ERK signaling is required for eye-specific retino-geniculate segregation

In the mammalian visual system, retinal ganglion cell (RGC) projections from each eye, initially intermixed within the dorsal-lateral geniculate nucleus (dLGN), become segregated during the early stages of development, occupying distinct eye-specific layers. Electrical activity has been suggested to play a role in this process; however, the cellular mechanisms underlying eye-specific segregation are not yet defined. It is known that electrical activity is among the strongest activators of the extracellular signal-regulated kinase (ERK) pathway. Moreover, the ERK pathway is involved in the plasticity of neural connections during development. We examine the role of ERK in the segregation of retinal afferents into eye-specific layers in the dLGN. The activation of this signaling cascade was selectively blocked along the retino-thalamic circuitry by specific inhibitors, and the distribution of RGC fibers in the dLGN was studied. Our results demonstrate that the blockade of ERK signaling prevents eye-specific segregation in the dLGN, providing evidence that ERK pathway is required for the proper development of retino-geniculate connections. Of particular interest is the finding that ERK mediates this process both at the retinal and geniculate level.     

Failure to maintain eye-specific segregation in nob, a mutant with abnormally patterned retinal activity

Axon terminals from the two eyes initially overlap in the dorsal-lateral geniculate nucleus (dLGN) but subsequently refine to occupy nonoverlapping territories. Retinal activity is required to establish and maintain this segregation. We show that despite the presence of retinal activity, segregated projections desegregate when the structure of activity is altered. Early in development, spontaneous retinal activity in the no b-wave (nob) mouse is indistinguishable from that of wild-type mice, and eye-specific segregation proceeds normally. But, around eye-opening, spontaneous and visually evoked activity in nob retinas become abnormal, coincident with a failure to preserve precise eye-specific territories. Dark-rearing studies suggest that altered visual experience is not responsible. Transgenic rescue of the mutated protein (nyctalopin) within nob retinal interneurons, without rescuing expression in either retinal projection neurons or their postsynaptic targets in the dLGN, restores spontaneous retinal activity patterns and prevents desegregation. Thus, normally structured spontaneous retinal activity stabilizes newly refined retinogeniculate circuitry.     

Epibatidine Blocks Eye-Specific Segregation in Ferret Dorsal Lateral Geniculate Nucleus during Stage III Retinal Waves

The segregation and maintenance of eye-specific inputs in the dorsal lateral geniculate nucleus (dLGN) during early postnatal development requires the patterned spontaneous activity of retinal waves. In contrast to the development of the mouse, ferret eye-specific segregation is not complete at the start of stage III glutamatergic retinal waves, and the remaining overlap is limited to the C/C1 lamina of the dLGN. To investigate the role of patterned spontaneous activity in this late segregation, we disrupted retinal waves pharmacologically for 5 day windows from postnatal day (P) 10 to P25. Multi-electrode array recordings of the retina in vitro reveal that the cholinergic agonist epibatidine disrupts correlated retinal activity during stage III waves. Epibatidine also prevents the segregation of eye-specific inputs in vivo during that period. Our results reveal a novel role for cholinergic influence on stage III retinal waves as an instructive signal for the continued segregation of eye-specific inputs in the ferret dLGN.     

Role of calcineurin in activity-dependent pattern formation in the dorsal lateral geniculate nucleus of the ferret

In the retinogeniculate pathway of the ferret, in addition to the separation of the inputs from the two eyes to form eye-specific layers, there is also an anatomical segregation of the terminal arbors of on-center retinal ganglion cells from the terminal arbors of off-center retinal ganglion cell axons to form on/off sublaminae. Sublamination normally occurs during postnatal weeks 3-4 and requires the activity of retinal afferents, N-methyl-D-aspartate receptors, nitric oxide synthase, and a target of nitric oxide, cyclic guanosine monophosphate. Calcineurin is a calcium/calmodulin dependent serine, threonine protein phosphatase suggested to mediate NMDA-receptor dependent synaptic plasticity in the hippocampus. We have examined whether calcineurin plays a role during on/off sublamination in the dorsal lateral geniculate nucleus (dLGN) of the ferret. Immunohistochemistry showed that calcineurin expression is transiently up-regulated in dLGN cells and neuropil during the period of on/off sublamination. A functional role for calcineurin during sublamination was investigated by blocking the enzyme locally via intracranial infusion of FK506. Treatment with FK506 during postnatal weeks 3-4 disrupted the appearance of sublaminae. These results suggest that calcineurin may play a role during this process of activity-dependent pattern formation in the visual pathway.     

Cannabinoid Receptor CB2 Modulates Axon Guidance

Navigation of retinal projections towards their targets is regulated by guidance molecules and growth cone transduction mechanisms. Here, we present in vitro and in vivo evidences that the cannabinoid receptor 2 (CB₂R) is expressed along the retino-thalamic pathway and exerts a modulatory action on axon guidance. These effects are specific to CB₂R since no changes were observed in mice where the gene coding for this receptor was altered (cnr2 ^−/−). The CB₂R induced morphological changes observed at the growth cone are PKA dependent and require the presence of the netrin-1 receptor, Deleted in Colorectal Cancer. Interfering with endogenous CB₂R signalling using pharmacological agents increased retinal axon length and induced aberrant projections. Additionally, cnr2 ^−/− mice showed abnormal eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus (dLGN) indicating CB₂R’s implication in retinothalamic development. Overall, this study demonstrates that the contribution of endocannabinoids to brain development is not solely mediated by CB₁R, but also involves CB₂R.     

The role of activity in development of the visual system

Neuronal activity is important for both the initial formation and the subsequent refinement of anatomical and physiological features of the mammalian visual system. Here we examine recent evidence concerning the role that spontaneous activity plays in axonal segregation, both of retinogeniculate afferents into eye-specific layers and of geniculocortical afferents into ocular dominance bands. We also assess the role of activity in the generation and plasticity of orientation selectivity in the primary visual cortex. Finally, we review recent challenges to textbook views on how inputs representing the two eyes interact during the critical period of visual cortical plasticity.     

Developmental remodeling of the retinogeniculate synapse

Anatomical rearrangement of retinogeniculate connections contributes to the refinement of synaptic circuits in the developing visual system, but the underlying changes in synaptic function are unclear. Here, we study such changes in mouse brain slices. Each geniculate cell receives a surprisingly large number of retinal inputs (>20) well after eye-specific zones are formed. All but one to three of these inputs are eliminated over a 3-week period spanning eye opening. Remaining inputs are strengthened approximately 50-fold, in part through an increase in quantal size, but primarily through an increase in the number of release sites. Changes in release probability do not contribute significantly. Thus, a redistribution of release sites from many inputs to few inputs at this late developmental stage contributes to the precise receptive fields of thalamic relay neurons.     

Mechanisms of eye-specific visual circuit development

Eye-specific visual connections are a prominent model system for exploring how precise circuits develop in the CNS and, in particular, for addressing the role of neural activity in synapse elimination and axon refinement. Recent experiments have identified the features of spontaneous retinal activity that mediate eye-specific retinogeniculate segregation, the synaptic events associated with this process, and the importance of axon guidance cues for organizing the overall layout of eye-specific maps. The classic model of ocular dominance column development, in which spontaneous retinal activity plays a crucial role, has also gained new support. Although many outstanding questions remain, the mechanisms that instruct eye-specific circuit development are becoming clear.     

Molecular guidance cues in the development of visual pathway

70%–80% of our sensory input comes from vision. Light hit the retina at the back of our eyes and the visual information is relayed into the dorsal lateral geniculate nuclei (dLGN) and primary visual cortex (V1) thereafter, constituting the image-forming visual circuit. Molecular cues are one of the key factors to guide the wiring and refinement of the image-forming visual circuit during pre- and post-embryonic stages. Distinct molecular cues are involved in different developmental stages and nucleus, suggesting diverse guidance mechanisms. In this review, we summarize molecular guidance cues throughout the image-forming visual circuit, including chiasm determination, eye-specific segregation and refinement in the dLGN, and at last the reciprocal connections between the dLGN and V1.     

The role of retinal waves and synaptic normalization in retinogeniculate development

The prenatal development of the cat retinogeniculate pathway is thought to involve activity-dependent mechanisms driven by spontaneous waves of retinal activity. The role of these waves upon the segregation of the dorsal lateral geniculate nucleus (LGN) into two eye-specific layers and the development of retinotopic mappings have previously been investigated in a computer model. Using this model, we examine three aspects of retinogeniculate development. First, the mapping of visual space across the whole network into projection columns is shown to be similar to the mapping found in the cat. Second, the simplicity of the model allows us to explore how different forms of synaptic normalization affect development. In comparison to most previous models of ocular dominance, we find that subtractive postsynaptic normalization is redundant and divisive presynaptic normalization is sufficient for normal development. Third, the model predicts that the more often one eye generates waves relative to the other eye, the more LGN units will monocularly respond to the more active eye. In the limit when one eye does not generate any waves, that eye totally disconnects from the LGN allowing the non-deprived eye to innervate all of the LGN. Thus, as well as accounting for normal retinogeniculate development, the model also predicts development under abnormal conditions which can be experimentally tested.     

Competitive interactions between retinal ganglion cells during prenatal development

In the mammalian visual system, retinal ganglion cell axons terminate within the LGN in a series of alternating eye-specific layers. These layers are not present initially during development. In the cat they emerge secondarily following a prenatal period in which originally intermixed inputs from the two eyes gradually segregate from each other to give rise to the characteristic set of layers by birth. Many lines of evidence suggest that activity-dependent competitive interactions between ganglion cell axons from the two eyes for LGN neurons play an important role in the final patterning of retinogeniculate connections. Studies of the branching patterns of individual ganglion cell axons suggest that during the period when inputs from the two eyes are intermixed, axons from one eye send side branches into territory later occupied exclusively by axons from the other eye. Ultrastructural studies indicate that these branches in fact are sites of synaptic contacts, which are later eliminated since the side branches disappear as axons form their mature terminal arbors in appropriate territory. In vitro microelectrode recordings from LGN neurons indicate that they can receive convergent synaptic excitation from electrical stimulation of the optic nerves before but not after the eye-specific layers form, suggesting that at least some of the synaptic contacts seen at the ultrastructural level are functonal. Finally, experiments in which tetrodotoxin was infused intracranially during the two week period during which the eye-specific layers normally form demonstrate that it is possible to prevent, or at least delay, the formation of the layers. Accordingly, individual axons fail to develop their restricted terminal arbor branching pattern and instead branch widely throughout the LGN. These results indicate that all of the machinery necessary for synaptic function and competition is present during fetal life. Moreover, it is highly likely that neuronal activity is required for the formation of the eye-specific layers. If so, then activity would have to be present in the form of spontaneously generated action potentials, since vision is not possible at these early ages. Thus, the functioning of the retinogeniculate system many weeks before it is put to the use for which it is ultimately designed may contribute to the final patterning of connections present in the adult.     

Eye-specific segregation of optic afferents in mammals,fish, and frogs: The role of activity

Eye-specific patches or stripes normally develop in the visual cortex and superior colliculus of many (but not all) mammals and are also formed, after surgically produced binocular innervation, in the optic tectum of fish and frogs. The segregation of ocular dominance patches or columns has been studied using a variety of anatomical pathway-tracing techniques, by electrophysiological recording of postsynaptic units or field potentials, and by the 2-deoxyglucose method following visual stimulation of only one eye. In the tectum of both fish and frogs and in the cortex and colliculus of mammals, eye-specific patches develop from initially diffuse, overlapping projections. Of the various mechanisms that might cause such segregation, the evidence favors an activity-dependent process that stabilizes synapses from the same eye because of their correlated activity. First, several environmental manipulations affect the segregation of afferents in visual cortex: strabismus and alternate monocular exposure apparently enhance segregation, whereas dark rearing slows the segregation process, and monocular deprivation causes the experienced eye to form larger patches at the expense of those of the deprived eye. Second, blocking activity in both eyes is effective in preventing the segregation both in the tectum of fish and frog and in the visual cortex of cat. With the eyes blocked, alternate stimulation of the optic nerves permits the segregation of ocular dominance, at least onto single cells in the cat visual cortex. These findings are discussed in terms of an activity-dependent stabilization of those synapses having correlated activity (those from neighboring ganglion cells within one eye) but not of those lacking correlated activity (those from left and right eyes). We suggest that the eye-specific patches represent a compromise between total segregation of the projections from the two eyes and the formation of a single continuous retinotopic map across the surface of the cortex or tectum.     

Abnormal functional organization in the dorsal lateral geniculate nucleus of mice lacking the beta 2 subunit of the nicotinic acetylcholine receptor

Spontaneous activity patterns in the developing retina appear important for the functional organization of the visual system. We show here that an absence of early retinal waves in mice lacking the beta2 subunit of the nicotinic acetylcholine receptor (nAChR) is associated with both gain and loss of functional organization in the dorsal lateral geniculate nucleus (dLGN). Anatomical studies show normal gross retinotopy in the beta2(-/-) dLGN but suggest reduced topographic precision in the retinogeniculate projection. Physiological recordings reveal normal topography in the dorsoventral visual axis but a lack of fine-scale mapping in the nasotemporal visual plane. In contrast, unlike wild-type mice, on- and off-center cells in the beta2(-/-) dLGN are spatially segregated. The presence of the beta2 subunit of the nAChR in the CNS is therefore important for normal functional organization in the retinogeniculate projection.     

Role of calcineurin in activity‐dependent pattern formation in the dorsal lateral geniculate nucleus of the ferret

In the retinogeniculate pathway of the ferret, in addition to the separation of the inputs from the two eyes to form eye‐specific layers, there is also an anatomical segregation of the terminal arbors of on‐center retinal ganglion cells from the terminal arbors of off‐center retinal ganglion cell axons to form on/off sublaminae. Sublamination normally occurs during postnatal weeks 3–4 and requires the activity of retinal afferents, N‐methyl‐D‐aspartate receptors, nitric oxide synthase, and a target of nitric oxide, cyclic guanosine monophosphate. Calcineurin is a calcium/calmodulin dependent serine, threonine protein phosphatase suggested to mediate NMDA‐receptor dependent synaptic plasticity in the hippocampus. We have examined whether calcineurin plays a role during on/off sublamination in the dorsal lateral geniculate nucleus (dLGN) of the ferret. Immunohistochemistry showed that calcineurin expression is transiently up‐regulated in dLGN cells and neuropil during the period of on/off sublamination. A functional role for calcineurin during sublamination was investigated by blocking the enzyme locally via intracranial infusion of FK506. Treatment with FK506 during postnatal weeks 3–4 disrupted the appearance of sublaminae. These results suggest that calcineurin may play a role during this process of activity‐dependent pattern formation in the visual pathway. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 153–162, 2003     

Diffuse and specific tectopulvinar terminals in the tree shrew: synapses, synapsins, and synaptic potentials

The pulvinar nucleus of the tree shrew receives both topographic (specific) and nontopographic (diffuse) projections from superior colliculus (SC), which form distinct synaptic arrangements. We characterized the physiological properties of these synapses and describe two distinct types of excitatory postsynaptic potentials (EPSPs) that correlate with structural properties of the specific and diffuse terminals. Synapses formed by specific terminals were found to be significantly longer than those formed by diffuse terminals. Stimulation of these two terminal types elicited two types of EPSPs that differed in their latency and threshold amplitudes. In addition, in response to repetitive stimulation (0.5-20 Hz) one type of EPSP displayed frequency-dependent depression whereas the amplitudes of the second type of EPSP were not changed by repetitive stimulation of up to 20 Hz. To relate these features to vesicle release, we compared the synapsin content of terminals in the pulvinar nucleus and the dorsal lateral geniculate (dLGN) by combining immunohistochemical staining for synapsin I or II with staining for the type 1 or type 2 vesicular glutamate transporters (markers for corticothalamic and tectothalamic/retinogeniculate terminals, respectively). We found that retinogeniculate terminals do not contain either synapsin I or synapsin II, corticothalamic terminals in the dLGN and pulvinar contain synapsin I, but not synapsin II, whereas tectopulvinar terminals contain both synapsin I and synapsin II. Finally, both types of EPSPs showed a graded increase in amplitude with increasing stimulation intensity, suggesting convergence; this was confirmed using a combination of anterograde tract tracing and immunocytochemistry. We suggest that the convergent synaptic arrangements, as well as the unique synapsin content of tectopulvinar terminals, allow them to relay a dynamic range of visual signals from the SC.     

The development of intrinsic excitability in mouse retinal ganglion cells

Activity-dependent refinement of synaptic connections occurs throughout the developing nervous system, including the visual system. Retinal ganglion cells (RGCs) overproduce synapses then refine them in an activity-dependent manner that segregates RGC connections into multicellular patterns, such as eye-specific regions and retinotopic maps. Ferrets additionally segregate ON and OFF retinogeniculate pathways in an activity-dependent manner. It was unknown whether differences in ON versus OFF intrinsic and spontaneous activity occur in postnatal mouse. The work reported here measured the intrinsic properties and spontaneous activity of morphologically identified postnatal mouse RGCs, and tested the hypothesis that mouse ON and OFF RGCs develop differences in spontaneous activity. We found developmental changes in resting potential, action potential threshold, depolarization to threshold, action potential width, action potential patterns, and maximal firing rates. These results are consistent with the maturation of the intrinsic properties of RGCs extending through the first three postnatal weeks. However, there were no differences among mouse ON, OFF, and multistratified RGCs in intrinsic excitability, spontaneous synaptic drive or spontaneous action potential patterns. The absence of differences between ON and OFF activity patterns is unlike the differences that arise in ferrets. In contrast to the ferret, the ON and OFF target neurons in the mouse are organized in a random pattern, not layers. This supports the hypothesis that the absence of systematic differences in activity results in the nonlayered distribution of retinogeniculate connections.     

An instructive role for patterned spontaneous retinal activity in mouse visual map development

Complex neural circuits in the mammalian brain develop through a combination of genetic instruction and activity-dependent refinement. The relative role of these factors and the form of neuronal activity responsible for circuit development is a matter of significant debate. In the mammalian visual system, retinal ganglion cell projections to the brain are mapped with respect to retinotopic location and eye of origin. We manipulated the pattern of spontaneous retinal waves present during development without changing overall activity levels through the transgenic expression of β2-nicotinic acetylcholine receptors in retinal ganglion cells of mice. We used this manipulation to demonstrate that spontaneous retinal activity is not just permissive, but instructive in the emergence of eye-specific segregation and retinotopic refinement in the mouse visual system. This suggests that specific patterns of spontaneous activity throughout the developing brain are essential in the emergence of specific and distinct patterns of neuronal connectivity.     

Patterns of correlated spontaneous bursting activity in the developing mammalian retina

The adult visual system is highly organized in its patterns of connectivity. Connections between the retina and its central target, the dorsal lateral geniculate nucleus (dLGN), are remodeled during development as inappropriate synaptic inputs are eliminated by a process that requires retinal activity. Multineuronal recordings of the neonatal ferret retina reveal that during the refinement period, retinal ganglion cells spontaneously display rhythmic bursting activity in which the bursts of neighboring cells are correlated by propagating excitatory waves. These spontaneous retinal waves have temporal and spatial properties that appear instructive for the refinement of the early patterns of retinogeniculate connections prior to visual stimulation.     

The segregation of inner and outer cells in porcine embryos follows a different pattern compared to the segregation in mouse embryos

The mammalian blastocyst consists of an inner cell mass (ICM) enclosed by the trophectoderm. The origin of these two cell populations lies in the segregation of inner and outer cells in the early morula. In the present study, the segregation of inner and outer cells has been studied in porcine embryos and is compared with segregation in mouse embryos. For this, nuclei of inner and outer cells were differentially labelled with two fluorochromes after partial complement-mediated lysis of the outer cells. In porcine and mouse embryos compaction and the first appearance of inner cells occur at different stages of development. In porcine embryos compaction was observed as early as the 4-cell stage, while in mouse embryos compaction occurred in the 8-cell stage. The first inner cells segregated in porcine embryos which were in the transition from four to eight cells and inner cells were added during two subsequent cell cycles. In mouse embryos inner cells segregated predominantly during the fourth cleavage division. From the results obtained we conclude that the segregation of inner and outer cells follows a different pattern in mouse and in porcine embryos.     

Serotonin and norepinephrine uptake in discrete brain regions during the pregnant mare serum (PMS) induced estrous cycle in the rat

The role of hypothalamic and limbic aminergic systems in neuroendocrine control in the rat has been investigated by measuring the temporal changes in serotonergic and noradrenergic neuronal uptake in a model of the estrous cycle. Using the pregnant mare serum (PMS) model of induced ovulation and an in vitro uptake system, serotonergic activity reached peak values (p = 0.01) in the suprachiasmatic nuclear region (SNR) and median eminence (ME) (p = 0.007) during the critical period for luteinizing hormone release. 5, 7-dihydroxytryptamine lesions of the SNR or ME regions inhibited PMS induced ovulation suggesting that at least in these two areas a certain amplitude of 5-HT activity may be required at some time prior to the critical period for LH release. Serotonergic uptake during diestrus in the median eminence showed maximum values during the mid-light phase suggesting a role for this region in ovulation control mechanisms prior to the proestrous critical period. Significant changes (p = 0.001) in serotonergic uptake were found in mid to later light diestrus in the amygdala. This pattern was repeated during proestrus and estrus only in the amygdala suggesting a daily pattern of serotonergic uptake during the estrous cycle. Significant changes (p = 0.001) in noradrenergic uptake only occurred in the amygdala during proestrus.