous work has demonstrated that this process is dependent on the activation of NMDA-receptors. Other studies have provided strong evidence that nitric oxide, a diffusible gas which is produced downstream of NMDA-receptor activation, acts as a retrograde messenger molecule to induce changes in pre-synaptic structures. In this article we review the evidence that nitric oxide plays a role in activity-dependent synaptic plasticity in the developing retinogeniculate pathway. The role of nitric oxide in other aspects of visual system development is also discussed.

The neuronal form of nitric oxide synthase is required for pattern formation by retinal afferents in the ferret lateral geniculate nucleus.

The ferret retinogeniculate projection undergoes activity-dependent refinement of connections that become restricted to eye specific laminae and On/Off sublaminae in the lateral geniculate nucleus (LGN). We have previously shown that the developmental process by which On/Off sublaminae form requires N-methyl-D-aspartate (NMDA) receptors and nitric oxide (NO). In this study, we investigate the role of the neuronal form of NO synthase (nNOS) in sublaminar refinement. This isoform of NOS may be coupled with NMDA receptors at postsynaptic sites. We found that nNOS is present in the developing LGN, and that blocking nNOS during development disrupts the formation of On/Off sublaminae. Endothelial NOS (eNOS) is not expressed in the LGN until after sublaminae have formed. These results suggest that the nNOS isoform is the predominant contributor of NO during development, and support the hypothesis that NO acts downstream of NMDA receptor activation to mediate activity-dependent changes in the patterning of connections in the LGN.

Blockade of afferent impulse activity disrupts on/off sublamination in the ferret lateral geniculate nucleus.

The ferret retinogeniculate projection is organized into on/off sublaminae within each eye-specific layer. The role of afferent activity in the formation of these sublaminae was examined. We report that when the activity of retinal ganglion cells is blocked with tetrodotoxin during postnatal weeks three and four, on/off sublamination is disrupted, supporting a role for afferent activity in the formation of this pattern.

Axon guidance in the auditory system: multiple functions of Eph receptors.

The neural pathways of the auditory system underlie our ability to detect sounds and to transform amplitude and frequency information into rich and meaningful perception. While it shares some organizational features with other sensory systems, the auditory system has some unique functions that impose special demands on precision in circuit assembly. In particular, the cochlear epithelium creates a frequency map rather than a space map, and specialized pathways extract information on interaural time and intensity differences to permit sound source localization. The assembly of auditory circuitry requires the coordinated function of multiple molecular cues. Eph receptors and their ephrin ligands constitute a large family of axon guidance molecules with developmentally regulated expression throughout the auditory system. Functional studies of Eph/ephrin signaling have revealed important roles at multiple levels of the auditory pathway, from the cochlea to the auditory cortex. These proteins provide graded cues used in establishing tonotopically ordered connections between auditory areas, as well as discrete cues that enable axons to form connections with appropriate postsynaptic partners within a target area. Throughout the auditory system, Eph proteins help to establish patterning in neural pathways during early development. This early targeting, which is further refined with neuronal activity, establishes the precision needed for auditory perception.

Distribution of glial cells in the auditory brainstem: normal development and effects of unilateral lesion.

Auditory brainstem networks facilitate sound source localization through binaural integration. A key component of this circuitry is the projection from the ventral cochlear nucleus (VCN) to the medial nucleus of the trapezoid body (MNTB), a relay nucleus that provides inhibition to the superior olivary complex. This strictly contralateral projection terminates in the large calyx of Held synapse. The formation of this pathway requires spatiotemporal coordination of cues that promote cell maturation, axon growth, and synaptogenesis. Here we have examined the emergence of distinct classes of glial cells, which are known to function in development and in response to injury. Immunofluorescence for several astrocyte markers revealed unique expression patterns. Aldehyde dehydrogenase 1 family member L1 (ALDH1L1) was expressed earliest in both nuclei, followed by S100ß, during the first postnatal week. Glial fibrillary acidic protein (GFAP) expression was seen in the second postnatal week. GFAP-positive cell bodies remained outside the boundaries of VCN and MNTB, with a limited number of labeled fibers penetrating into the margins of the nuclei. Oligodendrocyte transcription factor 2 (OLIG2) expression revealed the presence of oligodendrocytes in VCN and MNTB from birth until after hearing onset. In addition, ionized calcium binding adaptor molecule 1 (IBA1)-positive microglia were observed after the first postnatal week. Following hearing onset, all glial populations were found in MNTB. We then determined the distribution of glial cells following early (P2) unilateral cochlear removal, which results in formation of ectopic projections from the intact VCN to ipsilateral MNTB. We found that following perturbation, astrocytic markers showed expression near the ectopic ipsilateral calyx. Taken together, the developmental expression patterns are consistent with a role for glial cells in the maturation of the calyx of Held and suggest that these cells may have a similar role in maturation of lesion-induced connections.

Embryonic origins of auditory brain-stem nuclei in the chick hindbrain.

The auditory nuclei of the chick brain stem have distinct morphologies and highly specific synaptic connectivity. Nucleus magnocellularis (NM) and nucleus angularis receive tonotopically ordered cochlear input. NM in turn projects tonotopically to nucleus laminaris (NL), maintaining binaural specificity with projections to either dorsal or ventral NL dendrites. NM and NL arise from a common anlage, which differentiates as the cells migrate and acquire their mature morphologies. NM and NL cells are closely associated during embryogenesis and synapse formation. However, the morphologies of the nuclei and of the cells within the nuclei differ greatly between NM and NL. While later maturation of these nuclei has been described in considerable detail, relatively little is known about the early embryonic events that lead to the formation of these nuclei. We examined the embryonic origins of cells in brain-stem auditory nuclei with particular emphasis on NM and NL. Lipophilic dyes were injected into small regions of the embryonic hindbrain prior to the birth and migration of cells that contribute to these nuclei. We found that NM arises from rhombomeres r5, r6, and r7, and NL arises mostly from r5 with a few cells arising from r6. NM and NL thus have partially overlapping rhombomeres of origin. However, we found that the precursors for NM and NL are found in distinct regions within rhombomere 5, with NM precursors in medial regions and NL precursors in lateral regions. Our results do not support a lineage relationship between NM and NL cells and they suggest that NM and NL are specified prior to migration of precursors to the auditory anlage.

Maturation of fast and slow motor units during synapse elimination in the rabbit soleus muscle.

In adult mammalian skeletal muscles, fast and slow muscle fibers are selectively innervated by single inputs from corresponding motor neuron types, giving rise to fast and slow motor units. At birth, however, muscle fibers are polyinnervated, and connections between motor neurons and muscle fibers are not as specific as those found in adults. Excess synapses are removed during the first few postnatal weeks. In addition to changes in the degree of polyinnervation, motor unit types undergo maturation in their contractile properties. In this study, we have investigated the maturation of motor unit types during postnatal synapse elimination in the rabbit soleus muscle. The ratio of twitch tension to tetanic tension in a motor unit is an indication of its contractile type. Our results indicate that during synapse elimination, the twitch/tetanus ratios for fast motor units increase while those for slow motor units decrease. The ratio of motor unit tension at polyinnervated ages to that at singly innervated ages has previously been used to estimate the degree of polyinnervation for fast versus slow muscle fibers. We found that twitch and tetanic tension yield conflicting estimates of polyinnervation. This discrepancy was resolved on the basis of intracellular recordings of endplate potentials. Using latencies to endplate potentials as an indicator of muscle fiber type, we found that fast and slow muscle fibers are polyinnervated to a similar extent during both early and intermediate stages of synapse elimination, suggesting that specific tension, and not polyinnervation, changes differently in fast versus slow muscle fibers. These changes are consistent with those we found in twitch/tetanus ratios. Furthermore, these intracellular recordings suggest a high degree of specificity at birth, which is further refined during synapse elimination.

Experimentally induced retinal projections to the ferret auditory thalamus: development of clustered eye-specific patterns in a novel target.

We have examined the relative role of afferents and targets in pattern formation using a novel preparation, in which retinal projections in ferrets are induced to innervate the medial geniculate nucleus (MGN). We find that retinal projections to the MGN are arranged in scattered clusters. Clusters arising from the ipsilateral eye are frequently adjacent to, but spatially segregated from, clusters arising from the contralateral eye. Both clustering and eye-specific segregation in the MGN arise as a refinement of initially diffuse and overlapped projections. The shape, size, and orientation of retinal terminal clusters in the MGN closely match those of relay cell dendrites arrayed within fibrodendritic laminae in the MGN. We conclude that specific aspects of a projection system are regulated by afferents and others by targets. Clustering of retinal projections within the MGN and eye-specific segregation involve progressive remodeling of retinal axon arbors, over a time period that closely parallels pattern formation by retinal afferents within their normal target, the lateral geniculate nucleus (LGN). Thus, afferent-driven mechanisms are implicated in these events. However, the termination zones are aligned within the normal cellular organization of the MGN, which does not differentiate into eye-specific cell layers similar to the LGN. Thus, target-driven mechanisms are implicated in lamina formation and cellular differentiation.

A role for nitric oxide in the development of the ferret retinogeniculate projection.

The ferret retinogeniculate projection segregates into eye-specific layers during the first postnatal week and into ON/OFF sublaminae, which receive inputs from either on-center or off-center retinal ganglion cells, during the third and fourth postnatal weeks. The restriction of retinogeniculate axon arbors into eye-specific layers appears to depend on action potential activity () but does not require activation of NMDA receptors (). The formation of ON/OFF sublaminae is also activity-dependent and is disrupted by in vivo blockade of NMDA receptors (). To investigate a possible mechanism whereby blockade of postsynaptic NMDA receptors in the lateral geniculate nucleus (LGN) results in changes in the size and position of presynaptic axon arbors, we tested the role of the diffusible messenger nitric oxide (NO) in the development of the retinogeniculate pathway. We found previously that NO synthase (NOS) is transiently expressed in LGN cells during the refinement of retinogeniculate projections (). In this study, treatment with NG-nitro-L-arginine (L-NoArg), an arginine analog that inhibits NOS, during the third and fourth postnatal weeks resulted in an overall pattern of sublamination that was significantly reduced compared with normal and control animals. Single retinogeniculate axon arbors were located in the middle of eye-specific layers rather than toward the inner or outer half as in normal or control animals. The effect of NOS inhibition was not a consequence of the hypertensive effect of L-NoArg. In contrast to the effect of L-NoArg on the formation of ON/OFF sublaminae, treatment with L-NoArg during the first postnatal week did not disrupt the formation of eye-specific layers. Biochemical assays indicated significant inhibition of NOS during both treatment periods. These data suggest that NO acts together with NMDA receptors in activity-dependent refinement of connections during a specific phase of retinogeniculate development.